THE ROLE OF ANTI-MUumlLLERIAN HORMONE

IN ASSISTED REPRODUCTION IN WOMEN

A thesis submitted to the University of Manchester

for the degree of MD in the Faculty of Medical and Human

Sciences

2014

OYBEK RUSTAMOV

SCHOOL OF MEDICINE

2

TABLE OF CONTENTS Abstracthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip3 Publications arising from the thesishelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip5 Chapter 1 General Introduction amp Literature reviewhelliphelliphelliphelliphelliphelliphelliphellip8 Chapter 2 Evaluation of the Gen II AMH Assay between-sample variability

and assay- method comparabilityhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip43 21 Anti-Muumlllerian hormone serum levels and reproducibility in a large cohort of subjects suggest sample instabilityhelliphelliphelliphellip44 22 AMH Gen II assay A validation study of observed variability between repeated AMH measurementshelliphelliphelliphellip65

Chapter 3 The measurement of anti-Muumlllerian hormone a critical appraisalhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip78

Chapter 4 Extraction preparation and collation of datasets for the

assessment of the role of the markers of ovarian reserve in female reproduction and IVF treatmenthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip106

Chapter 5 Assessment of determinants of anti-Muumlllerian hormone in infertile womenhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip135

51 The effect of ethnicity BMI endometriosis and the causes of infertility on ovarian reservehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip136 52 The effect of salpingectomy ovarian cystectomy and unilateral salpingoopherectomy on ovarian reservehelliphelliphelliphellip167

Chapter 6 Assessment of determinants of oocyte number using large

retrospective data on IVF cycles and explorative study of the potential for optimization of AMH-tailored stratification of controlled ovarian hyperstimulationhellip187

Chapter 7 General Summaryhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip229 Authors and affiliationshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip245 Acknowledgmentshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip246

3

ABSTRACT The University of Manchester Dr Oybek Rustamov Degre MD Title The role of anti-Muumlllerian hormone in assisted reproduction in women Date 30 March 2014

Anti-Muumlllerian hormone appears to play central role in regulation of oocyte recruitment and folliculogenesis Serum AMH concentration was found to be one of the best predictors of ovarian performance in IVF treatment Consequently many fertility centres have introduced AMH for the assessment of ovarian reserve and as a tool for formulation of ovarian stimulation strategies in IVF However published evidence on reliability of AMH assay methods and the role of AMH-tailored individualisation of ovarian stimulation in IVF appear to be weak Consequently I decided to conduct a series of studies that directed towards an improvement of the scientific evidence in these areas of research

The studies on performance of Gen II AMH assay revealed the assay suffers from significant instability and provides erroneous results Consequently the manufacturer introduced a modification on assay method

In view of the observed issues with Gen II assay I conducted a critical appraisal of all published research on the previous and current assay methods that reported AMH variability assay method comparison and sample stability The literature indicated clinically important variability between AMH measurements in repeated samples which was reported to be more significant with Gen II assay The studies on between-assay conversion factors derived conflicting conclusions Correspondingly the review of studies on sample stability revealed conflicting reports on the stability of AMH under normal storage and processing conditions which was reported to be more significant issue in Gen II assay In view of above findings we concluded that AMH in serum may exhibit pre-analytical instability which may vary with assay method Therefore robust international standards for the development and validation of AMH assays are required In the analysis of determinants of ovarian reserve I evaluated the effect of ethnicity BMI endometriosis causes of infertility and reproductive surgery on AMH AFC and FSH measurements using data on a large cohort of infertile patients

Using robust multivariable regression analysis in a large cohort of IVF cycles I established the effect of age AMH AFC diagnosis attempt COS protocol changes gonadotrophin type USOR operator regime and initial dose of gonadotrophins on oocyte yield Then I examined effect of gonadotrophin dose and regime on total and mature oocyte numbers The study found that after adjustment for all above variables there was no increase in oocyte yield with increasing gonadotrophin dose categories beyond the very lowest doses This suggests that there may not be significant direct dose-response effect and consequently strict protocols for tailoring the initial dose of gonadotrophins may not necessarily optimize ovarian performance in IVF treatment

In summary studies described in this thesis have revealed instability of Gen II assay samples and raised awareness of the pitfalls of AMH measurements These studies have also demonstrated the effect of clinically measurable factors on ovarian reserve and provided data on the effect of AMH other patient characteristics and treatment interventions on oocyte yield in cycles of IVF Furthermore a robust database and statistical models have been developed which can be used in future studies on ovarian reserve and IVF treatment interventions

4

DECLARATION

No portion of the work referred to in the thesis has been submitted in support

of an application for another degree or qualification of this or any other

university or other institute of learning

COPYRIGHT STATEMENT

i The author of this thesis (including any appendices andor schedules to this

thesis) owns certain copyright or related rights in it (the ldquoCopyrightrdquo) and she

has given The University of Manchester certain rights to use such Copyright

including for administrative purposes

ii Copies of this thesis either in full or in extracts and whether in hard or

electronic copy may be made only in accordance with the Copyright Designs

and Patents Act 1988 (as amended) and regulations issued under it or where

appropriate in accordance with licensing agreements which the University has

from time to time This page must form part of any such copies made

iii The ownership of certain Copyright patents designs trade marks and

other intellectual property (the ldquoIntellectual Propertyrdquo) and any reproductions

of copyright works in the thesis for example graphs and tables

(ldquoReproductionsrdquo) which may be described in this thesis may not be owned

by the author and may be owned by third parties Such Intellectual Property

and Reproductions cannot and must not be made available for use without the

prior written permission of the owner(s) of the relevant Intellectual Property

andor Reproductions

iv Further information on the conditions under which disclosure publication

and commercialisation of this thesis the Copyright and any Intellectual

Property andor Reproductions described in it may take place is available in

the University IP Policy (see

httpdocumentsmanchesteracukDocuInfoaspxDocID=487) in any

relevant Thesis restriction declarations deposited in the University Library The

University Libraryrsquos regulations (see

httpwwwmanchesteracuklibraryaboutusregulations) and in The

Universityrsquos policy on Presentation of Theses

5

PUBLICATIONS ARISING FROM THE THESIS

Journal Articles

1 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo Philip W Pemberton

The measurement of Anti-Muumlllerian hormone a critical appraisal

The Journal of Clinical Endocrinology amp Metabolism 2014 Mar99(3)723-32

2A Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton Anti-Muumlllerian hormone poor assay reproducibility in a large

cohort of subjects suggests sample instability Human Reproduction 2012 Oct

27(10) 3085-91

2B Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton Human Reproduction Dec2012 Vol 27 Issue 12 p3641

6

Conference presentations

1 O Rustamov S Roberts C Fitzgerald

Ovarian endometrioma is associated with increased AMH levels

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2014 Munich

Poster Presentation

2 O Rustamov M Krishnan R Mathur S Roberts C Fitzgerald

The effect of BMI to the ovarian reserve

Annual Meeting of British Fertility Society January 2014 Sheffield

Oral presentation Dr O Rustamov

3 M Krishnan O Rustamov R Mathur S Roberts C Fitzgerald

The effect of the ethnicity to the ovarian reserve

Annual Meeting of British Fertility Society January 2014 Sheffield

Oral Presentation Dr M Krishnan

4 O Rustamov M Krishnan S Roberts C Fitzgerald

Reproductive surgery and ovarian reserve

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2013 London

Oral presentation Dr O Rustamov

5 C Fitzgerald O Rustamov P Pemberton A Smith A Yates M Krishnan

R Russell L Nardo SRoberts

AMH assays A review of the literature on assay method comparability

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2013 London

Oral presentation Dr C Fitzgerald

6 M Krishnan O Rustamov R Russell C Fitzgerald S Roberts

The role of the ethnicity and the body weight in determination of AMH levels

in infertile women

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2013 London

7

Poster presentation

7 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton

AMH Gen II assay - can we believe the measurements

8th Biennial Conference of UK Fertility Societies January 2013 Liverpool

Poster presentation

8 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton

Old and new AMH assays Can we rely on current conversion factor

8th Biennial Conference of UK Fertility Societies January 2013 Liverpool

Poster presentation

9 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton

Random AMH measurement is not reproducible

8th Biennial Conference of UK Fertility Societies January 2013 Liverpool

Poster presentation

10 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton

The reproducibility of serum Anti-Muumlllerian hormone AMH Gen II assay

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2012 Istanbul

Oral Presentation Dr O Rustamov

8

GENERAL INTRODUCTION

AND LITERATURE REVIEW

1

9

CONTENTS I LITERATURE REVIEWhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip10 GENERAL BACKGROUNDhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip10

1 OVARIAN RESERVEhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip12 11 Primordial Follicle Assemblyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip13 12 Oocyte recruitmenthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip14 13 Theory of neo-oogenesishelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip15 2 MARKERS OF OVARIAN RESERVEhelliphelliphelliphelliphelliphelliphelliphelliphelliphellip16 21 Ovarian reserve markers with limited clinical valuehelliphelliphelliphelliphellip16 213 Inhibin Bhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip16 214 Basal oestradiolhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip17 215 Dynamic testshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip17 216 Ovarian volumehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip18 22 Ovarian reserve markers in routine clinical usehelliphelliphelliphelliphelliphelliphellip18 221 Chronological agehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip18 222 Basal FSHhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip18 223 Antral follicle counthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip19 3 ANTI-MUumlLLERIAN HORMONEhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip20 31 Biology of anti-Muumlllerian hormonehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip20 311 The role of AMH in the ovaryhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip21 312 AMH in women with polycystic ovary syndromehelliphelliphelliphelliphellip22 32 AMH Assayhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip23 33 Variability of AMH measurementshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip24 34 Role of AMH in assessment of ovarian reservehelliphelliphelliphelliphelliphellip25 341 Prediction of poor and excessive ovarian response in IVFhelliphellip25 342 Prediction of live birth in cycles of IVFhelliphelliphelliphelliphelliphelliphelliphelliphellip26

3 5 Role of AMH in ovarian stimulation for cycles of IVFhelliphelliphelliphellip26

4 MULTIVARIATE TESTShelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip27

5 SUMMARYhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip28 II GENERAL INTRODUCTIONhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip29 REFERENCEShelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip31

10

I LITERATURE REVIEW GENERAL BACKGROUND

Infertility is a disease of the reproductive system defined by the failure to

achieve a pregnancy after 12 months of regular unprotected sexual intercourse

although the criteria for the duration vary between different countries (NICE

2013) Worldwide prevalence of infertility estimated to be around 724 million

couples and around 40 million of those seek medical care (Hull et al 1985) In

the UK 15 couples present with infertility with an annual incidence of 12

couples per 1000 general population (Scott et al 2009) The main causes of

infertility are tubal disease ovulatory disorders male factor and poor ovarian

reserve In a third of couples the cause of failure to achieve pregnancy is not

established which is known as unexplained infertility (NICE 2013) Effective

treatment options include improving lifestyle factors medical andor surgical

treatment of underlying pathology induction of ovulation and Assisted

Reproductive Technology (ART) Assisted Reproduction consist of

intrauterine insemination (IUI) and in vitro fertilisation (IVF) cycles with or

without introcytoplasmic sperm injection (ICSI) as well as treatment involving

donated gametes It is estimated that 75 of infertile couples presenting at

primary care centres in the UK are referred to fertility specialists based at

secondary or tertiary care centres and nearly 50 of those are subsequently

offered IVFICSI treatment (Scott et al 2009) This is supported by figures of

Human Fertility and Embryology Authority (HFEA) which indicates more

than 50000 IVF treatment cycles are performed in the UK annually (HFEA

2008)

An IVF treatment cycle involves a) pituitary down regulation b)

controlled ovarian stimulation c) oocyte recovery c) in vitro fertilisation of eggs

with sperm d) transfer of resulting embryo(s) back to uterus and c) luteal

phase support (NICE 2013) Prevention of premature surge of luteinising

hormone during controlled ovarian stimulation (COS) is achieved by pituitary

down regulation using either preparations of gonadotrophin releasing hormone

agonist which is widely known as ldquoAgonist cyclerdquo or gonadotrophin releasing

hormone antagonist which is known ldquoAntagonist cyclerdquo (Figure 1 and 2)

Controlled ovarian stimulation involves administration of gonadotrophins to

encourage the development of supernumerary preovulatory follicles followed

by administration of exogenous human chorionic gonadotropin (hCG) or

11

recombinant luteinising hormone (rLH) to assist in maturation of oocytes 34-

36 hours prior to egg collection which is usually conducted with guidance of

transvaginal ultrasound scanning Subject to sperm parameters the fertilisation

of oocytes is conducted by in vitro insemination or intracytoplasmic sperm

injection The resulting embryo(s) are cultured under strict laboratory

conditions and undergo regular qualitative and quantitative assessments before

transferring the best quality embryo(s) back into uterus during its cleavage

(Day 2 or Day 3) or blastocyst (Day 5 or Day 6) stage of development In

natural menstrual cycles under the influence of HCG progesterone secreted

by the ovarian corpus luteum ensures proliferative changes in the endometrium

providing the optimal environment for implantation of embryo(s) (van der

Linden et al 2011) However in IVF treatment cycles owing to pituitary down

regulation and lack of HCG progesterone levels are not in sufficiently high

concentration to ensure an adequate endometrial receptivity and therefore

exogenous analogues of this hormone is administered following transfer of

embryo(s) This is called ldquoluteal phase supportrdquo and in patients with viable

pregnancy usually lasts till 12th week of gestation when placenta starts

producing progesterone in sufficient quantities (van der Linden et al 2011)

In IVF programmes the ldquosuccessrdquo of the treatment often defined as

achieving a live birth following IVF cycle and expressed using Live Birth Rate

(LBR) In general success in IVF predominantly determined by womanrsquos age

cause(s) of infertility ovarian reserve previous reproductive history and

lifestyle factors (NICE 2013 Taylor 2003 Lintsen et al 2005) However

effectiveness of medical interventions as well as the quality of care play

important role in determining the outcome of IVF treatment This is evident

from significant variation in live birth rates among fertility clinics given for

instance in the UK LBR for women younger than 35 years of age after IVF

cycles varies from 15 to 61 (HFEA 2008 HFEA 2007) The provision of

effective interventions in both clinical and laboratory aspects of the care

appears to be the key in achieving high success rates Identification of patients

with sufficient ovarian reserve who benefit from IVF cycles followed by

providing optimal ovarian stimulation regimens may be useful in improving the

outcomes of IVF programmes According to HFEA data around 12 of IVF

cycles are cancelled due to poor or excessive ovarian response (Kurinczuk et al

2010) Availability of reliable markers for assessment of ovarian reserve and

tailoring ovarian stimulation regimens to the need of each individual patient

12

may improve selection of patients with sufficient ovarian reserve and reduce

the rate of cycle cancellation consequently improving the success of IVF

cycles (Yates et al 2011)

Assessment of ovarian reserve can be achieved using various biomarkers

and four of those are currently used by most clinics womanrsquos chronological

age (Age) serum follicle stimulating hormone (FSH) antral follicle count

(AFC) and serum anti-Muumlllerian hormone (AMH) More recently AMH has

been a focus of interest given it is the only available endocrine marker that is

suitable for direct assessment of the activity of ovarian follicles in their non-

cyclical stage development providing a window to FSH independent phase of

follicular recruitment Furthermore it appears to be reliable biomarker for a)

both the assessment of ovarian reserve and the optimisation of ovarian

stimulation regimens (Yates et al 2011 La Marca et al 2009) b) screening and

diagnosis of polycystic ovarian syndrome (PCOS) (Cook et al 2002) c)

monitoring of disease activity in women with a history of granulosa cell

tumours (Lane et al 1999) d) prediction of the age of diminished fertility and

the menopause (van Disseldorp et al 2008 Broer et al 2011) and finally (e)

assessment of the long term effect of chemotherapy on ovarian reserve

(Anderson 2011)

In this review I first discuss current knowledge on factors that

determine ovarian reserve including the formation and loss of oocyte pool

Then characteristics of the markers of ovarian reserve are reviewed Finally I

examine current understanding of biology of anti-Muumlllerian hormone and its

role in management of infertility

1 OVARIAN RESERVE

It is important to recognize that there is no universal definition for the

term ldquoovarian reserverdquo and the term can have various meanings depending on

the context in which it is used For instance the scientific literature describing

the biology of ovarian reserve usually refers to ldquothe total number of remaining

oocytes in the ovaries which consists of the number of resting primordial

follicles and growing primary pre-antral and antral folliclesrdquo (Gleicher et al

2011) In contrast the use of the term in the context of clinical studies may

refer to ldquoclinically measurable ovarian reserve established using available

biomarkers of ovarian reserverdquo For the purpose of clarity in this thesis the

13

term ldquoovarian reserverdquo refers to clinically measurable ovarian reserve whilst

true biological ovarian reserve will be termed ldquobiological ovarian reserverdquo

Recent studies have demonstrated that ovarian reserve is highly variable

between women due to the variation in the size of initial ovarian reserve at

birth as well as the rate of loss of ovarian reserve thereafter (Wallace et al

2010) Interestingly the rate of oocyte loss appears to be mainly determined by

the initial ovarian reserve which is believed to be facilitated by most potent

ovarian growth factor anti-Muumlllerian hormone Similarly the size of the initial

ovarian reserve is mainly underpinned by the rate of primordial follicle

assembly in the embryo which is also regulated by AMH Both primordial

follicle assembly and the rate of oocyte loss appear to be primarily under the

influence of genetic factors although developmental and environmental factors

are also believed to play a role (Nilsson et al 2010 Shuh-Huerta et al 2012)

11 Primordial follicle assembly

The process of assembly of primordial follicles in the female embryo

spans from the early embryonic to the early postnatal period and formation of

primordial follicles consists of following stages 1) primordial germ cell (PGC)

2) oogonia 3) primary oocyte and 4) primordial follicle In the human female

fetus around a hundred cells that differentiated from extra-embryonic

ectoderm form early PGCs on the yolk sac and migrate via hindgut to gonadal

ridges during 4th - 6th weeks of gestation (MC et al 1953 Donovan 1998) Once

arrived to the gonadal ridges these cells are called primary oogonia which

consequently undergo several rounds of mitotic division during 6th - 28th weeks

of gestation Interestingly the numbers of oogonia reach as high as six million

during its highest rate of mitotic division at around 20 weeks of gestation

Following the last round of mitotic division oogonia enter meiosis which

marks their new stage of development-primary oocyte Formation of

primordial follicles starts as early as at 8th week of gestation and is characterised

by meiosis of primary oocyte that arrest in diplotyne stage and surrounding of

the oocyte by somatic granulosa cell (Baker et al 1963 Maheshwari and Fowler

2010) Indeed the primordial follicle is the cardinal unit of the biological

ovarian reserve and therefore the rate of formation of primordial follicles is the

main determinant of initial biological ovarian reserve at birth

Interestingly the process of loss of oogonia and oocytes which is also

one of the main determinants of the initial ovarian reserve takes place

14

throughout the period of follicle assembly The formation of the granulosa cell

layer around the oocyte prevents the oocyte from subsequent atresia The

oocyte enveloped in a single layer of granulosa cells which is also known as

primordial follicle remains quiescent until recruitment of the follicle for

growth which may not take place for a number of decades after the formation

of a particular primordial follicle (Skinner 2005 Maheshwari and Fowler 2010)

12 Oocyte recruitment

Follicle growth in women consists of two stages a) the initial non-cyclical

recruitment of primordial follicles and the formation of a primary and a pre-

antral follicles and b) cyclical development of antral follicles with subsequent

selection of usually a single dominant follicle The initial recruitment of

primordial follicles is continuous non-cyclical process that starts as early as

from 18-20 weeks of gestation and lasts till the depletion of follicle pool which

later results in the menopause (McGee and Hsueh 2000) Transformation of

flat granulosa cells into cuboidal cells increases the diameter of the oocyte and

the formation of zona pellicuda completes the stage of formation of a primary

follicle During pre-antral stage oocytes increase in diameter and mitotic

division of granulose cells create a new layer of cells-theca cells The

mechanism of initial recruitment of oocytes is not well understood but it is

clear that the process is independent of influence of pituitary gonadotrophins

and appears to be governed by the genetically pre-programmed interaction of

the oocyte with local growth factors the most important of which appears to

be anti-Muumlllerian hormone and cytokines (McGee and Hsueh 2000)

The cyclical phase of development of oocytes is characterised by the

transformation of secondary follicle into antral follicle and subsequent growth

of antral follicles into pre-ovulatory stages In general the process of cyclic

recruitment starts from puberty under the influence of rising levels of pituitary

follicular stimulating hormone (FSH) During the antral stage oocyte increases

in size even further and the formation of a fluid filled space in follicle is

observed Under the influence of FSH luteinising hormone (LH) and local

growth factorsselection of a single dominant follicle occurs which followsby an

ovulation (McGee and Hsueh 2000)

Oocyte loss is a continuous process and occurs due to atresia of oocytes

during primary secondary and antral stages of development The rate of

oocyte loss appears to increase until the age of around 14 and declines

15

thereafter until the age of the menopause when around 1000 primordial

follicles remain (Hansen et al 2008 Oktem and Oktayl 2008) Furthermore by

the age of 30 years the average age at which women of western societies plan

to start a family around 90 of initial primordial follicles are lost which

illustrates that formation and maintenance of ovarian reserve is wasteful

process in humans (ONS 2012 Wallace and Kelsey 2010) As mentioned

above there is a wide individual variation in both sizes of initial primordial

follicular pool and the rate of oocyte loss which explains variation in the

reproductive lifespan in women Evidently the number of primordial follicles

at birth ranges between around 35000 to 25 million per ovary and similarly

the rate of oocyte loss during its peak at 14 years of age may range between

100 to 7500 primordial follicles per month which is believed to be inversely

proportional to initial size of primordial follicle pool (Wallace and Kelsey

2010)

13 Theory of neo-oogenesis

The traditional view of oogenesis states that the process of the creation

and the mitotic division of oogonia with subsequent formation of primordial

follicles takes place only during embryonic and foetal life (Zuckerman 1951)

According to this central theory of mammalian reproductive biology females

are born with a certain number of germ cells that is gradually lost but not

renewed during postnatal period However Johnson et al have recently

challenged this view and reported that adult mammalian ovary may possesses

mitotically active germ cells that continuously replenish the primordial follicle

pool (Johnson et al 2004) The group reported that ovaries of juvenile and

young adult mice contained large ovoid cells which resemble germ cells of

foetal mouse ovaries Interestingly immunohistochemical staining for a gene

which is expressed exclusively in germ cells have been reported to have

confirmed that these large ovoid cells were of germline lineage Furthermore

application of a mitotic germ cell toxicant busulphan appeared to have

eliminated primordial follicle reserve by early adulthood but did not induce

atresia suggesting the presence of proliferative germ cells in postnatal mouse

ovary (Johnson et al 2004 Bazer 2004) The study has generated enormous

amount of interest as well as debate among reproductive biologists (Notarianni

2011) Some other groups have also reported an evidence of postnatal

oogenesis (Pacchiarott et al 2010 Zou et al 2009 Bukovsky et al 2004)) while

16

others do not support the theory (Bristol-Gould et al 2006 Byskov et al 2005

Begum et al 2008) Furthermore some authors argued that adult mouse

germline stem cells exist and remain quiescent in physiologic conditions and

neo-oogenesis occurs only in response to ovotoxic damage (Tilly et al 2007 De

Felici 2010) Although consensus has yet to emerge to date there is no

conclusive evidence on validity of theory of neo-oogenesis

2 MARKERS FOR ASSESMENT OF OVARIAN RESERVE

Biological ovarian reserve is defined as the number of primordial and

growing follicles left in the ovary at any given time and therefore only

counting the number of primordial follicles by histological assessment can

accurately determine ovarian reserve which is clearly not feasible in clinical

setting However ovarian reserve can be estimated using various biomarkers

dynamic clinical tests and implied from the outcomes of ART cycles

Although a wide range of clinical (age ovarian response in previous IVF

cycles) biochemical (basal FSH Inhibin B basal oestradiol AMH) ultrasound

(ovarian volume antral follicle count (AFC)) and dynamic (clomiphene

challenge test exogenous FSH ovarian reserve test GnRH analogue

stimulating test) tests of ovarian reserve exist only a few of the markers are

reliable and practical enough to be of use in routine clinical practice In this

chapter first I discuss the research evidence on the assessment of the markers

andor tests of ovarian reserve that have limited clinical value Then I

evaluated more reliable markers that are in routine clinical use Age FSH

AFC and combination of these markers in multivariable tests Finally I

conducted detailed review of biology of AMH and the role AMH measurement

in the management of infertility

21 Ovarian reserve markers with limited clinical value

211 Inhibin B

Inhibins are members of TGFβ family and expressed in granulosa cells

of growing follicles Principal role of inhibins is thought to be the negative

feedback regulation of pituitary FSH secretion and therefore the serum level of

circulating hormone is believed to reflect the state of folliculogenesis

17

Consequently several groups have studied the role of serum Inhibin β in the

assessment of ovarian reserve Although initial reports were encouraging

(Seifer et al 1997) more robust studies demonstrated that serum Inhibin β was

less reliable than chronological age or basal FSH (Creus et al 2000 Urbancsek

2005) The systematic review of nine studies demonstrated that accuracy of the

Inhibin β test for predicting poor ovarian response and non-pregnancy in IVF

cycles was modest even at a very low threshold level (Broekmans et al 2006)

Therefore it is recommended that inhibin β at best can be used as only

screening test in the fertility centers where other more reliable markers are not

available (Broekmans et al 2006)

212 Basal oestradiol

Some studies suggested that elevated basal oestradiol levels indicate low

ovarian reserve and are associated with poor fertility prognosis (Johannes et al

1998 Licciardi and Rosenwaks 1995) Johannes et al demonstrated basal

oestradiol in conjunction with serum FSH is more reliable than serum FSH

alone in prediction of cycle cancellation due to the poor response in IVF cycles

(Johannes et al 1998) However there are no published data on the comparison

of basal oestradiol to more reliable markers such as AMH or antral follicle

count (AFC) Moreover a recent systematic review has demonstrated that

basal oestradiol has very low predictive value for poor response and has no

discriminatory power for accuracy of non-pregnancy prediction (Broekmans et

al 2006)

213 Dynamic tests of ovarian reserve

The dynamic tests of ovarian reserve are based on assessment of ovarian

response by measuring serum FSH and oestradiol levels following

administration of exogenous stimulation The following tests are reported in

literature Clomiphene Citrate Challenge Test (CCCT) Exogenous FSH

Ovarian Reserve Test (EFORT) and GnRH agonist stimulation test A recent

systematic review and meta-analysis on the accuracy of these tests showed that

none of them can adequately predict poor response or non-pregnancy in IVF

cycles and therefore are not recommended for use in routine clinical practice

(Maheshwari et al 2009)

18

214 Ovarian volume

There is some evidence that increased age is associated with decreased

ovarian volume and women with smaller ovaries are more likely to have

cancellation of their IVF cycles due to poor ovarian response (Syrop et al 1995

Syrop et al 1999 Templeton 1995) However a meta-analysis of the published

studies on the accuracy of ovarian volume as a predictor of poor response and

non-pregnancy in IVF cycles failed to demonstrate clinical usefulness of the

test and suggested the test is not reliable enough for use in a routine clinical

practice (Broekmans et al 2006)

22 Ovarian reserve markers in routine clinical use

221 Chronological age

Owing to the biological age-related decline of the quantity and arguably

the quality of oocytes the chronological age can be used as a marker of ovarian

reserve Studies have demonstrated that ovarian reserve (Wallace and Kelsey

2010 Kelsey 2011) natural fecundity (Islam et al 1989 and outcomes of ART

(Templeton et al 1996 van Kooij et al 1996) decline significantly from age of

35 when it is believed the ovarian reserve undergoes accelerated decline

Although there is a strong association between chronological age and reduction

in fertility evidently there is a significant variation in age-related ovarian

reserve indicating chronological age alone may not be sufficient to estimate the

individual womanrsquos ovarian reserve reliably (Broekmans et al 2006)

222 Basal FSH

Basal FSH was one of the first endocrine markers introduced in ART

programs and is still utilized in many fertility clinics albeit in conjunction with

other markers which are considered more reliable (Creus et al 2000) Secretion

of FSH is largely governed by the negative feedback effect of steroid

hormones primarily oestradiol and inhibins which are expressed in granulosa

cells of growing ovarian follicles Consequently decreased or diminished

recruitment of ovarian follicles is associated increased serum FSH

measurements and high particularly very high basal FSH reading is considered

as a good marker of very low or diminished ovarian reserve (Abdalla et al

2006) However unlike some other markers FSH measurements do not

appear to have discriminatory power for categorisation of patients to various

19

bands of ovarian reserve Given between-patient variability FSH measurement

(CV 30) is similar to its within-patient variability (27) stratification of

patients to various ranges of ovarian reserve does not appear to be feasible

(Rustamov et al 2011) Indeed a recent systematic review of 37 studies on the

prediction of poor response and non-pregnancy in IVF cycle has concluded

that basal FSH is an adequate test at very high threshold levels and therefore

has limited value in modern ART programs (Broekmans et al 2006)

223 Antral follicle count

Antral follicle count estimation involves ultrasound assessment of

ovaries between 2nd and 4th day of menstrual period and counting ldquofolliclesrdquo

which corresponds to antral stage of folliculogenesis (Broekmans et al 2010)

The test provides direct quantitative assessment of growing follicles and is

known as one of the most reliable markers of ovarian reserve (Broekmans et al

2006) AFC measurement has been reported as having a similar sensitivity and

specificity to AMH in prediction of poor and excessive ovarian response in

IVF cycles (Broekmans et al 2006 Broer et al 2010 Jayaprakasan et al 2010)

Given AFC measurement is available instantly and allows patients to be

counseled immediately the test eliminates the need for an additional patient

visit prior to IVF cycle However AFC is normally performed only in the early

follicular phase of the menstrual cycle given most published data on

measurement of AFC are based on studies that assessed antral follicles during

this stage of the cycle (Broekmans et al 2010a) Interestingly more recent

studies suggest that variability of AFC during menstrual cycle is small

particularly when follicles between 2-6mm are counted and therefore

assessment of AFC without account for the day of menstrual cycle may be

feasible (Deb et al 2013)

One of the main drawbacks of AFC is that the cut off levels for size of

counted follicles remains to be standardised (Broekmans 2010b) Initially

follicles of 2-10mm were introduced as the range for AFC and many studies

were based on this cut off Later counting follicles of 2-6mm was reported to

provide most accurate assessment of ovarian reserve (Jayaprakasan et al 2010b

Haadsma et al 2007) and therefore some newer studies are based on AFC

measurements that used this criterion Consequently direct comparison of the

outcomes of various studies on assessment of AFC requires careful analysis

20

3 ANTI-MUumlLLERIAN HORMONE

31 Biology of Anti-Muumlllerian hormone

AMH is a member of transforming growth factor β superfamily which

was discovered by Jost et al in 1947 and was initially known for its is role in

regression of Muumlllerian ducts in sex differentiation of the male embryo In

women AMH is believed to be solely produced by ovaries and expressed in

granulosa cells of growing follicles of 2-6 mm in size which corresponds to

primary pre-antral and early antral stage of follicular development Although

there has been a report of expression of AMH in endometrial cells to date

there is no other published evidence that supports this finding (Wang et al

2009) Indeed studies that evaluated half-life of AMH in serum have

demonstrated that in women who had bilateral salpingo-oopherectomy AMH

becomes undetectable within 3-5 days of following surgery suggesting ovaries

are the only source of secretion of AMH in appreciable quantity (La Marca et

al 2005b) Anti-Muumlllerian hormone is a dimeric glycoprotein which is

composed of a long N-terminus and short C-terminus and was believed to be

secreted in serum only in this dimeric form (AMH-N C)

Like other members of TGF-β family which includes inhibins activins

bone morphogenic proteins (BMPs) and growth and differentiation factors

(Massague et al 1990) AMH binds to two type of serinethreonine kinase

receptors referred to as type I and type II In order to activate AMH signaling

pathway both receptors have to form a heteromeric complex When AMH

binds to the type II (AMHR-II) receptor (Massague et al 2000) this will

phosphorylate and activate a type I receptor (ALK2 -3 andor -6) which

subsequently activates the SMAD pathway through phosphorylation of

SMAD 1 5 andor 8 These activated SMADs interact with SMAD4 and

translocate to the nucleus regulating the expression of different genes

inhibiting the recruitment of primordial follicles and reducing FSH sensitivity

in growing follicles In addition AMH receptors as well as the other members

of TGF-β family can activate MAPK and PI3KAKT pathways

Studies on AMHR II-deficient male mice demonstrated lack of

regression of Muumlllerian ducts suggesting that type II receptor is essential in

AMH signaling (Mishina et al 1996) Similarly Type I receptors which includes

three members of activin receptor-like kinase (ALK2 ALK3 and ALK6) also

appear to play an important role in the regression of Muumlllerian ducts although

21

the role of ALK 6 in AMH signaling appears not to be crucial (Visser 2003

Clarke et al 2001) The signal transduction pathway of AMH in the ovary is

largely not understood In postnatal mice ovary AMHR-II receptor was

expressed in both granulosa and theca cells of pre-antral and antral follicles

(Visser 2003) AMH type I receptors ALK 2 and ALK 3 is expressed in foetal

as well as adult mouse ovary while ALK 6 is expressed in only adult ovary

(Visser 2003)

311 The role of AMH in the ovary

In the mammalian ovary the role of AMH appears to be one of a

regulation of size of the primordial follicle pool by its inhibitory effect on the

formation as well as the growth of primordial follicles (Nilsson et al 2011) In

the embryonic mouse ovary AMH inhibits the initiation of the assembly of

follicles when the process of apoptosis of the majority of oocytes is observed

(Nilsson et al 2011) Consequently AMH reduces the rate of oocyte loss

which plays an important role in the determination of the size of initial follicle

pool Similarly in the adult mouse ovary AMH plays a central role in

maintaining the follicle pool AMH inhibits both the processes of the initial

(non-cyclical) recruitment of primordial follicles and subsequent FSH-

dependent cyclical growth of antral follicles (Figure 3) Inhibition of the initial

recruitment of a new cohort of follicles is believed to be achieved by a

paracrine negative feedback effect of the rising levels of AMH secreted from

already recruited growing follicles (Durlinger et al 1999) Durlinger et al

compared the complete follicle population of AMHnull mice and wild type

mice of different ages of 25 days 4 months old and 13 months old and found

that the ovaries of 25 day and 4 months old AMHnull females contained

significantly higher number of growing pre-antral and antral follicles but

significantly fewer primordial follicles compared to wild-type females

(Durlinger et al 1999) Interestingly almost no primordial follicles were

detected in 13 months old AMHnull mice ovaries suggesting AMH is a potent

inhibitor of the recruitment of primordial follicles and in the absence of AMH

ovaries undergo premature depletion of primordial follicles due to an

accelerated recruitment Subsequent study conducted by the group

demonstrated that in addition to its inhibitory effect to the resting follicles

AMH also suppresses the development of the growing follicles (Durlinger et al

2001 Durlinger et al 2002 Themmen 2005) It appears that AMH inhibits

22

FSH-induced follicle growth by reducing the sensitivity of growing follicles to

FSH which has been confirmed by in vivo as well as in vitro studies (Durlinger

et al 1999 Durlinger et al 2001) In the initial study the group observed that

despite lower levels of serum FSH concentration ovaries of AMHnull mice

contained more growing follicles than that of their wild-type littermates which

has been supported by the findings of subsequent in vitro study (Durlinger et al

1999) Addition of AMH to the culture inhibited FSH-induced follicle growth

of pre-antral mouse follicles due to reduction in granulosa cell proliferation

(Durlinger et al 2001)

In the human embryo the expression of AMH commences in the late

foetal life and can be detected only from 36 weeks of gestation (Rajpert-De et

al 1999 Lee et al 1996) Following a small decline in first two years of life

AMH levels gradually increase to peak at (mean 5 ngml) around age of 24

years In line with the pattern of oocyte loss serum hormone levels gradually

decline with increasing age and become undetectable around 5 years prior to

menopause (Kelsey et al 2011 Nelson et al 2011)

It has been suggested that anti-Muumlllerian hormone plays a central role in

determining the pace of recruitment of primordial follicles hence maintaining

the primordial follicle pool of postnatal mammalian ovary Consequently a

reduction in the concentration of circulating AMH signals the exhaustion of

the primordial follicle pool and the decline of ovarian function

312 AMH in women with polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) endocrine abnormality characterised

by increased ovarian androgen secretion infrequent ovulation and the

appearance of ldquopolycysticrdquo ovaries on ultrasound scan (Dunaif 1997 Homburg

et al 1993) It is the commonest endocrine abnormality in women of

reproductive age and affects around 15-20 of women PCOS is also one of

the main causes of anovulation and subsequent sub-fertility (Webber et al

2003) Although the role of anti-Muumlllerian hormone in the development of

PCOS is not fully understood it is becoming increasingly evident that the

hormone plays an important role in its pathogenesis (Pehlivanov et al 2011)

There is a strong association between serum AMH levels and PCOS and it

appears that women diagnosed with PCOS have two to three fold higher

serum AMH concentration compared to normo-ovulatory women (Cook et al

2002 Pigny et al 2003) Similarly women with PCOS are found to have

23

significantly higher number antral follicles Interestingly the expression of

AMH in granulosa cells of follicles were found to be 75 times higher in women

with PCOS compared to those without a the disease suggesting increased

serum AMH in PCOS may be due to increased secretion of hormone per

follicle rather than due to an increased number of antral follicles (Pellat et al

2007) High AMH concentrations may act as the main facilitator of abnormal

folliculogenesis in PCOS given the follicles appear to arrest when they reach

an antral stage (2-6mm) of development (Rajpert-De et al 1999) Indeed the

studies of Durlinger et al have demonstrated that AMH inhibits selection of

dominant follicle when follicles reach antral stage of development (Durlinger et

al 2001) Serum AMH levels appear to decrease with treatment of PCOS

which may play important role in restoration of ovulatory cycles Studies have

reported a significant reduction in serum concentration of AMH following

treatment of PCOS with metformin and laparoscopic ovarian diathermy (Falbo

et al 2010 Amer et al 2009 Elmashad 2011) Similarly reduction of BMI

following intensified endurance exercise training for treatment of PCOS may

also lead to a significant reduction in serum AMH levels (Moran et al 2011)

This suggests that there is strong association between serum concentration of

AMH and abnormal folliculogenesis in PCOS and therefore understanding the

molecular mechanisms of this interaction should be one of the priorities of

future research

32 AMH Assays

Enzyme-linked immunosorbent assay specific for measurement of anti-

Muumlllerian hormone was first developed in 1990 and was recognised as a

significant step in the assessment of ovarian reserve (Hudson et al 1990)

Subsequently a number of non-commercial immunoassays were developed

which were mainly used in research settings (Lee et al 1996) Later Diagnostic

Systems Ltd (DSL) and Immunotech Beckman Coulter Ltd (IOT) introduced

two commercial immunoassays for the routine clinical assessment of ovarian

reserve which are known as ldquofirst generation AMH assaysrdquo (Nelson and La

Marca 2011) These assays employed two different antibodies against AMH

and used different standards for calibration providing non-comparable

measurements (Nelson and La Marca 2011) Consequently several studies

attempted to develop a reliable between-assay conversion factor which

interestingly revealed from five-fold higher with the IOT assay to assay

24

equivalence causing significant impact to reliability of AMH measurements and

interpretation of research findings (Hehenkamp et al 2006 Freour et al 2007

Bersinger et al 2007 Taieb et al 2008 Lee et al 2011)

Later the manufacturer of IOT assay (Beckmann Coulter Ltd)

consolidated the manufacturer of the DSL assay (Diagnostic Systems

Laboratories Inc) and introduced a new assay ldquoGen II AMH assayrdquo which is

only available commercial immunoassay in most countries including the UK

AMH Gen II assay was developed using the antibodies derived from first

generation DSL assay and calibrated using the standards used for IOT assay

and was believed to be considerably more stable compared to the first

generation immunoassays providing more reliable measurements (Kumar et al

2010 Nelson and La Marca 2011) The manufacturer as well as initial external

validation study recommended when compared to old DSL assay AMH Gen

II assay provides around 40 higher measurements and therefore previously

reported DSL-based clinical cut-off levels for estimation of ovarian reserve

should be increased by 40 in order to use Gen II-based AMH results (Kumar

et al 2010 Wallace et al 2011 Nelson and La Marca 2011)

33 Variability of AMH measurements

It is generally believed that AMH values do not change throughout the

menstrual cycle and early studies reported that variation in AMH

measurements between repeated measurements of same patient was negligible

(van Disseldorp et al 2010 La Marca 2010) On the basis of these studies

sampling at a random time in the menstrual cycle was introduced as a method

for measurement of AMH in routine clinical practice However the

methodologies of some of these studies do not appear to be robust enough to

reliably estimate sample-to-sample variability of AMH which is mainly due to

small sample sizes (Rustamov et al 2011) Consequently in a recent study we

assessed sample-to-sample variability of AMH using DSL assay and found that

within-subject coefficient of variation (CV) of AMH between samples were as

high as 28 which cannot be attributed to any patient or cycle characteristics

(Rustamov et al 2011) Although there is no consensus in the causes of this

observed variability in AMH measurements we believe it is largely attributable

to instability of AMH samples given initial recruitment of primordial follicles

and growth of AMH producing pre-antral and antral follicles are continuous

process and therefore the true biological variation between samples is unlikely

25

to be high However given the importance of establishing true variability of

AMH in both understanding of the biology of hormone and clinical

application of the test future studies should be conducted to establish the

source of variability in the clinical samples

3 4 The role of AMH in the assessment of ovarian reserve

341 Prediction of poor and excessive ovarian response in cycles of

IVF

A number of studies have assessed the role of AMH in the prediction of

poor ovarian response in IVF cycles using first generation AMH assays and

found that AMH and AFC were the best predictors of poor ovarian response

compared to other markers of ovarian reserve Nardo et al showed that the

predictive value of AMH in receiver operating characteristic curve (ROC)

analysis was similar to (AUC 088) that of AFC (AUC 081) and found that

AMH cut offs of gt375 ngmL and lt10 ngmL would have modest

sensitivity and specificity in predicting the extremes of response (Nardo et al

2009) These findings were largely supported by subsequent prospective studies

and a systematic review (Nelson et al 2007 Jayaprakasan et al 2010 Broer et al

2011) Similarly comparison of chronological age basal FSH ovarian volume

AFC and AMH found that only AMH (AUC 090) and AFC (AUC 093) were

reliable predictors of poor ovarian response in cycles of IVF Subsequent

combination of the effect of AMH and AFC using multivariable regression

analysis did not improve the level of prediction of poor ovarian response

significantly (AUC 094) suggesting both AMH and AFC can be used as

independent markers (Jayaprakasan et al 2010)

Similarly most studies agree that AMH and AFC are the best predictors

of excessive ovarian response and ovarian hyperstimulation syndrome (OHSS)

compared to other clinical endocrine and ultrasound markers (Nardo et al

2009 Nelson et al 2007) Broer et al compared these two tests in systematic

review of 14 studies and reported that the summary estimates of the sensitivity

and the specificity for AMH were 82 and 76 respectively and for AFC 82

and 80 respectively (Broer et al 2011) Consequently the study concluded

that AMH and AFC were equally predictive and the difference in the predictive

value between the tests was not statistically significant

26

342 Prediction of live birth rate (LBR) in cycles of IVF

Lee at al reported that AMH and chronological age were more accurate

than basal FSH AFC BMI and causes of infertility in the prediction of live

birth rate (Lee et al 2009) Similarly La Marca et al suggested that odds of live

birth could be reliably predicted using AMH (La Marca et al 2010b) although

subsequent review of the study questioned strength of the evidence (Loh and

Maheshwari 2011)

A study conducted by Nelson et al found that higher AMH levels had

stronger association with increased live birth rate compared to age and FSH

(Nelson et al 2007) However the study also suggested that this association

was mainly confined in the women with low AMH levels and there was no

additional increase in live birth in women with AMH levels of higher than 710

pmolL This may suggest that achieving a live birth may be under the

influence of number of other factors and that markers of ovarian reserve alone

may not be able predict this outcome reliably

35 The role of AMH in individualisation of ovarian stimulation in

IVF cycles

Prediction of ovarian response to the stimulation of ovaries in cycles of

IVF plays an important role in the counseling of couples undergoing treatment

programmes and hence many clinical studies on AMH have focused on the

prognostic value of AMH measurements However data on using AMH as a

tool for improving the clinical outcomes in IVF cycles appear to be lacking

considering AMH may be useful tool in tailoring treatment strategies to an

individual patientrsquos ovarian reserve Unlike most other markers AMH has

discriminatory power in determining various degrees of ovarian reserve due to

significantly higher between patient (CV 94) variability compared to its

within-patient (CV 28) variation (Rustamov et al 2011) which allows

stratification of patients into various degrees of (eg low normal high) ovarian

reserve Subsequently most optimal ovarian stimulation protocol may be

established for each band of ovarian reserve Consequently reference ranges

on the basis of distribution of AMH in infertile women were developed which

were subsequently adopted by fertility clinics for a tailoring the mode of

27

ovarian stimulation and daily dose of gonadotrophins in IVF (The Doctors

Laboratory 2008 However currently available clinical reference ranges are

based on the first generation DSL assay and may not be reliably convertible to

currently available Gen II assay measurements (Wallace et al 2011) Indeed the

findings of the studies on comparability of the first generation AMH assays

suggest that establishing a reliable between assay conversion factor between

AMH assays may not be straightforward Furthermore the reference ranges

appear to reflect the distribution of AMH measurements within a specific

population and may therefore not be directly applicable for the prediction of

response to ovarian stimulation in IVF patients (The Doctors Laboratory

2008)

More importantly despite lack of good quality evidence on the

effectiveness of AMH-tailored ovarian stimulation protocols a number of

fertility clinics appear to have introduced various AMH-based COH protocols

in their IVF programs At present research evidence on AMH-tailored

ovarian stimulation in IVF is largely based on two retrospective studies

(Nelson et al 2009 Yates et al 2012) Both of these studies display considerable

methodological limitations including small sample size and centre-related or

period-related selection of their cohorts In this context AMH is used as a tool

for therapeutic intervention and therefore the research evidence should ideally

be derived from randomised controlled trials However recruitment of large

enough patients in IVF setting may take considerable time and resources In

the meantime given AMH-tailored ovarian stimulation has already been

introduced in clinical practice and there is urgent need for more reliable data

the studies with a larger cohorts and robust methodology should assess the role

of AMH in individualisation of ovarian stimulation in IVF treatment cycles

4 Multivariate models of assessment of ovarian reserve

In view of the fact there is not a single marker of ovarian reserve that

can accurately predict ovarian response various models for combination of

multiple ovarian markers have been developed (Verhagen et al 2008) A

number of studies reported that multivariate models are better predictors of

poor ovarian response in IVF compared to a single marker (Bancsi et al 2002

Balasch et al 1996 Creus et al 2000 Durmusoglu et al 2004) However a meta-

analysis showed that when compared to a single marker (AFC) multivariate

28

model has a similar accuracy in terms of prediction of poor ovarian response

(Verhagen et al 2008) In contrast a more recent study demonstrated that

multivariate score was superior to chronological age basal FSH or AFC alone

in predicting likelihood of poor ovarian response and clinical pregnancy

(Younis et al 2010) However the study did not include one of the most

reliable markers AMH in either arm necessitating further assessment of the

role of combined tests which include all reliable biomarkers

4 SUMMARY

During the last two decades a significant leap has been taken towards

understanding the biology of anti-Muumlllerian hormone and its role in female

reproduction (Durlinger et al 2002 Themmen et al 2005) Availability of

commercial AMH assays has resulted in significant increase in interest in the

role of the measurement of serum AMH in the assessment of ovarian reserve

which has been followed by the introduction of the test into routine clinical

practice (Nelson et al 2011) However more recent studies suggest that current

methodologies for the measurement of AMH may provide significant sampling

variability (Rustamov et al 2011) Furthermore the studies that compared first

generation commercial assay methods appear to provide non-reproducible

results suggesting there may be underlying issues with assay methodologies

(Lee et al 2011) Similarly despite lack of sufficient evidence in the role of

AMH in individualisation of ovarian stimulation protocols in IVF AMH-

tailored IVF protocols have been introduced in routine clinical practice of

many fertility clinics around the world

Consequently it appears that clinical application of AMH test has

surpassed the research evidence in some aspects of fertility treatment and

therefore future projects should be directed toward areas where gaps in

research evidence exist On the basis of the review of literature we believe that

evaluation of the performance of assay methods understanding the role of

AMH in assessment ovarian reserve and establishing its role in

individualisation of ovarian stimulation protocols should be research priority

29

II GENERAL INTRODUCTION

On the basis of the review of published literature I have identified that

the following areas of research on the clinical application of AMH in the

management of infertility requires further investigation 1) Within-patient

variability of measurement of AMH using Gen II assay method 2)

Establishment of clinically measurable determinants of AMH levels and 3) The

role of AMH in individualisation of ovarian stimulation in IVF treatment

cycles

In our previous study we estimated that there was significant sample-to-

sample variation (CV 28) in AMH measurements when the first generation

DSL assay was used (Rustamov et al 2011) The source of variability is likely to

be related to the assay method given that biological within-cycle variation of

AMH is believed to be small (La Marca et al 2006) Therefore assessment of

sample-to-sample variability of AMH using the newly introduced Gen II assay

which is believed to be significantly more stable and sensitive compared to that

of DSL assay should enable us to establish the measurement related variability

of AMH Furthermore given I am planning to use data from both DSL and

Gen II assays I need to establish between-assay conversion factor for these

assays using data on clinical samples

There appears to be a lack of good quality data on the effect of

ethnicity BMI causes of infertility reproductive history and reproductive

surgery on ovarian reserve Therefore I am planning to ascertain the role of

above factors on determination of ovarian reserve by analysing AMH

measurements of a large cohort of patients

There is a strong correlation between AMH and ovarian performance

in IVF treatment when conventional ovarian stimulation using GnRH agonist

regimens with a standard daily dose of gonadotrophins are used (Nelson et al

2007 Nardo et al 2007) Furthermore studies suggest tailoring the ovarian

stimulation protocols to AMH measurement may improve ovarian

performance and subsequently the success of IVF treatment (Nelson et al

2011 Yates et al 2012) However given methodologies of the published

studies the effectiveness of currently proposed AMH-tailored ovarian

stimulation protocols remains unknown Therefore I am planning to develop

individualised ovarian stimulation protocols by establishing the most optimal

mode of pituitary down regulation and starting dose of gonadotrophins for

30

each AMH cut-off bands using a robust research methodology However

development of individualised ovarian stimulation protocols on the basis of

retrospective data requires a reliable and validated database containing a large

number of observations In the IVF Department of St Maryrsquos Hospital we

have data on a large number of patients who underwent ovarian stimulation

following the introduction of AMH However the data on various aspects of

investigation and treatment of patients is stored in different clinical data

management systems and may not be easily linkable In addition it appears that

data on certain important variables (eg causes of infertility AFC) are available

only in the hospital records necessitating searching for data from the hospital

records of each patient Consequently I designed a project for building a

research database which will have comprehensive and validated datasets that

are necessary for investigation of the research questions of the MD

programme

In conclusion I am planning to conduct a series of studies to improve

the understanding of the role of AMH in the management of women with

infertility Specifically I am intending to evaluate 1) sample-to-sample variability

of Gen II AMH measurements 2) conversion factor between DSL and Gen II

assays in clinical samples 3) the effect of ethnicity BMI causes of infertility

endometriosis reproductive history and reproductive surgery to ovarian

reserve and explore AMH-tailored individualisation of ovarian stimulation in

IVF cycles

31

References

Abbeel E The Istanbul consensus workshop on embryo assessment proceedings of an expert meeting Human reproduction 2011 26 p 1270-83 Abdalla HT M Y Repeated testing of basal FSH levels has no predictive value for IVF outcome in women with elevated basal FSH Human reproduction 2006 21(1) p 171-4 Amer SA LT Ledger WL The value of measuring anti-Mullerian hormone in women with polycystic ovary syndrome undergoing laparoscopic ovarian diathermy Human reproduction 2009 24 p 2760-6 Anderson RA Pretreatment serum anti-Mullerian hormone predicts long-term ovarian function and bone mass after chemotherapy for early breast cancer J Clin Endocrinol Metab 2011961336-1343 Balaban B BD Calderoacuten G Catt J Conaghan J Cowan L Ebner T Gardner D Hardarson T Lundin K Cristina Magli M Mortimer D Mortimer S Munneacute S Royere D Scott L Smitz J Thornhill A van Blerkom J Van den Baker A quantitative and cytological study of germ cells in human ovaries Proc R Soc Lond B Biol Sci 1963 158 p 417-433 Balasch J CM Fabregues F Carmona F Casamitjana R Ascaso and VJ C Inhibin follicle-stimulating hormone and age as predictors of ovarian response in in vitro fertilization cycles stimulated with gonadotropin-releasing hormone agonist-gonadotropin treatment Am J Obstet Gynecol 1996 175 p 1226-1230 Bancsi LF BF Eiijekemans MJ at al Predictors of poor ovarian response in in vitro fertilisation a prospective study comparing basal markers of ovarian reserve Fertility and Sterility 2002 77 p 328-336 Bazer FW Strong science challenges conventional wisdom new perspectives on ovarian biology Reprod Biol Endocrinol 2004 2 p 28 Begum S VE Papaioannou and RG Gosden The oocyte population is not renewed in transplanted or irradiated adult ovaries Hum Reprod 2008 23(10) p 2326-30

Bersinger NA Wunder D Birkhaumluser MH Guibourdenche J Measurement of anti-mullerian hormone by Beckman Coulter ELISA and DSL ELISA in assisted reproduction differences between serum and follicular fluid Clin Chim Acta 2007384174ndash175 Bristol-Gould SK et al Fate of the initial follicle pool empirical and mathematical evidence supporting its sufficiency for adult fertility Dev Biol 2006 298(1) p 149-54 Broekmans FJ et al A systematic review of tests predicting ovarian reserve and IVF outcome Hum Reprod Update 2006 12(6) p 685-718

2

TABLE OF CONTENTS Abstracthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip3 Publications arising from the thesishelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip5 Chapter 1 General Introduction amp Literature reviewhelliphelliphelliphelliphelliphelliphelliphellip8 Chapter 2 Evaluation of the Gen II AMH Assay between-sample variability

and assay- method comparabilityhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip43 21 Anti-Muumlllerian hormone serum levels and reproducibility in a large cohort of subjects suggest sample instabilityhelliphelliphelliphellip44 22 AMH Gen II assay A validation study of observed variability between repeated AMH measurementshelliphelliphelliphellip65

Chapter 3 The measurement of anti-Muumlllerian hormone a critical appraisalhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip78

Chapter 4 Extraction preparation and collation of datasets for the

assessment of the role of the markers of ovarian reserve in female reproduction and IVF treatmenthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip106

Chapter 5 Assessment of determinants of anti-Muumlllerian hormone in infertile womenhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip135

51 The effect of ethnicity BMI endometriosis and the causes of infertility on ovarian reservehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip136 52 The effect of salpingectomy ovarian cystectomy and unilateral salpingoopherectomy on ovarian reservehelliphelliphelliphellip167

Chapter 6 Assessment of determinants of oocyte number using large

retrospective data on IVF cycles and explorative study of the potential for optimization of AMH-tailored stratification of controlled ovarian hyperstimulationhellip187

Chapter 7 General Summaryhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip229 Authors and affiliationshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip245 Acknowledgmentshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip246

3

ABSTRACT The University of Manchester Dr Oybek Rustamov Degre MD Title The role of anti-Muumlllerian hormone in assisted reproduction in women Date 30 March 2014

Anti-Muumlllerian hormone appears to play central role in regulation of oocyte recruitment and folliculogenesis Serum AMH concentration was found to be one of the best predictors of ovarian performance in IVF treatment Consequently many fertility centres have introduced AMH for the assessment of ovarian reserve and as a tool for formulation of ovarian stimulation strategies in IVF However published evidence on reliability of AMH assay methods and the role of AMH-tailored individualisation of ovarian stimulation in IVF appear to be weak Consequently I decided to conduct a series of studies that directed towards an improvement of the scientific evidence in these areas of research

The studies on performance of Gen II AMH assay revealed the assay suffers from significant instability and provides erroneous results Consequently the manufacturer introduced a modification on assay method

In view of the observed issues with Gen II assay I conducted a critical appraisal of all published research on the previous and current assay methods that reported AMH variability assay method comparison and sample stability The literature indicated clinically important variability between AMH measurements in repeated samples which was reported to be more significant with Gen II assay The studies on between-assay conversion factors derived conflicting conclusions Correspondingly the review of studies on sample stability revealed conflicting reports on the stability of AMH under normal storage and processing conditions which was reported to be more significant issue in Gen II assay In view of above findings we concluded that AMH in serum may exhibit pre-analytical instability which may vary with assay method Therefore robust international standards for the development and validation of AMH assays are required In the analysis of determinants of ovarian reserve I evaluated the effect of ethnicity BMI endometriosis causes of infertility and reproductive surgery on AMH AFC and FSH measurements using data on a large cohort of infertile patients

Using robust multivariable regression analysis in a large cohort of IVF cycles I established the effect of age AMH AFC diagnosis attempt COS protocol changes gonadotrophin type USOR operator regime and initial dose of gonadotrophins on oocyte yield Then I examined effect of gonadotrophin dose and regime on total and mature oocyte numbers The study found that after adjustment for all above variables there was no increase in oocyte yield with increasing gonadotrophin dose categories beyond the very lowest doses This suggests that there may not be significant direct dose-response effect and consequently strict protocols for tailoring the initial dose of gonadotrophins may not necessarily optimize ovarian performance in IVF treatment

In summary studies described in this thesis have revealed instability of Gen II assay samples and raised awareness of the pitfalls of AMH measurements These studies have also demonstrated the effect of clinically measurable factors on ovarian reserve and provided data on the effect of AMH other patient characteristics and treatment interventions on oocyte yield in cycles of IVF Furthermore a robust database and statistical models have been developed which can be used in future studies on ovarian reserve and IVF treatment interventions

4

DECLARATION

No portion of the work referred to in the thesis has been submitted in support

of an application for another degree or qualification of this or any other

university or other institute of learning

COPYRIGHT STATEMENT

i The author of this thesis (including any appendices andor schedules to this

thesis) owns certain copyright or related rights in it (the ldquoCopyrightrdquo) and she

has given The University of Manchester certain rights to use such Copyright

including for administrative purposes

ii Copies of this thesis either in full or in extracts and whether in hard or

electronic copy may be made only in accordance with the Copyright Designs

and Patents Act 1988 (as amended) and regulations issued under it or where

appropriate in accordance with licensing agreements which the University has

from time to time This page must form part of any such copies made

iii The ownership of certain Copyright patents designs trade marks and

other intellectual property (the ldquoIntellectual Propertyrdquo) and any reproductions

of copyright works in the thesis for example graphs and tables

(ldquoReproductionsrdquo) which may be described in this thesis may not be owned

by the author and may be owned by third parties Such Intellectual Property

and Reproductions cannot and must not be made available for use without the

prior written permission of the owner(s) of the relevant Intellectual Property

andor Reproductions

iv Further information on the conditions under which disclosure publication

and commercialisation of this thesis the Copyright and any Intellectual

Property andor Reproductions described in it may take place is available in

the University IP Policy (see

httpdocumentsmanchesteracukDocuInfoaspxDocID=487) in any

relevant Thesis restriction declarations deposited in the University Library The

University Libraryrsquos regulations (see

httpwwwmanchesteracuklibraryaboutusregulations) and in The

Universityrsquos policy on Presentation of Theses

5

PUBLICATIONS ARISING FROM THE THESIS

Journal Articles

1 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo Philip W Pemberton

The measurement of Anti-Muumlllerian hormone a critical appraisal

The Journal of Clinical Endocrinology amp Metabolism 2014 Mar99(3)723-32

2A Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton Anti-Muumlllerian hormone poor assay reproducibility in a large

cohort of subjects suggests sample instability Human Reproduction 2012 Oct

27(10) 3085-91

2B Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton Human Reproduction Dec2012 Vol 27 Issue 12 p3641

6

Conference presentations

1 O Rustamov S Roberts C Fitzgerald

Ovarian endometrioma is associated with increased AMH levels

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2014 Munich

Poster Presentation

2 O Rustamov M Krishnan R Mathur S Roberts C Fitzgerald

The effect of BMI to the ovarian reserve

Annual Meeting of British Fertility Society January 2014 Sheffield

Oral presentation Dr O Rustamov

3 M Krishnan O Rustamov R Mathur S Roberts C Fitzgerald

The effect of the ethnicity to the ovarian reserve

Annual Meeting of British Fertility Society January 2014 Sheffield

Oral Presentation Dr M Krishnan

4 O Rustamov M Krishnan S Roberts C Fitzgerald

Reproductive surgery and ovarian reserve

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2013 London

Oral presentation Dr O Rustamov

5 C Fitzgerald O Rustamov P Pemberton A Smith A Yates M Krishnan

R Russell L Nardo SRoberts

AMH assays A review of the literature on assay method comparability

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2013 London

Oral presentation Dr C Fitzgerald

6 M Krishnan O Rustamov R Russell C Fitzgerald S Roberts

The role of the ethnicity and the body weight in determination of AMH levels

in infertile women

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2013 London

7

Poster presentation

7 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton

AMH Gen II assay - can we believe the measurements

8th Biennial Conference of UK Fertility Societies January 2013 Liverpool

Poster presentation

8 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton

Old and new AMH assays Can we rely on current conversion factor

8th Biennial Conference of UK Fertility Societies January 2013 Liverpool

Poster presentation

9 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton

Random AMH measurement is not reproducible

8th Biennial Conference of UK Fertility Societies January 2013 Liverpool

Poster presentation

10 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton

The reproducibility of serum Anti-Muumlllerian hormone AMH Gen II assay

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2012 Istanbul

Oral Presentation Dr O Rustamov

8

GENERAL INTRODUCTION

AND LITERATURE REVIEW

1

9

CONTENTS I LITERATURE REVIEWhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip10 GENERAL BACKGROUNDhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip10

1 OVARIAN RESERVEhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip12 11 Primordial Follicle Assemblyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip13 12 Oocyte recruitmenthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip14 13 Theory of neo-oogenesishelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip15 2 MARKERS OF OVARIAN RESERVEhelliphelliphelliphelliphelliphelliphelliphelliphelliphellip16 21 Ovarian reserve markers with limited clinical valuehelliphelliphelliphelliphellip16 213 Inhibin Bhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip16 214 Basal oestradiolhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip17 215 Dynamic testshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip17 216 Ovarian volumehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip18 22 Ovarian reserve markers in routine clinical usehelliphelliphelliphelliphelliphelliphellip18 221 Chronological agehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip18 222 Basal FSHhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip18 223 Antral follicle counthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip19 3 ANTI-MUumlLLERIAN HORMONEhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip20 31 Biology of anti-Muumlllerian hormonehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip20 311 The role of AMH in the ovaryhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip21 312 AMH in women with polycystic ovary syndromehelliphelliphelliphelliphellip22 32 AMH Assayhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip23 33 Variability of AMH measurementshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip24 34 Role of AMH in assessment of ovarian reservehelliphelliphelliphelliphelliphellip25 341 Prediction of poor and excessive ovarian response in IVFhelliphellip25 342 Prediction of live birth in cycles of IVFhelliphelliphelliphelliphelliphelliphelliphelliphellip26

3 5 Role of AMH in ovarian stimulation for cycles of IVFhelliphelliphelliphellip26

4 MULTIVARIATE TESTShelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip27

5 SUMMARYhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip28 II GENERAL INTRODUCTIONhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip29 REFERENCEShelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip31

10

I LITERATURE REVIEW GENERAL BACKGROUND

Infertility is a disease of the reproductive system defined by the failure to

achieve a pregnancy after 12 months of regular unprotected sexual intercourse

although the criteria for the duration vary between different countries (NICE

2013) Worldwide prevalence of infertility estimated to be around 724 million

couples and around 40 million of those seek medical care (Hull et al 1985) In

the UK 15 couples present with infertility with an annual incidence of 12

couples per 1000 general population (Scott et al 2009) The main causes of

infertility are tubal disease ovulatory disorders male factor and poor ovarian

reserve In a third of couples the cause of failure to achieve pregnancy is not

established which is known as unexplained infertility (NICE 2013) Effective

treatment options include improving lifestyle factors medical andor surgical

treatment of underlying pathology induction of ovulation and Assisted

Reproductive Technology (ART) Assisted Reproduction consist of

intrauterine insemination (IUI) and in vitro fertilisation (IVF) cycles with or

without introcytoplasmic sperm injection (ICSI) as well as treatment involving

donated gametes It is estimated that 75 of infertile couples presenting at

primary care centres in the UK are referred to fertility specialists based at

secondary or tertiary care centres and nearly 50 of those are subsequently

offered IVFICSI treatment (Scott et al 2009) This is supported by figures of

Human Fertility and Embryology Authority (HFEA) which indicates more

than 50000 IVF treatment cycles are performed in the UK annually (HFEA

2008)

An IVF treatment cycle involves a) pituitary down regulation b)

controlled ovarian stimulation c) oocyte recovery c) in vitro fertilisation of eggs

with sperm d) transfer of resulting embryo(s) back to uterus and c) luteal

phase support (NICE 2013) Prevention of premature surge of luteinising

hormone during controlled ovarian stimulation (COS) is achieved by pituitary

down regulation using either preparations of gonadotrophin releasing hormone

agonist which is widely known as ldquoAgonist cyclerdquo or gonadotrophin releasing

hormone antagonist which is known ldquoAntagonist cyclerdquo (Figure 1 and 2)

Controlled ovarian stimulation involves administration of gonadotrophins to

encourage the development of supernumerary preovulatory follicles followed

by administration of exogenous human chorionic gonadotropin (hCG) or

11

recombinant luteinising hormone (rLH) to assist in maturation of oocytes 34-

36 hours prior to egg collection which is usually conducted with guidance of

transvaginal ultrasound scanning Subject to sperm parameters the fertilisation

of oocytes is conducted by in vitro insemination or intracytoplasmic sperm

injection The resulting embryo(s) are cultured under strict laboratory

conditions and undergo regular qualitative and quantitative assessments before

transferring the best quality embryo(s) back into uterus during its cleavage

(Day 2 or Day 3) or blastocyst (Day 5 or Day 6) stage of development In

natural menstrual cycles under the influence of HCG progesterone secreted

by the ovarian corpus luteum ensures proliferative changes in the endometrium

providing the optimal environment for implantation of embryo(s) (van der

Linden et al 2011) However in IVF treatment cycles owing to pituitary down

regulation and lack of HCG progesterone levels are not in sufficiently high

concentration to ensure an adequate endometrial receptivity and therefore

exogenous analogues of this hormone is administered following transfer of

embryo(s) This is called ldquoluteal phase supportrdquo and in patients with viable

pregnancy usually lasts till 12th week of gestation when placenta starts

producing progesterone in sufficient quantities (van der Linden et al 2011)

In IVF programmes the ldquosuccessrdquo of the treatment often defined as

achieving a live birth following IVF cycle and expressed using Live Birth Rate

(LBR) In general success in IVF predominantly determined by womanrsquos age

cause(s) of infertility ovarian reserve previous reproductive history and

lifestyle factors (NICE 2013 Taylor 2003 Lintsen et al 2005) However

effectiveness of medical interventions as well as the quality of care play

important role in determining the outcome of IVF treatment This is evident

from significant variation in live birth rates among fertility clinics given for

instance in the UK LBR for women younger than 35 years of age after IVF

cycles varies from 15 to 61 (HFEA 2008 HFEA 2007) The provision of

effective interventions in both clinical and laboratory aspects of the care

appears to be the key in achieving high success rates Identification of patients

with sufficient ovarian reserve who benefit from IVF cycles followed by

providing optimal ovarian stimulation regimens may be useful in improving the

outcomes of IVF programmes According to HFEA data around 12 of IVF

cycles are cancelled due to poor or excessive ovarian response (Kurinczuk et al

2010) Availability of reliable markers for assessment of ovarian reserve and

tailoring ovarian stimulation regimens to the need of each individual patient

12

may improve selection of patients with sufficient ovarian reserve and reduce

the rate of cycle cancellation consequently improving the success of IVF

cycles (Yates et al 2011)

Assessment of ovarian reserve can be achieved using various biomarkers

and four of those are currently used by most clinics womanrsquos chronological

age (Age) serum follicle stimulating hormone (FSH) antral follicle count

(AFC) and serum anti-Muumlllerian hormone (AMH) More recently AMH has

been a focus of interest given it is the only available endocrine marker that is

suitable for direct assessment of the activity of ovarian follicles in their non-

cyclical stage development providing a window to FSH independent phase of

follicular recruitment Furthermore it appears to be reliable biomarker for a)

both the assessment of ovarian reserve and the optimisation of ovarian

stimulation regimens (Yates et al 2011 La Marca et al 2009) b) screening and

diagnosis of polycystic ovarian syndrome (PCOS) (Cook et al 2002) c)

monitoring of disease activity in women with a history of granulosa cell

tumours (Lane et al 1999) d) prediction of the age of diminished fertility and

the menopause (van Disseldorp et al 2008 Broer et al 2011) and finally (e)

assessment of the long term effect of chemotherapy on ovarian reserve

(Anderson 2011)

In this review I first discuss current knowledge on factors that

determine ovarian reserve including the formation and loss of oocyte pool

Then characteristics of the markers of ovarian reserve are reviewed Finally I

examine current understanding of biology of anti-Muumlllerian hormone and its

role in management of infertility

1 OVARIAN RESERVE

It is important to recognize that there is no universal definition for the

term ldquoovarian reserverdquo and the term can have various meanings depending on

the context in which it is used For instance the scientific literature describing

the biology of ovarian reserve usually refers to ldquothe total number of remaining

oocytes in the ovaries which consists of the number of resting primordial

follicles and growing primary pre-antral and antral folliclesrdquo (Gleicher et al

2011) In contrast the use of the term in the context of clinical studies may

refer to ldquoclinically measurable ovarian reserve established using available

biomarkers of ovarian reserverdquo For the purpose of clarity in this thesis the

13

term ldquoovarian reserverdquo refers to clinically measurable ovarian reserve whilst

true biological ovarian reserve will be termed ldquobiological ovarian reserverdquo

Recent studies have demonstrated that ovarian reserve is highly variable

between women due to the variation in the size of initial ovarian reserve at

birth as well as the rate of loss of ovarian reserve thereafter (Wallace et al

2010) Interestingly the rate of oocyte loss appears to be mainly determined by

the initial ovarian reserve which is believed to be facilitated by most potent

ovarian growth factor anti-Muumlllerian hormone Similarly the size of the initial

ovarian reserve is mainly underpinned by the rate of primordial follicle

assembly in the embryo which is also regulated by AMH Both primordial

follicle assembly and the rate of oocyte loss appear to be primarily under the

influence of genetic factors although developmental and environmental factors

are also believed to play a role (Nilsson et al 2010 Shuh-Huerta et al 2012)

11 Primordial follicle assembly

The process of assembly of primordial follicles in the female embryo

spans from the early embryonic to the early postnatal period and formation of

primordial follicles consists of following stages 1) primordial germ cell (PGC)

2) oogonia 3) primary oocyte and 4) primordial follicle In the human female

fetus around a hundred cells that differentiated from extra-embryonic

ectoderm form early PGCs on the yolk sac and migrate via hindgut to gonadal

ridges during 4th - 6th weeks of gestation (MC et al 1953 Donovan 1998) Once

arrived to the gonadal ridges these cells are called primary oogonia which

consequently undergo several rounds of mitotic division during 6th - 28th weeks

of gestation Interestingly the numbers of oogonia reach as high as six million

during its highest rate of mitotic division at around 20 weeks of gestation

Following the last round of mitotic division oogonia enter meiosis which

marks their new stage of development-primary oocyte Formation of

primordial follicles starts as early as at 8th week of gestation and is characterised

by meiosis of primary oocyte that arrest in diplotyne stage and surrounding of

the oocyte by somatic granulosa cell (Baker et al 1963 Maheshwari and Fowler

2010) Indeed the primordial follicle is the cardinal unit of the biological

ovarian reserve and therefore the rate of formation of primordial follicles is the

main determinant of initial biological ovarian reserve at birth

Interestingly the process of loss of oogonia and oocytes which is also

one of the main determinants of the initial ovarian reserve takes place

14

throughout the period of follicle assembly The formation of the granulosa cell

layer around the oocyte prevents the oocyte from subsequent atresia The

oocyte enveloped in a single layer of granulosa cells which is also known as

primordial follicle remains quiescent until recruitment of the follicle for

growth which may not take place for a number of decades after the formation

of a particular primordial follicle (Skinner 2005 Maheshwari and Fowler 2010)

12 Oocyte recruitment

Follicle growth in women consists of two stages a) the initial non-cyclical

recruitment of primordial follicles and the formation of a primary and a pre-

antral follicles and b) cyclical development of antral follicles with subsequent

selection of usually a single dominant follicle The initial recruitment of

primordial follicles is continuous non-cyclical process that starts as early as

from 18-20 weeks of gestation and lasts till the depletion of follicle pool which

later results in the menopause (McGee and Hsueh 2000) Transformation of

flat granulosa cells into cuboidal cells increases the diameter of the oocyte and

the formation of zona pellicuda completes the stage of formation of a primary

follicle During pre-antral stage oocytes increase in diameter and mitotic

division of granulose cells create a new layer of cells-theca cells The

mechanism of initial recruitment of oocytes is not well understood but it is

clear that the process is independent of influence of pituitary gonadotrophins

and appears to be governed by the genetically pre-programmed interaction of

the oocyte with local growth factors the most important of which appears to

be anti-Muumlllerian hormone and cytokines (McGee and Hsueh 2000)

The cyclical phase of development of oocytes is characterised by the

transformation of secondary follicle into antral follicle and subsequent growth

of antral follicles into pre-ovulatory stages In general the process of cyclic

recruitment starts from puberty under the influence of rising levels of pituitary

follicular stimulating hormone (FSH) During the antral stage oocyte increases

in size even further and the formation of a fluid filled space in follicle is

observed Under the influence of FSH luteinising hormone (LH) and local

growth factorsselection of a single dominant follicle occurs which followsby an

ovulation (McGee and Hsueh 2000)

Oocyte loss is a continuous process and occurs due to atresia of oocytes

during primary secondary and antral stages of development The rate of

oocyte loss appears to increase until the age of around 14 and declines

15

thereafter until the age of the menopause when around 1000 primordial

follicles remain (Hansen et al 2008 Oktem and Oktayl 2008) Furthermore by

the age of 30 years the average age at which women of western societies plan

to start a family around 90 of initial primordial follicles are lost which

illustrates that formation and maintenance of ovarian reserve is wasteful

process in humans (ONS 2012 Wallace and Kelsey 2010) As mentioned

above there is a wide individual variation in both sizes of initial primordial

follicular pool and the rate of oocyte loss which explains variation in the

reproductive lifespan in women Evidently the number of primordial follicles

at birth ranges between around 35000 to 25 million per ovary and similarly

the rate of oocyte loss during its peak at 14 years of age may range between

100 to 7500 primordial follicles per month which is believed to be inversely

proportional to initial size of primordial follicle pool (Wallace and Kelsey

2010)

13 Theory of neo-oogenesis

The traditional view of oogenesis states that the process of the creation

and the mitotic division of oogonia with subsequent formation of primordial

follicles takes place only during embryonic and foetal life (Zuckerman 1951)

According to this central theory of mammalian reproductive biology females

are born with a certain number of germ cells that is gradually lost but not

renewed during postnatal period However Johnson et al have recently

challenged this view and reported that adult mammalian ovary may possesses

mitotically active germ cells that continuously replenish the primordial follicle

pool (Johnson et al 2004) The group reported that ovaries of juvenile and

young adult mice contained large ovoid cells which resemble germ cells of

foetal mouse ovaries Interestingly immunohistochemical staining for a gene

which is expressed exclusively in germ cells have been reported to have

confirmed that these large ovoid cells were of germline lineage Furthermore

application of a mitotic germ cell toxicant busulphan appeared to have

eliminated primordial follicle reserve by early adulthood but did not induce

atresia suggesting the presence of proliferative germ cells in postnatal mouse

ovary (Johnson et al 2004 Bazer 2004) The study has generated enormous

amount of interest as well as debate among reproductive biologists (Notarianni

2011) Some other groups have also reported an evidence of postnatal

oogenesis (Pacchiarott et al 2010 Zou et al 2009 Bukovsky et al 2004)) while

16

others do not support the theory (Bristol-Gould et al 2006 Byskov et al 2005

Begum et al 2008) Furthermore some authors argued that adult mouse

germline stem cells exist and remain quiescent in physiologic conditions and

neo-oogenesis occurs only in response to ovotoxic damage (Tilly et al 2007 De

Felici 2010) Although consensus has yet to emerge to date there is no

conclusive evidence on validity of theory of neo-oogenesis

2 MARKERS FOR ASSESMENT OF OVARIAN RESERVE

Biological ovarian reserve is defined as the number of primordial and

growing follicles left in the ovary at any given time and therefore only

counting the number of primordial follicles by histological assessment can

accurately determine ovarian reserve which is clearly not feasible in clinical

setting However ovarian reserve can be estimated using various biomarkers

dynamic clinical tests and implied from the outcomes of ART cycles

Although a wide range of clinical (age ovarian response in previous IVF

cycles) biochemical (basal FSH Inhibin B basal oestradiol AMH) ultrasound

(ovarian volume antral follicle count (AFC)) and dynamic (clomiphene

challenge test exogenous FSH ovarian reserve test GnRH analogue

stimulating test) tests of ovarian reserve exist only a few of the markers are

reliable and practical enough to be of use in routine clinical practice In this

chapter first I discuss the research evidence on the assessment of the markers

andor tests of ovarian reserve that have limited clinical value Then I

evaluated more reliable markers that are in routine clinical use Age FSH

AFC and combination of these markers in multivariable tests Finally I

conducted detailed review of biology of AMH and the role AMH measurement

in the management of infertility

21 Ovarian reserve markers with limited clinical value

211 Inhibin B

Inhibins are members of TGFβ family and expressed in granulosa cells

of growing follicles Principal role of inhibins is thought to be the negative

feedback regulation of pituitary FSH secretion and therefore the serum level of

circulating hormone is believed to reflect the state of folliculogenesis

17

Consequently several groups have studied the role of serum Inhibin β in the

assessment of ovarian reserve Although initial reports were encouraging

(Seifer et al 1997) more robust studies demonstrated that serum Inhibin β was

less reliable than chronological age or basal FSH (Creus et al 2000 Urbancsek

2005) The systematic review of nine studies demonstrated that accuracy of the

Inhibin β test for predicting poor ovarian response and non-pregnancy in IVF

cycles was modest even at a very low threshold level (Broekmans et al 2006)

Therefore it is recommended that inhibin β at best can be used as only

screening test in the fertility centers where other more reliable markers are not

available (Broekmans et al 2006)

212 Basal oestradiol

Some studies suggested that elevated basal oestradiol levels indicate low

ovarian reserve and are associated with poor fertility prognosis (Johannes et al

1998 Licciardi and Rosenwaks 1995) Johannes et al demonstrated basal

oestradiol in conjunction with serum FSH is more reliable than serum FSH

alone in prediction of cycle cancellation due to the poor response in IVF cycles

(Johannes et al 1998) However there are no published data on the comparison

of basal oestradiol to more reliable markers such as AMH or antral follicle

count (AFC) Moreover a recent systematic review has demonstrated that

basal oestradiol has very low predictive value for poor response and has no

discriminatory power for accuracy of non-pregnancy prediction (Broekmans et

al 2006)

213 Dynamic tests of ovarian reserve

The dynamic tests of ovarian reserve are based on assessment of ovarian

response by measuring serum FSH and oestradiol levels following

administration of exogenous stimulation The following tests are reported in

literature Clomiphene Citrate Challenge Test (CCCT) Exogenous FSH

Ovarian Reserve Test (EFORT) and GnRH agonist stimulation test A recent

systematic review and meta-analysis on the accuracy of these tests showed that

none of them can adequately predict poor response or non-pregnancy in IVF

cycles and therefore are not recommended for use in routine clinical practice

(Maheshwari et al 2009)

18

214 Ovarian volume

There is some evidence that increased age is associated with decreased

ovarian volume and women with smaller ovaries are more likely to have

cancellation of their IVF cycles due to poor ovarian response (Syrop et al 1995

Syrop et al 1999 Templeton 1995) However a meta-analysis of the published

studies on the accuracy of ovarian volume as a predictor of poor response and

non-pregnancy in IVF cycles failed to demonstrate clinical usefulness of the

test and suggested the test is not reliable enough for use in a routine clinical

practice (Broekmans et al 2006)

22 Ovarian reserve markers in routine clinical use

221 Chronological age

Owing to the biological age-related decline of the quantity and arguably

the quality of oocytes the chronological age can be used as a marker of ovarian

reserve Studies have demonstrated that ovarian reserve (Wallace and Kelsey

2010 Kelsey 2011) natural fecundity (Islam et al 1989 and outcomes of ART

(Templeton et al 1996 van Kooij et al 1996) decline significantly from age of

35 when it is believed the ovarian reserve undergoes accelerated decline

Although there is a strong association between chronological age and reduction

in fertility evidently there is a significant variation in age-related ovarian

reserve indicating chronological age alone may not be sufficient to estimate the

individual womanrsquos ovarian reserve reliably (Broekmans et al 2006)

222 Basal FSH

Basal FSH was one of the first endocrine markers introduced in ART

programs and is still utilized in many fertility clinics albeit in conjunction with

other markers which are considered more reliable (Creus et al 2000) Secretion

of FSH is largely governed by the negative feedback effect of steroid

hormones primarily oestradiol and inhibins which are expressed in granulosa

cells of growing ovarian follicles Consequently decreased or diminished

recruitment of ovarian follicles is associated increased serum FSH

measurements and high particularly very high basal FSH reading is considered

as a good marker of very low or diminished ovarian reserve (Abdalla et al

2006) However unlike some other markers FSH measurements do not

appear to have discriminatory power for categorisation of patients to various

19

bands of ovarian reserve Given between-patient variability FSH measurement

(CV 30) is similar to its within-patient variability (27) stratification of

patients to various ranges of ovarian reserve does not appear to be feasible

(Rustamov et al 2011) Indeed a recent systematic review of 37 studies on the

prediction of poor response and non-pregnancy in IVF cycle has concluded

that basal FSH is an adequate test at very high threshold levels and therefore

has limited value in modern ART programs (Broekmans et al 2006)

223 Antral follicle count

Antral follicle count estimation involves ultrasound assessment of

ovaries between 2nd and 4th day of menstrual period and counting ldquofolliclesrdquo

which corresponds to antral stage of folliculogenesis (Broekmans et al 2010)

The test provides direct quantitative assessment of growing follicles and is

known as one of the most reliable markers of ovarian reserve (Broekmans et al

2006) AFC measurement has been reported as having a similar sensitivity and

specificity to AMH in prediction of poor and excessive ovarian response in

IVF cycles (Broekmans et al 2006 Broer et al 2010 Jayaprakasan et al 2010)

Given AFC measurement is available instantly and allows patients to be

counseled immediately the test eliminates the need for an additional patient

visit prior to IVF cycle However AFC is normally performed only in the early

follicular phase of the menstrual cycle given most published data on

measurement of AFC are based on studies that assessed antral follicles during

this stage of the cycle (Broekmans et al 2010a) Interestingly more recent

studies suggest that variability of AFC during menstrual cycle is small

particularly when follicles between 2-6mm are counted and therefore

assessment of AFC without account for the day of menstrual cycle may be

feasible (Deb et al 2013)

One of the main drawbacks of AFC is that the cut off levels for size of

counted follicles remains to be standardised (Broekmans 2010b) Initially

follicles of 2-10mm were introduced as the range for AFC and many studies

were based on this cut off Later counting follicles of 2-6mm was reported to

provide most accurate assessment of ovarian reserve (Jayaprakasan et al 2010b

Haadsma et al 2007) and therefore some newer studies are based on AFC

measurements that used this criterion Consequently direct comparison of the

outcomes of various studies on assessment of AFC requires careful analysis

20

3 ANTI-MUumlLLERIAN HORMONE

31 Biology of Anti-Muumlllerian hormone

AMH is a member of transforming growth factor β superfamily which

was discovered by Jost et al in 1947 and was initially known for its is role in

regression of Muumlllerian ducts in sex differentiation of the male embryo In

women AMH is believed to be solely produced by ovaries and expressed in

granulosa cells of growing follicles of 2-6 mm in size which corresponds to

primary pre-antral and early antral stage of follicular development Although

there has been a report of expression of AMH in endometrial cells to date

there is no other published evidence that supports this finding (Wang et al

2009) Indeed studies that evaluated half-life of AMH in serum have

demonstrated that in women who had bilateral salpingo-oopherectomy AMH

becomes undetectable within 3-5 days of following surgery suggesting ovaries

are the only source of secretion of AMH in appreciable quantity (La Marca et

al 2005b) Anti-Muumlllerian hormone is a dimeric glycoprotein which is

composed of a long N-terminus and short C-terminus and was believed to be

secreted in serum only in this dimeric form (AMH-N C)

Like other members of TGF-β family which includes inhibins activins

bone morphogenic proteins (BMPs) and growth and differentiation factors

(Massague et al 1990) AMH binds to two type of serinethreonine kinase

receptors referred to as type I and type II In order to activate AMH signaling

pathway both receptors have to form a heteromeric complex When AMH

binds to the type II (AMHR-II) receptor (Massague et al 2000) this will

phosphorylate and activate a type I receptor (ALK2 -3 andor -6) which

subsequently activates the SMAD pathway through phosphorylation of

SMAD 1 5 andor 8 These activated SMADs interact with SMAD4 and

translocate to the nucleus regulating the expression of different genes

inhibiting the recruitment of primordial follicles and reducing FSH sensitivity

in growing follicles In addition AMH receptors as well as the other members

of TGF-β family can activate MAPK and PI3KAKT pathways

Studies on AMHR II-deficient male mice demonstrated lack of

regression of Muumlllerian ducts suggesting that type II receptor is essential in

AMH signaling (Mishina et al 1996) Similarly Type I receptors which includes

three members of activin receptor-like kinase (ALK2 ALK3 and ALK6) also

appear to play an important role in the regression of Muumlllerian ducts although

21

the role of ALK 6 in AMH signaling appears not to be crucial (Visser 2003

Clarke et al 2001) The signal transduction pathway of AMH in the ovary is

largely not understood In postnatal mice ovary AMHR-II receptor was

expressed in both granulosa and theca cells of pre-antral and antral follicles

(Visser 2003) AMH type I receptors ALK 2 and ALK 3 is expressed in foetal

as well as adult mouse ovary while ALK 6 is expressed in only adult ovary

(Visser 2003)

311 The role of AMH in the ovary

In the mammalian ovary the role of AMH appears to be one of a

regulation of size of the primordial follicle pool by its inhibitory effect on the

formation as well as the growth of primordial follicles (Nilsson et al 2011) In

the embryonic mouse ovary AMH inhibits the initiation of the assembly of

follicles when the process of apoptosis of the majority of oocytes is observed

(Nilsson et al 2011) Consequently AMH reduces the rate of oocyte loss

which plays an important role in the determination of the size of initial follicle

pool Similarly in the adult mouse ovary AMH plays a central role in

maintaining the follicle pool AMH inhibits both the processes of the initial

(non-cyclical) recruitment of primordial follicles and subsequent FSH-

dependent cyclical growth of antral follicles (Figure 3) Inhibition of the initial

recruitment of a new cohort of follicles is believed to be achieved by a

paracrine negative feedback effect of the rising levels of AMH secreted from

already recruited growing follicles (Durlinger et al 1999) Durlinger et al

compared the complete follicle population of AMHnull mice and wild type

mice of different ages of 25 days 4 months old and 13 months old and found

that the ovaries of 25 day and 4 months old AMHnull females contained

significantly higher number of growing pre-antral and antral follicles but

significantly fewer primordial follicles compared to wild-type females

(Durlinger et al 1999) Interestingly almost no primordial follicles were

detected in 13 months old AMHnull mice ovaries suggesting AMH is a potent

inhibitor of the recruitment of primordial follicles and in the absence of AMH

ovaries undergo premature depletion of primordial follicles due to an

accelerated recruitment Subsequent study conducted by the group

demonstrated that in addition to its inhibitory effect to the resting follicles

AMH also suppresses the development of the growing follicles (Durlinger et al

2001 Durlinger et al 2002 Themmen 2005) It appears that AMH inhibits

22

FSH-induced follicle growth by reducing the sensitivity of growing follicles to

FSH which has been confirmed by in vivo as well as in vitro studies (Durlinger

et al 1999 Durlinger et al 2001) In the initial study the group observed that

despite lower levels of serum FSH concentration ovaries of AMHnull mice

contained more growing follicles than that of their wild-type littermates which

has been supported by the findings of subsequent in vitro study (Durlinger et al

1999) Addition of AMH to the culture inhibited FSH-induced follicle growth

of pre-antral mouse follicles due to reduction in granulosa cell proliferation

(Durlinger et al 2001)

In the human embryo the expression of AMH commences in the late

foetal life and can be detected only from 36 weeks of gestation (Rajpert-De et

al 1999 Lee et al 1996) Following a small decline in first two years of life

AMH levels gradually increase to peak at (mean 5 ngml) around age of 24

years In line with the pattern of oocyte loss serum hormone levels gradually

decline with increasing age and become undetectable around 5 years prior to

menopause (Kelsey et al 2011 Nelson et al 2011)

It has been suggested that anti-Muumlllerian hormone plays a central role in

determining the pace of recruitment of primordial follicles hence maintaining

the primordial follicle pool of postnatal mammalian ovary Consequently a

reduction in the concentration of circulating AMH signals the exhaustion of

the primordial follicle pool and the decline of ovarian function

312 AMH in women with polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) endocrine abnormality characterised

by increased ovarian androgen secretion infrequent ovulation and the

appearance of ldquopolycysticrdquo ovaries on ultrasound scan (Dunaif 1997 Homburg

et al 1993) It is the commonest endocrine abnormality in women of

reproductive age and affects around 15-20 of women PCOS is also one of

the main causes of anovulation and subsequent sub-fertility (Webber et al

2003) Although the role of anti-Muumlllerian hormone in the development of

PCOS is not fully understood it is becoming increasingly evident that the

hormone plays an important role in its pathogenesis (Pehlivanov et al 2011)

There is a strong association between serum AMH levels and PCOS and it

appears that women diagnosed with PCOS have two to three fold higher

serum AMH concentration compared to normo-ovulatory women (Cook et al

2002 Pigny et al 2003) Similarly women with PCOS are found to have

23

significantly higher number antral follicles Interestingly the expression of

AMH in granulosa cells of follicles were found to be 75 times higher in women

with PCOS compared to those without a the disease suggesting increased

serum AMH in PCOS may be due to increased secretion of hormone per

follicle rather than due to an increased number of antral follicles (Pellat et al

2007) High AMH concentrations may act as the main facilitator of abnormal

folliculogenesis in PCOS given the follicles appear to arrest when they reach

an antral stage (2-6mm) of development (Rajpert-De et al 1999) Indeed the

studies of Durlinger et al have demonstrated that AMH inhibits selection of

dominant follicle when follicles reach antral stage of development (Durlinger et

al 2001) Serum AMH levels appear to decrease with treatment of PCOS

which may play important role in restoration of ovulatory cycles Studies have

reported a significant reduction in serum concentration of AMH following

treatment of PCOS with metformin and laparoscopic ovarian diathermy (Falbo

et al 2010 Amer et al 2009 Elmashad 2011) Similarly reduction of BMI

following intensified endurance exercise training for treatment of PCOS may

also lead to a significant reduction in serum AMH levels (Moran et al 2011)

This suggests that there is strong association between serum concentration of

AMH and abnormal folliculogenesis in PCOS and therefore understanding the

molecular mechanisms of this interaction should be one of the priorities of

future research

32 AMH Assays

Enzyme-linked immunosorbent assay specific for measurement of anti-

Muumlllerian hormone was first developed in 1990 and was recognised as a

significant step in the assessment of ovarian reserve (Hudson et al 1990)

Subsequently a number of non-commercial immunoassays were developed

which were mainly used in research settings (Lee et al 1996) Later Diagnostic

Systems Ltd (DSL) and Immunotech Beckman Coulter Ltd (IOT) introduced

two commercial immunoassays for the routine clinical assessment of ovarian

reserve which are known as ldquofirst generation AMH assaysrdquo (Nelson and La

Marca 2011) These assays employed two different antibodies against AMH

and used different standards for calibration providing non-comparable

measurements (Nelson and La Marca 2011) Consequently several studies

attempted to develop a reliable between-assay conversion factor which

interestingly revealed from five-fold higher with the IOT assay to assay

24

equivalence causing significant impact to reliability of AMH measurements and

interpretation of research findings (Hehenkamp et al 2006 Freour et al 2007

Bersinger et al 2007 Taieb et al 2008 Lee et al 2011)

Later the manufacturer of IOT assay (Beckmann Coulter Ltd)

consolidated the manufacturer of the DSL assay (Diagnostic Systems

Laboratories Inc) and introduced a new assay ldquoGen II AMH assayrdquo which is

only available commercial immunoassay in most countries including the UK

AMH Gen II assay was developed using the antibodies derived from first

generation DSL assay and calibrated using the standards used for IOT assay

and was believed to be considerably more stable compared to the first

generation immunoassays providing more reliable measurements (Kumar et al

2010 Nelson and La Marca 2011) The manufacturer as well as initial external

validation study recommended when compared to old DSL assay AMH Gen

II assay provides around 40 higher measurements and therefore previously

reported DSL-based clinical cut-off levels for estimation of ovarian reserve

should be increased by 40 in order to use Gen II-based AMH results (Kumar

et al 2010 Wallace et al 2011 Nelson and La Marca 2011)

33 Variability of AMH measurements

It is generally believed that AMH values do not change throughout the

menstrual cycle and early studies reported that variation in AMH

measurements between repeated measurements of same patient was negligible

(van Disseldorp et al 2010 La Marca 2010) On the basis of these studies

sampling at a random time in the menstrual cycle was introduced as a method

for measurement of AMH in routine clinical practice However the

methodologies of some of these studies do not appear to be robust enough to

reliably estimate sample-to-sample variability of AMH which is mainly due to

small sample sizes (Rustamov et al 2011) Consequently in a recent study we

assessed sample-to-sample variability of AMH using DSL assay and found that

within-subject coefficient of variation (CV) of AMH between samples were as

high as 28 which cannot be attributed to any patient or cycle characteristics

(Rustamov et al 2011) Although there is no consensus in the causes of this

observed variability in AMH measurements we believe it is largely attributable

to instability of AMH samples given initial recruitment of primordial follicles

and growth of AMH producing pre-antral and antral follicles are continuous

process and therefore the true biological variation between samples is unlikely

25

to be high However given the importance of establishing true variability of

AMH in both understanding of the biology of hormone and clinical

application of the test future studies should be conducted to establish the

source of variability in the clinical samples

3 4 The role of AMH in the assessment of ovarian reserve

341 Prediction of poor and excessive ovarian response in cycles of

IVF

A number of studies have assessed the role of AMH in the prediction of

poor ovarian response in IVF cycles using first generation AMH assays and

found that AMH and AFC were the best predictors of poor ovarian response

compared to other markers of ovarian reserve Nardo et al showed that the

predictive value of AMH in receiver operating characteristic curve (ROC)

analysis was similar to (AUC 088) that of AFC (AUC 081) and found that

AMH cut offs of gt375 ngmL and lt10 ngmL would have modest

sensitivity and specificity in predicting the extremes of response (Nardo et al

2009) These findings were largely supported by subsequent prospective studies

and a systematic review (Nelson et al 2007 Jayaprakasan et al 2010 Broer et al

2011) Similarly comparison of chronological age basal FSH ovarian volume

AFC and AMH found that only AMH (AUC 090) and AFC (AUC 093) were

reliable predictors of poor ovarian response in cycles of IVF Subsequent

combination of the effect of AMH and AFC using multivariable regression

analysis did not improve the level of prediction of poor ovarian response

significantly (AUC 094) suggesting both AMH and AFC can be used as

independent markers (Jayaprakasan et al 2010)

Similarly most studies agree that AMH and AFC are the best predictors

of excessive ovarian response and ovarian hyperstimulation syndrome (OHSS)

compared to other clinical endocrine and ultrasound markers (Nardo et al

2009 Nelson et al 2007) Broer et al compared these two tests in systematic

review of 14 studies and reported that the summary estimates of the sensitivity

and the specificity for AMH were 82 and 76 respectively and for AFC 82

and 80 respectively (Broer et al 2011) Consequently the study concluded

that AMH and AFC were equally predictive and the difference in the predictive

value between the tests was not statistically significant

26

342 Prediction of live birth rate (LBR) in cycles of IVF

Lee at al reported that AMH and chronological age were more accurate

than basal FSH AFC BMI and causes of infertility in the prediction of live

birth rate (Lee et al 2009) Similarly La Marca et al suggested that odds of live

birth could be reliably predicted using AMH (La Marca et al 2010b) although

subsequent review of the study questioned strength of the evidence (Loh and

Maheshwari 2011)

A study conducted by Nelson et al found that higher AMH levels had

stronger association with increased live birth rate compared to age and FSH

(Nelson et al 2007) However the study also suggested that this association

was mainly confined in the women with low AMH levels and there was no

additional increase in live birth in women with AMH levels of higher than 710

pmolL This may suggest that achieving a live birth may be under the

influence of number of other factors and that markers of ovarian reserve alone

may not be able predict this outcome reliably

35 The role of AMH in individualisation of ovarian stimulation in

IVF cycles

Prediction of ovarian response to the stimulation of ovaries in cycles of

IVF plays an important role in the counseling of couples undergoing treatment

programmes and hence many clinical studies on AMH have focused on the

prognostic value of AMH measurements However data on using AMH as a

tool for improving the clinical outcomes in IVF cycles appear to be lacking

considering AMH may be useful tool in tailoring treatment strategies to an

individual patientrsquos ovarian reserve Unlike most other markers AMH has

discriminatory power in determining various degrees of ovarian reserve due to

significantly higher between patient (CV 94) variability compared to its

within-patient (CV 28) variation (Rustamov et al 2011) which allows

stratification of patients into various degrees of (eg low normal high) ovarian

reserve Subsequently most optimal ovarian stimulation protocol may be

established for each band of ovarian reserve Consequently reference ranges

on the basis of distribution of AMH in infertile women were developed which

were subsequently adopted by fertility clinics for a tailoring the mode of

27

ovarian stimulation and daily dose of gonadotrophins in IVF (The Doctors

Laboratory 2008 However currently available clinical reference ranges are

based on the first generation DSL assay and may not be reliably convertible to

currently available Gen II assay measurements (Wallace et al 2011) Indeed the

findings of the studies on comparability of the first generation AMH assays

suggest that establishing a reliable between assay conversion factor between

AMH assays may not be straightforward Furthermore the reference ranges

appear to reflect the distribution of AMH measurements within a specific

population and may therefore not be directly applicable for the prediction of

response to ovarian stimulation in IVF patients (The Doctors Laboratory

2008)

More importantly despite lack of good quality evidence on the

effectiveness of AMH-tailored ovarian stimulation protocols a number of

fertility clinics appear to have introduced various AMH-based COH protocols

in their IVF programs At present research evidence on AMH-tailored

ovarian stimulation in IVF is largely based on two retrospective studies

(Nelson et al 2009 Yates et al 2012) Both of these studies display considerable

methodological limitations including small sample size and centre-related or

period-related selection of their cohorts In this context AMH is used as a tool

for therapeutic intervention and therefore the research evidence should ideally

be derived from randomised controlled trials However recruitment of large

enough patients in IVF setting may take considerable time and resources In

the meantime given AMH-tailored ovarian stimulation has already been

introduced in clinical practice and there is urgent need for more reliable data

the studies with a larger cohorts and robust methodology should assess the role

of AMH in individualisation of ovarian stimulation in IVF treatment cycles

4 Multivariate models of assessment of ovarian reserve

In view of the fact there is not a single marker of ovarian reserve that

can accurately predict ovarian response various models for combination of

multiple ovarian markers have been developed (Verhagen et al 2008) A

number of studies reported that multivariate models are better predictors of

poor ovarian response in IVF compared to a single marker (Bancsi et al 2002

Balasch et al 1996 Creus et al 2000 Durmusoglu et al 2004) However a meta-

analysis showed that when compared to a single marker (AFC) multivariate

28

model has a similar accuracy in terms of prediction of poor ovarian response

(Verhagen et al 2008) In contrast a more recent study demonstrated that

multivariate score was superior to chronological age basal FSH or AFC alone

in predicting likelihood of poor ovarian response and clinical pregnancy

(Younis et al 2010) However the study did not include one of the most

reliable markers AMH in either arm necessitating further assessment of the

role of combined tests which include all reliable biomarkers

4 SUMMARY

During the last two decades a significant leap has been taken towards

understanding the biology of anti-Muumlllerian hormone and its role in female

reproduction (Durlinger et al 2002 Themmen et al 2005) Availability of

commercial AMH assays has resulted in significant increase in interest in the

role of the measurement of serum AMH in the assessment of ovarian reserve

which has been followed by the introduction of the test into routine clinical

practice (Nelson et al 2011) However more recent studies suggest that current

methodologies for the measurement of AMH may provide significant sampling

variability (Rustamov et al 2011) Furthermore the studies that compared first

generation commercial assay methods appear to provide non-reproducible

results suggesting there may be underlying issues with assay methodologies

(Lee et al 2011) Similarly despite lack of sufficient evidence in the role of

AMH in individualisation of ovarian stimulation protocols in IVF AMH-

tailored IVF protocols have been introduced in routine clinical practice of

many fertility clinics around the world

Consequently it appears that clinical application of AMH test has

surpassed the research evidence in some aspects of fertility treatment and

therefore future projects should be directed toward areas where gaps in

research evidence exist On the basis of the review of literature we believe that

evaluation of the performance of assay methods understanding the role of

AMH in assessment ovarian reserve and establishing its role in

individualisation of ovarian stimulation protocols should be research priority

29

II GENERAL INTRODUCTION

On the basis of the review of published literature I have identified that

the following areas of research on the clinical application of AMH in the

management of infertility requires further investigation 1) Within-patient

variability of measurement of AMH using Gen II assay method 2)

Establishment of clinically measurable determinants of AMH levels and 3) The

role of AMH in individualisation of ovarian stimulation in IVF treatment

cycles

In our previous study we estimated that there was significant sample-to-

sample variation (CV 28) in AMH measurements when the first generation

DSL assay was used (Rustamov et al 2011) The source of variability is likely to

be related to the assay method given that biological within-cycle variation of

AMH is believed to be small (La Marca et al 2006) Therefore assessment of

sample-to-sample variability of AMH using the newly introduced Gen II assay

which is believed to be significantly more stable and sensitive compared to that

of DSL assay should enable us to establish the measurement related variability

of AMH Furthermore given I am planning to use data from both DSL and

Gen II assays I need to establish between-assay conversion factor for these

assays using data on clinical samples

There appears to be a lack of good quality data on the effect of

ethnicity BMI causes of infertility reproductive history and reproductive

surgery on ovarian reserve Therefore I am planning to ascertain the role of

above factors on determination of ovarian reserve by analysing AMH

measurements of a large cohort of patients

There is a strong correlation between AMH and ovarian performance

in IVF treatment when conventional ovarian stimulation using GnRH agonist

regimens with a standard daily dose of gonadotrophins are used (Nelson et al

2007 Nardo et al 2007) Furthermore studies suggest tailoring the ovarian

stimulation protocols to AMH measurement may improve ovarian

performance and subsequently the success of IVF treatment (Nelson et al

2011 Yates et al 2012) However given methodologies of the published

studies the effectiveness of currently proposed AMH-tailored ovarian

stimulation protocols remains unknown Therefore I am planning to develop

individualised ovarian stimulation protocols by establishing the most optimal

mode of pituitary down regulation and starting dose of gonadotrophins for

30

each AMH cut-off bands using a robust research methodology However

development of individualised ovarian stimulation protocols on the basis of

retrospective data requires a reliable and validated database containing a large

number of observations In the IVF Department of St Maryrsquos Hospital we

have data on a large number of patients who underwent ovarian stimulation

following the introduction of AMH However the data on various aspects of

investigation and treatment of patients is stored in different clinical data

management systems and may not be easily linkable In addition it appears that

data on certain important variables (eg causes of infertility AFC) are available

only in the hospital records necessitating searching for data from the hospital

records of each patient Consequently I designed a project for building a

research database which will have comprehensive and validated datasets that

are necessary for investigation of the research questions of the MD

programme

In conclusion I am planning to conduct a series of studies to improve

the understanding of the role of AMH in the management of women with

infertility Specifically I am intending to evaluate 1) sample-to-sample variability

of Gen II AMH measurements 2) conversion factor between DSL and Gen II

assays in clinical samples 3) the effect of ethnicity BMI causes of infertility

endometriosis reproductive history and reproductive surgery to ovarian

reserve and explore AMH-tailored individualisation of ovarian stimulation in

IVF cycles

31

References

Abbeel E The Istanbul consensus workshop on embryo assessment proceedings of an expert meeting Human reproduction 2011 26 p 1270-83 Abdalla HT M Y Repeated testing of basal FSH levels has no predictive value for IVF outcome in women with elevated basal FSH Human reproduction 2006 21(1) p 171-4 Amer SA LT Ledger WL The value of measuring anti-Mullerian hormone in women with polycystic ovary syndrome undergoing laparoscopic ovarian diathermy Human reproduction 2009 24 p 2760-6 Anderson RA Pretreatment serum anti-Mullerian hormone predicts long-term ovarian function and bone mass after chemotherapy for early breast cancer J Clin Endocrinol Metab 2011961336-1343 Balaban B BD Calderoacuten G Catt J Conaghan J Cowan L Ebner T Gardner D Hardarson T Lundin K Cristina Magli M Mortimer D Mortimer S Munneacute S Royere D Scott L Smitz J Thornhill A van Blerkom J Van den Baker A quantitative and cytological study of germ cells in human ovaries Proc R Soc Lond B Biol Sci 1963 158 p 417-433 Balasch J CM Fabregues F Carmona F Casamitjana R Ascaso and VJ C Inhibin follicle-stimulating hormone and age as predictors of ovarian response in in vitro fertilization cycles stimulated with gonadotropin-releasing hormone agonist-gonadotropin treatment Am J Obstet Gynecol 1996 175 p 1226-1230 Bancsi LF BF Eiijekemans MJ at al Predictors of poor ovarian response in in vitro fertilisation a prospective study comparing basal markers of ovarian reserve Fertility and Sterility 2002 77 p 328-336 Bazer FW Strong science challenges conventional wisdom new perspectives on ovarian biology Reprod Biol Endocrinol 2004 2 p 28 Begum S VE Papaioannou and RG Gosden The oocyte population is not renewed in transplanted or irradiated adult ovaries Hum Reprod 2008 23(10) p 2326-30

Bersinger NA Wunder D Birkhaumluser MH Guibourdenche J Measurement of anti-mullerian hormone by Beckman Coulter ELISA and DSL ELISA in assisted reproduction differences between serum and follicular fluid Clin Chim Acta 2007384174ndash175 Bristol-Gould SK et al Fate of the initial follicle pool empirical and mathematical evidence supporting its sufficiency for adult fertility Dev Biol 2006 298(1) p 149-54 Broekmans FJ et al A systematic review of tests predicting ovarian reserve and IVF outcome Hum Reprod Update 2006 12(6) p 685-718

3

ABSTRACT The University of Manchester Dr Oybek Rustamov Degre MD Title The role of anti-Muumlllerian hormone in assisted reproduction in women Date 30 March 2014

Anti-Muumlllerian hormone appears to play central role in regulation of oocyte recruitment and folliculogenesis Serum AMH concentration was found to be one of the best predictors of ovarian performance in IVF treatment Consequently many fertility centres have introduced AMH for the assessment of ovarian reserve and as a tool for formulation of ovarian stimulation strategies in IVF However published evidence on reliability of AMH assay methods and the role of AMH-tailored individualisation of ovarian stimulation in IVF appear to be weak Consequently I decided to conduct a series of studies that directed towards an improvement of the scientific evidence in these areas of research

The studies on performance of Gen II AMH assay revealed the assay suffers from significant instability and provides erroneous results Consequently the manufacturer introduced a modification on assay method

In view of the observed issues with Gen II assay I conducted a critical appraisal of all published research on the previous and current assay methods that reported AMH variability assay method comparison and sample stability The literature indicated clinically important variability between AMH measurements in repeated samples which was reported to be more significant with Gen II assay The studies on between-assay conversion factors derived conflicting conclusions Correspondingly the review of studies on sample stability revealed conflicting reports on the stability of AMH under normal storage and processing conditions which was reported to be more significant issue in Gen II assay In view of above findings we concluded that AMH in serum may exhibit pre-analytical instability which may vary with assay method Therefore robust international standards for the development and validation of AMH assays are required In the analysis of determinants of ovarian reserve I evaluated the effect of ethnicity BMI endometriosis causes of infertility and reproductive surgery on AMH AFC and FSH measurements using data on a large cohort of infertile patients

Using robust multivariable regression analysis in a large cohort of IVF cycles I established the effect of age AMH AFC diagnosis attempt COS protocol changes gonadotrophin type USOR operator regime and initial dose of gonadotrophins on oocyte yield Then I examined effect of gonadotrophin dose and regime on total and mature oocyte numbers The study found that after adjustment for all above variables there was no increase in oocyte yield with increasing gonadotrophin dose categories beyond the very lowest doses This suggests that there may not be significant direct dose-response effect and consequently strict protocols for tailoring the initial dose of gonadotrophins may not necessarily optimize ovarian performance in IVF treatment

In summary studies described in this thesis have revealed instability of Gen II assay samples and raised awareness of the pitfalls of AMH measurements These studies have also demonstrated the effect of clinically measurable factors on ovarian reserve and provided data on the effect of AMH other patient characteristics and treatment interventions on oocyte yield in cycles of IVF Furthermore a robust database and statistical models have been developed which can be used in future studies on ovarian reserve and IVF treatment interventions

4

DECLARATION

No portion of the work referred to in the thesis has been submitted in support

of an application for another degree or qualification of this or any other

university or other institute of learning

COPYRIGHT STATEMENT

i The author of this thesis (including any appendices andor schedules to this

thesis) owns certain copyright or related rights in it (the ldquoCopyrightrdquo) and she

has given The University of Manchester certain rights to use such Copyright

including for administrative purposes

ii Copies of this thesis either in full or in extracts and whether in hard or

electronic copy may be made only in accordance with the Copyright Designs

and Patents Act 1988 (as amended) and regulations issued under it or where

appropriate in accordance with licensing agreements which the University has

from time to time This page must form part of any such copies made

iii The ownership of certain Copyright patents designs trade marks and

other intellectual property (the ldquoIntellectual Propertyrdquo) and any reproductions

of copyright works in the thesis for example graphs and tables

(ldquoReproductionsrdquo) which may be described in this thesis may not be owned

by the author and may be owned by third parties Such Intellectual Property

and Reproductions cannot and must not be made available for use without the

prior written permission of the owner(s) of the relevant Intellectual Property

andor Reproductions

iv Further information on the conditions under which disclosure publication

and commercialisation of this thesis the Copyright and any Intellectual

Property andor Reproductions described in it may take place is available in

the University IP Policy (see

httpdocumentsmanchesteracukDocuInfoaspxDocID=487) in any

relevant Thesis restriction declarations deposited in the University Library The

University Libraryrsquos regulations (see

httpwwwmanchesteracuklibraryaboutusregulations) and in The

Universityrsquos policy on Presentation of Theses

5

PUBLICATIONS ARISING FROM THE THESIS

Journal Articles

1 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo Philip W Pemberton

The measurement of Anti-Muumlllerian hormone a critical appraisal

The Journal of Clinical Endocrinology amp Metabolism 2014 Mar99(3)723-32

2A Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton Anti-Muumlllerian hormone poor assay reproducibility in a large

cohort of subjects suggests sample instability Human Reproduction 2012 Oct

27(10) 3085-91

2B Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton Human Reproduction Dec2012 Vol 27 Issue 12 p3641

6

Conference presentations

1 O Rustamov S Roberts C Fitzgerald

Ovarian endometrioma is associated with increased AMH levels

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2014 Munich

Poster Presentation

2 O Rustamov M Krishnan R Mathur S Roberts C Fitzgerald

The effect of BMI to the ovarian reserve

Annual Meeting of British Fertility Society January 2014 Sheffield

Oral presentation Dr O Rustamov

3 M Krishnan O Rustamov R Mathur S Roberts C Fitzgerald

The effect of the ethnicity to the ovarian reserve

Annual Meeting of British Fertility Society January 2014 Sheffield

Oral Presentation Dr M Krishnan

4 O Rustamov M Krishnan S Roberts C Fitzgerald

Reproductive surgery and ovarian reserve

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2013 London

Oral presentation Dr O Rustamov

5 C Fitzgerald O Rustamov P Pemberton A Smith A Yates M Krishnan

R Russell L Nardo SRoberts

AMH assays A review of the literature on assay method comparability

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2013 London

Oral presentation Dr C Fitzgerald

6 M Krishnan O Rustamov R Russell C Fitzgerald S Roberts

The role of the ethnicity and the body weight in determination of AMH levels

in infertile women

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2013 London

7

Poster presentation

7 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton

AMH Gen II assay - can we believe the measurements

8th Biennial Conference of UK Fertility Societies January 2013 Liverpool

Poster presentation

8 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton

Old and new AMH assays Can we rely on current conversion factor

8th Biennial Conference of UK Fertility Societies January 2013 Liverpool

Poster presentation

9 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton

Random AMH measurement is not reproducible

8th Biennial Conference of UK Fertility Societies January 2013 Liverpool

Poster presentation

10 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton

The reproducibility of serum Anti-Muumlllerian hormone AMH Gen II assay

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2012 Istanbul

Oral Presentation Dr O Rustamov

8

GENERAL INTRODUCTION

AND LITERATURE REVIEW

1

9

CONTENTS I LITERATURE REVIEWhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip10 GENERAL BACKGROUNDhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip10

1 OVARIAN RESERVEhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip12 11 Primordial Follicle Assemblyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip13 12 Oocyte recruitmenthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip14 13 Theory of neo-oogenesishelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip15 2 MARKERS OF OVARIAN RESERVEhelliphelliphelliphelliphelliphelliphelliphelliphelliphellip16 21 Ovarian reserve markers with limited clinical valuehelliphelliphelliphelliphellip16 213 Inhibin Bhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip16 214 Basal oestradiolhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip17 215 Dynamic testshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip17 216 Ovarian volumehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip18 22 Ovarian reserve markers in routine clinical usehelliphelliphelliphelliphelliphelliphellip18 221 Chronological agehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip18 222 Basal FSHhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip18 223 Antral follicle counthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip19 3 ANTI-MUumlLLERIAN HORMONEhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip20 31 Biology of anti-Muumlllerian hormonehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip20 311 The role of AMH in the ovaryhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip21 312 AMH in women with polycystic ovary syndromehelliphelliphelliphelliphellip22 32 AMH Assayhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip23 33 Variability of AMH measurementshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip24 34 Role of AMH in assessment of ovarian reservehelliphelliphelliphelliphelliphellip25 341 Prediction of poor and excessive ovarian response in IVFhelliphellip25 342 Prediction of live birth in cycles of IVFhelliphelliphelliphelliphelliphelliphelliphelliphellip26

3 5 Role of AMH in ovarian stimulation for cycles of IVFhelliphelliphelliphellip26

4 MULTIVARIATE TESTShelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip27

5 SUMMARYhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip28 II GENERAL INTRODUCTIONhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip29 REFERENCEShelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip31

10

I LITERATURE REVIEW GENERAL BACKGROUND

Infertility is a disease of the reproductive system defined by the failure to

achieve a pregnancy after 12 months of regular unprotected sexual intercourse

although the criteria for the duration vary between different countries (NICE

2013) Worldwide prevalence of infertility estimated to be around 724 million

couples and around 40 million of those seek medical care (Hull et al 1985) In

the UK 15 couples present with infertility with an annual incidence of 12

couples per 1000 general population (Scott et al 2009) The main causes of

infertility are tubal disease ovulatory disorders male factor and poor ovarian

reserve In a third of couples the cause of failure to achieve pregnancy is not

established which is known as unexplained infertility (NICE 2013) Effective

treatment options include improving lifestyle factors medical andor surgical

treatment of underlying pathology induction of ovulation and Assisted

Reproductive Technology (ART) Assisted Reproduction consist of

intrauterine insemination (IUI) and in vitro fertilisation (IVF) cycles with or

without introcytoplasmic sperm injection (ICSI) as well as treatment involving

donated gametes It is estimated that 75 of infertile couples presenting at

primary care centres in the UK are referred to fertility specialists based at

secondary or tertiary care centres and nearly 50 of those are subsequently

offered IVFICSI treatment (Scott et al 2009) This is supported by figures of

Human Fertility and Embryology Authority (HFEA) which indicates more

than 50000 IVF treatment cycles are performed in the UK annually (HFEA

2008)

An IVF treatment cycle involves a) pituitary down regulation b)

controlled ovarian stimulation c) oocyte recovery c) in vitro fertilisation of eggs

with sperm d) transfer of resulting embryo(s) back to uterus and c) luteal

phase support (NICE 2013) Prevention of premature surge of luteinising

hormone during controlled ovarian stimulation (COS) is achieved by pituitary

down regulation using either preparations of gonadotrophin releasing hormone

agonist which is widely known as ldquoAgonist cyclerdquo or gonadotrophin releasing

hormone antagonist which is known ldquoAntagonist cyclerdquo (Figure 1 and 2)

Controlled ovarian stimulation involves administration of gonadotrophins to

encourage the development of supernumerary preovulatory follicles followed

by administration of exogenous human chorionic gonadotropin (hCG) or

11

recombinant luteinising hormone (rLH) to assist in maturation of oocytes 34-

36 hours prior to egg collection which is usually conducted with guidance of

transvaginal ultrasound scanning Subject to sperm parameters the fertilisation

of oocytes is conducted by in vitro insemination or intracytoplasmic sperm

injection The resulting embryo(s) are cultured under strict laboratory

conditions and undergo regular qualitative and quantitative assessments before

transferring the best quality embryo(s) back into uterus during its cleavage

(Day 2 or Day 3) or blastocyst (Day 5 or Day 6) stage of development In

natural menstrual cycles under the influence of HCG progesterone secreted

by the ovarian corpus luteum ensures proliferative changes in the endometrium

providing the optimal environment for implantation of embryo(s) (van der

Linden et al 2011) However in IVF treatment cycles owing to pituitary down

regulation and lack of HCG progesterone levels are not in sufficiently high

concentration to ensure an adequate endometrial receptivity and therefore

exogenous analogues of this hormone is administered following transfer of

embryo(s) This is called ldquoluteal phase supportrdquo and in patients with viable

pregnancy usually lasts till 12th week of gestation when placenta starts

producing progesterone in sufficient quantities (van der Linden et al 2011)

In IVF programmes the ldquosuccessrdquo of the treatment often defined as

achieving a live birth following IVF cycle and expressed using Live Birth Rate

(LBR) In general success in IVF predominantly determined by womanrsquos age

cause(s) of infertility ovarian reserve previous reproductive history and

lifestyle factors (NICE 2013 Taylor 2003 Lintsen et al 2005) However

effectiveness of medical interventions as well as the quality of care play

important role in determining the outcome of IVF treatment This is evident

from significant variation in live birth rates among fertility clinics given for

instance in the UK LBR for women younger than 35 years of age after IVF

cycles varies from 15 to 61 (HFEA 2008 HFEA 2007) The provision of

effective interventions in both clinical and laboratory aspects of the care

appears to be the key in achieving high success rates Identification of patients

with sufficient ovarian reserve who benefit from IVF cycles followed by

providing optimal ovarian stimulation regimens may be useful in improving the

outcomes of IVF programmes According to HFEA data around 12 of IVF

cycles are cancelled due to poor or excessive ovarian response (Kurinczuk et al

2010) Availability of reliable markers for assessment of ovarian reserve and

tailoring ovarian stimulation regimens to the need of each individual patient

12

may improve selection of patients with sufficient ovarian reserve and reduce

the rate of cycle cancellation consequently improving the success of IVF

cycles (Yates et al 2011)

Assessment of ovarian reserve can be achieved using various biomarkers

and four of those are currently used by most clinics womanrsquos chronological

age (Age) serum follicle stimulating hormone (FSH) antral follicle count

(AFC) and serum anti-Muumlllerian hormone (AMH) More recently AMH has

been a focus of interest given it is the only available endocrine marker that is

suitable for direct assessment of the activity of ovarian follicles in their non-

cyclical stage development providing a window to FSH independent phase of

follicular recruitment Furthermore it appears to be reliable biomarker for a)

both the assessment of ovarian reserve and the optimisation of ovarian

stimulation regimens (Yates et al 2011 La Marca et al 2009) b) screening and

diagnosis of polycystic ovarian syndrome (PCOS) (Cook et al 2002) c)

monitoring of disease activity in women with a history of granulosa cell

tumours (Lane et al 1999) d) prediction of the age of diminished fertility and

the menopause (van Disseldorp et al 2008 Broer et al 2011) and finally (e)

assessment of the long term effect of chemotherapy on ovarian reserve

(Anderson 2011)

In this review I first discuss current knowledge on factors that

determine ovarian reserve including the formation and loss of oocyte pool

Then characteristics of the markers of ovarian reserve are reviewed Finally I

examine current understanding of biology of anti-Muumlllerian hormone and its

role in management of infertility

1 OVARIAN RESERVE

It is important to recognize that there is no universal definition for the

term ldquoovarian reserverdquo and the term can have various meanings depending on

the context in which it is used For instance the scientific literature describing

the biology of ovarian reserve usually refers to ldquothe total number of remaining

oocytes in the ovaries which consists of the number of resting primordial

follicles and growing primary pre-antral and antral folliclesrdquo (Gleicher et al

2011) In contrast the use of the term in the context of clinical studies may

refer to ldquoclinically measurable ovarian reserve established using available

biomarkers of ovarian reserverdquo For the purpose of clarity in this thesis the

13

term ldquoovarian reserverdquo refers to clinically measurable ovarian reserve whilst

true biological ovarian reserve will be termed ldquobiological ovarian reserverdquo

Recent studies have demonstrated that ovarian reserve is highly variable

between women due to the variation in the size of initial ovarian reserve at

birth as well as the rate of loss of ovarian reserve thereafter (Wallace et al

2010) Interestingly the rate of oocyte loss appears to be mainly determined by

the initial ovarian reserve which is believed to be facilitated by most potent

ovarian growth factor anti-Muumlllerian hormone Similarly the size of the initial

ovarian reserve is mainly underpinned by the rate of primordial follicle

assembly in the embryo which is also regulated by AMH Both primordial

follicle assembly and the rate of oocyte loss appear to be primarily under the

influence of genetic factors although developmental and environmental factors

are also believed to play a role (Nilsson et al 2010 Shuh-Huerta et al 2012)

11 Primordial follicle assembly

The process of assembly of primordial follicles in the female embryo

spans from the early embryonic to the early postnatal period and formation of

primordial follicles consists of following stages 1) primordial germ cell (PGC)

2) oogonia 3) primary oocyte and 4) primordial follicle In the human female

fetus around a hundred cells that differentiated from extra-embryonic

ectoderm form early PGCs on the yolk sac and migrate via hindgut to gonadal

ridges during 4th - 6th weeks of gestation (MC et al 1953 Donovan 1998) Once

arrived to the gonadal ridges these cells are called primary oogonia which

consequently undergo several rounds of mitotic division during 6th - 28th weeks

of gestation Interestingly the numbers of oogonia reach as high as six million

during its highest rate of mitotic division at around 20 weeks of gestation

Following the last round of mitotic division oogonia enter meiosis which

marks their new stage of development-primary oocyte Formation of

primordial follicles starts as early as at 8th week of gestation and is characterised

by meiosis of primary oocyte that arrest in diplotyne stage and surrounding of

the oocyte by somatic granulosa cell (Baker et al 1963 Maheshwari and Fowler

2010) Indeed the primordial follicle is the cardinal unit of the biological

ovarian reserve and therefore the rate of formation of primordial follicles is the

main determinant of initial biological ovarian reserve at birth

Interestingly the process of loss of oogonia and oocytes which is also

one of the main determinants of the initial ovarian reserve takes place

14

throughout the period of follicle assembly The formation of the granulosa cell

layer around the oocyte prevents the oocyte from subsequent atresia The

oocyte enveloped in a single layer of granulosa cells which is also known as

primordial follicle remains quiescent until recruitment of the follicle for

growth which may not take place for a number of decades after the formation

of a particular primordial follicle (Skinner 2005 Maheshwari and Fowler 2010)

12 Oocyte recruitment

Follicle growth in women consists of two stages a) the initial non-cyclical

recruitment of primordial follicles and the formation of a primary and a pre-

antral follicles and b) cyclical development of antral follicles with subsequent

selection of usually a single dominant follicle The initial recruitment of

primordial follicles is continuous non-cyclical process that starts as early as

from 18-20 weeks of gestation and lasts till the depletion of follicle pool which

later results in the menopause (McGee and Hsueh 2000) Transformation of

flat granulosa cells into cuboidal cells increases the diameter of the oocyte and

the formation of zona pellicuda completes the stage of formation of a primary

follicle During pre-antral stage oocytes increase in diameter and mitotic

division of granulose cells create a new layer of cells-theca cells The

mechanism of initial recruitment of oocytes is not well understood but it is

clear that the process is independent of influence of pituitary gonadotrophins

and appears to be governed by the genetically pre-programmed interaction of

the oocyte with local growth factors the most important of which appears to

be anti-Muumlllerian hormone and cytokines (McGee and Hsueh 2000)

The cyclical phase of development of oocytes is characterised by the

transformation of secondary follicle into antral follicle and subsequent growth

of antral follicles into pre-ovulatory stages In general the process of cyclic

recruitment starts from puberty under the influence of rising levels of pituitary

follicular stimulating hormone (FSH) During the antral stage oocyte increases

in size even further and the formation of a fluid filled space in follicle is

observed Under the influence of FSH luteinising hormone (LH) and local

growth factorsselection of a single dominant follicle occurs which followsby an

ovulation (McGee and Hsueh 2000)

Oocyte loss is a continuous process and occurs due to atresia of oocytes

during primary secondary and antral stages of development The rate of

oocyte loss appears to increase until the age of around 14 and declines

15

thereafter until the age of the menopause when around 1000 primordial

follicles remain (Hansen et al 2008 Oktem and Oktayl 2008) Furthermore by

the age of 30 years the average age at which women of western societies plan

to start a family around 90 of initial primordial follicles are lost which

illustrates that formation and maintenance of ovarian reserve is wasteful

process in humans (ONS 2012 Wallace and Kelsey 2010) As mentioned

above there is a wide individual variation in both sizes of initial primordial

follicular pool and the rate of oocyte loss which explains variation in the

reproductive lifespan in women Evidently the number of primordial follicles

at birth ranges between around 35000 to 25 million per ovary and similarly

the rate of oocyte loss during its peak at 14 years of age may range between

100 to 7500 primordial follicles per month which is believed to be inversely

proportional to initial size of primordial follicle pool (Wallace and Kelsey

2010)

13 Theory of neo-oogenesis

The traditional view of oogenesis states that the process of the creation

and the mitotic division of oogonia with subsequent formation of primordial

follicles takes place only during embryonic and foetal life (Zuckerman 1951)

According to this central theory of mammalian reproductive biology females

are born with a certain number of germ cells that is gradually lost but not

renewed during postnatal period However Johnson et al have recently

challenged this view and reported that adult mammalian ovary may possesses

mitotically active germ cells that continuously replenish the primordial follicle

pool (Johnson et al 2004) The group reported that ovaries of juvenile and

young adult mice contained large ovoid cells which resemble germ cells of

foetal mouse ovaries Interestingly immunohistochemical staining for a gene

which is expressed exclusively in germ cells have been reported to have

confirmed that these large ovoid cells were of germline lineage Furthermore

application of a mitotic germ cell toxicant busulphan appeared to have

eliminated primordial follicle reserve by early adulthood but did not induce

atresia suggesting the presence of proliferative germ cells in postnatal mouse

ovary (Johnson et al 2004 Bazer 2004) The study has generated enormous

amount of interest as well as debate among reproductive biologists (Notarianni

2011) Some other groups have also reported an evidence of postnatal

oogenesis (Pacchiarott et al 2010 Zou et al 2009 Bukovsky et al 2004)) while

16

others do not support the theory (Bristol-Gould et al 2006 Byskov et al 2005

Begum et al 2008) Furthermore some authors argued that adult mouse

germline stem cells exist and remain quiescent in physiologic conditions and

neo-oogenesis occurs only in response to ovotoxic damage (Tilly et al 2007 De

Felici 2010) Although consensus has yet to emerge to date there is no

conclusive evidence on validity of theory of neo-oogenesis

2 MARKERS FOR ASSESMENT OF OVARIAN RESERVE

Biological ovarian reserve is defined as the number of primordial and

growing follicles left in the ovary at any given time and therefore only

counting the number of primordial follicles by histological assessment can

accurately determine ovarian reserve which is clearly not feasible in clinical

setting However ovarian reserve can be estimated using various biomarkers

dynamic clinical tests and implied from the outcomes of ART cycles

Although a wide range of clinical (age ovarian response in previous IVF

cycles) biochemical (basal FSH Inhibin B basal oestradiol AMH) ultrasound

(ovarian volume antral follicle count (AFC)) and dynamic (clomiphene

challenge test exogenous FSH ovarian reserve test GnRH analogue

stimulating test) tests of ovarian reserve exist only a few of the markers are

reliable and practical enough to be of use in routine clinical practice In this

chapter first I discuss the research evidence on the assessment of the markers

andor tests of ovarian reserve that have limited clinical value Then I

evaluated more reliable markers that are in routine clinical use Age FSH

AFC and combination of these markers in multivariable tests Finally I

conducted detailed review of biology of AMH and the role AMH measurement

in the management of infertility

21 Ovarian reserve markers with limited clinical value

211 Inhibin B

Inhibins are members of TGFβ family and expressed in granulosa cells

of growing follicles Principal role of inhibins is thought to be the negative

feedback regulation of pituitary FSH secretion and therefore the serum level of

circulating hormone is believed to reflect the state of folliculogenesis

17

Consequently several groups have studied the role of serum Inhibin β in the

assessment of ovarian reserve Although initial reports were encouraging

(Seifer et al 1997) more robust studies demonstrated that serum Inhibin β was

less reliable than chronological age or basal FSH (Creus et al 2000 Urbancsek

2005) The systematic review of nine studies demonstrated that accuracy of the

Inhibin β test for predicting poor ovarian response and non-pregnancy in IVF

cycles was modest even at a very low threshold level (Broekmans et al 2006)

Therefore it is recommended that inhibin β at best can be used as only

screening test in the fertility centers where other more reliable markers are not

available (Broekmans et al 2006)

212 Basal oestradiol

Some studies suggested that elevated basal oestradiol levels indicate low

ovarian reserve and are associated with poor fertility prognosis (Johannes et al

1998 Licciardi and Rosenwaks 1995) Johannes et al demonstrated basal

oestradiol in conjunction with serum FSH is more reliable than serum FSH

alone in prediction of cycle cancellation due to the poor response in IVF cycles

(Johannes et al 1998) However there are no published data on the comparison

of basal oestradiol to more reliable markers such as AMH or antral follicle

count (AFC) Moreover a recent systematic review has demonstrated that

basal oestradiol has very low predictive value for poor response and has no

discriminatory power for accuracy of non-pregnancy prediction (Broekmans et

al 2006)

213 Dynamic tests of ovarian reserve

The dynamic tests of ovarian reserve are based on assessment of ovarian

response by measuring serum FSH and oestradiol levels following

administration of exogenous stimulation The following tests are reported in

literature Clomiphene Citrate Challenge Test (CCCT) Exogenous FSH

Ovarian Reserve Test (EFORT) and GnRH agonist stimulation test A recent

systematic review and meta-analysis on the accuracy of these tests showed that

none of them can adequately predict poor response or non-pregnancy in IVF

cycles and therefore are not recommended for use in routine clinical practice

(Maheshwari et al 2009)

18

214 Ovarian volume

There is some evidence that increased age is associated with decreased

ovarian volume and women with smaller ovaries are more likely to have

cancellation of their IVF cycles due to poor ovarian response (Syrop et al 1995

Syrop et al 1999 Templeton 1995) However a meta-analysis of the published

studies on the accuracy of ovarian volume as a predictor of poor response and

non-pregnancy in IVF cycles failed to demonstrate clinical usefulness of the

test and suggested the test is not reliable enough for use in a routine clinical

practice (Broekmans et al 2006)

22 Ovarian reserve markers in routine clinical use

221 Chronological age

Owing to the biological age-related decline of the quantity and arguably

the quality of oocytes the chronological age can be used as a marker of ovarian

reserve Studies have demonstrated that ovarian reserve (Wallace and Kelsey

2010 Kelsey 2011) natural fecundity (Islam et al 1989 and outcomes of ART

(Templeton et al 1996 van Kooij et al 1996) decline significantly from age of

35 when it is believed the ovarian reserve undergoes accelerated decline

Although there is a strong association between chronological age and reduction

in fertility evidently there is a significant variation in age-related ovarian

reserve indicating chronological age alone may not be sufficient to estimate the

individual womanrsquos ovarian reserve reliably (Broekmans et al 2006)

222 Basal FSH

Basal FSH was one of the first endocrine markers introduced in ART

programs and is still utilized in many fertility clinics albeit in conjunction with

other markers which are considered more reliable (Creus et al 2000) Secretion

of FSH is largely governed by the negative feedback effect of steroid

hormones primarily oestradiol and inhibins which are expressed in granulosa

cells of growing ovarian follicles Consequently decreased or diminished

recruitment of ovarian follicles is associated increased serum FSH

measurements and high particularly very high basal FSH reading is considered

as a good marker of very low or diminished ovarian reserve (Abdalla et al

2006) However unlike some other markers FSH measurements do not

appear to have discriminatory power for categorisation of patients to various

19

bands of ovarian reserve Given between-patient variability FSH measurement

(CV 30) is similar to its within-patient variability (27) stratification of

patients to various ranges of ovarian reserve does not appear to be feasible

(Rustamov et al 2011) Indeed a recent systematic review of 37 studies on the

prediction of poor response and non-pregnancy in IVF cycle has concluded

that basal FSH is an adequate test at very high threshold levels and therefore

has limited value in modern ART programs (Broekmans et al 2006)

223 Antral follicle count

Antral follicle count estimation involves ultrasound assessment of

ovaries between 2nd and 4th day of menstrual period and counting ldquofolliclesrdquo

which corresponds to antral stage of folliculogenesis (Broekmans et al 2010)

The test provides direct quantitative assessment of growing follicles and is

known as one of the most reliable markers of ovarian reserve (Broekmans et al

2006) AFC measurement has been reported as having a similar sensitivity and

specificity to AMH in prediction of poor and excessive ovarian response in

IVF cycles (Broekmans et al 2006 Broer et al 2010 Jayaprakasan et al 2010)

Given AFC measurement is available instantly and allows patients to be

counseled immediately the test eliminates the need for an additional patient

visit prior to IVF cycle However AFC is normally performed only in the early

follicular phase of the menstrual cycle given most published data on

measurement of AFC are based on studies that assessed antral follicles during

this stage of the cycle (Broekmans et al 2010a) Interestingly more recent

studies suggest that variability of AFC during menstrual cycle is small

particularly when follicles between 2-6mm are counted and therefore

assessment of AFC without account for the day of menstrual cycle may be

feasible (Deb et al 2013)

One of the main drawbacks of AFC is that the cut off levels for size of

counted follicles remains to be standardised (Broekmans 2010b) Initially

follicles of 2-10mm were introduced as the range for AFC and many studies

were based on this cut off Later counting follicles of 2-6mm was reported to

provide most accurate assessment of ovarian reserve (Jayaprakasan et al 2010b

Haadsma et al 2007) and therefore some newer studies are based on AFC

measurements that used this criterion Consequently direct comparison of the

outcomes of various studies on assessment of AFC requires careful analysis

20

3 ANTI-MUumlLLERIAN HORMONE

31 Biology of Anti-Muumlllerian hormone

AMH is a member of transforming growth factor β superfamily which

was discovered by Jost et al in 1947 and was initially known for its is role in

regression of Muumlllerian ducts in sex differentiation of the male embryo In

women AMH is believed to be solely produced by ovaries and expressed in

granulosa cells of growing follicles of 2-6 mm in size which corresponds to

primary pre-antral and early antral stage of follicular development Although

there has been a report of expression of AMH in endometrial cells to date

there is no other published evidence that supports this finding (Wang et al

2009) Indeed studies that evaluated half-life of AMH in serum have

demonstrated that in women who had bilateral salpingo-oopherectomy AMH

becomes undetectable within 3-5 days of following surgery suggesting ovaries

are the only source of secretion of AMH in appreciable quantity (La Marca et

al 2005b) Anti-Muumlllerian hormone is a dimeric glycoprotein which is

composed of a long N-terminus and short C-terminus and was believed to be

secreted in serum only in this dimeric form (AMH-N C)

Like other members of TGF-β family which includes inhibins activins

bone morphogenic proteins (BMPs) and growth and differentiation factors

(Massague et al 1990) AMH binds to two type of serinethreonine kinase

receptors referred to as type I and type II In order to activate AMH signaling

pathway both receptors have to form a heteromeric complex When AMH

binds to the type II (AMHR-II) receptor (Massague et al 2000) this will

phosphorylate and activate a type I receptor (ALK2 -3 andor -6) which

subsequently activates the SMAD pathway through phosphorylation of

SMAD 1 5 andor 8 These activated SMADs interact with SMAD4 and

translocate to the nucleus regulating the expression of different genes

inhibiting the recruitment of primordial follicles and reducing FSH sensitivity

in growing follicles In addition AMH receptors as well as the other members

of TGF-β family can activate MAPK and PI3KAKT pathways

Studies on AMHR II-deficient male mice demonstrated lack of

regression of Muumlllerian ducts suggesting that type II receptor is essential in

AMH signaling (Mishina et al 1996) Similarly Type I receptors which includes

three members of activin receptor-like kinase (ALK2 ALK3 and ALK6) also

appear to play an important role in the regression of Muumlllerian ducts although

21

the role of ALK 6 in AMH signaling appears not to be crucial (Visser 2003

Clarke et al 2001) The signal transduction pathway of AMH in the ovary is

largely not understood In postnatal mice ovary AMHR-II receptor was

expressed in both granulosa and theca cells of pre-antral and antral follicles

(Visser 2003) AMH type I receptors ALK 2 and ALK 3 is expressed in foetal

as well as adult mouse ovary while ALK 6 is expressed in only adult ovary

(Visser 2003)

311 The role of AMH in the ovary

In the mammalian ovary the role of AMH appears to be one of a

regulation of size of the primordial follicle pool by its inhibitory effect on the

formation as well as the growth of primordial follicles (Nilsson et al 2011) In

the embryonic mouse ovary AMH inhibits the initiation of the assembly of

follicles when the process of apoptosis of the majority of oocytes is observed

(Nilsson et al 2011) Consequently AMH reduces the rate of oocyte loss

which plays an important role in the determination of the size of initial follicle

pool Similarly in the adult mouse ovary AMH plays a central role in

maintaining the follicle pool AMH inhibits both the processes of the initial

(non-cyclical) recruitment of primordial follicles and subsequent FSH-

dependent cyclical growth of antral follicles (Figure 3) Inhibition of the initial

recruitment of a new cohort of follicles is believed to be achieved by a

paracrine negative feedback effect of the rising levels of AMH secreted from

already recruited growing follicles (Durlinger et al 1999) Durlinger et al

compared the complete follicle population of AMHnull mice and wild type

mice of different ages of 25 days 4 months old and 13 months old and found

that the ovaries of 25 day and 4 months old AMHnull females contained

significantly higher number of growing pre-antral and antral follicles but

significantly fewer primordial follicles compared to wild-type females

(Durlinger et al 1999) Interestingly almost no primordial follicles were

detected in 13 months old AMHnull mice ovaries suggesting AMH is a potent

inhibitor of the recruitment of primordial follicles and in the absence of AMH

ovaries undergo premature depletion of primordial follicles due to an

accelerated recruitment Subsequent study conducted by the group

demonstrated that in addition to its inhibitory effect to the resting follicles

AMH also suppresses the development of the growing follicles (Durlinger et al

2001 Durlinger et al 2002 Themmen 2005) It appears that AMH inhibits

22

FSH-induced follicle growth by reducing the sensitivity of growing follicles to

FSH which has been confirmed by in vivo as well as in vitro studies (Durlinger

et al 1999 Durlinger et al 2001) In the initial study the group observed that

despite lower levels of serum FSH concentration ovaries of AMHnull mice

contained more growing follicles than that of their wild-type littermates which

has been supported by the findings of subsequent in vitro study (Durlinger et al

1999) Addition of AMH to the culture inhibited FSH-induced follicle growth

of pre-antral mouse follicles due to reduction in granulosa cell proliferation

(Durlinger et al 2001)

In the human embryo the expression of AMH commences in the late

foetal life and can be detected only from 36 weeks of gestation (Rajpert-De et

al 1999 Lee et al 1996) Following a small decline in first two years of life

AMH levels gradually increase to peak at (mean 5 ngml) around age of 24

years In line with the pattern of oocyte loss serum hormone levels gradually

decline with increasing age and become undetectable around 5 years prior to

menopause (Kelsey et al 2011 Nelson et al 2011)

It has been suggested that anti-Muumlllerian hormone plays a central role in

determining the pace of recruitment of primordial follicles hence maintaining

the primordial follicle pool of postnatal mammalian ovary Consequently a

reduction in the concentration of circulating AMH signals the exhaustion of

the primordial follicle pool and the decline of ovarian function

312 AMH in women with polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) endocrine abnormality characterised

by increased ovarian androgen secretion infrequent ovulation and the

appearance of ldquopolycysticrdquo ovaries on ultrasound scan (Dunaif 1997 Homburg

et al 1993) It is the commonest endocrine abnormality in women of

reproductive age and affects around 15-20 of women PCOS is also one of

the main causes of anovulation and subsequent sub-fertility (Webber et al

2003) Although the role of anti-Muumlllerian hormone in the development of

PCOS is not fully understood it is becoming increasingly evident that the

hormone plays an important role in its pathogenesis (Pehlivanov et al 2011)

There is a strong association between serum AMH levels and PCOS and it

appears that women diagnosed with PCOS have two to three fold higher

serum AMH concentration compared to normo-ovulatory women (Cook et al

2002 Pigny et al 2003) Similarly women with PCOS are found to have

23

significantly higher number antral follicles Interestingly the expression of

AMH in granulosa cells of follicles were found to be 75 times higher in women

with PCOS compared to those without a the disease suggesting increased

serum AMH in PCOS may be due to increased secretion of hormone per

follicle rather than due to an increased number of antral follicles (Pellat et al

2007) High AMH concentrations may act as the main facilitator of abnormal

folliculogenesis in PCOS given the follicles appear to arrest when they reach

an antral stage (2-6mm) of development (Rajpert-De et al 1999) Indeed the

studies of Durlinger et al have demonstrated that AMH inhibits selection of

dominant follicle when follicles reach antral stage of development (Durlinger et

al 2001) Serum AMH levels appear to decrease with treatment of PCOS

which may play important role in restoration of ovulatory cycles Studies have

reported a significant reduction in serum concentration of AMH following

treatment of PCOS with metformin and laparoscopic ovarian diathermy (Falbo

et al 2010 Amer et al 2009 Elmashad 2011) Similarly reduction of BMI

following intensified endurance exercise training for treatment of PCOS may

also lead to a significant reduction in serum AMH levels (Moran et al 2011)

This suggests that there is strong association between serum concentration of

AMH and abnormal folliculogenesis in PCOS and therefore understanding the

molecular mechanisms of this interaction should be one of the priorities of

future research

32 AMH Assays

Enzyme-linked immunosorbent assay specific for measurement of anti-

Muumlllerian hormone was first developed in 1990 and was recognised as a

significant step in the assessment of ovarian reserve (Hudson et al 1990)

Subsequently a number of non-commercial immunoassays were developed

which were mainly used in research settings (Lee et al 1996) Later Diagnostic

Systems Ltd (DSL) and Immunotech Beckman Coulter Ltd (IOT) introduced

two commercial immunoassays for the routine clinical assessment of ovarian

reserve which are known as ldquofirst generation AMH assaysrdquo (Nelson and La

Marca 2011) These assays employed two different antibodies against AMH

and used different standards for calibration providing non-comparable

measurements (Nelson and La Marca 2011) Consequently several studies

attempted to develop a reliable between-assay conversion factor which

interestingly revealed from five-fold higher with the IOT assay to assay

24

equivalence causing significant impact to reliability of AMH measurements and

interpretation of research findings (Hehenkamp et al 2006 Freour et al 2007

Bersinger et al 2007 Taieb et al 2008 Lee et al 2011)

Later the manufacturer of IOT assay (Beckmann Coulter Ltd)

consolidated the manufacturer of the DSL assay (Diagnostic Systems

Laboratories Inc) and introduced a new assay ldquoGen II AMH assayrdquo which is

only available commercial immunoassay in most countries including the UK

AMH Gen II assay was developed using the antibodies derived from first

generation DSL assay and calibrated using the standards used for IOT assay

and was believed to be considerably more stable compared to the first

generation immunoassays providing more reliable measurements (Kumar et al

2010 Nelson and La Marca 2011) The manufacturer as well as initial external

validation study recommended when compared to old DSL assay AMH Gen

II assay provides around 40 higher measurements and therefore previously

reported DSL-based clinical cut-off levels for estimation of ovarian reserve

should be increased by 40 in order to use Gen II-based AMH results (Kumar

et al 2010 Wallace et al 2011 Nelson and La Marca 2011)

33 Variability of AMH measurements

It is generally believed that AMH values do not change throughout the

menstrual cycle and early studies reported that variation in AMH

measurements between repeated measurements of same patient was negligible

(van Disseldorp et al 2010 La Marca 2010) On the basis of these studies

sampling at a random time in the menstrual cycle was introduced as a method

for measurement of AMH in routine clinical practice However the

methodologies of some of these studies do not appear to be robust enough to

reliably estimate sample-to-sample variability of AMH which is mainly due to

small sample sizes (Rustamov et al 2011) Consequently in a recent study we

assessed sample-to-sample variability of AMH using DSL assay and found that

within-subject coefficient of variation (CV) of AMH between samples were as

high as 28 which cannot be attributed to any patient or cycle characteristics

(Rustamov et al 2011) Although there is no consensus in the causes of this

observed variability in AMH measurements we believe it is largely attributable

to instability of AMH samples given initial recruitment of primordial follicles

and growth of AMH producing pre-antral and antral follicles are continuous

process and therefore the true biological variation between samples is unlikely

25

to be high However given the importance of establishing true variability of

AMH in both understanding of the biology of hormone and clinical

application of the test future studies should be conducted to establish the

source of variability in the clinical samples

3 4 The role of AMH in the assessment of ovarian reserve

341 Prediction of poor and excessive ovarian response in cycles of

IVF

A number of studies have assessed the role of AMH in the prediction of

poor ovarian response in IVF cycles using first generation AMH assays and

found that AMH and AFC were the best predictors of poor ovarian response

compared to other markers of ovarian reserve Nardo et al showed that the

predictive value of AMH in receiver operating characteristic curve (ROC)

analysis was similar to (AUC 088) that of AFC (AUC 081) and found that

AMH cut offs of gt375 ngmL and lt10 ngmL would have modest

sensitivity and specificity in predicting the extremes of response (Nardo et al

2009) These findings were largely supported by subsequent prospective studies

and a systematic review (Nelson et al 2007 Jayaprakasan et al 2010 Broer et al

2011) Similarly comparison of chronological age basal FSH ovarian volume

AFC and AMH found that only AMH (AUC 090) and AFC (AUC 093) were

reliable predictors of poor ovarian response in cycles of IVF Subsequent

combination of the effect of AMH and AFC using multivariable regression

analysis did not improve the level of prediction of poor ovarian response

significantly (AUC 094) suggesting both AMH and AFC can be used as

independent markers (Jayaprakasan et al 2010)

Similarly most studies agree that AMH and AFC are the best predictors

of excessive ovarian response and ovarian hyperstimulation syndrome (OHSS)

compared to other clinical endocrine and ultrasound markers (Nardo et al

2009 Nelson et al 2007) Broer et al compared these two tests in systematic

review of 14 studies and reported that the summary estimates of the sensitivity

and the specificity for AMH were 82 and 76 respectively and for AFC 82

and 80 respectively (Broer et al 2011) Consequently the study concluded

that AMH and AFC were equally predictive and the difference in the predictive

value between the tests was not statistically significant

26

342 Prediction of live birth rate (LBR) in cycles of IVF

Lee at al reported that AMH and chronological age were more accurate

than basal FSH AFC BMI and causes of infertility in the prediction of live

birth rate (Lee et al 2009) Similarly La Marca et al suggested that odds of live

birth could be reliably predicted using AMH (La Marca et al 2010b) although

subsequent review of the study questioned strength of the evidence (Loh and

Maheshwari 2011)

A study conducted by Nelson et al found that higher AMH levels had

stronger association with increased live birth rate compared to age and FSH

(Nelson et al 2007) However the study also suggested that this association

was mainly confined in the women with low AMH levels and there was no

additional increase in live birth in women with AMH levels of higher than 710

pmolL This may suggest that achieving a live birth may be under the

influence of number of other factors and that markers of ovarian reserve alone

may not be able predict this outcome reliably

35 The role of AMH in individualisation of ovarian stimulation in

IVF cycles

Prediction of ovarian response to the stimulation of ovaries in cycles of

IVF plays an important role in the counseling of couples undergoing treatment

programmes and hence many clinical studies on AMH have focused on the

prognostic value of AMH measurements However data on using AMH as a

tool for improving the clinical outcomes in IVF cycles appear to be lacking

considering AMH may be useful tool in tailoring treatment strategies to an

individual patientrsquos ovarian reserve Unlike most other markers AMH has

discriminatory power in determining various degrees of ovarian reserve due to

significantly higher between patient (CV 94) variability compared to its

within-patient (CV 28) variation (Rustamov et al 2011) which allows

stratification of patients into various degrees of (eg low normal high) ovarian

reserve Subsequently most optimal ovarian stimulation protocol may be

established for each band of ovarian reserve Consequently reference ranges

on the basis of distribution of AMH in infertile women were developed which

were subsequently adopted by fertility clinics for a tailoring the mode of

27

ovarian stimulation and daily dose of gonadotrophins in IVF (The Doctors

Laboratory 2008 However currently available clinical reference ranges are

based on the first generation DSL assay and may not be reliably convertible to

currently available Gen II assay measurements (Wallace et al 2011) Indeed the

findings of the studies on comparability of the first generation AMH assays

suggest that establishing a reliable between assay conversion factor between

AMH assays may not be straightforward Furthermore the reference ranges

appear to reflect the distribution of AMH measurements within a specific

population and may therefore not be directly applicable for the prediction of

response to ovarian stimulation in IVF patients (The Doctors Laboratory

2008)

More importantly despite lack of good quality evidence on the

effectiveness of AMH-tailored ovarian stimulation protocols a number of

fertility clinics appear to have introduced various AMH-based COH protocols

in their IVF programs At present research evidence on AMH-tailored

ovarian stimulation in IVF is largely based on two retrospective studies

(Nelson et al 2009 Yates et al 2012) Both of these studies display considerable

methodological limitations including small sample size and centre-related or

period-related selection of their cohorts In this context AMH is used as a tool

for therapeutic intervention and therefore the research evidence should ideally

be derived from randomised controlled trials However recruitment of large

enough patients in IVF setting may take considerable time and resources In

the meantime given AMH-tailored ovarian stimulation has already been

introduced in clinical practice and there is urgent need for more reliable data

the studies with a larger cohorts and robust methodology should assess the role

of AMH in individualisation of ovarian stimulation in IVF treatment cycles

4 Multivariate models of assessment of ovarian reserve

In view of the fact there is not a single marker of ovarian reserve that

can accurately predict ovarian response various models for combination of

multiple ovarian markers have been developed (Verhagen et al 2008) A

number of studies reported that multivariate models are better predictors of

poor ovarian response in IVF compared to a single marker (Bancsi et al 2002

Balasch et al 1996 Creus et al 2000 Durmusoglu et al 2004) However a meta-

analysis showed that when compared to a single marker (AFC) multivariate

28

model has a similar accuracy in terms of prediction of poor ovarian response

(Verhagen et al 2008) In contrast a more recent study demonstrated that

multivariate score was superior to chronological age basal FSH or AFC alone

in predicting likelihood of poor ovarian response and clinical pregnancy

(Younis et al 2010) However the study did not include one of the most

reliable markers AMH in either arm necessitating further assessment of the

role of combined tests which include all reliable biomarkers

4 SUMMARY

During the last two decades a significant leap has been taken towards

understanding the biology of anti-Muumlllerian hormone and its role in female

reproduction (Durlinger et al 2002 Themmen et al 2005) Availability of

commercial AMH assays has resulted in significant increase in interest in the

role of the measurement of serum AMH in the assessment of ovarian reserve

which has been followed by the introduction of the test into routine clinical

practice (Nelson et al 2011) However more recent studies suggest that current

methodologies for the measurement of AMH may provide significant sampling

variability (Rustamov et al 2011) Furthermore the studies that compared first

generation commercial assay methods appear to provide non-reproducible

results suggesting there may be underlying issues with assay methodologies

(Lee et al 2011) Similarly despite lack of sufficient evidence in the role of

AMH in individualisation of ovarian stimulation protocols in IVF AMH-

tailored IVF protocols have been introduced in routine clinical practice of

many fertility clinics around the world

Consequently it appears that clinical application of AMH test has

surpassed the research evidence in some aspects of fertility treatment and

therefore future projects should be directed toward areas where gaps in

research evidence exist On the basis of the review of literature we believe that

evaluation of the performance of assay methods understanding the role of

AMH in assessment ovarian reserve and establishing its role in

individualisation of ovarian stimulation protocols should be research priority

29

II GENERAL INTRODUCTION

On the basis of the review of published literature I have identified that

the following areas of research on the clinical application of AMH in the

management of infertility requires further investigation 1) Within-patient

variability of measurement of AMH using Gen II assay method 2)

Establishment of clinically measurable determinants of AMH levels and 3) The

role of AMH in individualisation of ovarian stimulation in IVF treatment

cycles

In our previous study we estimated that there was significant sample-to-

sample variation (CV 28) in AMH measurements when the first generation

DSL assay was used (Rustamov et al 2011) The source of variability is likely to

be related to the assay method given that biological within-cycle variation of

AMH is believed to be small (La Marca et al 2006) Therefore assessment of

sample-to-sample variability of AMH using the newly introduced Gen II assay

which is believed to be significantly more stable and sensitive compared to that

of DSL assay should enable us to establish the measurement related variability

of AMH Furthermore given I am planning to use data from both DSL and

Gen II assays I need to establish between-assay conversion factor for these

assays using data on clinical samples

There appears to be a lack of good quality data on the effect of

ethnicity BMI causes of infertility reproductive history and reproductive

surgery on ovarian reserve Therefore I am planning to ascertain the role of

above factors on determination of ovarian reserve by analysing AMH

measurements of a large cohort of patients

There is a strong correlation between AMH and ovarian performance

in IVF treatment when conventional ovarian stimulation using GnRH agonist

regimens with a standard daily dose of gonadotrophins are used (Nelson et al

2007 Nardo et al 2007) Furthermore studies suggest tailoring the ovarian

stimulation protocols to AMH measurement may improve ovarian

performance and subsequently the success of IVF treatment (Nelson et al

2011 Yates et al 2012) However given methodologies of the published

studies the effectiveness of currently proposed AMH-tailored ovarian

stimulation protocols remains unknown Therefore I am planning to develop

individualised ovarian stimulation protocols by establishing the most optimal

mode of pituitary down regulation and starting dose of gonadotrophins for

30

each AMH cut-off bands using a robust research methodology However

development of individualised ovarian stimulation protocols on the basis of

retrospective data requires a reliable and validated database containing a large

number of observations In the IVF Department of St Maryrsquos Hospital we

have data on a large number of patients who underwent ovarian stimulation

following the introduction of AMH However the data on various aspects of

investigation and treatment of patients is stored in different clinical data

management systems and may not be easily linkable In addition it appears that

data on certain important variables (eg causes of infertility AFC) are available

only in the hospital records necessitating searching for data from the hospital

records of each patient Consequently I designed a project for building a

research database which will have comprehensive and validated datasets that

are necessary for investigation of the research questions of the MD

programme

In conclusion I am planning to conduct a series of studies to improve

the understanding of the role of AMH in the management of women with

infertility Specifically I am intending to evaluate 1) sample-to-sample variability

of Gen II AMH measurements 2) conversion factor between DSL and Gen II

assays in clinical samples 3) the effect of ethnicity BMI causes of infertility

endometriosis reproductive history and reproductive surgery to ovarian

reserve and explore AMH-tailored individualisation of ovarian stimulation in

IVF cycles

31

References

Abbeel E The Istanbul consensus workshop on embryo assessment proceedings of an expert meeting Human reproduction 2011 26 p 1270-83 Abdalla HT M Y Repeated testing of basal FSH levels has no predictive value for IVF outcome in women with elevated basal FSH Human reproduction 2006 21(1) p 171-4 Amer SA LT Ledger WL The value of measuring anti-Mullerian hormone in women with polycystic ovary syndrome undergoing laparoscopic ovarian diathermy Human reproduction 2009 24 p 2760-6 Anderson RA Pretreatment serum anti-Mullerian hormone predicts long-term ovarian function and bone mass after chemotherapy for early breast cancer J Clin Endocrinol Metab 2011961336-1343 Balaban B BD Calderoacuten G Catt J Conaghan J Cowan L Ebner T Gardner D Hardarson T Lundin K Cristina Magli M Mortimer D Mortimer S Munneacute S Royere D Scott L Smitz J Thornhill A van Blerkom J Van den Baker A quantitative and cytological study of germ cells in human ovaries Proc R Soc Lond B Biol Sci 1963 158 p 417-433 Balasch J CM Fabregues F Carmona F Casamitjana R Ascaso and VJ C Inhibin follicle-stimulating hormone and age as predictors of ovarian response in in vitro fertilization cycles stimulated with gonadotropin-releasing hormone agonist-gonadotropin treatment Am J Obstet Gynecol 1996 175 p 1226-1230 Bancsi LF BF Eiijekemans MJ at al Predictors of poor ovarian response in in vitro fertilisation a prospective study comparing basal markers of ovarian reserve Fertility and Sterility 2002 77 p 328-336 Bazer FW Strong science challenges conventional wisdom new perspectives on ovarian biology Reprod Biol Endocrinol 2004 2 p 28 Begum S VE Papaioannou and RG Gosden The oocyte population is not renewed in transplanted or irradiated adult ovaries Hum Reprod 2008 23(10) p 2326-30

Bersinger NA Wunder D Birkhaumluser MH Guibourdenche J Measurement of anti-mullerian hormone by Beckman Coulter ELISA and DSL ELISA in assisted reproduction differences between serum and follicular fluid Clin Chim Acta 2007384174ndash175 Bristol-Gould SK et al Fate of the initial follicle pool empirical and mathematical evidence supporting its sufficiency for adult fertility Dev Biol 2006 298(1) p 149-54 Broekmans FJ et al A systematic review of tests predicting ovarian reserve and IVF outcome Hum Reprod Update 2006 12(6) p 685-718

4

DECLARATION

No portion of the work referred to in the thesis has been submitted in support

of an application for another degree or qualification of this or any other

university or other institute of learning

COPYRIGHT STATEMENT

i The author of this thesis (including any appendices andor schedules to this

thesis) owns certain copyright or related rights in it (the ldquoCopyrightrdquo) and she

has given The University of Manchester certain rights to use such Copyright

including for administrative purposes

ii Copies of this thesis either in full or in extracts and whether in hard or

electronic copy may be made only in accordance with the Copyright Designs

and Patents Act 1988 (as amended) and regulations issued under it or where

appropriate in accordance with licensing agreements which the University has

from time to time This page must form part of any such copies made

iii The ownership of certain Copyright patents designs trade marks and

other intellectual property (the ldquoIntellectual Propertyrdquo) and any reproductions

of copyright works in the thesis for example graphs and tables

(ldquoReproductionsrdquo) which may be described in this thesis may not be owned

by the author and may be owned by third parties Such Intellectual Property

and Reproductions cannot and must not be made available for use without the

prior written permission of the owner(s) of the relevant Intellectual Property

andor Reproductions

iv Further information on the conditions under which disclosure publication

and commercialisation of this thesis the Copyright and any Intellectual

Property andor Reproductions described in it may take place is available in

the University IP Policy (see

httpdocumentsmanchesteracukDocuInfoaspxDocID=487) in any

relevant Thesis restriction declarations deposited in the University Library The

University Libraryrsquos regulations (see

httpwwwmanchesteracuklibraryaboutusregulations) and in The

Universityrsquos policy on Presentation of Theses

5

PUBLICATIONS ARISING FROM THE THESIS

Journal Articles

1 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo Philip W Pemberton

The measurement of Anti-Muumlllerian hormone a critical appraisal

The Journal of Clinical Endocrinology amp Metabolism 2014 Mar99(3)723-32

2A Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton Anti-Muumlllerian hormone poor assay reproducibility in a large

cohort of subjects suggests sample instability Human Reproduction 2012 Oct

27(10) 3085-91

2B Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton Human Reproduction Dec2012 Vol 27 Issue 12 p3641

6

Conference presentations

1 O Rustamov S Roberts C Fitzgerald

Ovarian endometrioma is associated with increased AMH levels

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2014 Munich

Poster Presentation

2 O Rustamov M Krishnan R Mathur S Roberts C Fitzgerald

The effect of BMI to the ovarian reserve

Annual Meeting of British Fertility Society January 2014 Sheffield

Oral presentation Dr O Rustamov

3 M Krishnan O Rustamov R Mathur S Roberts C Fitzgerald

The effect of the ethnicity to the ovarian reserve

Annual Meeting of British Fertility Society January 2014 Sheffield

Oral Presentation Dr M Krishnan

4 O Rustamov M Krishnan S Roberts C Fitzgerald

Reproductive surgery and ovarian reserve

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2013 London

Oral presentation Dr O Rustamov

5 C Fitzgerald O Rustamov P Pemberton A Smith A Yates M Krishnan

R Russell L Nardo SRoberts

AMH assays A review of the literature on assay method comparability

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2013 London

Oral presentation Dr C Fitzgerald

6 M Krishnan O Rustamov R Russell C Fitzgerald S Roberts

The role of the ethnicity and the body weight in determination of AMH levels

in infertile women

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2013 London

7

Poster presentation

7 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton

AMH Gen II assay - can we believe the measurements

8th Biennial Conference of UK Fertility Societies January 2013 Liverpool

Poster presentation

8 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton

Old and new AMH assays Can we rely on current conversion factor

8th Biennial Conference of UK Fertility Societies January 2013 Liverpool

Poster presentation

9 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton

Random AMH measurement is not reproducible

8th Biennial Conference of UK Fertility Societies January 2013 Liverpool

Poster presentation

10 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton

The reproducibility of serum Anti-Muumlllerian hormone AMH Gen II assay

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2012 Istanbul

Oral Presentation Dr O Rustamov

8

GENERAL INTRODUCTION

AND LITERATURE REVIEW

1

9

CONTENTS I LITERATURE REVIEWhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip10 GENERAL BACKGROUNDhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip10

1 OVARIAN RESERVEhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip12 11 Primordial Follicle Assemblyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip13 12 Oocyte recruitmenthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip14 13 Theory of neo-oogenesishelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip15 2 MARKERS OF OVARIAN RESERVEhelliphelliphelliphelliphelliphelliphelliphelliphelliphellip16 21 Ovarian reserve markers with limited clinical valuehelliphelliphelliphelliphellip16 213 Inhibin Bhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip16 214 Basal oestradiolhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip17 215 Dynamic testshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip17 216 Ovarian volumehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip18 22 Ovarian reserve markers in routine clinical usehelliphelliphelliphelliphelliphelliphellip18 221 Chronological agehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip18 222 Basal FSHhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip18 223 Antral follicle counthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip19 3 ANTI-MUumlLLERIAN HORMONEhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip20 31 Biology of anti-Muumlllerian hormonehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip20 311 The role of AMH in the ovaryhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip21 312 AMH in women with polycystic ovary syndromehelliphelliphelliphelliphellip22 32 AMH Assayhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip23 33 Variability of AMH measurementshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip24 34 Role of AMH in assessment of ovarian reservehelliphelliphelliphelliphelliphellip25 341 Prediction of poor and excessive ovarian response in IVFhelliphellip25 342 Prediction of live birth in cycles of IVFhelliphelliphelliphelliphelliphelliphelliphelliphellip26

3 5 Role of AMH in ovarian stimulation for cycles of IVFhelliphelliphelliphellip26

4 MULTIVARIATE TESTShelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip27

5 SUMMARYhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip28 II GENERAL INTRODUCTIONhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip29 REFERENCEShelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip31

10

I LITERATURE REVIEW GENERAL BACKGROUND

Infertility is a disease of the reproductive system defined by the failure to

achieve a pregnancy after 12 months of regular unprotected sexual intercourse

although the criteria for the duration vary between different countries (NICE

2013) Worldwide prevalence of infertility estimated to be around 724 million

couples and around 40 million of those seek medical care (Hull et al 1985) In

the UK 15 couples present with infertility with an annual incidence of 12

couples per 1000 general population (Scott et al 2009) The main causes of

infertility are tubal disease ovulatory disorders male factor and poor ovarian

reserve In a third of couples the cause of failure to achieve pregnancy is not

established which is known as unexplained infertility (NICE 2013) Effective

treatment options include improving lifestyle factors medical andor surgical

treatment of underlying pathology induction of ovulation and Assisted

Reproductive Technology (ART) Assisted Reproduction consist of

intrauterine insemination (IUI) and in vitro fertilisation (IVF) cycles with or

without introcytoplasmic sperm injection (ICSI) as well as treatment involving

donated gametes It is estimated that 75 of infertile couples presenting at

primary care centres in the UK are referred to fertility specialists based at

secondary or tertiary care centres and nearly 50 of those are subsequently

offered IVFICSI treatment (Scott et al 2009) This is supported by figures of

Human Fertility and Embryology Authority (HFEA) which indicates more

than 50000 IVF treatment cycles are performed in the UK annually (HFEA

2008)

An IVF treatment cycle involves a) pituitary down regulation b)

controlled ovarian stimulation c) oocyte recovery c) in vitro fertilisation of eggs

with sperm d) transfer of resulting embryo(s) back to uterus and c) luteal

phase support (NICE 2013) Prevention of premature surge of luteinising

hormone during controlled ovarian stimulation (COS) is achieved by pituitary

down regulation using either preparations of gonadotrophin releasing hormone

agonist which is widely known as ldquoAgonist cyclerdquo or gonadotrophin releasing

hormone antagonist which is known ldquoAntagonist cyclerdquo (Figure 1 and 2)

Controlled ovarian stimulation involves administration of gonadotrophins to

encourage the development of supernumerary preovulatory follicles followed

by administration of exogenous human chorionic gonadotropin (hCG) or

11

recombinant luteinising hormone (rLH) to assist in maturation of oocytes 34-

36 hours prior to egg collection which is usually conducted with guidance of

transvaginal ultrasound scanning Subject to sperm parameters the fertilisation

of oocytes is conducted by in vitro insemination or intracytoplasmic sperm

injection The resulting embryo(s) are cultured under strict laboratory

conditions and undergo regular qualitative and quantitative assessments before

transferring the best quality embryo(s) back into uterus during its cleavage

(Day 2 or Day 3) or blastocyst (Day 5 or Day 6) stage of development In

natural menstrual cycles under the influence of HCG progesterone secreted

by the ovarian corpus luteum ensures proliferative changes in the endometrium

providing the optimal environment for implantation of embryo(s) (van der

Linden et al 2011) However in IVF treatment cycles owing to pituitary down

regulation and lack of HCG progesterone levels are not in sufficiently high

concentration to ensure an adequate endometrial receptivity and therefore

exogenous analogues of this hormone is administered following transfer of

embryo(s) This is called ldquoluteal phase supportrdquo and in patients with viable

pregnancy usually lasts till 12th week of gestation when placenta starts

producing progesterone in sufficient quantities (van der Linden et al 2011)

In IVF programmes the ldquosuccessrdquo of the treatment often defined as

achieving a live birth following IVF cycle and expressed using Live Birth Rate

(LBR) In general success in IVF predominantly determined by womanrsquos age

cause(s) of infertility ovarian reserve previous reproductive history and

lifestyle factors (NICE 2013 Taylor 2003 Lintsen et al 2005) However

effectiveness of medical interventions as well as the quality of care play

important role in determining the outcome of IVF treatment This is evident

from significant variation in live birth rates among fertility clinics given for

instance in the UK LBR for women younger than 35 years of age after IVF

cycles varies from 15 to 61 (HFEA 2008 HFEA 2007) The provision of

effective interventions in both clinical and laboratory aspects of the care

appears to be the key in achieving high success rates Identification of patients

with sufficient ovarian reserve who benefit from IVF cycles followed by

providing optimal ovarian stimulation regimens may be useful in improving the

outcomes of IVF programmes According to HFEA data around 12 of IVF

cycles are cancelled due to poor or excessive ovarian response (Kurinczuk et al

2010) Availability of reliable markers for assessment of ovarian reserve and

tailoring ovarian stimulation regimens to the need of each individual patient

12

may improve selection of patients with sufficient ovarian reserve and reduce

the rate of cycle cancellation consequently improving the success of IVF

cycles (Yates et al 2011)

Assessment of ovarian reserve can be achieved using various biomarkers

and four of those are currently used by most clinics womanrsquos chronological

age (Age) serum follicle stimulating hormone (FSH) antral follicle count

(AFC) and serum anti-Muumlllerian hormone (AMH) More recently AMH has

been a focus of interest given it is the only available endocrine marker that is

suitable for direct assessment of the activity of ovarian follicles in their non-

cyclical stage development providing a window to FSH independent phase of

follicular recruitment Furthermore it appears to be reliable biomarker for a)

both the assessment of ovarian reserve and the optimisation of ovarian

stimulation regimens (Yates et al 2011 La Marca et al 2009) b) screening and

diagnosis of polycystic ovarian syndrome (PCOS) (Cook et al 2002) c)

monitoring of disease activity in women with a history of granulosa cell

tumours (Lane et al 1999) d) prediction of the age of diminished fertility and

the menopause (van Disseldorp et al 2008 Broer et al 2011) and finally (e)

assessment of the long term effect of chemotherapy on ovarian reserve

(Anderson 2011)

In this review I first discuss current knowledge on factors that

determine ovarian reserve including the formation and loss of oocyte pool

Then characteristics of the markers of ovarian reserve are reviewed Finally I

examine current understanding of biology of anti-Muumlllerian hormone and its

role in management of infertility

1 OVARIAN RESERVE

It is important to recognize that there is no universal definition for the

term ldquoovarian reserverdquo and the term can have various meanings depending on

the context in which it is used For instance the scientific literature describing

the biology of ovarian reserve usually refers to ldquothe total number of remaining

oocytes in the ovaries which consists of the number of resting primordial

follicles and growing primary pre-antral and antral folliclesrdquo (Gleicher et al

2011) In contrast the use of the term in the context of clinical studies may

refer to ldquoclinically measurable ovarian reserve established using available

biomarkers of ovarian reserverdquo For the purpose of clarity in this thesis the

13

term ldquoovarian reserverdquo refers to clinically measurable ovarian reserve whilst

true biological ovarian reserve will be termed ldquobiological ovarian reserverdquo

Recent studies have demonstrated that ovarian reserve is highly variable

between women due to the variation in the size of initial ovarian reserve at

birth as well as the rate of loss of ovarian reserve thereafter (Wallace et al

2010) Interestingly the rate of oocyte loss appears to be mainly determined by

the initial ovarian reserve which is believed to be facilitated by most potent

ovarian growth factor anti-Muumlllerian hormone Similarly the size of the initial

ovarian reserve is mainly underpinned by the rate of primordial follicle

assembly in the embryo which is also regulated by AMH Both primordial

follicle assembly and the rate of oocyte loss appear to be primarily under the

influence of genetic factors although developmental and environmental factors

are also believed to play a role (Nilsson et al 2010 Shuh-Huerta et al 2012)

11 Primordial follicle assembly

The process of assembly of primordial follicles in the female embryo

spans from the early embryonic to the early postnatal period and formation of

primordial follicles consists of following stages 1) primordial germ cell (PGC)

2) oogonia 3) primary oocyte and 4) primordial follicle In the human female

fetus around a hundred cells that differentiated from extra-embryonic

ectoderm form early PGCs on the yolk sac and migrate via hindgut to gonadal

ridges during 4th - 6th weeks of gestation (MC et al 1953 Donovan 1998) Once

arrived to the gonadal ridges these cells are called primary oogonia which

consequently undergo several rounds of mitotic division during 6th - 28th weeks

of gestation Interestingly the numbers of oogonia reach as high as six million

during its highest rate of mitotic division at around 20 weeks of gestation

Following the last round of mitotic division oogonia enter meiosis which

marks their new stage of development-primary oocyte Formation of

primordial follicles starts as early as at 8th week of gestation and is characterised

by meiosis of primary oocyte that arrest in diplotyne stage and surrounding of

the oocyte by somatic granulosa cell (Baker et al 1963 Maheshwari and Fowler

2010) Indeed the primordial follicle is the cardinal unit of the biological

ovarian reserve and therefore the rate of formation of primordial follicles is the

main determinant of initial biological ovarian reserve at birth

Interestingly the process of loss of oogonia and oocytes which is also

one of the main determinants of the initial ovarian reserve takes place

14

throughout the period of follicle assembly The formation of the granulosa cell

layer around the oocyte prevents the oocyte from subsequent atresia The

oocyte enveloped in a single layer of granulosa cells which is also known as

primordial follicle remains quiescent until recruitment of the follicle for

growth which may not take place for a number of decades after the formation

of a particular primordial follicle (Skinner 2005 Maheshwari and Fowler 2010)

12 Oocyte recruitment

Follicle growth in women consists of two stages a) the initial non-cyclical

recruitment of primordial follicles and the formation of a primary and a pre-

antral follicles and b) cyclical development of antral follicles with subsequent

selection of usually a single dominant follicle The initial recruitment of

primordial follicles is continuous non-cyclical process that starts as early as

from 18-20 weeks of gestation and lasts till the depletion of follicle pool which

later results in the menopause (McGee and Hsueh 2000) Transformation of

flat granulosa cells into cuboidal cells increases the diameter of the oocyte and

the formation of zona pellicuda completes the stage of formation of a primary

follicle During pre-antral stage oocytes increase in diameter and mitotic

division of granulose cells create a new layer of cells-theca cells The

mechanism of initial recruitment of oocytes is not well understood but it is

clear that the process is independent of influence of pituitary gonadotrophins

and appears to be governed by the genetically pre-programmed interaction of

the oocyte with local growth factors the most important of which appears to

be anti-Muumlllerian hormone and cytokines (McGee and Hsueh 2000)

The cyclical phase of development of oocytes is characterised by the

transformation of secondary follicle into antral follicle and subsequent growth

of antral follicles into pre-ovulatory stages In general the process of cyclic

recruitment starts from puberty under the influence of rising levels of pituitary

follicular stimulating hormone (FSH) During the antral stage oocyte increases

in size even further and the formation of a fluid filled space in follicle is

observed Under the influence of FSH luteinising hormone (LH) and local

growth factorsselection of a single dominant follicle occurs which followsby an

ovulation (McGee and Hsueh 2000)

Oocyte loss is a continuous process and occurs due to atresia of oocytes

during primary secondary and antral stages of development The rate of

oocyte loss appears to increase until the age of around 14 and declines

15

thereafter until the age of the menopause when around 1000 primordial

follicles remain (Hansen et al 2008 Oktem and Oktayl 2008) Furthermore by

the age of 30 years the average age at which women of western societies plan

to start a family around 90 of initial primordial follicles are lost which

illustrates that formation and maintenance of ovarian reserve is wasteful

process in humans (ONS 2012 Wallace and Kelsey 2010) As mentioned

above there is a wide individual variation in both sizes of initial primordial

follicular pool and the rate of oocyte loss which explains variation in the

reproductive lifespan in women Evidently the number of primordial follicles

at birth ranges between around 35000 to 25 million per ovary and similarly

the rate of oocyte loss during its peak at 14 years of age may range between

100 to 7500 primordial follicles per month which is believed to be inversely

proportional to initial size of primordial follicle pool (Wallace and Kelsey

2010)

13 Theory of neo-oogenesis

The traditional view of oogenesis states that the process of the creation

and the mitotic division of oogonia with subsequent formation of primordial

follicles takes place only during embryonic and foetal life (Zuckerman 1951)

According to this central theory of mammalian reproductive biology females

are born with a certain number of germ cells that is gradually lost but not

renewed during postnatal period However Johnson et al have recently

challenged this view and reported that adult mammalian ovary may possesses

mitotically active germ cells that continuously replenish the primordial follicle

pool (Johnson et al 2004) The group reported that ovaries of juvenile and

young adult mice contained large ovoid cells which resemble germ cells of

foetal mouse ovaries Interestingly immunohistochemical staining for a gene

which is expressed exclusively in germ cells have been reported to have

confirmed that these large ovoid cells were of germline lineage Furthermore

application of a mitotic germ cell toxicant busulphan appeared to have

eliminated primordial follicle reserve by early adulthood but did not induce

atresia suggesting the presence of proliferative germ cells in postnatal mouse

ovary (Johnson et al 2004 Bazer 2004) The study has generated enormous

amount of interest as well as debate among reproductive biologists (Notarianni

2011) Some other groups have also reported an evidence of postnatal

oogenesis (Pacchiarott et al 2010 Zou et al 2009 Bukovsky et al 2004)) while

16

others do not support the theory (Bristol-Gould et al 2006 Byskov et al 2005

Begum et al 2008) Furthermore some authors argued that adult mouse

germline stem cells exist and remain quiescent in physiologic conditions and

neo-oogenesis occurs only in response to ovotoxic damage (Tilly et al 2007 De

Felici 2010) Although consensus has yet to emerge to date there is no

conclusive evidence on validity of theory of neo-oogenesis

2 MARKERS FOR ASSESMENT OF OVARIAN RESERVE

Biological ovarian reserve is defined as the number of primordial and

growing follicles left in the ovary at any given time and therefore only

counting the number of primordial follicles by histological assessment can

accurately determine ovarian reserve which is clearly not feasible in clinical

setting However ovarian reserve can be estimated using various biomarkers

dynamic clinical tests and implied from the outcomes of ART cycles

Although a wide range of clinical (age ovarian response in previous IVF

cycles) biochemical (basal FSH Inhibin B basal oestradiol AMH) ultrasound

(ovarian volume antral follicle count (AFC)) and dynamic (clomiphene

challenge test exogenous FSH ovarian reserve test GnRH analogue

stimulating test) tests of ovarian reserve exist only a few of the markers are

reliable and practical enough to be of use in routine clinical practice In this

chapter first I discuss the research evidence on the assessment of the markers

andor tests of ovarian reserve that have limited clinical value Then I

evaluated more reliable markers that are in routine clinical use Age FSH

AFC and combination of these markers in multivariable tests Finally I

conducted detailed review of biology of AMH and the role AMH measurement

in the management of infertility

21 Ovarian reserve markers with limited clinical value

211 Inhibin B

Inhibins are members of TGFβ family and expressed in granulosa cells

of growing follicles Principal role of inhibins is thought to be the negative

feedback regulation of pituitary FSH secretion and therefore the serum level of

circulating hormone is believed to reflect the state of folliculogenesis

17

Consequently several groups have studied the role of serum Inhibin β in the

assessment of ovarian reserve Although initial reports were encouraging

(Seifer et al 1997) more robust studies demonstrated that serum Inhibin β was

less reliable than chronological age or basal FSH (Creus et al 2000 Urbancsek

2005) The systematic review of nine studies demonstrated that accuracy of the

Inhibin β test for predicting poor ovarian response and non-pregnancy in IVF

cycles was modest even at a very low threshold level (Broekmans et al 2006)

Therefore it is recommended that inhibin β at best can be used as only

screening test in the fertility centers where other more reliable markers are not

available (Broekmans et al 2006)

212 Basal oestradiol

Some studies suggested that elevated basal oestradiol levels indicate low

ovarian reserve and are associated with poor fertility prognosis (Johannes et al

1998 Licciardi and Rosenwaks 1995) Johannes et al demonstrated basal

oestradiol in conjunction with serum FSH is more reliable than serum FSH

alone in prediction of cycle cancellation due to the poor response in IVF cycles

(Johannes et al 1998) However there are no published data on the comparison

of basal oestradiol to more reliable markers such as AMH or antral follicle

count (AFC) Moreover a recent systematic review has demonstrated that

basal oestradiol has very low predictive value for poor response and has no

discriminatory power for accuracy of non-pregnancy prediction (Broekmans et

al 2006)

213 Dynamic tests of ovarian reserve

The dynamic tests of ovarian reserve are based on assessment of ovarian

response by measuring serum FSH and oestradiol levels following

administration of exogenous stimulation The following tests are reported in

literature Clomiphene Citrate Challenge Test (CCCT) Exogenous FSH

Ovarian Reserve Test (EFORT) and GnRH agonist stimulation test A recent

systematic review and meta-analysis on the accuracy of these tests showed that

none of them can adequately predict poor response or non-pregnancy in IVF

cycles and therefore are not recommended for use in routine clinical practice

(Maheshwari et al 2009)

18

214 Ovarian volume

There is some evidence that increased age is associated with decreased

ovarian volume and women with smaller ovaries are more likely to have

cancellation of their IVF cycles due to poor ovarian response (Syrop et al 1995

Syrop et al 1999 Templeton 1995) However a meta-analysis of the published

studies on the accuracy of ovarian volume as a predictor of poor response and

non-pregnancy in IVF cycles failed to demonstrate clinical usefulness of the

test and suggested the test is not reliable enough for use in a routine clinical

practice (Broekmans et al 2006)

22 Ovarian reserve markers in routine clinical use

221 Chronological age

Owing to the biological age-related decline of the quantity and arguably

the quality of oocytes the chronological age can be used as a marker of ovarian

reserve Studies have demonstrated that ovarian reserve (Wallace and Kelsey

2010 Kelsey 2011) natural fecundity (Islam et al 1989 and outcomes of ART

(Templeton et al 1996 van Kooij et al 1996) decline significantly from age of

35 when it is believed the ovarian reserve undergoes accelerated decline

Although there is a strong association between chronological age and reduction

in fertility evidently there is a significant variation in age-related ovarian

reserve indicating chronological age alone may not be sufficient to estimate the

individual womanrsquos ovarian reserve reliably (Broekmans et al 2006)

222 Basal FSH

Basal FSH was one of the first endocrine markers introduced in ART

programs and is still utilized in many fertility clinics albeit in conjunction with

other markers which are considered more reliable (Creus et al 2000) Secretion

of FSH is largely governed by the negative feedback effect of steroid

hormones primarily oestradiol and inhibins which are expressed in granulosa

cells of growing ovarian follicles Consequently decreased or diminished

recruitment of ovarian follicles is associated increased serum FSH

measurements and high particularly very high basal FSH reading is considered

as a good marker of very low or diminished ovarian reserve (Abdalla et al

2006) However unlike some other markers FSH measurements do not

appear to have discriminatory power for categorisation of patients to various

19

bands of ovarian reserve Given between-patient variability FSH measurement

(CV 30) is similar to its within-patient variability (27) stratification of

patients to various ranges of ovarian reserve does not appear to be feasible

(Rustamov et al 2011) Indeed a recent systematic review of 37 studies on the

prediction of poor response and non-pregnancy in IVF cycle has concluded

that basal FSH is an adequate test at very high threshold levels and therefore

has limited value in modern ART programs (Broekmans et al 2006)

223 Antral follicle count

Antral follicle count estimation involves ultrasound assessment of

ovaries between 2nd and 4th day of menstrual period and counting ldquofolliclesrdquo

which corresponds to antral stage of folliculogenesis (Broekmans et al 2010)

The test provides direct quantitative assessment of growing follicles and is

known as one of the most reliable markers of ovarian reserve (Broekmans et al

2006) AFC measurement has been reported as having a similar sensitivity and

specificity to AMH in prediction of poor and excessive ovarian response in

IVF cycles (Broekmans et al 2006 Broer et al 2010 Jayaprakasan et al 2010)

Given AFC measurement is available instantly and allows patients to be

counseled immediately the test eliminates the need for an additional patient

visit prior to IVF cycle However AFC is normally performed only in the early

follicular phase of the menstrual cycle given most published data on

measurement of AFC are based on studies that assessed antral follicles during

this stage of the cycle (Broekmans et al 2010a) Interestingly more recent

studies suggest that variability of AFC during menstrual cycle is small

particularly when follicles between 2-6mm are counted and therefore

assessment of AFC without account for the day of menstrual cycle may be

feasible (Deb et al 2013)

One of the main drawbacks of AFC is that the cut off levels for size of

counted follicles remains to be standardised (Broekmans 2010b) Initially

follicles of 2-10mm were introduced as the range for AFC and many studies

were based on this cut off Later counting follicles of 2-6mm was reported to

provide most accurate assessment of ovarian reserve (Jayaprakasan et al 2010b

Haadsma et al 2007) and therefore some newer studies are based on AFC

measurements that used this criterion Consequently direct comparison of the

outcomes of various studies on assessment of AFC requires careful analysis

20

3 ANTI-MUumlLLERIAN HORMONE

31 Biology of Anti-Muumlllerian hormone

AMH is a member of transforming growth factor β superfamily which

was discovered by Jost et al in 1947 and was initially known for its is role in

regression of Muumlllerian ducts in sex differentiation of the male embryo In

women AMH is believed to be solely produced by ovaries and expressed in

granulosa cells of growing follicles of 2-6 mm in size which corresponds to

primary pre-antral and early antral stage of follicular development Although

there has been a report of expression of AMH in endometrial cells to date

there is no other published evidence that supports this finding (Wang et al

2009) Indeed studies that evaluated half-life of AMH in serum have

demonstrated that in women who had bilateral salpingo-oopherectomy AMH

becomes undetectable within 3-5 days of following surgery suggesting ovaries

are the only source of secretion of AMH in appreciable quantity (La Marca et

al 2005b) Anti-Muumlllerian hormone is a dimeric glycoprotein which is

composed of a long N-terminus and short C-terminus and was believed to be

secreted in serum only in this dimeric form (AMH-N C)

Like other members of TGF-β family which includes inhibins activins

bone morphogenic proteins (BMPs) and growth and differentiation factors

(Massague et al 1990) AMH binds to two type of serinethreonine kinase

receptors referred to as type I and type II In order to activate AMH signaling

pathway both receptors have to form a heteromeric complex When AMH

binds to the type II (AMHR-II) receptor (Massague et al 2000) this will

phosphorylate and activate a type I receptor (ALK2 -3 andor -6) which

subsequently activates the SMAD pathway through phosphorylation of

SMAD 1 5 andor 8 These activated SMADs interact with SMAD4 and

translocate to the nucleus regulating the expression of different genes

inhibiting the recruitment of primordial follicles and reducing FSH sensitivity

in growing follicles In addition AMH receptors as well as the other members

of TGF-β family can activate MAPK and PI3KAKT pathways

Studies on AMHR II-deficient male mice demonstrated lack of

regression of Muumlllerian ducts suggesting that type II receptor is essential in

AMH signaling (Mishina et al 1996) Similarly Type I receptors which includes

three members of activin receptor-like kinase (ALK2 ALK3 and ALK6) also

appear to play an important role in the regression of Muumlllerian ducts although

21

the role of ALK 6 in AMH signaling appears not to be crucial (Visser 2003

Clarke et al 2001) The signal transduction pathway of AMH in the ovary is

largely not understood In postnatal mice ovary AMHR-II receptor was

expressed in both granulosa and theca cells of pre-antral and antral follicles

(Visser 2003) AMH type I receptors ALK 2 and ALK 3 is expressed in foetal

as well as adult mouse ovary while ALK 6 is expressed in only adult ovary

(Visser 2003)

311 The role of AMH in the ovary

In the mammalian ovary the role of AMH appears to be one of a

regulation of size of the primordial follicle pool by its inhibitory effect on the

formation as well as the growth of primordial follicles (Nilsson et al 2011) In

the embryonic mouse ovary AMH inhibits the initiation of the assembly of

follicles when the process of apoptosis of the majority of oocytes is observed

(Nilsson et al 2011) Consequently AMH reduces the rate of oocyte loss

which plays an important role in the determination of the size of initial follicle

pool Similarly in the adult mouse ovary AMH plays a central role in

maintaining the follicle pool AMH inhibits both the processes of the initial

(non-cyclical) recruitment of primordial follicles and subsequent FSH-

dependent cyclical growth of antral follicles (Figure 3) Inhibition of the initial

recruitment of a new cohort of follicles is believed to be achieved by a

paracrine negative feedback effect of the rising levels of AMH secreted from

already recruited growing follicles (Durlinger et al 1999) Durlinger et al

compared the complete follicle population of AMHnull mice and wild type

mice of different ages of 25 days 4 months old and 13 months old and found

that the ovaries of 25 day and 4 months old AMHnull females contained

significantly higher number of growing pre-antral and antral follicles but

significantly fewer primordial follicles compared to wild-type females

(Durlinger et al 1999) Interestingly almost no primordial follicles were

detected in 13 months old AMHnull mice ovaries suggesting AMH is a potent

inhibitor of the recruitment of primordial follicles and in the absence of AMH

ovaries undergo premature depletion of primordial follicles due to an

accelerated recruitment Subsequent study conducted by the group

demonstrated that in addition to its inhibitory effect to the resting follicles

AMH also suppresses the development of the growing follicles (Durlinger et al

2001 Durlinger et al 2002 Themmen 2005) It appears that AMH inhibits

22

FSH-induced follicle growth by reducing the sensitivity of growing follicles to

FSH which has been confirmed by in vivo as well as in vitro studies (Durlinger

et al 1999 Durlinger et al 2001) In the initial study the group observed that

despite lower levels of serum FSH concentration ovaries of AMHnull mice

contained more growing follicles than that of their wild-type littermates which

has been supported by the findings of subsequent in vitro study (Durlinger et al

1999) Addition of AMH to the culture inhibited FSH-induced follicle growth

of pre-antral mouse follicles due to reduction in granulosa cell proliferation

(Durlinger et al 2001)

In the human embryo the expression of AMH commences in the late

foetal life and can be detected only from 36 weeks of gestation (Rajpert-De et

al 1999 Lee et al 1996) Following a small decline in first two years of life

AMH levels gradually increase to peak at (mean 5 ngml) around age of 24

years In line with the pattern of oocyte loss serum hormone levels gradually

decline with increasing age and become undetectable around 5 years prior to

menopause (Kelsey et al 2011 Nelson et al 2011)

It has been suggested that anti-Muumlllerian hormone plays a central role in

determining the pace of recruitment of primordial follicles hence maintaining

the primordial follicle pool of postnatal mammalian ovary Consequently a

reduction in the concentration of circulating AMH signals the exhaustion of

the primordial follicle pool and the decline of ovarian function

312 AMH in women with polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) endocrine abnormality characterised

by increased ovarian androgen secretion infrequent ovulation and the

appearance of ldquopolycysticrdquo ovaries on ultrasound scan (Dunaif 1997 Homburg

et al 1993) It is the commonest endocrine abnormality in women of

reproductive age and affects around 15-20 of women PCOS is also one of

the main causes of anovulation and subsequent sub-fertility (Webber et al

2003) Although the role of anti-Muumlllerian hormone in the development of

PCOS is not fully understood it is becoming increasingly evident that the

hormone plays an important role in its pathogenesis (Pehlivanov et al 2011)

There is a strong association between serum AMH levels and PCOS and it

appears that women diagnosed with PCOS have two to three fold higher

serum AMH concentration compared to normo-ovulatory women (Cook et al

2002 Pigny et al 2003) Similarly women with PCOS are found to have

23

significantly higher number antral follicles Interestingly the expression of

AMH in granulosa cells of follicles were found to be 75 times higher in women

with PCOS compared to those without a the disease suggesting increased

serum AMH in PCOS may be due to increased secretion of hormone per

follicle rather than due to an increased number of antral follicles (Pellat et al

2007) High AMH concentrations may act as the main facilitator of abnormal

folliculogenesis in PCOS given the follicles appear to arrest when they reach

an antral stage (2-6mm) of development (Rajpert-De et al 1999) Indeed the

studies of Durlinger et al have demonstrated that AMH inhibits selection of

dominant follicle when follicles reach antral stage of development (Durlinger et

al 2001) Serum AMH levels appear to decrease with treatment of PCOS

which may play important role in restoration of ovulatory cycles Studies have

reported a significant reduction in serum concentration of AMH following

treatment of PCOS with metformin and laparoscopic ovarian diathermy (Falbo

et al 2010 Amer et al 2009 Elmashad 2011) Similarly reduction of BMI

following intensified endurance exercise training for treatment of PCOS may

also lead to a significant reduction in serum AMH levels (Moran et al 2011)

This suggests that there is strong association between serum concentration of

AMH and abnormal folliculogenesis in PCOS and therefore understanding the

molecular mechanisms of this interaction should be one of the priorities of

future research

32 AMH Assays

Enzyme-linked immunosorbent assay specific for measurement of anti-

Muumlllerian hormone was first developed in 1990 and was recognised as a

significant step in the assessment of ovarian reserve (Hudson et al 1990)

Subsequently a number of non-commercial immunoassays were developed

which were mainly used in research settings (Lee et al 1996) Later Diagnostic

Systems Ltd (DSL) and Immunotech Beckman Coulter Ltd (IOT) introduced

two commercial immunoassays for the routine clinical assessment of ovarian

reserve which are known as ldquofirst generation AMH assaysrdquo (Nelson and La

Marca 2011) These assays employed two different antibodies against AMH

and used different standards for calibration providing non-comparable

measurements (Nelson and La Marca 2011) Consequently several studies

attempted to develop a reliable between-assay conversion factor which

interestingly revealed from five-fold higher with the IOT assay to assay

24

equivalence causing significant impact to reliability of AMH measurements and

interpretation of research findings (Hehenkamp et al 2006 Freour et al 2007

Bersinger et al 2007 Taieb et al 2008 Lee et al 2011)

Later the manufacturer of IOT assay (Beckmann Coulter Ltd)

consolidated the manufacturer of the DSL assay (Diagnostic Systems

Laboratories Inc) and introduced a new assay ldquoGen II AMH assayrdquo which is

only available commercial immunoassay in most countries including the UK

AMH Gen II assay was developed using the antibodies derived from first

generation DSL assay and calibrated using the standards used for IOT assay

and was believed to be considerably more stable compared to the first

generation immunoassays providing more reliable measurements (Kumar et al

2010 Nelson and La Marca 2011) The manufacturer as well as initial external

validation study recommended when compared to old DSL assay AMH Gen

II assay provides around 40 higher measurements and therefore previously

reported DSL-based clinical cut-off levels for estimation of ovarian reserve

should be increased by 40 in order to use Gen II-based AMH results (Kumar

et al 2010 Wallace et al 2011 Nelson and La Marca 2011)

33 Variability of AMH measurements

It is generally believed that AMH values do not change throughout the

menstrual cycle and early studies reported that variation in AMH

measurements between repeated measurements of same patient was negligible

(van Disseldorp et al 2010 La Marca 2010) On the basis of these studies

sampling at a random time in the menstrual cycle was introduced as a method

for measurement of AMH in routine clinical practice However the

methodologies of some of these studies do not appear to be robust enough to

reliably estimate sample-to-sample variability of AMH which is mainly due to

small sample sizes (Rustamov et al 2011) Consequently in a recent study we

assessed sample-to-sample variability of AMH using DSL assay and found that

within-subject coefficient of variation (CV) of AMH between samples were as

high as 28 which cannot be attributed to any patient or cycle characteristics

(Rustamov et al 2011) Although there is no consensus in the causes of this

observed variability in AMH measurements we believe it is largely attributable

to instability of AMH samples given initial recruitment of primordial follicles

and growth of AMH producing pre-antral and antral follicles are continuous

process and therefore the true biological variation between samples is unlikely

25

to be high However given the importance of establishing true variability of

AMH in both understanding of the biology of hormone and clinical

application of the test future studies should be conducted to establish the

source of variability in the clinical samples

3 4 The role of AMH in the assessment of ovarian reserve

341 Prediction of poor and excessive ovarian response in cycles of

IVF

A number of studies have assessed the role of AMH in the prediction of

poor ovarian response in IVF cycles using first generation AMH assays and

found that AMH and AFC were the best predictors of poor ovarian response

compared to other markers of ovarian reserve Nardo et al showed that the

predictive value of AMH in receiver operating characteristic curve (ROC)

analysis was similar to (AUC 088) that of AFC (AUC 081) and found that

AMH cut offs of gt375 ngmL and lt10 ngmL would have modest

sensitivity and specificity in predicting the extremes of response (Nardo et al

2009) These findings were largely supported by subsequent prospective studies

and a systematic review (Nelson et al 2007 Jayaprakasan et al 2010 Broer et al

2011) Similarly comparison of chronological age basal FSH ovarian volume

AFC and AMH found that only AMH (AUC 090) and AFC (AUC 093) were

reliable predictors of poor ovarian response in cycles of IVF Subsequent

combination of the effect of AMH and AFC using multivariable regression

analysis did not improve the level of prediction of poor ovarian response

significantly (AUC 094) suggesting both AMH and AFC can be used as

independent markers (Jayaprakasan et al 2010)

Similarly most studies agree that AMH and AFC are the best predictors

of excessive ovarian response and ovarian hyperstimulation syndrome (OHSS)

compared to other clinical endocrine and ultrasound markers (Nardo et al

2009 Nelson et al 2007) Broer et al compared these two tests in systematic

review of 14 studies and reported that the summary estimates of the sensitivity

and the specificity for AMH were 82 and 76 respectively and for AFC 82

and 80 respectively (Broer et al 2011) Consequently the study concluded

that AMH and AFC were equally predictive and the difference in the predictive

value between the tests was not statistically significant

26

342 Prediction of live birth rate (LBR) in cycles of IVF

Lee at al reported that AMH and chronological age were more accurate

than basal FSH AFC BMI and causes of infertility in the prediction of live

birth rate (Lee et al 2009) Similarly La Marca et al suggested that odds of live

birth could be reliably predicted using AMH (La Marca et al 2010b) although

subsequent review of the study questioned strength of the evidence (Loh and

Maheshwari 2011)

A study conducted by Nelson et al found that higher AMH levels had

stronger association with increased live birth rate compared to age and FSH

(Nelson et al 2007) However the study also suggested that this association

was mainly confined in the women with low AMH levels and there was no

additional increase in live birth in women with AMH levels of higher than 710

pmolL This may suggest that achieving a live birth may be under the

influence of number of other factors and that markers of ovarian reserve alone

may not be able predict this outcome reliably

35 The role of AMH in individualisation of ovarian stimulation in

IVF cycles

Prediction of ovarian response to the stimulation of ovaries in cycles of

IVF plays an important role in the counseling of couples undergoing treatment

programmes and hence many clinical studies on AMH have focused on the

prognostic value of AMH measurements However data on using AMH as a

tool for improving the clinical outcomes in IVF cycles appear to be lacking

considering AMH may be useful tool in tailoring treatment strategies to an

individual patientrsquos ovarian reserve Unlike most other markers AMH has

discriminatory power in determining various degrees of ovarian reserve due to

significantly higher between patient (CV 94) variability compared to its

within-patient (CV 28) variation (Rustamov et al 2011) which allows

stratification of patients into various degrees of (eg low normal high) ovarian

reserve Subsequently most optimal ovarian stimulation protocol may be

established for each band of ovarian reserve Consequently reference ranges

on the basis of distribution of AMH in infertile women were developed which

were subsequently adopted by fertility clinics for a tailoring the mode of

27

ovarian stimulation and daily dose of gonadotrophins in IVF (The Doctors

Laboratory 2008 However currently available clinical reference ranges are

based on the first generation DSL assay and may not be reliably convertible to

currently available Gen II assay measurements (Wallace et al 2011) Indeed the

findings of the studies on comparability of the first generation AMH assays

suggest that establishing a reliable between assay conversion factor between

AMH assays may not be straightforward Furthermore the reference ranges

appear to reflect the distribution of AMH measurements within a specific

population and may therefore not be directly applicable for the prediction of

response to ovarian stimulation in IVF patients (The Doctors Laboratory

2008)

More importantly despite lack of good quality evidence on the

effectiveness of AMH-tailored ovarian stimulation protocols a number of

fertility clinics appear to have introduced various AMH-based COH protocols

in their IVF programs At present research evidence on AMH-tailored

ovarian stimulation in IVF is largely based on two retrospective studies

(Nelson et al 2009 Yates et al 2012) Both of these studies display considerable

methodological limitations including small sample size and centre-related or

period-related selection of their cohorts In this context AMH is used as a tool

for therapeutic intervention and therefore the research evidence should ideally

be derived from randomised controlled trials However recruitment of large

enough patients in IVF setting may take considerable time and resources In

the meantime given AMH-tailored ovarian stimulation has already been

introduced in clinical practice and there is urgent need for more reliable data

the studies with a larger cohorts and robust methodology should assess the role

of AMH in individualisation of ovarian stimulation in IVF treatment cycles

4 Multivariate models of assessment of ovarian reserve

In view of the fact there is not a single marker of ovarian reserve that

can accurately predict ovarian response various models for combination of

multiple ovarian markers have been developed (Verhagen et al 2008) A

number of studies reported that multivariate models are better predictors of

poor ovarian response in IVF compared to a single marker (Bancsi et al 2002

Balasch et al 1996 Creus et al 2000 Durmusoglu et al 2004) However a meta-

analysis showed that when compared to a single marker (AFC) multivariate

28

model has a similar accuracy in terms of prediction of poor ovarian response

(Verhagen et al 2008) In contrast a more recent study demonstrated that

multivariate score was superior to chronological age basal FSH or AFC alone

in predicting likelihood of poor ovarian response and clinical pregnancy

(Younis et al 2010) However the study did not include one of the most

reliable markers AMH in either arm necessitating further assessment of the

role of combined tests which include all reliable biomarkers

4 SUMMARY

During the last two decades a significant leap has been taken towards

understanding the biology of anti-Muumlllerian hormone and its role in female

reproduction (Durlinger et al 2002 Themmen et al 2005) Availability of

commercial AMH assays has resulted in significant increase in interest in the

role of the measurement of serum AMH in the assessment of ovarian reserve

which has been followed by the introduction of the test into routine clinical

practice (Nelson et al 2011) However more recent studies suggest that current

methodologies for the measurement of AMH may provide significant sampling

variability (Rustamov et al 2011) Furthermore the studies that compared first

generation commercial assay methods appear to provide non-reproducible

results suggesting there may be underlying issues with assay methodologies

(Lee et al 2011) Similarly despite lack of sufficient evidence in the role of

AMH in individualisation of ovarian stimulation protocols in IVF AMH-

tailored IVF protocols have been introduced in routine clinical practice of

many fertility clinics around the world

Consequently it appears that clinical application of AMH test has

surpassed the research evidence in some aspects of fertility treatment and

therefore future projects should be directed toward areas where gaps in

research evidence exist On the basis of the review of literature we believe that

evaluation of the performance of assay methods understanding the role of

AMH in assessment ovarian reserve and establishing its role in

individualisation of ovarian stimulation protocols should be research priority

29

II GENERAL INTRODUCTION

On the basis of the review of published literature I have identified that

the following areas of research on the clinical application of AMH in the

management of infertility requires further investigation 1) Within-patient

variability of measurement of AMH using Gen II assay method 2)

Establishment of clinically measurable determinants of AMH levels and 3) The

role of AMH in individualisation of ovarian stimulation in IVF treatment

cycles

In our previous study we estimated that there was significant sample-to-

sample variation (CV 28) in AMH measurements when the first generation

DSL assay was used (Rustamov et al 2011) The source of variability is likely to

be related to the assay method given that biological within-cycle variation of

AMH is believed to be small (La Marca et al 2006) Therefore assessment of

sample-to-sample variability of AMH using the newly introduced Gen II assay

which is believed to be significantly more stable and sensitive compared to that

of DSL assay should enable us to establish the measurement related variability

of AMH Furthermore given I am planning to use data from both DSL and

Gen II assays I need to establish between-assay conversion factor for these

assays using data on clinical samples

There appears to be a lack of good quality data on the effect of

ethnicity BMI causes of infertility reproductive history and reproductive

surgery on ovarian reserve Therefore I am planning to ascertain the role of

above factors on determination of ovarian reserve by analysing AMH

measurements of a large cohort of patients

There is a strong correlation between AMH and ovarian performance

in IVF treatment when conventional ovarian stimulation using GnRH agonist

regimens with a standard daily dose of gonadotrophins are used (Nelson et al

2007 Nardo et al 2007) Furthermore studies suggest tailoring the ovarian

stimulation protocols to AMH measurement may improve ovarian

performance and subsequently the success of IVF treatment (Nelson et al

2011 Yates et al 2012) However given methodologies of the published

studies the effectiveness of currently proposed AMH-tailored ovarian

stimulation protocols remains unknown Therefore I am planning to develop

individualised ovarian stimulation protocols by establishing the most optimal

mode of pituitary down regulation and starting dose of gonadotrophins for

30

each AMH cut-off bands using a robust research methodology However

development of individualised ovarian stimulation protocols on the basis of

retrospective data requires a reliable and validated database containing a large

number of observations In the IVF Department of St Maryrsquos Hospital we

have data on a large number of patients who underwent ovarian stimulation

following the introduction of AMH However the data on various aspects of

investigation and treatment of patients is stored in different clinical data

management systems and may not be easily linkable In addition it appears that

data on certain important variables (eg causes of infertility AFC) are available

only in the hospital records necessitating searching for data from the hospital

records of each patient Consequently I designed a project for building a

research database which will have comprehensive and validated datasets that

are necessary for investigation of the research questions of the MD

programme

In conclusion I am planning to conduct a series of studies to improve

the understanding of the role of AMH in the management of women with

infertility Specifically I am intending to evaluate 1) sample-to-sample variability

of Gen II AMH measurements 2) conversion factor between DSL and Gen II

assays in clinical samples 3) the effect of ethnicity BMI causes of infertility

endometriosis reproductive history and reproductive surgery to ovarian

reserve and explore AMH-tailored individualisation of ovarian stimulation in

IVF cycles

31

References

Abbeel E The Istanbul consensus workshop on embryo assessment proceedings of an expert meeting Human reproduction 2011 26 p 1270-83 Abdalla HT M Y Repeated testing of basal FSH levels has no predictive value for IVF outcome in women with elevated basal FSH Human reproduction 2006 21(1) p 171-4 Amer SA LT Ledger WL The value of measuring anti-Mullerian hormone in women with polycystic ovary syndrome undergoing laparoscopic ovarian diathermy Human reproduction 2009 24 p 2760-6 Anderson RA Pretreatment serum anti-Mullerian hormone predicts long-term ovarian function and bone mass after chemotherapy for early breast cancer J Clin Endocrinol Metab 2011961336-1343 Balaban B BD Calderoacuten G Catt J Conaghan J Cowan L Ebner T Gardner D Hardarson T Lundin K Cristina Magli M Mortimer D Mortimer S Munneacute S Royere D Scott L Smitz J Thornhill A van Blerkom J Van den Baker A quantitative and cytological study of germ cells in human ovaries Proc R Soc Lond B Biol Sci 1963 158 p 417-433 Balasch J CM Fabregues F Carmona F Casamitjana R Ascaso and VJ C Inhibin follicle-stimulating hormone and age as predictors of ovarian response in in vitro fertilization cycles stimulated with gonadotropin-releasing hormone agonist-gonadotropin treatment Am J Obstet Gynecol 1996 175 p 1226-1230 Bancsi LF BF Eiijekemans MJ at al Predictors of poor ovarian response in in vitro fertilisation a prospective study comparing basal markers of ovarian reserve Fertility and Sterility 2002 77 p 328-336 Bazer FW Strong science challenges conventional wisdom new perspectives on ovarian biology Reprod Biol Endocrinol 2004 2 p 28 Begum S VE Papaioannou and RG Gosden The oocyte population is not renewed in transplanted or irradiated adult ovaries Hum Reprod 2008 23(10) p 2326-30

Bersinger NA Wunder D Birkhaumluser MH Guibourdenche J Measurement of anti-mullerian hormone by Beckman Coulter ELISA and DSL ELISA in assisted reproduction differences between serum and follicular fluid Clin Chim Acta 2007384174ndash175 Bristol-Gould SK et al Fate of the initial follicle pool empirical and mathematical evidence supporting its sufficiency for adult fertility Dev Biol 2006 298(1) p 149-54 Broekmans FJ et al A systematic review of tests predicting ovarian reserve and IVF outcome Hum Reprod Update 2006 12(6) p 685-718

5

PUBLICATIONS ARISING FROM THE THESIS

Journal Articles

1 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo Philip W Pemberton

The measurement of Anti-Muumlllerian hormone a critical appraisal

The Journal of Clinical Endocrinology amp Metabolism 2014 Mar99(3)723-32

2A Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton Anti-Muumlllerian hormone poor assay reproducibility in a large

cohort of subjects suggests sample instability Human Reproduction 2012 Oct

27(10) 3085-91

2B Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton Human Reproduction Dec2012 Vol 27 Issue 12 p3641

6

Conference presentations

1 O Rustamov S Roberts C Fitzgerald

Ovarian endometrioma is associated with increased AMH levels

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2014 Munich

Poster Presentation

2 O Rustamov M Krishnan R Mathur S Roberts C Fitzgerald

The effect of BMI to the ovarian reserve

Annual Meeting of British Fertility Society January 2014 Sheffield

Oral presentation Dr O Rustamov

3 M Krishnan O Rustamov R Mathur S Roberts C Fitzgerald

The effect of the ethnicity to the ovarian reserve

Annual Meeting of British Fertility Society January 2014 Sheffield

Oral Presentation Dr M Krishnan

4 O Rustamov M Krishnan S Roberts C Fitzgerald

Reproductive surgery and ovarian reserve

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2013 London

Oral presentation Dr O Rustamov

5 C Fitzgerald O Rustamov P Pemberton A Smith A Yates M Krishnan

R Russell L Nardo SRoberts

AMH assays A review of the literature on assay method comparability

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2013 London

Oral presentation Dr C Fitzgerald

6 M Krishnan O Rustamov R Russell C Fitzgerald S Roberts

The role of the ethnicity and the body weight in determination of AMH levels

in infertile women

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2013 London

7

Poster presentation

7 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton

AMH Gen II assay - can we believe the measurements

8th Biennial Conference of UK Fertility Societies January 2013 Liverpool

Poster presentation

8 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton

Old and new AMH assays Can we rely on current conversion factor

8th Biennial Conference of UK Fertility Societies January 2013 Liverpool

Poster presentation

9 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton

Random AMH measurement is not reproducible

8th Biennial Conference of UK Fertility Societies January 2013 Liverpool

Poster presentation

10 Oybek Rustamov Alexander Smith Stephen A Roberts Allen P Yates

Cheryl Fitzgerald Monica Krishnan Luciano G Nardo and Philip W

Pemberton

The reproducibility of serum Anti-Muumlllerian hormone AMH Gen II assay

Annual Meeting of European Society of Human Reproduction and

Embryology (ESHRE) July 2012 Istanbul

Oral Presentation Dr O Rustamov

8

GENERAL INTRODUCTION

AND LITERATURE REVIEW

1

9

CONTENTS I LITERATURE REVIEWhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip10 GENERAL BACKGROUNDhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip10

1 OVARIAN RESERVEhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip12 11 Primordial Follicle Assemblyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip13 12 Oocyte recruitmenthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip14 13 Theory of neo-oogenesishelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip15 2 MARKERS OF OVARIAN RESERVEhelliphelliphelliphelliphelliphelliphelliphelliphelliphellip16 21 Ovarian reserve markers with limited clinical valuehelliphelliphelliphelliphellip16 213 Inhibin Bhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip16 214 Basal oestradiolhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip17 215 Dynamic testshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip17 216 Ovarian volumehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip18 22 Ovarian reserve markers in routine clinical usehelliphelliphelliphelliphelliphelliphellip18 221 Chronological agehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip18 222 Basal FSHhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip18 223 Antral follicle counthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip19 3 ANTI-MUumlLLERIAN HORMONEhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip20 31 Biology of anti-Muumlllerian hormonehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip20 311 The role of AMH in the ovaryhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip21 312 AMH in women with polycystic ovary syndromehelliphelliphelliphelliphellip22 32 AMH Assayhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip23 33 Variability of AMH measurementshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip24 34 Role of AMH in assessment of ovarian reservehelliphelliphelliphelliphelliphellip25 341 Prediction of poor and excessive ovarian response in IVFhelliphellip25 342 Prediction of live birth in cycles of IVFhelliphelliphelliphelliphelliphelliphelliphelliphellip26

3 5 Role of AMH in ovarian stimulation for cycles of IVFhelliphelliphelliphellip26

4 MULTIVARIATE TESTShelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip27

5 SUMMARYhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip28 II GENERAL INTRODUCTIONhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip29 REFERENCEShelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip31

10

I LITERATURE REVIEW GENERAL BACKGROUND

Infertility is a disease of the reproductive system defined by the failure to

achieve a pregnancy after 12 months of regular unprotected sexual intercourse

although the criteria for the duration vary between different countries (NICE

2013) Worldwide prevalence of infertility estimated to be around 724 million

couples and around 40 million of those seek medical care (Hull et al 1985) In

the UK 15 couples present with infertility with an annual incidence of 12

couples per 1000 general population (Scott et al 2009) The main causes of

infertility are tubal disease ovulatory disorders male factor and poor ovarian

reserve In a third of couples the cause of failure to achieve pregnancy is not

established which is known as unexplained infertility (NICE 2013) Effective

treatment options include improving lifestyle factors medical andor surgical

treatment of underlying pathology induction of ovulation and Assisted

Reproductive Technology (ART) Assisted Reproduction consist of

intrauterine insemination (IUI) and in vitro fertilisation (IVF) cycles with or

without introcytoplasmic sperm injection (ICSI) as well as treatment involving

donated gametes It is estimated that 75 of infertile couples presenting at

primary care centres in the UK are referred to fertility specialists based at

secondary or tertiary care centres and nearly 50 of those are subsequently

offered IVFICSI treatment (Scott et al 2009) This is supported by figures of

Human Fertility and Embryology Authority (HFEA) which indicates more

than 50000 IVF treatment cycles are performed in the UK annually (HFEA

2008)

An IVF treatment cycle involves a) pituitary down regulation b)

controlled ovarian stimulation c) oocyte recovery c) in vitro fertilisation of eggs

with sperm d) transfer of resulting embryo(s) back to uterus and c) luteal

phase support (NICE 2013) Prevention of premature surge of luteinising

hormone during controlled ovarian stimulation (COS) is achieved by pituitary

down regulation using either preparations of gonadotrophin releasing hormone

agonist which is widely known as ldquoAgonist cyclerdquo or gonadotrophin releasing

hormone antagonist which is known ldquoAntagonist cyclerdquo (Figure 1 and 2)

Controlled ovarian stimulation involves administration of gonadotrophins to

encourage the development of supernumerary preovulatory follicles followed

by administration of exogenous human chorionic gonadotropin (hCG) or

11

recombinant luteinising hormone (rLH) to assist in maturation of oocytes 34-

36 hours prior to egg collection which is usually conducted with guidance of

transvaginal ultrasound scanning Subject to sperm parameters the fertilisation

of oocytes is conducted by in vitro insemination or intracytoplasmic sperm

injection The resulting embryo(s) are cultured under strict laboratory

conditions and undergo regular qualitative and quantitative assessments before

transferring the best quality embryo(s) back into uterus during its cleavage

(Day 2 or Day 3) or blastocyst (Day 5 or Day 6) stage of development In

natural menstrual cycles under the influence of HCG progesterone secreted

by the ovarian corpus luteum ensures proliferative changes in the endometrium

providing the optimal environment for implantation of embryo(s) (van der

Linden et al 2011) However in IVF treatment cycles owing to pituitary down

regulation and lack of HCG progesterone levels are not in sufficiently high

concentration to ensure an adequate endometrial receptivity and therefore

exogenous analogues of this hormone is administered following transfer of

embryo(s) This is called ldquoluteal phase supportrdquo and in patients with viable

pregnancy usually lasts till 12th week of gestation when placenta starts

producing progesterone in sufficient quantities (van der Linden et al 2011)

In IVF programmes the ldquosuccessrdquo of the treatment often defined as

achieving a live birth following IVF cycle and expressed using Live Birth Rate

(LBR) In general success in IVF predominantly determined by womanrsquos age

cause(s) of infertility ovarian reserve previous reproductive history and

lifestyle factors (NICE 2013 Taylor 2003 Lintsen et al 2005) However

effectiveness of medical interventions as well as the quality of care play

important role in determining the outcome of IVF treatment This is evident

from significant variation in live birth rates among fertility clinics given for

instance in the UK LBR for women younger than 35 years of age after IVF

cycles varies from 15 to 61 (HFEA 2008 HFEA 2007) The provision of

effective interventions in both clinical and laboratory aspects of the care

appears to be the key in achieving high success rates Identification of patients

with sufficient ovarian reserve who benefit from IVF cycles followed by

providing optimal ovarian stimulation regimens may be useful in improving the

outcomes of IVF programmes According to HFEA data around 12 of IVF

cycles are cancelled due to poor or excessive ovarian response (Kurinczuk et al

2010) Availability of reliable markers for assessment of ovarian reserve and

tailoring ovarian stimulation regimens to the need of each individual patient

12

may improve selection of patients with sufficient ovarian reserve and reduce

the rate of cycle cancellation consequently improving the success of IVF

cycles (Yates et al 2011)

Assessment of ovarian reserve can be achieved using various biomarkers

and four of those are currently used by most clinics womanrsquos chronological

age (Age) serum follicle stimulating hormone (FSH) antral follicle count

(AFC) and serum anti-Muumlllerian hormone (AMH) More recently AMH has

been a focus of interest given it is the only available endocrine marker that is

suitable for direct assessment of the activity of ovarian follicles in their non-

cyclical stage development providing a window to FSH independent phase of

follicular recruitment Furthermore it appears to be reliable biomarker for a)

both the assessment of ovarian reserve and the optimisation of ovarian

stimulation regimens (Yates et al 2011 La Marca et al 2009) b) screening and

diagnosis of polycystic ovarian syndrome (PCOS) (Cook et al 2002) c)

monitoring of disease activity in women with a history of granulosa cell

tumours (Lane et al 1999) d) prediction of the age of diminished fertility and

the menopause (van Disseldorp et al 2008 Broer et al 2011) and finally (e)

assessment of the long term effect of chemotherapy on ovarian reserve

(Anderson 2011)

In this review I first discuss current knowledge on factors that

determine ovarian reserve including the formation and loss of oocyte pool

Then characteristics of the markers of ovarian reserve are reviewed Finally I

examine current understanding of biology of anti-Muumlllerian hormone and its

role in management of infertility

1 OVARIAN RESERVE

It is important to recognize that there is no universal definition for the

term ldquoovarian reserverdquo and the term can have various meanings depending on

the context in which it is used For instance the scientific literature describing

the biology of ovarian reserve usually refers to ldquothe total number of remaining

oocytes in the ovaries which consists of the number of resting primordial

follicles and growing primary pre-antral and antral folliclesrdquo (Gleicher et al

2011) In contrast the use of the term in the context of clinical studies may

refer to ldquoclinically measurable ovarian reserve established using available

biomarkers of ovarian reserverdquo For the purpose of clarity in this thesis the

13

term ldquoovarian reserverdquo refers to clinically measurable ovarian reserve whilst

true biological ovarian reserve will be termed ldquobiological ovarian reserverdquo

Recent studies have demonstrated that ovarian reserve is highly variable

between women due to the variation in the size of initial ovarian reserve at

birth as well as the rate of loss of ovarian reserve thereafter (Wallace et al

2010) Interestingly the rate of oocyte loss appears to be mainly determined by

the initial ovarian reserve which is believed to be facilitated by most potent

ovarian growth factor anti-Muumlllerian hormone Similarly the size of the initial

ovarian reserve is mainly underpinned by the rate of primordial follicle

assembly in the embryo which is also regulated by AMH Both primordial

follicle assembly and the rate of oocyte loss appear to be primarily under the

influence of genetic factors although developmental and environmental factors

are also believed to play a role (Nilsson et al 2010 Shuh-Huerta et al 2012)

11 Primordial follicle assembly

The process of assembly of primordial follicles in the female embryo

spans from the early embryonic to the early postnatal period and formation of

primordial follicles consists of following stages 1) primordial germ cell (PGC)

2) oogonia 3) primary oocyte and 4) primordial follicle In the human female

fetus around a hundred cells that differentiated from extra-embryonic

ectoderm form early PGCs on the yolk sac and migrate via hindgut to gonadal

ridges during 4th - 6th weeks of gestation (MC et al 1953 Donovan 1998) Once

arrived to the gonadal ridges these cells are called primary oogonia which

consequently undergo several rounds of mitotic division during 6th - 28th weeks

of gestation Interestingly the numbers of oogonia reach as high as six million

during its highest rate of mitotic division at around 20 weeks of gestation

Following the last round of mitotic division oogonia enter meiosis which

marks their new stage of development-primary oocyte Formation of

primordial follicles starts as early as at 8th week of gestation and is characterised

by meiosis of primary oocyte that arrest in diplotyne stage and surrounding of

the oocyte by somatic granulosa cell (Baker et al 1963 Maheshwari and Fowler

2010) Indeed the primordial follicle is the cardinal unit of the biological

ovarian reserve and therefore the rate of formation of primordial follicles is the

main determinant of initial biological ovarian reserve at birth

Interestingly the process of loss of oogonia and oocytes which is also

one of the main determinants of the initial ovarian reserve takes place

14

throughout the period of follicle assembly The formation of the granulosa cell

layer around the oocyte prevents the oocyte from subsequent atresia The

oocyte enveloped in a single layer of granulosa cells which is also known as

primordial follicle remains quiescent until recruitment of the follicle for

growth which may not take place for a number of decades after the formation

of a particular primordial follicle (Skinner 2005 Maheshwari and Fowler 2010)

12 Oocyte recruitment

Follicle growth in women consists of two stages a) the initial non-cyclical

recruitment of primordial follicles and the formation of a primary and a pre-

antral follicles and b) cyclical development of antral follicles with subsequent

selection of usually a single dominant follicle The initial recruitment of

primordial follicles is continuous non-cyclical process that starts as early as

from 18-20 weeks of gestation and lasts till the depletion of follicle pool which

later results in the menopause (McGee and Hsueh 2000) Transformation of

flat granulosa cells into cuboidal cells increases the diameter of the oocyte and

the formation of zona pellicuda completes the stage of formation of a primary

follicle During pre-antral stage oocytes increase in diameter and mitotic

division of granulose cells create a new layer of cells-theca cells The

mechanism of initial recruitment of oocytes is not well understood but it is

clear that the process is independent of influence of pituitary gonadotrophins

and appears to be governed by the genetically pre-programmed interaction of

the oocyte with local growth factors the most important of which appears to

be anti-Muumlllerian hormone and cytokines (McGee and Hsueh 2000)

The cyclical phase of development of oocytes is characterised by the

transformation of secondary follicle into antral follicle and subsequent growth

of antral follicles into pre-ovulatory stages In general the process of cyclic

recruitment starts from puberty under the influence of rising levels of pituitary

follicular stimulating hormone (FSH) During the antral stage oocyte increases

in size even further and the formation of a fluid filled space in follicle is

observed Under the influence of FSH luteinising hormone (LH) and local

growth factorsselection of a single dominant follicle occurs which followsby an

ovulation (McGee and Hsueh 2000)

Oocyte loss is a continuous process and occurs due to atresia of oocytes

during primary secondary and antral stages of development The rate of

oocyte loss appears to increase until the age of around 14 and declines

15

thereafter until the age of the menopause when around 1000 primordial

follicles remain (Hansen et al 2008 Oktem and Oktayl 2008) Furthermore by

the age of 30 years the average age at which women of western societies plan

to start a family around 90 of initial primordial follicles are lost which

illustrates that formation and maintenance of ovarian reserve is wasteful

process in humans (ONS 2012 Wallace and Kelsey 2010) As mentioned

above there is a wide individual variation in both sizes of initial primordial

follicular pool and the rate of oocyte loss which explains variation in the

reproductive lifespan in women Evidently the number of primordial follicles

at birth ranges between around 35000 to 25 million per ovary and similarly

the rate of oocyte loss during its peak at 14 years of age may range between

100 to 7500 primordial follicles per month which is believed to be inversely

proportional to initial size of primordial follicle pool (Wallace and Kelsey

2010)

13 Theory of neo-oogenesis

The traditional view of oogenesis states that the process of the creation

and the mitotic division of oogonia with subsequent formation of primordial

follicles takes place only during embryonic and foetal life (Zuckerman 1951)

According to this central theory of mammalian reproductive biology females

are born with a certain number of germ cells that is gradually lost but not

renewed during postnatal period However Johnson et al have recently

challenged this view and reported that adult mammalian ovary may possesses

mitotically active germ cells that continuously replenish the primordial follicle

pool (Johnson et al 2004) The group reported that ovaries of juvenile and

young adult mice contained large ovoid cells which resemble germ cells of

foetal mouse ovaries Interestingly immunohistochemical staining for a gene

which is expressed exclusively in germ cells have been reported to have

confirmed that these large ovoid cells were of germline lineage Furthermore

application of a mitotic germ cell toxicant busulphan appeared to have

eliminated primordial follicle reserve by early adulthood but did not induce

atresia suggesting the presence of proliferative germ cells in postnatal mouse

ovary (Johnson et al 2004 Bazer 2004) The study has generated enormous

amount of interest as well as debate among reproductive biologists (Notarianni

2011) Some other groups have also reported an evidence of postnatal

oogenesis (Pacchiarott et al 2010 Zou et al 2009 Bukovsky et al 2004)) while

16

others do not support the theory (Bristol-Gould et al 2006 Byskov et al 2005

Begum et al 2008) Furthermore some authors argued that adult mouse

germline stem cells exist and remain quiescent in physiologic conditions and

neo-oogenesis occurs only in response to ovotoxic damage (Tilly et al 2007 De

Felici 2010) Although consensus has yet to emerge to date there is no

conclusive evidence on validity of theory of neo-oogenesis

2 MARKERS FOR ASSESMENT OF OVARIAN RESERVE

Biological ovarian reserve is defined as the number of primordial and

growing follicles left in the ovary at any given time and therefore only

counting the number of primordial follicles by histological assessment can

accurately determine ovarian reserve which is clearly not feasible in clinical

setting However ovarian reserve can be estimated using various biomarkers

dynamic clinical tests and implied from the outcomes of ART cycles

Although a wide range of clinical (age ovarian response in previous IVF

cycles) biochemical (basal FSH Inhibin B basal oestradiol AMH) ultrasound

(ovarian volume antral follicle count (AFC)) and dynamic (clomiphene

challenge test exogenous FSH ovarian reserve test GnRH analogue

stimulating test) tests of ovarian reserve exist only a few of the markers are

reliable and practical enough to be of use in routine clinical practice In this

chapter first I discuss the research evidence on the assessment of the markers

andor tests of ovarian reserve that have limited clinical value Then I

evaluated more reliable markers that are in routine clinical use Age FSH

AFC and combination of these markers in multivariable tests Finally I

conducted detailed review of biology of AMH and the role AMH measurement

in the management of infertility

21 Ovarian reserve markers with limited clinical value

211 Inhibin B

Inhibins are members of TGFβ family and expressed in granulosa cells

of growing follicles Principal role of inhibins is thought to be the negative

feedback regulation of pituitary FSH secretion and therefore the serum level of

circulating hormone is believed to reflect the state of folliculogenesis

17

Consequently several groups have studied the role of serum Inhibin β in the

assessment of ovarian reserve Although initial reports were encouraging

(Seifer et al 1997) more robust studies demonstrated that serum Inhibin β was

less reliable than chronological age or basal FSH (Creus et al 2000 Urbancsek

2005) The systematic review of nine studies demonstrated that accuracy of the

Inhibin β test for predicting poor ovarian response and non-pregnancy in IVF

cycles was modest even at a very low threshold level (Broekmans et al 2006)

Therefore it is recommended that inhibin β at best can be used as only

screening test in the fertility centers where other more reliable markers are not

available (Broekmans et al 2006)

212 Basal oestradiol

Some studies suggested that elevated basal oestradiol levels indicate low

ovarian reserve and are associated with poor fertility prognosis (Johannes et al

1998 Licciardi and Rosenwaks 1995) Johannes et al demonstrated basal

oestradiol in conjunction with serum FSH is more reliable than serum FSH

alone in prediction of cycle cancellation due to the poor response in IVF cycles

(Johannes et al 1998) However there are no published data on the comparison

of basal oestradiol to more reliable markers such as AMH or antral follicle

count (AFC) Moreover a recent systematic review has demonstrated that

basal oestradiol has very low predictive value for poor response and has no

discriminatory power for accuracy of non-pregnancy prediction (Broekmans et

al 2006)

213 Dynamic tests of ovarian reserve

The dynamic tests of ovarian reserve are based on assessment of ovarian

response by measuring serum FSH and oestradiol levels following

administration of exogenous stimulation The following tests are reported in

literature Clomiphene Citrate Challenge Test (CCCT) Exogenous FSH

Ovarian Reserve Test (EFORT) and GnRH agonist stimulation test A recent

systematic review and meta-analysis on the accuracy of these tests showed that

none of them can adequately predict poor response or non-pregnancy in IVF

cycles and therefore are not recommended for use in routine clinical practice

(Maheshwari et al 2009)

18

214 Ovarian volume

There is some evidence that increased age is associated with decreased

ovarian volume and women with smaller ovaries are more likely to have

cancellation of their IVF cycles due to poor ovarian response (Syrop et al 1995

Syrop et al 1999 Templeton 1995) However a meta-analysis of the published

studies on the accuracy of ovarian volume as a predictor of poor response and

non-pregnancy in IVF cycles failed to demonstrate clinical usefulness of the

test and suggested the test is not reliable enough for use in a routine clinical

practice (Broekmans et al 2006)

22 Ovarian reserve markers in routine clinical use

221 Chronological age

Owing to the biological age-related decline of the quantity and arguably

the quality of oocytes the chronological age can be used as a marker of ovarian

reserve Studies have demonstrated that ovarian reserve (Wallace and Kelsey

2010 Kelsey 2011) natural fecundity (Islam et al 1989 and outcomes of ART

(Templeton et al 1996 van Kooij et al 1996) decline significantly from age of

35 when it is believed the ovarian reserve undergoes accelerated decline

Although there is a strong association between chronological age and reduction

in fertility evidently there is a significant variation in age-related ovarian

reserve indicating chronological age alone may not be sufficient to estimate the

individual womanrsquos ovarian reserve reliably (Broekmans et al 2006)

222 Basal FSH

Basal FSH was one of the first endocrine markers introduced in ART

programs and is still utilized in many fertility clinics albeit in conjunction with

other markers which are considered more reliable (Creus et al 2000) Secretion

of FSH is largely governed by the negative feedback effect of steroid

hormones primarily oestradiol and inhibins which are expressed in granulosa

cells of growing ovarian follicles Consequently decreased or diminished

recruitment of ovarian follicles is associated increased serum FSH

measurements and high particularly very high basal FSH reading is considered

as a good marker of very low or diminished ovarian reserve (Abdalla et al

2006) However unlike some other markers FSH measurements do not

appear to have discriminatory power for categorisation of patients to various

19

bands of ovarian reserve Given between-patient variability FSH measurement

(CV 30) is similar to its within-patient variability (27) stratification of

patients to various ranges of ovarian reserve does not appear to be feasible

(Rustamov et al 2011) Indeed a recent systematic review of 37 studies on the

prediction of poor response and non-pregnancy in IVF cycle has concluded

that basal FSH is an adequate test at very high threshold levels and therefore

has limited value in modern ART programs (Broekmans et al 2006)

223 Antral follicle count

Antral follicle count estimation involves ultrasound assessment of

ovaries between 2nd and 4th day of menstrual period and counting ldquofolliclesrdquo

which corresponds to antral stage of folliculogenesis (Broekmans et al 2010)

The test provides direct quantitative assessment of growing follicles and is

known as one of the most reliable markers of ovarian reserve (Broekmans et al

2006) AFC measurement has been reported as having a similar sensitivity and

specificity to AMH in prediction of poor and excessive ovarian response in

IVF cycles (Broekmans et al 2006 Broer et al 2010 Jayaprakasan et al 2010)

Given AFC measurement is available instantly and allows patients to be

counseled immediately the test eliminates the need for an additional patient

visit prior to IVF cycle However AFC is normally performed only in the early

follicular phase of the menstrual cycle given most published data on

measurement of AFC are based on studies that assessed antral follicles during

this stage of the cycle (Broekmans et al 2010a) Interestingly more recent

studies suggest that variability of AFC during menstrual cycle is small

particularly when follicles between 2-6mm are counted and therefore

assessment of AFC without account for the day of menstrual cycle may be

feasible (Deb et al 2013)

One of the main drawbacks of AFC is that the cut off levels for size of

counted follicles remains to be standardised (Broekmans 2010b) Initially

follicles of 2-10mm were introduced as the range for AFC and many studies

were based on this cut off Later counting follicles of 2-6mm was reported to

provide most accurate assessment of ovarian reserve (Jayaprakasan et al 2010b

Haadsma et al 2007) and therefore some newer studies are based on AFC

measurements that used this criterion Consequently direct comparison of the

outcomes of various studies on assessment of AFC requires careful analysis

20

3 ANTI-MUumlLLERIAN HORMONE

31 Biology of Anti-Muumlllerian hormone

AMH is a member of transforming growth factor β superfamily which

was discovered by Jost et al in 1947 and was initially known for its is role in

regression of Muumlllerian ducts in sex differentiation of the male embryo In

women AMH is believed to be solely produced by ovaries and expressed in

granulosa cells of growing follicles of 2-6 mm in size which corresponds to

primary pre-antral and early antral stage of follicular development Although

there has been a report of expression of AMH in endometrial cells to date

there is no other published evidence that supports this finding (Wang et al

2009) Indeed studies that evaluated half-life of AMH in serum have

demonstrated that in women who had bilateral salpingo-oopherectomy AMH

becomes undetectable within 3-5 days of following surgery suggesting ovaries

are the only source of secretion of AMH in appreciable quantity (La Marca et

al 2005b) Anti-Muumlllerian hormone is a dimeric glycoprotein which is

composed of a long N-terminus and short C-terminus and was believed to be

secreted in serum only in this dimeric form (AMH-N C)

Like other members of TGF-β family which includes inhibins activins

bone morphogenic proteins (BMPs) and growth and differentiation factors

(Massague et al 1990) AMH binds to two type of serinethreonine kinase

receptors referred to as type I and type II In order to activate AMH signaling

pathway both receptors have to form a heteromeric complex When AMH

binds to the type II (AMHR-II) receptor (Massague et al 2000) this will

phosphorylate and activate a type I receptor (ALK2 -3 andor -6) which

subsequently activates the SMAD pathway through phosphorylation of

SMAD 1 5 andor 8 These activated SMADs interact with SMAD4 and

translocate to the nucleus regulating the expression of different genes

inhibiting the recruitment of primordial follicles and reducing FSH sensitivity

in growing follicles In addition AMH receptors as well as the other members

of TGF-β family can activate MAPK and PI3KAKT pathways

Studies on AMHR II-deficient male mice demonstrated lack of

regression of Muumlllerian ducts suggesting that type II receptor is essential in

AMH signaling (Mishina et al 1996) Similarly Type I receptors which includes

three members of activin receptor-like kinase (ALK2 ALK3 and ALK6) also

appear to play an important role in the regression of Muumlllerian ducts although

21

the role of ALK 6 in AMH signaling appears not to be crucial (Visser 2003

Clarke et al 2001) The signal transduction pathway of AMH in the ovary is

largely not understood In postnatal mice ovary AMHR-II receptor was

expressed in both granulosa and theca cells of pre-antral and antral follicles

(Visser 2003) AMH type I receptors ALK 2 and ALK 3 is expressed in foetal

as well as adult mouse ovary while ALK 6 is expressed in only adult ovary

(Visser 2003)

311 The role of AMH in the ovary

In the mammalian ovary the role of AMH appears to be one of a

regulation of size of the primordial follicle pool by its inhibitory effect on the

formation as well as the growth of primordial follicles (Nilsson et al 2011) In

the embryonic mouse ovary AMH inhibits the initiation of the assembly of

follicles when the process of apoptosis of the majority of oocytes is observed

(Nilsson et al 2011) Consequently AMH reduces the rate of oocyte loss

which plays an important role in the determination of the size of initial follicle

pool Similarly in the adult mouse ovary AMH plays a central role in

maintaining the follicle pool AMH inhibits both the processes of the initial

(non-cyclical) recruitment of primordial follicles and subsequent FSH-

dependent cyclical growth of antral follicles (Figure 3) Inhibition of the initial

recruitment of a new cohort of follicles is believed to be achieved by a

paracrine negative feedback effect of the rising levels of AMH secreted from

already recruited growing follicles (Durlinger et al 1999) Durlinger et al

compared the complete follicle population of AMHnull mice and wild type

mice of different ages of 25 days 4 months old and 13 months old and found

that the ovaries of 25 day and 4 months old AMHnull females contained

significantly higher number of growing pre-antral and antral follicles but

significantly fewer primordial follicles compared to wild-type females

(Durlinger et al 1999) Interestingly almost no primordial follicles were

detected in 13 months old AMHnull mice ovaries suggesting AMH is a potent

inhibitor of the recruitment of primordial follicles and in the absence of AMH

ovaries undergo premature depletion of primordial follicles due to an

accelerated recruitment Subsequent study conducted by the group

demonstrated that in addition to its inhibitory effect to the resting follicles

AMH also suppresses the development of the growing follicles (Durlinger et al

2001 Durlinger et al 2002 Themmen 2005) It appears that AMH inhibits

22

FSH-induced follicle growth by reducing the sensitivity of growing follicles to

FSH which has been confirmed by in vivo as well as in vitro studies (Durlinger

et al 1999 Durlinger et al 2001) In the initial study the group observed that

despite lower levels of serum FSH concentration ovaries of AMHnull mice

contained more growing follicles than that of their wild-type littermates which

has been supported by the findings of subsequent in vitro study (Durlinger et al

1999) Addition of AMH to the culture inhibited FSH-induced follicle growth

of pre-antral mouse follicles due to reduction in granulosa cell proliferation

(Durlinger et al 2001)

In the human embryo the expression of AMH commences in the late

foetal life and can be detected only from 36 weeks of gestation (Rajpert-De et

al 1999 Lee et al 1996) Following a small decline in first two years of life

AMH levels gradually increase to peak at (mean 5 ngml) around age of 24

years In line with the pattern of oocyte loss serum hormone levels gradually

decline with increasing age and become undetectable around 5 years prior to

menopause (Kelsey et al 2011 Nelson et al 2011)

It has been suggested that anti-Muumlllerian hormone plays a central role in

determining the pace of recruitment of primordial follicles hence maintaining

the primordial follicle pool of postnatal mammalian ovary Consequently a

reduction in the concentration of circulating AMH signals the exhaustion of

the primordial follicle pool and the decline of ovarian function

312 AMH in women with polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) endocrine abnormality characterised

by increased ovarian androgen secretion infrequent ovulation and the

appearance of ldquopolycysticrdquo ovaries on ultrasound scan (Dunaif 1997 Homburg

et al 1993) It is the commonest endocrine abnormality in women of

reproductive age and affects around 15-20 of women PCOS is also one of

the main causes of anovulation and subsequent sub-fertility (Webber et al

2003) Although the role of anti-Muumlllerian hormone in the development of

PCOS is not fully understood it is becoming increasingly evident that the

hormone plays an important role in its pathogenesis (Pehlivanov et al 2011)

There is a strong association between serum AMH levels and PCOS and it

appears that women diagnosed with PCOS have two to three fold higher

serum AMH concentration compared to normo-ovulatory women (Cook et al

2002 Pigny et al 2003) Similarly women with PCOS are found to have

23

significantly higher number antral follicles Interestingly the expression of

AMH in granulosa cells of follicles were found to be 75 times higher in women

with PCOS compared to those without a the disease suggesting increased

serum AMH in PCOS may be due to increased secretion of hormone per

follicle rather than due to an increased number of antral follicles (Pellat et al

2007) High AMH concentrations may act as the main facilitator of abnormal

folliculogenesis in PCOS given the follicles appear to arrest when they reach

an antral stage (2-6mm) of development (Rajpert-De et al 1999) Indeed the

studies of Durlinger et al have demonstrated that AMH inhibits selection of

dominant follicle when follicles reach antral stage of development (Durlinger et

al 2001) Serum AMH levels appear to decrease with treatment of PCOS

which may play important role in restoration of ovulatory cycles Studies have

reported a significant reduction in serum concentration of AMH following

treatment of PCOS with metformin and laparoscopic ovarian diathermy (Falbo

et al 2010 Amer et al 2009 Elmashad 2011) Similarly reduction of BMI

following intensified endurance exercise training for treatment of PCOS may

also lead to a significant reduction in serum AMH levels (Moran et al 2011)

This suggests that there is strong association between serum concentration of

AMH and abnormal folliculogenesis in PCOS and therefore understanding the

molecular mechanisms of this interaction should be one of the priorities of

future research

32 AMH Assays

Enzyme-linked immunosorbent assay specific for measurement of anti-

Muumlllerian hormone was first developed in 1990 and was recognised as a

significant step in the assessment of ovarian reserve (Hudson et al 1990)

Subsequently a number of non-commercial immunoassays were developed

which were mainly used in research settings (Lee et al 1996) Later Diagnostic

Systems Ltd (DSL) and Immunotech Beckman Coulter Ltd (IOT) introduced

two commercial immunoassays for the routine clinical assessment of ovarian

reserve which are known as ldquofirst generation AMH assaysrdquo (Nelson and La

Marca 2011) These assays employed two different antibodies against AMH

and used different standards for calibration providing non-comparable

measurements (Nelson and La Marca 2011) Consequently several studies

attempted to develop a reliable between-assay conversion factor which

interestingly revealed from five-fold higher with the IOT assay to assay

24

equivalence causing significant impact to reliability of AMH measurements and

interpretation of research findings (Hehenkamp et al 2006 Freour et al 2007

Bersinger et al 2007 Taieb et al 2008 Lee et al 2011)

Later the manufacturer of IOT assay (Beckmann Coulter Ltd)

consolidated the manufacturer of the DSL assay (Diagnostic Systems

Laboratories Inc) and introduced a new assay ldquoGen II AMH assayrdquo which is

only available commercial immunoassay in most countries including the UK

AMH Gen II assay was developed using the antibodies derived from first

generation DSL assay and calibrated using the standards used for IOT assay

and was believed to be considerably more stable compared to the first

generation immunoassays providing more reliable measurements (Kumar et al

2010 Nelson and La Marca 2011) The manufacturer as well as initial external

validation study recommended when compared to old DSL assay AMH Gen

II assay provides around 40 higher measurements and therefore previously

reported DSL-based clinical cut-off levels for estimation of ovarian reserve

should be increased by 40 in order to use Gen II-based AMH results (Kumar

et al 2010 Wallace et al 2011 Nelson and La Marca 2011)

33 Variability of AMH measurements

It is generally believed that AMH values do not change throughout the

menstrual cycle and early studies reported that variation in AMH

measurements between repeated measurements of same patient was negligible

(van Disseldorp et al 2010 La Marca 2010) On the basis of these studies

sampling at a random time in the menstrual cycle was introduced as a method

for measurement of AMH in routine clinical practice However the

methodologies of some of these studies do not appear to be robust enough to

reliably estimate sample-to-sample variability of AMH which is mainly due to

small sample sizes (Rustamov et al 2011) Consequently in a recent study we

assessed sample-to-sample variability of AMH using DSL assay and found that

within-subject coefficient of variation (CV) of AMH between samples were as

high as 28 which cannot be attributed to any patient or cycle characteristics

(Rustamov et al 2011) Although there is no consensus in the causes of this

observed variability in AMH measurements we believe it is largely attributable

to instability of AMH samples given initial recruitment of primordial follicles

and growth of AMH producing pre-antral and antral follicles are continuous

process and therefore the true biological variation between samples is unlikely

25

to be high However given the importance of establishing true variability of

AMH in both understanding of the biology of hormone and clinical

application of the test future studies should be conducted to establish the

source of variability in the clinical samples

3 4 The role of AMH in the assessment of ovarian reserve

341 Prediction of poor and excessive ovarian response in cycles of

IVF

A number of studies have assessed the role of AMH in the prediction of

poor ovarian response in IVF cycles using first generation AMH assays and

found that AMH and AFC were the best predictors of poor ovarian response

compared to other markers of ovarian reserve Nardo et al showed that the

predictive value of AMH in receiver operating characteristic curve (ROC)

analysis was similar to (AUC 088) that of AFC (AUC 081) and found that

AMH cut offs of gt375 ngmL and lt10 ngmL would have modest

sensitivity and specificity in predicting the extremes of response (Nardo et al

2009) These findings were largely supported by subsequent prospective studies

and a systematic review (Nelson et al 2007 Jayaprakasan et al 2010 Broer et al

2011) Similarly comparison of chronological age basal FSH ovarian volume

AFC and AMH found that only AMH (AUC 090) and AFC (AUC 093) were

reliable predictors of poor ovarian response in cycles of IVF Subsequent

combination of the effect of AMH and AFC using multivariable regression

analysis did not improve the level of prediction of poor ovarian response

significantly (AUC 094) suggesting both AMH and AFC can be used as

independent markers (Jayaprakasan et al 2010)

Similarly most studies agree that AMH and AFC are the best predictors

of excessive ovarian response and ovarian hyperstimulation syndrome (OHSS)

compared to other clinical endocrine and ultrasound markers (Nardo et al

2009 Nelson et al 2007) Broer et al compared these two tests in systematic

review of 14 studies and reported that the summary estimates of the sensitivity

and the specificity for AMH were 82 and 76 respectively and for AFC 82

and 80 respectively (Broer et al 2011) Consequently the study concluded

that AMH and AFC were equally predictive and the difference in the predictive

value between the tests was not statistically significant

26

342 Prediction of live birth rate (LBR) in cycles of IVF

Lee at al reported that AMH and chronological age were more accurate

than basal FSH AFC BMI and causes of infertility in the prediction of live

birth rate (Lee et al 2009) Similarly La Marca et al suggested that odds of live

birth could be reliably predicted using AMH (La Marca et al 2010b) although

subsequent review of the study questioned strength of the evidence (Loh and

Maheshwari 2011)

A study conducted by Nelson et al found that higher AMH levels had

stronger association with increased live birth rate compared to age and FSH

(Nelson et al 2007) However the study also suggested that this association

was mainly confined in the women with low AMH levels and there was no

additional increase in live birth in women with AMH levels of higher than 710

pmolL This may suggest that achieving a live birth may be under the

influence of number of other factors and that markers of ovarian reserve alone

may not be able predict this outcome reliably

35 The role of AMH in individualisation of ovarian stimulation in

IVF cycles

Prediction of ovarian response to the stimulation of ovaries in cycles of

IVF plays an important role in the counseling of couples undergoing treatment

programmes and hence many clinical studies on AMH have focused on the

prognostic value of AMH measurements However data on using AMH as a

tool for improving the clinical outcomes in IVF cycles appear to be lacking

considering AMH may be useful tool in tailoring treatment strategies to an

individual patientrsquos ovarian reserve Unlike most other markers AMH has

discriminatory power in determining various degrees of ovarian reserve due to

significantly higher between patient (CV 94) variability compared to its

within-patient (CV 28) variation (Rustamov et al 2011) which allows

stratification of patients into various degrees of (eg low normal high) ovarian

reserve Subsequently most optimal ovarian stimulation protocol may be

established for each band of ovarian reserve Consequently reference ranges

on the basis of distribution of AMH in infertile women were developed which

were subsequently adopted by fertility clinics for a tailoring the mode of

27

ovarian stimulation and daily dose of gonadotrophins in IVF (The Doctors

Laboratory 2008 However currently available clinical reference ranges are

based on the first generation DSL assay and may not be reliably convertible to

currently available Gen II assay measurements (Wallace et al 2011) Indeed the

findings of the studies on comparability of the first generation AMH assays

suggest that establishing a reliable between assay conversion factor between

AMH assays may not be straightforward Furthermore the reference ranges

appear to reflect the distribution of AMH measurements within a specific

population and may therefore not be directly applicable for the prediction of

response to ovarian stimulation in IVF patients (The Doctors Laboratory

2008)

More importantly despite lack of good quality evidence on the

effectiveness of AMH-tailored ovarian stimulation protocols a number of

fertility clinics appear to have introduced various AMH-based COH protocols

in their IVF programs At present research evidence on AMH-tailored

ovarian stimulation in IVF is largely based on two retrospective studies

(Nelson et al 2009 Yates et al 2012) Both of these studies display considerable

methodological limitations including small sample size and centre-related or

period-related selection of their cohorts In this context AMH is used as a tool

for therapeutic intervention and therefore the research evidence should ideally

be derived from randomised controlled trials However recruitment of large

enough patients in IVF setting may take considerable time and resources In

the meantime given AMH-tailored ovarian stimulation has already been

introduced in clinical practice and there is urgent need for more reliable data

the studies with a larger cohorts and robust methodology should assess the role

of AMH in individualisation of ovarian stimulation in IVF treatment cycles

4 Multivariate models of assessment of ovarian reserve

In view of the fact there is not a single marker of ovarian reserve that

can accurately predict ovarian response various models for combination of

multiple ovarian markers have been developed (Verhagen et al 2008) A

number of studies reported that multivariate models are better predictors of

poor ovarian response in IVF compared to a single marker (Bancsi et al 2002

Balasch et al 1996 Creus et al 2000 Durmusoglu et al 2004) However a meta-

analysis showed that when compared to a single marker (AFC) multivariate

28

model has a similar accuracy in terms of prediction of poor ovarian response

(Verhagen et al 2008) In contrast a more recent study demonstrated that

multivariate score was superior to chronological age basal FSH or AFC alone

in predicting likelihood of poor ovarian response and clinical pregnancy

(Younis et al 2010) However the study did not include one of the most

reliable markers AMH in either arm necessitating further assessment of the

role of combined tests which include all reliable biomarkers

4 SUMMARY

During the last two decades a significant leap has been taken towards

understanding the biology of anti-Muumlllerian hormone and its role in female

reproduction (Durlinger et al 2002 Themmen et al 2005) Availability of

commercial AMH assays has resulted in significant increase in interest in the

role of the measurement of serum AMH in the assessment of ovarian reserve

which has been followed by the introduction of the test into routine clinical

practice (Nelson et al 2011) However more recent studies suggest that current

methodologies for the measurement of AMH may provide significant sampling

variability (Rustamov et al 2011) Furthermore the studies that compared first

generation commercial assay methods appear to provide non-reproducible

results suggesting there may be underlying issues with assay methodologies

(Lee et al 2011) Similarly despite lack of sufficient evidence in the role of

AMH in individualisation of ovarian stimulation protocols in IVF AMH-

tailored IVF protocols have been introduced in routine clinical practice of

many fertility clinics around the world

Consequently it appears that clinical application of AMH test has

surpassed the research evidence in some aspects of fertility treatment and

therefore future projects should be directed toward areas where gaps in

research evidence exist On the basis of the review of literature we believe that

evaluation of the performance of assay methods understanding the role of

AMH in assessment ovarian reserve and establishing its role in

individualisation of ovarian stimulation protocols should be research priority

29

II GENERAL INTRODUCTION

On the basis of the review of published literature I have identified that

the following areas of research on the clinical application of AMH in the

management of infertility requires further investigation 1) Within-patient

variability of measurement of AMH using Gen II assay method 2)

Establishment of clinically measurable determinants of AMH levels and 3) The

role of AMH in individualisation of ovarian stimulation in IVF treatment

cycles

In our previous study we estimated that there was significant sample-to-

sample variation (CV 28) in AMH measurements when the first generation

DSL assay was used (Rustamov et al 2011) The source of variability is likely to

be related to the assay method given that biological within-cycle variation of

AMH is believed to be small (La Marca et al 2006) Therefore assessment of

sample-to-sample variability of AMH using the newly introduced Gen II assay

which is believed to be significantly more stable and sensitive compared to that

of DSL assay should enable us to establish the measurement related variability

of AMH Furthermore given I am planning to use data from both DSL and

Gen II assays I need to establish between-assay conversion factor for these

assays using data on clinical samples

There appears to be a lack of good quality data on the effect of

ethnicity BMI causes of infertility reproductive history and reproductive

surgery on ovarian reserve Therefore I am planning to ascertain the role of

above factors on determination of ovarian reserve by analysing AMH

measurements of a large cohort of patients

There is a strong correlation between AMH and ovarian performance

in IVF treatment when conventional ovarian stimulation using GnRH agonist

regimens with a standard daily dose of gonadotrophins are used (Nelson et al

2007 Nardo et al 2007) Furthermore studies suggest tailoring the ovarian

stimulation protocols to AMH measurement may improve ovarian

performance and subsequently the success of IVF treatment (Nelson et al

2011 Yates et al 2012) However given methodologies of the published

studies the effectiveness of currently proposed AMH-tailored ovarian

stimulation protocols remains unknown Therefore I am planning to develop

individualised ovarian stimulation protocols by establishing the most optimal

mode of pituitary down regulation and starting dose of gonadotrophins for

30

each AMH cut-off bands using a robust research methodology However

development of individualised ovarian stimulation protocols on the basis of

retrospective data requires a reliable and validated database containing a large

number of observations In the IVF Department of St Maryrsquos Hospital we

have data on a large number of patients who underwent ovarian stimulation

following the introduction of AMH However the data on various aspects of

investigation and treatment of patients is stored in different clinical data

management systems and may not be easily linkable In addition it appears that

data on certain important variables (eg causes of infertility AFC) are available

only in the hospital records necessitating searching for data from the hospital

records of each patient Consequently I designed a project for building a

research database which will have comprehensive and validated datasets that

are necessary for investigation of the research questions of the MD

programme

In conclusion I am planning to conduct a series of studies to improve

the understanding of the role of AMH in the management of women with

infertility Specifically I am intending to evaluate 1) sample-to-sample variability

of Gen II AMH measurements 2) conversion factor between DSL and Gen II

assays in clinical samples 3) the effect of ethnicity BMI causes of infertility

endometriosis reproductive history and reproductive surgery to ovarian

reserve and explore AMH-tailored individualisation of ovarian stimulation in

IVF cycles

31

References

Abbeel E The Istanbul consensus workshop on embryo assessment proceedings of an expert meeting Human reproduction 2011 26 p 1270-83 Abdalla HT M Y Repeated testing of basal FSH levels has no predictive value for IVF outcome in women with elevated basal FSH Human reproduction 2006 21(1) p 171-4 Amer SA LT Ledger WL The value of measuring anti-Mullerian hormone in women with polycystic ovary syndrome undergoing laparoscopic ovarian diathermy Human reproduction 2009 24 p 2760-6 Anderson RA Pretreatment serum anti-Mullerian hormone predicts long-term ovarian function and bone mass after chemotherapy for early breast cancer J Clin Endocrinol Metab 2011961336-1343 Balaban B BD Calderoacuten G Catt J Conaghan J Cowan L Ebner T Gardner D Hardarson T Lundin K Cristina Magli M Mortimer D Mortimer S Munneacute S Royere D Scott L Smitz J Thornhill A van Blerkom J Van den Baker A quantitative and cytological study of germ cells in human ovaries Proc R Soc Lond B Biol Sci 1963 158 p 417-433 Balasch J CM Fabregues F Carmona F Casamitjana R Ascaso and VJ C Inhibin follicle-stimulating hormone and age as predictors of ovarian response in in vitro fertilization cycles stimulated with gonadotropin-releasing hormone agonist-gonadotropin treatment Am J Obstet Gynecol 1996 175 p 1226-1230 Bancsi LF BF Eiijekemans MJ at al Predictors of poor ovarian response in in vitro fertilisation a prospective study comparing basal markers of ovarian reserve Fertility and Sterility 2002 77 p 328-336 Bazer FW Strong science challenges conventional wisdom new perspectives on ovarian biology Reprod Biol Endocrinol 2004 2 p 28 Begum S VE Papaioannou and RG Gosden The oocyte population is not renewed in transplanted or irradiated adult ovaries Hum Reprod 2008 23(10) p 2326-30

Bersinger NA Wunder D Birkhaumluser MH Guibourdenche J Measurement of anti-mullerian hormone by Beckman Coulter ELISA and DSL ELISA in assisted reproduction differences between serum and follicular fluid Clin Chim Acta 2007384174ndash175 Bristol-Gould SK et al Fate of the initial follicle pool empirical and mathematical evidence supporting its sufficiency for adult fertility Dev Biol 2006 298(1) p 149-54 Broekmans FJ et al A systematic review of tests predicting ovarian reserve and IVF outcome Hum Reprod Update 2006 12(6) p 685-718