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Accepted Manuscript

Natural hormone replacement therapy with a functioning ovary afterthe menopause: dream or reality?

Jacques Donnez , Marie-Madeleine Dolmans

PII: S1472-6483(18)30325-0DOI: 10.1016/j.rbmo.2018.05.018Reference: RBMO 1963

To appear in: The End-to-end Journal

Received date: 31 January 2018Revised date: 28 May 2018Accepted date: 29 May 2018

Please cite this article as: Jacques Donnez , Marie-Madeleine Dolmans , Natural hormone replace-ment therapy with a functioning ovary after the menopause: dream or reality?, The End-to-end Journal(2018), doi: 10.1016/j.rbmo.2018.05.018

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Short title: Natural hormone replacement therapy

Natural hormone replacement therapy with a functioning ovary after the menopause:

dream or reality?

Jacques Donnez,a Marie-Madeleine Dolmans,

b, c

aSociété de Recherche pour l'Infertilité, Avenue Grandchamp 143, 1150 Brussels, Belgium

Brussels, Belgium

bPôle de Gynécologie, Institut de Recherche Expérimentale et Clinique (IREC), Université

Catholique de Louvain, Brussels, Belgium

cGynecology Department, Cliniques Universitaires Saint Luc, Avenue Hippocrate 10, 1200

Brussels, Belgium

Corresponding author.

E-mail address: jacques.donnez@gmail.com (J. Donnez).

Key message

Cryopreservation of ovarian tissue at a young age followed by reimplantation may restore

long-term ovarian endocrine function that can persist for more than 7 years (even more if the

procedure is repeated) and prevent menopause-related conditions.

Abstract

At the dawn of humanity, it was rare to live beyond the age of 35 years, so the ovary was

intended to function for a woman’s entire life. Nowadays, it is not unusual for women to live

into their 80s. This means that many of them spend 30–40% of their lives in the menopause at

increased risk of various conditions associated with an absence of oestrogens (cardiovascular

disease, bone mineral density loss). Reimplantation of frozen–thawed ovarian tissue is able to

restore long-term ovarian endocrine function that can persist for more than 7 years (12 years if

the procedure is repeated). If ovarian tissue reimplantation is capable of restoring ovarian

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activity after menopause induced by chemotherapy, radiotherapy, surgery, or a combination

of all three, why not propose it to recover sex steroid secretion after natural menopause and

prevent menopause-related conditions in the ageing population? In this application, the graft

site could be outside the pelvic cavity, e.g., forearm or rectus muscle. Could ovarian tissue

freezing at a young age followed by reimplantation upon reaching menopause be the anti-

ageing therapy of the future? Sufficient existing evidence now surely merits serious debate.

KEYWORDS: hormone replacement therapy, ovary, ovarian tissue reimplantation,

menopause

<A>The ovary from birth to menopause

During fetal life, 100–2000 primordial germ cells enter a massive proliferation process. By

mid-gestation, there are several million potential oocytes, but 85% of them are lost before

birth. After birth, the number of primordial follicles, also known as non-growing follicles,

decreases year on year until puberty, even in the absence of ovarian activity (Wallace and

Kelsey, 2012) (Figure 1).

Of around 1 million oocytes per ovary at birth, only 450 are actually used. Indeed, the decline

in follicle numbers continues throughout reproductive life, during which time about 450

monthly ovulatory cycles occur, with most follicles undergoing atresia (degeneration and

resorption) during their growth phase. Cyclic folliculogenesis and ovulation, associated with

massive follicular atresia and ageing-induced apoptosis, result in ovarian atrophy and reduced

fertility (Wallace and Kelsey, 2012; Donnez and Dolmans, 2013).

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Among numerous mechanisms proposed to explain decreased fertility in women aged over 40

years, poor oocyte quality, characterized by abnormalities in the meiotic spindle, chromosome

misalignment and shortened telomeres, is the most frequently cited (Liu and Keefe, 2002; Liu

and Li, 2010; Donnez and Dolmans, 2013). Depletion of the ovarian reserve at a young age

may also be the consequence of medical treatment, such as chemotherapy with or without

radiotherapy (Donnez and Dolmans, 2013; 2017a).

Ovarian surgery for severe and recurrent endometriosis or recurrent ovarian cysts is another

common cause of ovarian reserve decline, as are known risk factors for premature menopause

(Turner syndrome, family history) (Donnez et al., 2012; Donnez and Dolmans, 2013; 2017a).

At menopause, follicular density is low. About 1500 primordial follicles remain, but most are

inactive (Wallace and Kelsey 2012). According to Amundsen and Diers (1970) and Lobo et

al. (2013), the age of menopause has changed very little over the centuries, whereas life

expectancy has increased (Amundsen and Diers, 1970; 1973; Lobo, 2013).

<A>The facts

When humans first walked this earth, life expectancy rarely exceeded 35 years and the ovary

was intended to go on working for an entire lifetime. These days, it is not unusual to live

beyond 80 years, begging the question: is it possible for natural ovaries, each containing

several million oocytes at mid-gestation, to continue functioning until death?

A better way of life and improved health care were able to boost life expectancy in the space

of only 1 century, from 48.3 years in 1900 to 80 years in 2000, essentially thanks to advances

in public health measures and efforts. In the 20th century, despite a brief dip caused by the

1918 flu pandemic (Figure 2) (Smith and Bradshaw, 2006), the average lifespan increased by

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more than 30 years (in the USA and the Western world). It is estimated that 50% of all girls

born today will live to over 100 years of age in many countries. The consequence of this

extended longevity is that many women will spend even greater proportions of their lives in

the menopause, exposed to an elevated risk of diseases linked to the absence of oestrogens,

such as cardiovascular disease and bone mineral density loss (Lobo, 2013; 2017).

<A>Does hormone replacement therapy alleviate menopausal symptoms?

In the 1980s, several studies (including meta-analyses) suggested that hormone replacement

therapy (HRT) could be beneficial for preventing osteoporosis, coronary heart disease and

dementia, leading to decreased mortality. In 1992, the American College of Physicians

recommended HRT for preventing coronary disease (American College of Physicians, 1992).

In the early 2000s, however several randomized trials (Women’s Health Initiative [WHI]

[Rossouw et al., 2002], Million Women Study, Lancet, 2003) suggested that the risks,

including for breast cancer, outweighed any benefits. This unfortunately led to new

recommendations being made, resulting in a 50% drop in HRT use (Lobo, 2013). For this

reason, in 2013, Lobo asked a crucial question: ‘Where are we, 10 years after the WHI?’ He

used age stratification, reanalysed data from the original randomized trials, along with more

recent studies, and found that women aged 50–59-years or within 10 years of menopause

taking HRT had lower rates of coronary heart disease and all-cause mortality, and not did

show any increase in perceived risks, including for breast cancer compared with women

taking HRT (Lobo, 2013; 2016; 2017; Lobo et al. 2016).

When cumulative data from the group aged 50–59 years taking part in the conjugated equine

oestrogens alone trial of the WHI were analysed after 13 years, the relative risk for coronary

heart disease was 0.65 (0.44 to 0.96), for breast cancer 0.76 (0.52 to 1.11) and for total

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mortality 0.78 (0.59 to 1.03) (Lobo et al. 2016; Lobo, 2017). Hormone replacement therapy is

also known to decrease the incidence of menopausal symptoms and risk of osteoporotic

fractures, improving quality of life.

Hence, the risk–benefit balance is positive for HRT use, with risks considered rare in healthy

women aged 50–60 years, something known as the ‘timing hypothesis’ (Lobo, 2016; 2017).

As suggested by Lobo et al. (2016) (Figure 3), having already come full circle with HRT

since its introduction, we should now be looking at using it in the context of a general

prevention strategy for women approaching menopause.

<A>Ovarian tissue reimplantation as potential HRT

Only two studies have reported data on ovarian tissue reimplantation as potential HRT. The

first, by Callejo et al. (2001), described a series of three patients undergoing hysterectomy

and bilateral oophorectomy, followed by immediate reimplantation of ovarian cortical tissue

in the abdominal wall. Ovarian secretion was observed 4–5 months after surgery in two of the

three cases, but only for a ‘short reproductive span’. The authors concluded that it is unlikely

that heterotopic grafts would have the longevity to be an adequate substitute for HRT after

normal menopause. Clearly, their conclusion cannot be generalized, as the women involved

were aged 45–49 years and their ovarian reserve was already extremely depleted.

The second study, by Kiran et al. (2005), is a case report. A 44-year-old woman operated on

for uterine fibroids received an ovarian tissue graft (10 cortical strips) implanted in the

Pfannenstiel incision site above the rectus abdominis fascia. Folliculogenesis was still evident

by ultrasonography 18 months later, as were low LH and FSH levels, despite having shown

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an increase at 12 and 15 months of follow-up. No further details on this case have been

reported.

<A>Duration of ovarian activity after reimplantation of frozen ovarian tissue in case of

‘iatrogenic’ menopause

<B>Donnez and Dolmans

In our series, we observed restoration of ovarian activity with resumption of menses in 100%

of cases when primordial follicles were present in frozen biopsies (Donnez and Dolmans,

2015b). In an earlier series, ovarian activity failed to resume in three patients with no follicles

in their grafted tissue (Donnez and Dolmans, 2013; 2015a), highlighting the importance of an

intact follicular reserve.

The long-term duration of ovarian function in a series of five women who underwent ovarian

tissue cryopreservation before the age of 22 years (median 19 years), and reimplantation some

years, later is shown in Figure 4. Ovarian activity was restored for a period of 6–7 years. By

repeating the procedure, this can be extended to over 12 years (Donnez and Dolmans, 2015b)

(Figure 4).

The ovaries of a newborn girl contain an average of 1 million primordial follicles, dropping to

100,000 at 20 years of age and 65,000 at 25 years (95% prediction interval 7700–546,000),

according to Wallace and Kelsey (2010). Ideally, ovarian biopsies should be taken when

follicular density is high (between the ages of 20 and 25 years) because, as demonstrated by

our team, ovarian function can then be restored for long periods of time (Donnez and

Dolmans, 2015b).

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<B>Andersen and Kristensen

In a recent paper, Andersen and Kristensen (2015) reported that four patients underwent their

first tissue transplantation procedure more than 10 years ago in their centre. Two of them

experienced ovarian activity for 6–7 years, similar to results obtained in our series. The other

two underwent a second transplantation and still have functioning ovaries 11 years after the

initial procedure.

<B>Kim

In a study by Kim (2012), four patients aged between 27 and 37 years had their ovarian tissue

cryopreserved between 1999 and 2004. Reimplantation was carried out between 2001 and

2011. Ovarian tissue slices were grafted to a heterotopic site (between the rectus muscle and

rectus sheath) and long-term follow-up was initiated (Kim, 2012). Recovery of ovarian

function was achieved but was relatively short (3–6 months). The patients underwent a second

transplantation. In one woman, low FSH and high oestradiol levels proved that the graft was

still functioning after 7 years. This clearly shows that, if the goal is restoring ovarian function

but not fertility, a heterotopic location (forearm, abdominal wall) could be an easy and

effective site for reimplantation (Kim, 2014).

According to Kim (2012; 2014), the heterotopic graft produced progesterone secretion.

A recent paper (Damásio et al., 2016) reported that heterotopic transplantation was able to

preserve ovarian follicle integrity in an animal model, but the manuscript failed to report

endocrine secretion. In a study by Suzuki et al. (2012), it was reported that heterotopic

autografts can give rise to long-term ovarian function, with progesterone values over 10

mg/ml during the luteal phase. Progesterone secretion was also documented in another animal

model (Lee et al., 2017) after autografting of vitrified ovarian tissue to a heterotopic site.

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<A>Could removal of biopsies or ovaries cause any harm?

Removal of five to six biopsies of 1 cm in length and 5 mm in width will have little if any

effect on fertility or age of menopause. Even removal of a whole ovary is known to have a

negligible effect. Indeed, it is now well proven that women with one ovary remain as fertile as

women with both (Lass et al., 1997; Blanco et al., 2001). Two studies (Yasui et al., 2012;

Bjelland et al. 2014) have also shown that onset of menopause is only marginally affected in

women with only one ovary, who tend to start their menopause around 1 year earlier. We

may, therefore, state with some degree of certainty that removal of less than 30% of one ovary

has a minimal effect on the ovarian reserve and subsequent follicular recruitment (Donnez and

Dolmans, 2017a).

<A>The future

In 2015, two different teams (Andersen and Kristensen, 2015; Donnez and Dolmans 2015b)

demonstrated that long-term endocrine function could persist for more than 7 years (12 years

with a repeat procedure) after frozen–thawed ovarian tissue reimplantation (Figure 4). They

suggested that restoration of endocrine function could prevent menopause-related conditions,

such as osteoporosis and other complaints in an ageing population.

Therefore, having established that ovarian tissue reimplantation is able to restore ovarian

activity after induced menopause, why not propose it to restore sex steroid secretion after

natural menopause? In this instance, the graft site could be heterotopic, namely outside the

pelvic cavity, e.g. the forearm, rectus muscle, as the goal here is not fertility restoration

(Figure 5). This makes the procedure less invasive, potentially achievable under local

anaesthesia, and feasible even if severe pelvic adhesions are present (Table 1). As suggested

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by Andersen and Kristensen (2015), this approach should initially be considered in women

with ovarian tissue already frozen. Undoubtedly, this application will become increasingly

widespread in the future (Donnez and Dolmans, 2017).

When the implants stop functioning, surgery may be repeated and endocrine function restored

for longer. Owing to improvements in new techniques favourably affecting the time needed

for revascularization, follicle loss rates may be reduced and the benefits of grafting might be

seen sooner and for longer periods (Manavella et al., 2017).

Questions surrounding possible risks and uncertainties will, of course, be raised and must be

satisfactorily addressed (Andersen and Kristensen, 2015; Donnez and Dolmans, 2015b), but

some remain unanswered at present. Is the endometrium adequately protected by progesterone

secretion? Is progesterone secreted by ovarian tissue grafted to a heterotopic site? Kim (2012;

2014) has the most extensive experience in heterotopic grafting and has confirmed that

progesterone secretion was similar to that observed in the case of orthotopic grafting.

Moreover, a progesterone level of 5 mg/ml for 10 days is enough to induce differentiation to

the endometrial secretory phase, and this level is easily achieved even after heterotopic

grafting. Heterotopic transplantation is not optimal for oocyte quality, but corpora lutea may

develop in heterotopic sites. Stern et al. (2013; 2014) reported that heterotopic grafts, while

not optimal, nevertheless allow the potential for pregnancy and live birth. Other authors

(Rosendahl et al., 2006; Demeestere et al., 2009) have also demonstrated ovarian activity and

progesterone secretion after heterografting of human ovarian tissue. Other issues, however,

are an even greater cause of concern and need to be thoroughly investigated.

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Is there an increased risk of cancer after tissue reimplantation? Although we are unable to

offer any conclusive response at this stage, the debate should at least be opened, taking into

account the well-balanced benefits of HRT. Would postponing the age of menopause increase

the risk of breast cancer? The question is appropriate, but the risks attributed to HRT have

been overestimated (Sitruk-Ware, 2007). According to a recent paper by Lobo (2017), the

risk–benefit balance of HRT use in healthy young women aged 50–59 years or within 10

years of menopause shows lower rates of all-cause mortality, without any increase in breast

cancer. Moreover, although HRT should be primarily oestrogen-based, no particular HRT

regimen has emerged as the frontrunner (Lobo, 2017).

Ovarian neoplasms originating from ovarian tissue grafted beneath the skin are the subject of

the most heated debate related to the procedure. It should be stressed, however, that follow-up

of implants placed under the skin is straightforward (Table 1), and resection is possible if

implants require excision.

When evaluating the cancer risk of grafted tissue, it is logical to compare values with those of

a normal intra-abdominal ovary. Furthermore, if the biopsy was taken at the age of 20–25

years, the risk of developing ovarian cancer over a 10-year period is low. Indeed, this risk

increases essentially after the natural menopause, when the ovary is more than 50 years old.

An alternative way of avoiding the threat of ovarian cancer involves isolating follicles,

transferring them inside a specially created scaffold (artificial ovary), and then reimplanting

this scaffold in the forearm or abdominal wall (Figure 5).

In conclusion, if ovarian tissue freezing and reimplantation can restore ovarian hormone

function in case of iatrogenic menopause, why not consider it for naturally occurring

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menopause (Donnez and Dolmans, 2017)? In response to the opinion paper by Andersen and

Kristensen (2015a), Von Wolff et al. (2015) ask the question: ‘is it really more advantageous

for women’s health than menopausal hormone replacement therapy?’ They argue that women

without a uterus should be given conventional oestrogen therapy, whereas transplantation of

frozen–thawed ovarian tissue may be beneficial to patients with an intact uterus, as there is a

need for progesterone. They also claim that no studies support grafting of ovarian tissue as a

way of postponing menopause.

In their subsequent response, Andersen and Kristensen (2015b) emphasize their central

message, maintaining that individualization is key and that new diagnostic tools should be

considered when choosing the type of HRT and avoiding menopausal effects. Strong familial

predisposition to osteoporosis, previous hysterectomy, age at menopause and many other

factors need to be considered in such individualized therapy.

Clinical trials to investigate this approach are clearly needed, but we should bear in mind that

most natural oestrogens are produced by the ovary itself, and not by a pharmaceutical

company (Donnez and Dolmans, 2017b).

In another letter to the editor, Patrizio and Caplan (2015) concluded that both medical and

ethical issues must be fully addressed before this alternative can be offered as potential

menopause treatment. They stressed the key moral challenge regarding efficacy, questioning

the capacity of ovarian tissue harvested at a young age to maintain its own ‘youth’ and not

adapt to the actual age of the patient, ceasing to function much earlier than anticipated. We

may quell such misgivings, having proved that ovarian function can be maintained for 6–7

years or more, and that the procedure, being easily repeatable (at least once), can extend

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ovarian function for over 12 years (Andersen and Kristensen 2015a; 2015b; Donnez and

Dolmans, 2015b).

We acknowledge that numerous issues must still be satisfactorily resolved, and that animal

research and human investigations should be conducted according to morally responsible

standards. Nevertheless, we need to establish whether ovarian tissue freezing at a young age

followed by reimplantation at menopause could indeed be the anti-ageing therapy of the

future. We believe we now have enough evidence to initiate a meaningful debate on this

subject.

Acknowledgements

The authors thank Guillaume E Courtoy for his input to the figures. They also thank Mira

Hryniuk, BA, for reviewing the English language of the manuscript and Deborah Godefroidt

for her administrative help.

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Table 1 – Advantages of heterotopic transplantation to restore ovarian function.

Advantages

Less invasive procedure

Repeat transplantations possible

Feasible even with severe pelvic adhesions

Preferred method for restoration of ovarian

function (not fertility)

Allows close monitoring for potential

recurrence of malignancy in grafts

Figure legend

Figure 1 – The ovarian reserve throughout a woman’s life, from conception to age 55 years.

Only around 1000 follicles remain at menopause. Adapted from Wallace WH and Kelsey

TW. PLOS ONE 27, e8772, 2010; published under an open-access license by PLoS. Donnez J

and Dolmans MM propose ovarian tissue reimplantation when women reach the menopause.

Ideally, their tissue should have been removed and frozen at the age of 20–25 years. Adapted

from Wallace and Keysey, PLOS ONE, 2010.

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Figure 2 – Life expectancy in the US (based on National Vital Statistics Reports [Volume 58,

Number 21] and Smith and Bradshaw, 2006). Adapted from National Vital Statistics Reports

Volume 58, number 21, and Smith and Bradshaw, Demography, 2006.

Figure 3 – Lobo’s full circle (Lobo, Nature Reviews Endocrinology, 2017), giving rise to the

timing hypothesis. Adapted from Lobo, Nature Reviews Endocrinology, 2017. HRT, hormone

replacement therapy; MWS, Million Women Study; WHI, Women’s Health Initiative.

Figure 4 – Long-term duration of ovarian function after orthotopic transplantation in a series

of five women who had their ovarian tissue cryopreserved aged younger than 22 years.

Adapted from Donnez and Dolmans, Journal of Assisted Reproduction and Genetics, 2015,

and New England Journal of Medicine, 2017.

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Figure 5 – Ovarian tissue freezing followed by reimplantation upon reaching menopause.

Ovarian tissue may be grafted to a heterotopic site in the form of ovarian tissue strips, or

inside an artificial ovary containing isolated primordial follicles (Based on Donnez and

Dolmans, New England Journal of Medicine, 2017). Adapted from Donnez and Dolmans,

New England Journal of Medicine, 2017.

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Declaration

JD has been a member of the Scientific Advisory Board (SAB) of PregLem SA since 2007.

He receives grants and fees for lectures and coverage of travel expenses to investigator

meetings of PEARL studies from the Gedeon Richter Group. He was the principal

investigator of the four PEARL trials. MMD has no conflict of interest in relation to the

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paper. Her salary is partially (50%) paid by the Fonds National de la Recherche Scientifique

de Belgique-FNRS (grant number 5/4/150/5).

Author biography

Jacques Donnez was appointed Professor Emeritus at the Catholic University of Louvain in

2012. He has published over 600 original articles in peer-review journals. He was the first

President of the International Society for Fertility Preservation (to end 2010) and was elected

to the Royal Belgian Academy of Medicine in 2009.