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

September 2018Volume 37, Issue 3, Pages 359–366 Jacques Donnez'Correspondence information about the author Jacques DonnezEmail the author Jacques Donnez , Marie-Madeleine Dolmans PlumX Metrics DOI: https://doi.org/10.1016/j.rbmo.2018.05.018 | 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 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, Menopause, Ovarian tissue reimplantation, Ovary + Key message 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). Fig 1 Opens large image 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. View Large Image | View Hi-Res Image | Download PowerPoint Slide 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). 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, Donnez and Dolmans, 2017a; 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, Donnez and Dolmans, 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, Amundsen and Diers, 1973, Lobo, 2013). 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 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, Lobo, 2017). Fig 2 Opens large image 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. View Large Image | View Hi-Res Image | Download PowerPoint Slide 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; Beral et al., 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, Lobo, 2016, Lobo, 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 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, Lobo, 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. Fig 3 Opens large image 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. View Large Image | View Hi-Res Image | Download PowerPoint Slide 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 an increase at 12 and 15 months of follow-up. No further details on this case have been reported. Duration of ovarian activity after reimplantation of frozen ovarian tissue in case of ‘iatrogenic’ menopause 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). Fig 4 Opens large image 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. View Large Image | View Hi-Res Image | Download PowerPoint Slide 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). 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. 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, Kim, 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. 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). The future In 2015, two different teams (Andersen and Kristensen, 2015a, 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 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). Fig 5 Opens large image 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. View Large Image | View Hi-Res Image | Download PowerPoint Slide Table 1Advantages of heterotopic transplantation to restore ovarian functionLess 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 View Table in HTML 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, 2015a, 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, Kim, 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, Stern et al., 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. 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 (Chiti et al., 2017) (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 menopause (Amorim et al., 2003; 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 ovarian function for over 12 years (Andersen and Kristensen, 2015a, Andersen and Kristensen, 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. References American College of Physicians. Guidelines for counseling postmenopausal women about preventive hormone therapy. Ann. Intern. Med. 1992; 117: 1038–1041 View in Article | Google Scholar Amundsen, D.W. and Diers, C.L. The age of menopause in classical Greece and Rome. 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