Age-related fertility decline: is there a role for elective ovarian tissue cryopreservation?

Over the last 50 years, societal perceptions and cultural reproductive norms have evolved significantly. The development of gender equality and improved women’s rights have enhanced professional and educational opportunities, financial independence and empowerment for women. This has resulted in a shift of reproductive aspirations and plans, as exemplified by the increasing age of first-time motherhood observed amongst women in the European Union (EU), from 28.8 years old in 2013 to 29.3 in 2018 ( Eurostat, 2020 ). This deferment of childbearing years has significant reproductive implications. The progressive reduction in number of primordial follicles causes depletion of ovarian reserve in an exponential fashion from the age of 37 years onwards ( Devesa et al. , 2018 ). This results not only in a reduction in quantity of oocytes but also a deterioration in oocyte quality, thereby potentiating risk of aneuploidy ( Hassold and Hunt, 2001 ). Clinically, this exhibits itself as reduced fecundity and an increased risk of miscarriage; from 10% in the second decade of life, to 53% in those over 45 years old ( Magnus et al. , 2019 ). Advanced age is also associated with an increased incidence of uterine pathology, including adenomyosis, commonly observed in women aged 40–49 years old ( Naftalin et al. , 2012 ) and leiomyomas, which are associated with unfavourable reproductive outcomes and increased obstetric complications ( Olive and Pritts, 2010 ). Delaying motherhood thereby, results in inevitable and often untreatable age-related consequences, which if not pre-empted and actioned, may result in involuntary childlessness, or an inability to meet reproductive aspirations. It is therefore unsurprising that as the age of first-time motherhood has increased, microsimulation models used to estimate the rates of permanent involuntary childlessness amongst six European countries, have demonstrated that overall rates have doubled since the 1970’s, with an increase of 2.5% observed in Sweden, 3% in Austria, Netherlands, Czech Republic and West Germany and 4% in Spain ( Te Velde et al. , 2012 ). Furthermore, the risk of involuntary childlessness in women aged over 40 years is 3% higher than in women under 30 years old (33% versus 36%, respectively) ( Steenhof and De Jong, 2000 ; Te Velde et al. , 2012 ). Advancements in artifical reproductive technology (ART) have provided women the opportunity to overcome such challenges by utilizing oocyte donation for in vitro fertilisation (IVF) cycles. Although this enables the experience of gestation, it denies the opportunity for biologically related offspring. Women wishing to preserve their fertility to mitigate the impact of age-related fertility decline (ARFD) can now undergo elective oocyte cryopreservation (EOC). Whilst this allows women the opportunity to extend their reproductive years, it does not guarantee future livebirths. Whilst ovarian cortex cryopreservation has been used to preserve reproductive potential in women for medical reasons, such as undergoing gonadotoxic chemotherapy, it has not yet been used in the context of ARFD. The aim of this article is to critically appraise EOC as an option for women wishing to preserve their fertility to prevent ARFD and to introduce the prospect of elective ovarian cortex cryopreservation as an alternative.

No funding was required for this paper.

An alternative method of fertility preservation for ARFD is ovarian tissue cryopreservation (OTC) ( Martinez, 2017 ). OTC involves laparoscopic resection of ovarian tissue, either from the ovarian cortex containing primordial follicles or whole ovary; followed by cryopreservation ( Salama and Woodruff, 2015 ). The concept was proposed initially to mitigate the risk of secondary premature ovarian insufficiency (POI) in women undergoing gonadotoxic chemotherapy and to preserve fertility in pre- and peripubertal girls, in whom OC is not possible ( Salama and Woodruff, 2015 ; Jensen et al. , 2017b ). Figures from national databases suggest that based on a population of 500 million in the European Union, between 2500 and 6500 OTC procedures take place in Europe per year ( Van der Ven et al. , 2016 ). Given the increasing use and acceptance, it is no longer considered experimental in patients at risk of iatrogenic ovarian failure according to criteria by the European Society of Human Reproduction and Embryology (ESHRE) ( Provoost et al. , 2014 ). Following the success of OTC as an established method of fertility preservation in women with cancer, it has evolved further as a technique for women undergoing treatment with a high or intermediate risk of POI due to benign conditions ( Jadoul et al. , 2017 ; Lotz et al. , 2019 ). This includes autoimmune, haematological or medical illness treated by cytotoxic agents, presence of bilateral ovarian tumours and severe recurrent ovarian endometriosis ( Jadoul et al. , 2017 ; Lotz et al. , 2019 ). Multiple centres have performed frozen-thawed orthotopic ovarian tissue transplantation worldwide. The thawed or warmed tissue is transplanted into either the broad ligament, the remaining ovary or ovarian fossae ( Jensen et al. , 2017a ). Following transplantation, restoration of endocrine function is dependent upon various factors at the time of OTC, including the age of the woman, the follicular density and quality of the graft tissue ( Takae and Suzuki, 2019 ). The procedure is deemed successful when both return of menstruation and follicular growth is observed. A recent meta-analysis highlighted that ovarian endocrine function was restored in 85.2% (n = 309) of women receiving transplanted tissue ( Pacheco and Oktay, 2017 ), and in a separate study of 800 women, amongst 44 women who underwent ovarian tissue reimplantation following retrieval, 98% (n = 43) had resumed or improved ovarian function ( Diaz-Garcia et al. , 2018 ). Reasons for unsuccessful return of ovarian function have been reported as inadequate quantity of ovarian tissue cryopreserved, or when the procedure was performed at an advanced age ( Pacheco and Oktay, 2017 ). The mean duration of ovarian function has been demonstrated to be 5 years, although normal graft function can be maintained up to 10 years later ( Donnez et al. , 2015 ; Takae and Suzuki, 2019 ). In addition, outcomes have been shown to be similar between both fresh and frozen grafts, with comparable ovarian function observed after 2 years of follow-up ( Silber et al. , 2015 ; Sheshpari et al. , 2019 ). The pregnancy rate following orthotopic transplantation was reportedly between 27% and 37% ( Bedaiwy et al. , 2008 ; Jensen et al. , 2015 ; Silber, 2016 ; Van der Ven et al. , 2016 ) compared to 26% in a study of 285 women who underwent frozen-thawed ovarian tissue transplantation. ( Dolmans et al. , 2021 ). As the method of cryopreservation and surgical techniques have been optimized ( Beckmann et al. , 2019 ), more recent reports from three major centres from Tel Aviv, Brussels and St Louis have published pregnancy rates of 50% and LBRs of 41% amongst a cohort of 60 patients ( Shapira et al. , 2020 ). Much like OC, the ovarian reserve and genetic quality of the oocyte is dependent on the age at the time of cryopreservation, and thus an independent predictive factor for pregnancy ( Rozen et al. , 2021 ), with the highest success rates observed in women aged 34 years or younger ( Lotz et al. , 2019 ). Further data suggests that the pregnancy rate when OTC was performed at the following ages: 40 years old, were 41%, 33%, 18% and 0%, respectively ( Van der Ven et al. , 2016 ). These findings are also consistent with data from one of the largest national fertility databases, which deduced that OTC should only be performed in women ≤40 years old ( Beckmann et al. , 2018 ). Various studies also report a LBR between 21.6% and 30% amongst women undergoing OTC ( Dolmans et al. , 2009 ; Andersen, 2015 ; Lotz et al. , 2016 ; Meirow et al. , 2016 ; Van der Ven et al. , 2016 ). An overall trend for lower LBRs associated with OTC may be attributed to the impaired folliculogenesis observed, causing disruption between the granulosa cells and oocytes, subsequently resulting in reduced oocyte maturity, poor fertilization rates and inadequate embryo quality ( Dolmans et al. , 2009 ). However, as with most novel therapies, it is expected that further advancement will improve outcomes, particularly as novel cryopreservation regimens are developed. Although literature reports more than 130 livebirths following OTC since 2017 ( Donnez and Dolmans, 2017 ; Lotz et al. , 2019 ; Oktay et al. , 2021 ), the figure is now likely to be more than 200 ( Dolmans et al. , 2020 ). The largest systematic review of 210 recipients reported that 70% of all pregnancies were achieved spontaneously (n = 84), whereas 30% (n = 36) were following IVF ( Sheshpari et al. , 2019 ). Furthermore, in a study of 285 women; from 106 who conceived, 63% (n = 67) did so naturally whilst 37% (n = 39) conceived through IVF ( Dolmans et al. , 2021 ). Women can also achieve multiple pregnancies from the same graft, with some cases reporting >3 pregnancies in the same woman ( Jensen et al. , 2015 ). Data extrapolated from various national databases suggests that the majority of pregnancies following OTC were carried to term with positive perinatal outcomes ( Pacheco and Oktay, 2017 ; Jensen et al. , 2017a ). A congenital abnormality rate of 1.2% has been reported, which is comparable to the general population ( Pacheco and Oktay, 2017 ). It is important to consider that the majority of data regarding reproductive outcomes following ovarian tissue transplantation were taken from women who had undergone chemotherapy or radiotherapy for malignant pathology or had POI (78% versus 20%, respectively); and therefore likely had an existing degree of ovarian insufficiency prior to transplantation ( Sheshpari et al. , 2019 ). For the purpose of fertility preservation, the number of follicles restored during the freeze-thaw stage is important ( Rozen et al. , 2021 ), and for that to be achieved, at least one-half to two-thirds of the ovarian cortex is usually harvested ( Meirow, 2008 ). In such instances, a follicle survival rate as high as 84% from frozen-thawed tissue has been described ( Kristensen et al. , 2018 ), and a follicular density of 89% has been retained following implantation of paired fresh samples ( Christianson et al. , 2021 ). One of the current challenges of OTC is optimizing survival of the follicular pool within the ovarian graft. Significant follicle demise occurs secondary to the exposure of hypoxia. Transplantation onto the vascular pelvic structures, is dependent on the process of neovascularization which occurs during the first 10 days post-implantation ( Li et al. , 2014 ). Inadequate neovascularization results in oxygen-derived free radicals and lipid peroxidation, which triggers ischaemic reperfusion injury within the ovarian tissue ( Takae and Suzuki, 2019 ). The initial phase of ischaemia can be associated with loss of the follicular reserve by up to 60%, which can subsequently impact ovarian reserve and longevity of the graft ( Kim et al. , 2004 ; Gavish et al. , 2014 ; Oktay et al. , 2021 ). Various methods have been described to reduce the risk of post-implantation graft hypoxia, such as using the isoform of vascular endothelial growth factor 165 within a collagen matrix to encapsulate the ovarian tissue ( Henry et al. , 2015 ). This has been demonstrated to result in earlier revascularization and improved angiogenesis of the graft in the first 3 days post-implantation ( Henry et al. , 2015 ). Furthermore, anti-apoptotic agents such as Sphingosine-1-phosphate (S1P), an endogenous phospholipid messenger, significantly accelerates revascularization of the ovarian grafts to 2–3 days and doubles the microvascular density ( Li et al. , 2014 ). This results in reduced tissue hypoxia and apoptosis of follicular cells, thus improving overall success of the transplantation ( Soleimani et al. , 2011 ). Plasma levels of S1P are significantly higher in younger women and synthesis has been shown to be directly associated with oestrogen levels ( Guo et al. , 2014 ). Therefore, if elective OTC (EOTC) is undertaken in young healthy women, improved outcomes and greater graft longevity could potentially be observed, when compared with women who have undergone the procedure for medical pathology. A second cause of follicular demise is the process of cryopreservation itself, which promotes uncontrolled follicular activation of primordial follicles, also known as follicular burnout ( Masciangelo et al. , 2019 ). The administration of recombinant anti-Müllerian hormone, has been shown to inhibit initiation of primordial follicle recruitment in mice studies, which prevents ovarian reserve depletion and subsequent follicular burnout ( Kano et al. , 2017 ). Further animal studies have proposed the use of adipose-derived stem cells, whereby a mean survival rate of 62% was reported one week following transplantation ( Manavella et al. , 2018 ). Moreover, the application of microsurgical scissors has been shown to preserve the total number of follicles, but to the detriment of triggering follicular abnormalities including stromal death ( Herraiz et al. , 2020 ). During the process of vitrification, solutions consisting of a high concentration of cryoprotectant agents (CPAs) and high viscosity are used in order to protect the tissue and cells from dehydration or changes in temperature ( Leonel et al. , 2019 ; Shahsavari et al. , 2020 ). The most commonly used CPA’s in vitrification of ovarian tissue includes dimethyl sulfoxide (DMSO), ethylene glycol (EG), sucrose and 1-2-propanediol (PrOH) ( Leonel et al. , 2019 ). However, when used for a prolonged period of time, detrimental impairment of the tissue can occur in addition to cytotoxicity. Studies suggest that enhanced outcomes with a survival of more than 90% intact follicles, can be achieved when a combination of DMSO in low concentration (27%) is used with EG and other CPAs ( El Cury-Silva et al. , 2021 ). Even higher rates (98%) of normal follicles following cryopreservation are observed when a combination of 27% of EG and 27% glycerol are used with non-permeable synthetic polymers ( El Cury-Silva et al. , 2021 ). Thus, it is feasible for vitrification techniques to preserve the integrity of the majority of follicles ( El Cury-Silva et al. , 2021 ).

The authors have no conflicts of interests to declare.

For women who wish to preserve or extend their reproductive potential to prevent or restore their fertility following ARFD, EOTC may offer an alternative option to EOC. Similar to the motives for undergoing EOC, women who do not plan on having children until a time when their reproductive potential has started to deteriorate could consider EOTC. Women with normal endocrine function and appropriate ovarian reserve would be suitable to undergo the procedure at a time when age and follicular density are optimal, following extensive counselling and with the understanding that outcomes will be related to age at EOTC. The same surgical technique should be used as is currently utilized for OTC for medical indications. Once the circumstances of women who choose to undergo EOTC change to an extent where conception is desired, if the remaining ovarian reserve has physiologically deteriorated, reimplantation of the cryopreserved ovarian tissue could be undertaken, thereby restoring or enhancing their reproductive potential. Consideration of the potential risks and benefits is essential in such a novel approach. When evaluating the safety of OTC, primary risk includes undergoing at least two laparoscopic procedures; retrieval and implantation of ovarian tissue. The complication rate so far reported in 1302 women who underwent retrieval and implantation was 0.2% (n = 2) and 0.07% (n = 1), respectively ( Beckmann et al. , 2018 ). A separate analysis of 476 women identified no cases of significant surgical adverse events ( Dolmans et al. , 2013 ). Therefore, the overall surgical risks are similar, if not smaller, compared to laparoscopic surgery performed for other benign pathology ( Lotz et al. , 2019 ). In a study of 90 women who underwent laparoscopic salpingo-ovariolysis, 40.2% developed moderate to severe adhesion reformation identified during early second look laparoscopy ( Alborzi et al. , 2003 ). Whilst no correlation has been identified between self-reported pain, physical or emotional scores with the presence or absence of pelvic adhesions identified during diagnostic laparoscopy ( Cheong et al. , 2018 ), adhesions are associated with increased risk of infertility ( Vrijland et al. , 2003 ), which would be counterproductive in a procedure intended to preserve and restore reproductive potential. In EOTC, iatrogenic POI is a risk factor following resection of substantial volumes of ovarian tissue. Therefore, individualized assessment including consideration of age and pre-existing ovarian reserve should be determined when deciding how much ovarian tissue to resect ( Oktay et al. , 2021 ). Evidence suggests removal of <30% of ovarian tissue does not have a significant impact upon ovarian reserve ( Vuković et al. , 2019 ). Data can also be extrapolated from outcomes following unilateral oophorectomy (UO); where in a study of more than 23 000 women, menopause was brought forward by only 1 year ( Bjelland et al. , 2014 ). Another study demonstrated that when UO was performed at 20, 30 and 45 years of age, it was associated with onset of menopause at 44.7, 46.3 and 48.7 years old, respectively ( Rosendahl et al. , 2017 ). In the eventuality of POI following EOTC, premature reimplantation could be undertaken, or alternatively hormone replacement therapy (HRT) used until reimplantation was considered at a time when conception was subsequently desired. Consideration is also required for the potential impact upon reproductive potential following EOTC. Data can be inferred from a study of women who underwent UO, whereby no impact on conception rates, both spontaneously and following assisted conception, was demonstrated ( Lass, 1999 ). When compared with EOC, EOTC offers a great advantage of the possibility of spontaneous conception. This is exemplified by a study comparing OTC with OC, whereby almost half of the OTC patients conceived naturally ( Diaz-Garcia et al. , 2018 ). The potential for natural conception would likely have significant psychological, emotional and economic advantages, whilst reserving the option of IVF, if necessary. Although EOTC provides the opportunity for spontaneous conception, much like EOC, it may not guarantee future offspring, particularly as reproductive outcomes are also dependent on paternal factors, such as age. This is important considering the mean paternal age has also increased globally, from 27.4 to 30.9 years observed in America ( Khandwala et al. , 2017 ; Bergh et al. , 2019 ), and from 29.2 in 1980 to 32.1 over the last four decades in England and Wales (Birth Statistics, 2007). In a recent systematic review, both the livebirth and pregnancy rate were increased when the male age was ≤40 years old in autologous oocyte cycles, and the miscarriage rate more likely when the male was >40 years old ( Morris et al. , 2020 ). Paternal age should therefore also be considered in the management of ARFD. Moreover, OTC provides the opportunity to preserve hundreds of primordial follicles at once ( Lotz et al. , 2019 ), thereby not restricting women to a finite number of oocytes cryopreserved, which is a known limitation of EOC. Interestingly, a recent cost-analysis study of women undergoing onco-fertility treatment in America, demonstrated that OC was more costly than OTC ($16 588 versus $10 032, respectively) ( Chung et al. , 2021 ). In a prospective study comparing the efficacy of oocyte vitrification vs OTC in women undergoing gonadotoxic treatments, higher LBRs per patient were observed in the OC group, although there was no statistical significance between the groups (32.6% versus 18.2%, respectively) ( Diaz-Garcia et al. , 2018 ). Furthermore, a sensitivity analysis reported no successful pregnancies in women who underwent OTC above the age of 36, compared to a 30% pregnancy rate in women undergoing oocyte vitrification above the same age ( Diaz-Garcia et al. , 2018 ). Studies so far have reported an average storage time of 9.1 years, with an upper range of 17.9 years, which resulted in a 98% follicle survival rate following OTC ( Kristensen et al. , 2018 ). Should EOTC therefore subsequently transcend into clinical practice, updated legislation is essential to ensure tissue is not implanted for fertility restoration purposes in women outside of their natural reproductive years. As such, limiting the age at reimplantation to a maximum of 45 years may be an appropriate compromise, although further ethical reflection and debate is needed. In addition, if a woman decides not to use her stored ovarian tissue to extend her reproductive potential, it could instead be used later in life to alleviate menopausal symptoms, as a method of cell tissue HRT ( Kristensen and Andersen, 2018 ). If the tissue is used for this purpose, permanent contraception such as concomitant bilateral tubal occlusion would be essential, to prevent unwanted pregnancies outside of physiological reproductive years.

L.S.K. drafted and revised the article for important intellectual content. S.S., N.G., H.O., T.B.-M., F.S., J.Y., M.-Y.T., J.N., J.B.N. and P.H. revised the article for important intellectual content. C.D.-G. provided substantial contribution to the analysis and interpretation of evidence and revised the manuscript critically for important intellectual content. B.P.J. conceived the idea of the manuscript, helped revise the article and provided final approval of the version to be published.

The clinical application of OTC is undoubtedly feasible as a method of fertility preservation for medical indications and with more than 200 reported livebirths, it is no longer considered an experimental procedure. In the context of the societal trend of women delaying motherhood, the impact of ARFD is becoming increasingly prevalent, often resulting in involuntary childlessness or failure to meet reproductive aspirations. Women can now electively cryopreserve oocytes, however not without risks, including those associated with COS and being restricted to store a finite number of oocytes giving a reasonable probability of achieving a livebirth based on the woman’s age. As established from the evidence provided herein, EOTC could provide an alternative option to EOC, which overcomes some of these challenges, by facilitating spontaneous conception and not being curtailed by a limited number of oocytes for cryopreservation. However, given the novelty of this technology, further research, ethical reflection and legislative reform is required to help determine the suitability, cost-effectiveness, reproductive efficacy and sustainability of this procedure in the context of ARFD.