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Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=igye20 Gynecological Endocrinology ISSN: 0951-3590 (Print) 1473-0766 (Online) Journal homepage: https://www.tandfonline.com/loi/igye20 Relationship between serum anti-Mullerian hormone and intrafollicular AMH levels in PCOS women M. Stracquadanio, L. Ciotta & M. A. Palumbo To cite this article: M. Stracquadanio, L. Ciotta & M. A. Palumbo (2018) Relationship between serum anti-Mullerian hormone and intrafollicular AMH levels in PCOS women, Gynecological Endocrinology, 34:3, 223-228, DOI: 10.1080/09513590.2017.1381838 To link to this article: https://doi.org/10.1080/09513590.2017.1381838 Published online: 23 Sep 2017. Submit your article to this journal Article views: 331 View Crossmark data Citing articles: 1 View citing articles

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Full Terms & Conditions of access and use can be found athttps://www.tandfonline.com/action/journalInformation?journalCode=igye20

Gynecological Endocrinology

ISSN: 0951-3590 (Print) 1473-0766 (Online) Journal homepage: https://www.tandfonline.com/loi/igye20

Relationship between serum anti-Mullerianhormone and intrafollicular AMH levels in PCOSwomen

M. Stracquadanio, L. Ciotta & M. A. Palumbo

To cite this article: M. Stracquadanio, L. Ciotta & M. A. Palumbo (2018) Relationship betweenserum anti-Mullerian hormone and intrafollicular AMH levels in PCOS women, GynecologicalEndocrinology, 34:3, 223-228, DOI: 10.1080/09513590.2017.1381838

To link to this article: https://doi.org/10.1080/09513590.2017.1381838

Published online: 23 Sep 2017.

Submit your article to this journal

Article views: 331

View Crossmark data

Citing articles: 1 View citing articles

PCOS: SERUM AMH AND INTRAFOLLICULAR AMH LEVELS

Relationship between serum anti-Mullerian hormone and intrafollicular AMH levelsin PCOS women

M. Stracquadanio, L. Ciotta and M. A. Palumbo

Institute of Obstetric and Gynecological Pathology, Santo Bambino Hospital, University of Catania, Catania, Italy

ABSTRACTPolycystic ovary syndrome is a complex disease characterized by various endocrine disorders that are thepotential cause of anovulation and hyperandrogenism. Anti-M€ullerian hormone expression is suspected tobe overexpressed in PCOS granulosa cells. AMH acts as a regulator of folliculogenesis: it is produced bythe granulosa cells of follicles from the stage of the primary follicle to the initial formation of the antrum.Serum and intrafollicular AMH levels are elevated in patients with PCOS due to increased number of smallfollicles and an increased secretion within each of these small follicles. This excess of AMH is strongly sus-pected to play a role in the characteristic follicular arrest of PCOS, through a negative action on aromataseexpression and on FSH action. Value above 5 ng/ml or 35pmol/l might be considered as a diagnostic cri-terion for PCOS. The aim of our study is to demonstrate the presence of higher AMH serum levels andhigher AMH intrafollicular fluid level of PCOS patients, undergone to IVF cycles, compared to normovula-tory patients. The results clearly indicate that blood and intrafollicular AMH levels are significantly higherin PCOS women comparing to the normovulatory population. Serum AMH level appears to be a good pre-dictive marker for the risk ovarian hyperstimulation syndrome: thus, its evaluation should be recom-mended before starting a controlled ovarian stimulation for IVF.

ARTICLE HISTORYReceived 28 March 2017Revised 27 August 2017Accepted 16 September 2017Published online 25 Septem-ber 2017

KEYWORDSPCOS; AMH; IVF; OHSS

Introduction

Polycystic ovary syndrome (PCOS) is a complex disease charac-terized by various endocrine disorders that are the potentialcause of anovulation and hyperandrogenism.

This heterogeneous syndrome affects about 5–10% of repro-ductive-age female population, and it can be considered the mostcommon endocrine disorder affecting women during reproduct-ive life [1].

PCOS is a multifactorial polygenic disease: genes involved ininsulin-resistance, genes that encode inflammatory factors andgenes that regulate steroidogenesis and folliculogenesis areinterested.

Chronic anovulation is often manifested as oligomenorrhea;anovulatory cycles may lead to dysfunctional uterine bleedingand decreased fertility. Cutaneous manifestations of hyperandro-genemia include hirsutism, acne, and male-pattern hair loss(androgenic alopecia), whereas acanthosis nigricans is a cutane-ous marker of hyperinsulinemia [2].

Anti-M€ullerian hormone (AMH) is a glycoprotein hormonewith a molecular weight of 140 kDa that belongs to the super-family of the transforming growth factor beta (TGF-b) [3].

AMH acts as a regulator of folliculogenesis: it is produced bythe granulosa cells of follicles from the stage of the primary fol-licle to the initial formation of the antrum. Its production beginsin the perinatal period, it increases gradually until puberty,remaining stable in the reproductive period and it becomes verylow after the menopause [4]. It is predominantly produced bypre-antral and antral follicles (diameter<4mm), and the produc-tion decreases during the process of final maturation and luteini-zation. AMH contributes to the initial recruitment and selection

of the dominant follicle. AMH is produced by growing folliclesin the ovary, according to two mechanisms of action:

� Inhibition of the initial recruitment of follicles [5];� Inhibition of growth hormone (FSH-dependent) and

selection of pre-antral and small antral follicles [6].Serum AMH level has been proposed as a test of ovarian

reserve: unlike other blood tests, such as FSH and estradiol,which must be dosed in the very early days of the cycle, theAMH remains constant during all the phases of the menstrualcycle. Its decrease to minimal levels might correlate with areduced number of ovarian follicles, while its abnormal increasecorrelates to an excess of follicles in precocious maturation stage,as in PCOS.

There are lots of scientific evidences supporting the thesis thatin the PCOS, in addition to intrinsic thecal dysregulation leadingto hyperandrogenism, a granulosa cell (GC) dysregulation mayoccur: AMH expression is suspected to be overexpressed inPCOS granulosa cells [7].

Follicular fluid (FF) is a mixture of chemical constituents,comprising a variety of different proteins as well as growth fac-tors, reactive oxygen species, anti-apoptotic factors, fatty acids,sugars, and hormones [8]. Among them, AMH seems to be themost prominent targets for reproductive health research [9,10].

Intrafollicular and serum AMH levels are elevated in patientswith PCOS due to increased number of small follicles and anincreased secretion within each of these small follicles. Thisexcess of AMH is strongly suspected to play a role in the charac-teristic follicular arrest of PCOS, through a negative action onaromatase expression and on FSH action. Value above 5 ng/ml or35 pmol/l might be considered as a diagnostic criterion for PCOS[11,12].

CONTACT Mariagrazia Stracquadanio [email protected] Istituto di Patologia Ostetrica e Ginecologica, Ospedale Santo Bambino, Catania� 2017 Informa UK Limited, trading as Taylor & Francis Group

GYNECOLOGICAL ENDOCRINOLOGY, 2018VOL. 34, NO. 3, 223–228https://doi.org/10.1080/09513590.2017.1381838

The positive correlation between high AMH values and PCOSwas also put in evidence in a work of Tomova, that showed howAMH value decreases after treatment with metformin, the mostused insulin-sensitizing agent administered to correct the meta-bolic aspect of the syndrome [13].

Thus, AMH serum levels could be also used as a prognosticfactor of metformin therapy in clinical practice [14].

Indeed, insulin is one of the causes of increased AMH inPCOS: hyperinsulinemia may stimulate the development ofantral follicles and improve the sensitivity of granulosa cells toFSH, thus increasing the number of follicles and ovarian vol-ume [15].

AMH positively correlates with ovarian volume and peripheralfollicle distribution, especially in young PCOS adolescents [16].

Particularly, AMH serum levels were significantly higher inPCOS patients with hyperandrogenism. Li et al. assert that thediagnostic power of AMH is limited when used to predictpatients without hyperandrogenism [17].

Intraovarian hyperandrogenism may cause follicular arrestand follicles’ excess with a consequent increased intraovarianAMH level; thus, elevated AMH in PCOS may be due toincreased follicle numbers rather than increased production byeach follicle [18].

This research point is controversial: in fact, other authorsdemonstrated that an ovulatory PCOS groups had lower concen-trations of AMH than those who were equally hyperandrogenicbut anovulatory, which supports the hypothesis that increasedAMH is due to increased production of AMH by eachfollicle [19].

On the other hand, Rosenfield noticed that AMH levels areindependently related to ovarian androgenic function and poly-cystic ovaries: very high AMH levels are specific but not sensitivefor PCOS. In the absence of hyperandrogenism, moderate AMHelevation in women with normal-variant polycystic ovaries seemsto indicate just an enlarged oocyte pool [20].

A serum AMH�35 pmol/l (or�5 ng/mL) appears to be moresensitive and specific than a USS follicle count>19 in PCOSwomen [21], but there is not yet an international consensus thatvalidates the threshold for AMH. In a recent study, Lin et al.have divided patients in reproductive age into three groups: highAMH (>11 ng/mL), moderate AMH (4–11 ng/mL), and lowAMH (<4 ng/mL). As the AMH level increased, the prevalenceof PCOS increased significantly from 21% in the low-AMHgroup to 37% in the moderate-AMH group, up to 80% in thehigh-AMH group [22].

As shown above, values of serum AMH have been deeplyinvestigated [23,24], but there is less data regarding the behaviorof AMH concentrations in FF, since FF has usually been desig-nated as waste product during oocyte collection for IVF treat-ment [10]. Recent studies revealed the importance of FF inoocyte development [25], providing information on FF, oocytequality and fertilization. Information on individual follicularAMH concentrations within one stimulated IVF cycles is still notavailable, although it is tempting to speculate that individual fol-licular AMH could be a potential predictor of fertilization successin IVF treatments [10,26].

Mean follicular AMH showed to be negatively correlated withage, which is in line with the overall AMH decrease in patientswith increasing age [10].

A very recent study demonstrated that individual follicularAMH concentrations reflected serum AMH values of a stimu-lated cycle during IVF treatment and that follicular AMH con-centrations did not significantly differ within one patient [10].

The aim of our study is to demonstrate the presence of higherAMH serum levels and higher AMH intrafollicular fluid level ofPCOS patients, undergone to IVF cycles, compared to normovu-latory patients.

Materials and methods

All the patients enrolled in this study attended the ‘ReproductiveDiseases Outpatient Clinic’ at the Institute of Obstetric andGynecological Pathology (‘Santo Bambino’ Hospital, Catania)from January 2016 to February 2017.

In the 12-month enrollment phase a total of 110 women withinfertility history were selected: 55 PCOS patients (Group A) and55 normovulatory women with tubaric disease (such as PID out-comes) or male infertility factors, which constituted the ‘ControlGroup B’.

Group A patients characteristics were:� Patient’s age >20 but <35 years old (in order that AMH

value would not be affected by a high maternal age);� BMI <30 (it is an internal criterion for all the patients of

our ART center, because it is related to better outcomes);� Ethnicity: all the patients were Caucasian females of

Sicilian ancestry. Thus, patients from Bangladesh, Japan,China, India, USA and Northern Europe were excluded.

� Patients not on any medications for menstrual disorders(oral contraceptives, metformin, inositol) or other pathol-ogies (hypertension, dyslipidemia or any other medical orpsychiatric illness) for at least 3 months before studyenrollment;

� Diagnosis of PCOS, according to Rotterdam Criteria [27];� Exclusion of other endocrine diseases (hyperprolactine-

mia, thyroid dysfunctions, Cushing’s syndrome, congeni-tal adrenal hyperplasia or androgen-secreting tumors).

Inclusion criteria for Group B patients were:� Patient’s age >20 but <35 years old;� BMI <30;� Ethnicity: all the patients were Caucasian females of

Sicilian ancestry. Thus, patients from Bangladesh, Japan,China, India, USA and Northern Europe were excluded.

� Patients not on any medications for menstrual disorders(oral contraceptives, metformin, inositol) or other pathol-ogies (hypertension, dyslipidemia or any other medical orpsychiatric illness) for at least 3 months before studyenrollment;

� Presence of both ovaries;� Exclusion of PCOS, hyperprolactinemia, thyroid

dysfunctions, endometriosis, POI (premature ovarianinsufficiency) and other menstrual irregularities (hypo-thalamic amenorrhea, idiopatic oligomenhorrea orpolymenhorrea).

AMH serum levels were determined by AMH Gen II Elisa(enzymatically amplified two-site immunoassay) during the thirdday of menstrual bleeding: the analytical sensitivity of this assayis 0.14 ng/mL.

All the patients followed the long-down regulation protocol:the women were injected leuprorelin acetate 0.2ml every dayfrom the 21st day of their menstrual cycle. Controlled ovarianhyperstimulation (COH) was performed by administration ofrecombinant FSH after pituitary suppression was reached, withan initial dose of 125 IU/daily. Follicle development was moni-tored with serial ultrasonographic examinations and serum estra-diol measurements. Afterwards, 5000–1000 IU of hCG wereadministered when the follicles were 18mm in diameter.

224 M. STRACQUADANIO ET AL.

Transvaginal ultrasound-guided follicular aspiration (oocytes’pick-up) was performed 34–36 h later.

The FF from more than one follicle was gently aspirated andsubsequently frozen at �20 �C for AMH measurement using anultrasensitive enzyme-linked immunosorbent assay. The lowerdetection limit was estimated at 0.1 ng/mL. To avoid possiblebias due to FF volume variability, AMH concentrations in the FFwere adjusted to its protein content by the Bradford assay.

The differences between the groups regarding age and BMIwere determined through the Mann–Whitney U test; to compareserum and intrafollicular AMH levels of the two groups theT-Test for two independent means was used.

Results

The mean age of Group A was 28 y ± 4m, whereas the mean ageof Group B was 27 y ± 1m; the mean BMI value was 27 ± 2 inboth groups. Thus, there were no statistically significant differen-ces between the control and the PCOS group in age and BMI(Table 1).

Serum AMH levels were significantly increased in PCOSgroup (p< .05): the mean value was 9.4 ± 3.3 ng/mL vs.3.25 ± 2.62 ng/mL. Even intrafollicular AMH levels were higher inPCOS groups (p< .05): the mean value was 3.25 ± 2.62 ng/g pro-tein vs. 2.8 ± 1.8 ng/g protein (Table 1) (Figure 1).

Furthermore, our data revealed that 60% of PCOS patientshave a moderate serum AMH value between 4 and 11 ng/mL,23.5% has high blood AMH levels and only 16.5% showed a nor-mal value <4 ng/mL (Figure 2).

Group B showed completely different AMH levelspercentages:

< 4 ng/mL: 81%4–11 ng/mL: 19%>11 ng/mL: 0%.From a clinical point of view, it is important to underline that

the only cases of ovarian hyperstimulation syndrome (OHSS)occurred in the high-serum AMH PCOS group: 8 out of 13patients of this group (61%).

Discussion

The serum levels of AMH reflect the size of the ovarian follicularpool [28]. The main function of AMH seems to be the inhibitionof the early stages of follicular development and of the FSH-dependent selection progress. In ovaries, in fact, AMH inhibitsprimordial follicle recruitment [29], meiosis II [30], granulosacell division and progesterone production [31]. AMH concentra-tions in FF is inversely correlated with granulosa cell prolifer-ation, although normal physiology is disrupted with advancingage and in PCOS [32,33].

Serum AMH assessment has demonstrated its utility in thetreatment of infertility; but the absence of an international stand-ard for serum AMH assay and the inability to define clearthresholds makes application of serum AMH valuation more dif-ficult [34].

AMH could be used as a menstrual cycle-independent markerof ovarian response to controlled ovarian stimulation [35]. AMHappears to correspond well with antral follicle counts and ovarianresponse to hyperstimulation in in vitro fertilization (IVF)[36,37] and to be useful for predicting ovarian response inwomen undergoing IVF treatment [38–40].

One of the main advantages of AMH measurement in IVFtreatment, comparing to other markers of ovarian reserve, mightbe its low inter- and intra-cycle variability [41–43]. Moreover,serum AMH level is independent from the hypothalamic pituit-ary axis and it is not modified in pathologies such as hyperpro-lactinemia and functional hypothalamic amenorrhea [44].

Indeed, the increased serum basal AMH concentrations inPCOS have been explained by the increased number of smallovarian follicles responsible for AMH secretion [45].Additionally, these high AMH levels are probably related to thefollicular arrest, during the selection process of the dominant fol-licle, through a negative interaction between AMH and FSH [46].Moreover, the demonstration that intrafollicular AMH levels aregreater in women with PCOS compared to the controls, maysuggest increased ‘per follicle’ AMH secretion in PCOS [47].

Some studies report higher intra-follicular levels of AMH inPCOS women compared to controls, indicating that AMH-excesscould result from overactive follicles [48–50]. However, highintra-follicular levels of AMH do not necessarily imply increasedrelease of AMH from the follicles [48].

Intra-follicular AMH levels were found to be 75-fold higher inanovulatory PCOS-women and intra-follicular AMH decreasedwith increasing follicle-size [49]: this indicates that follicles ofwomen with PCOM synthetize more AMH than those of womenwith normal ovary morphology [49].

In PCOS-women referred to routine laparoscopy or laparot-omy, intra-follicular AMH-levels were 60-fold higher than serumAMH-levels [50]. Intra-follicular AMH levels in anovulatory

Table 1. Patients’ characteristics and AMH values in Group A and B.

Group A Group B p Value

Women’s age (years) 28y ± 4 27y ± 1 NSBMI (kg/m2) 27 ± 2 27 ± 2 NSAMH (blood levels, ng/mL) 9.4 ± 3.3 3.25 ± 2.62 .00001AMH (intrafollicular, ng/g protein) 8.2 ± 1.6 2.8 ± 1.8 .001

Figure 1. Mean serum and FF (follicular fluid) AMH levels in PCOS and controlgroups.

Figure 2. AMH serum levels – percentage of distribution in PCOS group.

GYNECOLOGICAL ENDOCRINOLOGY 225

PCOS women were 6-fold higher than in eumenorrhoicwomen. Serum levels of AMH were highly correlated to theintra-follicular levels in PCOS-women, in contrast to eumenno-rhoic controls [50].

The aim of this study was to prove the validity of serumAMH levels as an important marker for PCOS diagnosis, con-firming its high levels also in the FF of PCOS women.

In summary, the results clearly indicate that blood and intra-follicular AMH levels are significantly higher in PCOS womencomparing to the normovulatory population.

Our data confirm those of a recent Chinese study that dem-onstrated that FF AMH and serum AMH levels in patients withPCOS were significantly higher than those in non-PCOS patients.Additionally, the antral follicular count and the number ofoocytes retrieved from patients with PCOS were significantlyhigher than those obtained from control patients. These resultssuggested that basal serum AMH values reflect the quantity ofoocytes in ovaries [51].

Serum AMH level appears to be a good predictive marker forthe risk OHSS, as demonstrated in the meta-analysis of Broeret al. [52,53].

It is well known that the response of PCOS women togonadotropin stimulation differs significantly from that of nor-mal ovaries: it is defined ‘explosive’ and it is responsible for thehigher risk of canceled cycles and/or for OHSS [15,54].

It was shown that during ovarian stimulation, E2 productionand E2-to-A ratio are higher in patients with PCOS who haveelevated insulin levels than in normo-insulinemic women [15].Increased insulin levels involve greater ovarian endocrine andmorphologic responses to FSH-induced ovulation, which predis-pose to OHSS.

Therefore, it seems that the typical response of the polycysticovary to exogenous gonadotropin therapy is related to increasedplasma concentrations of insulin [55].

Hyperinsulinaemia is known to be present in anovulatorywomen more than ovulatory women [56] and a direct correlationwas found between serum AMH and insulin insensitivity [57,58].Hyperinsulinaemia increases androgen production and the raisedAMH concentrations may be secondary to an effect of insulin onandrogen levels [59].

Several studies had been performed to demonstrate a potentialrelationship between insulin resistance and hyper-AMH.

Caglar et al. [60] designed a study to evaluate the correlationbetween AMH levels and insulin-resistance (IR) in normal-weight PCOS patients. Although the PCOS patients were notsubdivided according to different phenotypes, no correlation wasfound between AMH levels and IR. Tian and his Chinese group[61] conducted a more comprehensive study involving morepatients, and according to the results, no correlation wasobserved between AMH and indices of IR among all phenotypesof PCOS cases.

Conversely, an Italian study [57] found a significant positiveassociation between AMH and IR, as well as an associationbetween androgens and AMH in women with PCOS.

Another recent study [62] found a significant relationshipbetween AMH levels and PCOS in patients both with and withoutIR: it was suggested that women with PCOS, especially thosewith IR, may have higher serum AMH levels due to a greaterrelease of inflammatory factors into the systemic circulation, thusaffecting AMH production in the ovaries. Consistent withthese studies, a recent Turkish study found a significant positivecorrelation between AMH and IR in adolescent patients withPCOS [63].

Therefore, serum AMH levels evaluation should be recom-mended before starting a controlled ovarian stimulation for IVFnot only for confirming the PCOS diagnosis, but also for theright management of patients in order to choose the appropriatedose of gonadotropins and avoid cases of hyperstimulation.

Indeed, according to our experience, PCOS patients withserum AMH levels >11 ng/mL should start COH with a verylow dose of gonadotropins (e.g. 75 IU daily instead of 125 IUper day).

Whether the conventional long protocol can be replaced bythe antagonist protocol in IVF for patients with PCOS needs tobe confirmed by further studies [51].

However, even if serum AMH determination may be a helpfultool in the prediction of the ovarian response to gonadotropinsin PCOS, there is actually no consensus on the threshold for theAMH values [3].

Intrafollicular AMH level might be a good marker for oocytequantity but not for their quality [41] and this criticism could bean interesting starting point for a future study through the evalu-ation of oocyte’s quality, embryo’s grade, fertilization andimplantation rate in a larger PCOS population.

Follicular AMH concentrations are positively associated withembryo implantation when measuring the AMH concentrationsin the dominant follicle [64]. Studies suggested that higher fol-licular AMH values positively correlated with fertilization [65]and implied higher chances for pregnancy [66].

However, other studies in literature have shown that fertiliza-tion rate, high quality grade embryo, and clinical pregnancy ratesare unchanged by AMH levels [41,64,67–72].

Disclosure statement

The authors declare that there is no conflict of interest regarding thepublication of this paper.

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