Delaying Reproductive Aging by Ovarian Tissue Cryopreservation and Transplantation: Is it Prime Time?

Petrokovski and Zharov recently reported their experience of five women who underwent heterotopic OT to the armpit for managing symptomatic menopause [ 28 ]. Ovarian wedge resections amounting to 20–25% of an ovary were obtained from women at the mean age of 28.2 ± 4.3 years during routine obstetrical or gynecological procedures. These women then underwent autotransplantation of their tissues at the mean age of 47.4 ± 5.1 years, after experiencing natural menopause. Of the five transplants, four functioned, while one had to be removed due to site infection. The time to function for the graft was 9 ± 2.6 weeks. All four women reported acceptable or excellent results within the 6 months of follow-up. At the time of the report, all four grafts had been functioning for at least 6 months [ 28 ]. Although encouraging, these data are preliminary, and a larger number of women with longer follow-up will be needed to reach valid conclusions.

The future success of elective ovarian tissue harvesting depends on advances in cryopreservation, thawing, and transplantation techniques. There has already been significant progress in these areas. As discussed earlier, the freezing and thawing process typically results in the loss of a small percentage of follicles. Nevertheless, the original slow-freezing and rapid-thawing approach has improved over the past two decades. Furthermore, preliminary data have given promising results with an open and closed system specifically designed for ovarian issue vitrification [ 29 ]. Another area of progress is with vascularization-enhancing approaches. OT is performed akin to skin grafting, where ovarian cortical strips are attached to vascular pelvic structures with full revascularization spontaneously occurring over 10 days [ 21 ]. During this period, ischemia may result in the loss of around two-thirds of the pre-existing primordial follicle reserve. Therefore, the most significant improvement in OT success would likely come from approaches that can enhance this vascularization process. Sphingosine-1-phosphate (S1P) is a naturally occurring phospholipid messenger previously tested for preserving human ovarian primordial follicle reserve against chemotherapy-induced death [ 30 ]. However, it might also have effects on endothelial cell migration. Thus, Oktay and colleagues hypothesized that S1P could improve the revascularization process and primordial follicle survival in OT. They found that S1P significantly accelerated the revascularization of ovarian grafts, allowing full revascularization to occur in 2–3 days instead of 10 days in the controls. It also resulted in the doubling of the microvascular density by the tenth day of administration compared with controls. This resulted in significantly reduced tissue hypoxia, improved stromal cell survival, and reduced primordial follicle apoptosis [ 21 ]. While S1P is not approved for clinical use, its synthetic analog FTY-720 has been approved to treat multiple sclerosis [ 31 ] [fingolimod (Gilenya™), Novartis]. However, in the aforementioned xenograft study, FTY-720 paradoxically resulted in reduced vascularization, possibly due to the use of high doses that downregulated receptor function. Hence, further dose-finding studies are needed in human ovarian xenograft models before this drug can be tested in clinical trials to improve OT longevity [ 21 ]. There have been other attempts at improving ovarian graft longevity with stem cells, growth factors [erythropoietin (EPO), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF)], antioxidants [vitamin E, N -acetyl cysteine (NAC), and melatonin], and androgens, among others [ 32 – 47 ]. These are summarized in Table 2 . Since these approaches are not at the translational stage, they are not detailed further here. Another improvement could come from the utility of neovascularizing membranes [ 48 , 49 ]. Alloderm is a decellularized extracellular tissue matrix (ECM) scaffold widely used in clinical practice to repair tissue defects, reconstruction, and bridging neovascularization from adjacent tissue edges, especially in the dental field and to repair skin wounds. Alloderm is immunologically inert and allows cell proliferation and migration from the recipient’s tissues [ 48 ]. In previous basic science experiments, it was shown that ECM is critical in primordial follicle growth and development [ 49 , 50 ]. In subsequent studies, it was surmised that the ECM scaffold may enhance ovarian graft revascularization and function [ 4 ]. In initial studies where the utility of the ECM scaffold was combined with the robot-assisted laparoscopic techniques, a larger than expected amount of follicle growth in ovarian grafts upon stimulation was found [ 4 ]. The method resulted in multiple live births in women who initially underwent these procedures. While these preliminary developments are promising, work to enhance ovarian cryopreservation and autotransplantation is continuing and further research will be needed to determine the best practices to restore ovarian function by these procedures.

One of the critical questions in determining the feasibility of elective ovarian tissue freezing to delay and extend reproductive potential is whether tissue harvesting can trigger premature ovarian insufficiency (POI). In a healthy woman, inducing POI by a procedure that is intended to delay menopause is of more significant concern. There are currently no studies reporting on the impact of ovarian tissue harvesting for fertility preservation in healthy women; however, there have been several studies looking at the impact of ovarian surgery on the age at natural menopause ( Table 1 ). Unfortunately, most of the studies are limited by the fact that the age at unilateral oophorectomy was not specified. In a cross-sectional study of 24 152 Japanese nurses who underwent unilateral oophorectomy (ULO), the cohort experienced menopause at a mean age of 50.9 versus 52.1 years in the control group with an odds ratio (OR) of POI of 3.32 (1.42–7.77) [ 12 ]. Another retrospective study found similar findings, with the mean age at menopause for the study group being 49.59 versus 50.66 years in the controls [OR for POI: 1.27; 95% confidence interval (CI): 1.14–1.41] [ 13 ]. A retrospective study considering the beginning of perimenopause as the primary endpoint found that perimenopause began 1 year earlier (age 47 versus 48 years in the controls) in women undergoing ULO, with an OR of 1.93 (95% CI: 1.13–3.31) [ 14 ]. In another retrospective study in Black women, the OR of experiencing earlier menopause was 1.17 (95% CI: 0.82–1.66) after ULO [ 15 ]. In particular, there was a stronger association between ULO and age at menopause among younger women [hazard ratio (HR): 2.00; 95% CI: 1.09–3.68), who had undergone ovariectomy at an earlier age than the average cohort in that study [ 15 ]. None of these studies provided data on the risk of POI based on age at ULO. There have been only two studies that attempted to provide age-specific risk information. A retrospective cohort study found a linear correlation between the age at ULO and age at natural menopause [ 16 ]. The age at menopause was 44.7, 46.3, and 48.7 years when ULO was performed at 20, 30, and 45 years of age, respectively [ 16 ]. Only one study investigated a young population [ 17 ]. In that study, the risk of POI was analyzed after ULO in a cohort of survivors of childhood cancer. The study found that the mean menopause age was 7 years earlier with an OR for POI of 6.3 (95% CI: 3.3–12.2) in those who had ULO versus the controls [ 17 ]. Given that the likelihood of success with ovarian autotransplantation (OT) is significantly lower with tissue cryopreserved after the age of 35 [ 18 ], studies by Rosendahl et al. [ 16 ] and Thomas-Teinturier et al. [ 17 ] may be more applicable to the population of women who may be best suited for elective ovarian tissue freezing. However, these limited data suggest that the younger the age at which the ovarian tissue were harvested, the higher the number of years lost from the reproductive period. This then raises the concern that elective ovarian tissue harvesting may result in earlier menopause in healthy women. However, the previously described studies were based on ULO. In general, an entire ovary is removed for cryopreservation given the high risk of ovarian insufficiency after cancer treatment and to compensate for the inefficiencies of the cryopreservation and transplantation processes i . However, removing an entire ovary may not be justified or medically necessary in the elective setting. Given the data limitations, we do not know the amount of ovarian tissue that can be safely harvested at a specific age without significantly altering the age at natural menopause. Considering that ovarian tissue harvesting procedures are not as common as other reproductive procedures, it may not be feasible to obtain such age-specific information in the near future. Nevertheless, existing data from healthy women suggest that, even though the removal of an entire ovary causes earlier menopause, it does not result in POI (menopause under 40 years of age).

Cryopreserved ovarian tissue transplantation can restore ovarian function and fertility in postmenopausal women [ 6 ]. This has been widely demonstrated in postmenopausal women after gonadotoxic treatments. However, postponing menopause with ovarian tissue transplantation could mean extending the fertility period to extreme ages, and this has a tremendous ethical impact. Some authors have already highlighted a possible solution to this ethical problem by proposing subcutaneous ovarian tissue transplantation [ 7 ]. In addition to preventing the possibility of spontaneous pregnancies, this type of transplant is less invasive than the orthotopic transplant procedure and could also respond to another ethical issue, that of surgical risk, if indeed the advantages brought by the transplantation of ovarian tissue outweigh such risks. However, to obtain ovarian tissue for cryopreservation, abdominal surgery for an ovarian biopsy is required. Women who could achieve a favorable benefit-risk profile are those who must undergo abdominal surgery for other reasons [ 7 ]. Many of the effects of menopause can be counteracted with less invasive methods, such as HRT [ 51 ]. However, this type of treatment was stopped by many women after the Women’s Health Initiative study reported an increased risk of breast cancer [ 52 ]. It was then shown that those data were inappropriately extrapolated from women in the advanced postmenopausal phase (age >60 years) and that there were instead benefits in terms of the prevention of coronary heart disease and mortality when HRT was initiated near the onset of menopause [ 53 , 54 ]. Although the advantage of ovarian tissue transplantation is the production of endogenous hormones rather than the intake of pharmaceutical hormones, the impact of endogenous hormones after ovarian tissue transplantation on women’s health has not yet been studied. Additionally, no studies have been performed to compare the health of postmenopausal women after ovarian tissue transplantation and women taking HRT therapy. Estrogen-only therapy reduces cardiovascular risks more when initiated during early postmenopause compared with combination therapy [ 55 ]. Since women who have undergone a hysterectomy can receive estrogen-only treatment, such patients are likely not to benefit from ovarian tissue transplant. Moreover, if delaying menopause can lead to alleviation of climacteric symptoms and a decrease in cardiovascular and osteoporosis risks [ 56 ], it can also lead to an increased risk of breast cancer. In fact, late menopause (>55 years) has been associated with an increased risk of breast cancer [ 57 ].

Building on prior animal studies on cryopreservation and transplantation [ 1 ], in 1999, Oktay et al. performed the first reported autotransplantation with previously cryopreserved ovarian tissue to restore ovarian function. In that particular case, the purpose of the procedure was to alleviate menopausal symptoms that were not aided by hormone replacement therapy (HRT) [ 2 ]. Given that the procedure successfully restored ovarian endocrine function and follicle development, efforts since then have focused on improving ovarian cryopreservation and transplantation techniques to preserve fertility in patients with cancer. As a result of the efforts of an international body of clinicians and scientists, ovarian cryopreservation and transplantation techniques evolved, culminating in worldwide live birth rates of ~40% per woman [ 3 ]. The success rate is higher in experienced centers, possibly because of the advent of improved surgical approaches, such as robot-assisted techniques with utility of human extracellular matrix and perioperative pharmacological support [ 4 , 5 ]. The culmination of these advances led the American Society of Reproductive Medicine to recently remove ovarian tissue cryopreservation and transplantation from the experimental category for fertility preservation purposes [ 6 ]. This is an excellent development for young survivors of cancer because ovarian tissue harvesting for cryopreservation restores ovarian function with spontaneous conception in most women attempting pregnancy. The success of ovarian tissue cryopreservation and transplantation has resulted in speculations that it could also be used to postpone childbearing and/or delay menopause in healthy women without any medical indications for this procedure [ 7 ]. At least two-thirds of women who receive autologous ovarian tissue cryopreservation have ‘menopause reversal’ [ 3 ] and some preliminary experience indicates nearly a 100% success rate [ 8 ]. Given this progress, here we review the medical facts as well as unknowns about the efficiency of ovarian tissue cryopreservation and transplantation, especially when there is no imminent danger to a female’s fertility from medical causes. This is by and large a risk-benefit assessment, as is the case for all elective procedures. To be able to render this risk-benefit assessment one needs to carefully analyze the efficiency of ovarian cryopreservation and transplantation in preserving primordial follicle reserves, the impact of ovarian tissue harvesting on a healthy woman’s age at natural menopause, and the current and future prospects of the technology. Herein we discuss these issues.

Clinical ovarian tissue cryopreservation and transplantation, beginning with the first successful procedure in 1999, opened a new era in fertility preservation and are no longer considered experimental [ 6 ]. The procedures provide high live birth rates when utilized for women under the age of 35 years and also restore ovarian endocrine function in most women of the same age. However, less certain is the longevity of endocrine function and whether the estrogen production from these autotransplants provides a steady and reliable source of hormone replacement without the need for supplementation with progesterone. A key question remains whether the reduction of the length of reproductive life by the partial or total removal of an ovary is matched by the length of endocrine function gained after autotransplantation. Unless there is a ‘zero’ or positive balance, ovarian cryopreservation for future HRT purposes may not be feasible. Moreover, to prevent menopause-related diseases, the ovarian transplant should provide a prompt endocrine restoration with a steady hormonal release over time [ 56 ]. It takes 3–6 months from the autotransplantation to endocrine function and, thus, if a woman is already experiencing menopausal symptoms, she will not receive an immediate benefit. However, continual advances in ovarian cryopreservation, thawing, and transplantation techniques may enable women to achieve more extended ovarian function with less tissue. It is also uncertain whether a clinically useful amount of ovarian tissue can be harvested from healthy women to prolong reproductive life span and delay menopause. At the heart of this issue is the insufficiency of data in determining the minimum amount of ovarian tissue that can be safely removed at a given age without inducing earlier menopause. While studies with patients with cancer, who are typically aged <25 years, suggest that years of ovarian function can be obtained with ovarian cortex from approximately one-third of an ovary, data for women between the ages of 25 and 40 years are scarce. Based on data accumulated from patients with cancer over a decade of experience, we surmise that cortex from one-third of the ovarian surface may provide sufficient primordial follicles to ensure adequate time for spontaneous pregnancy research or in vitro fertilization treatments. However, this recommendation should be individualized based on ovarian reserve assessments and age. In fact, when the ovarian cortex is removed in old age, the number of primordial follicles available after transplantation is lower and this may not guarantee adequate hormone production to overcome menopausal disorders. There is a significant and accelerated decline in oocyte quality after the age of 37 [ 58 ] and the ovarian reserve is significantly diminished after the age of 40 [ 58 ]. Pregnancy outcome data from patients with cancer thus far showed that the oldest age at which the ovarian tissue can be cryopreserved and successfully result in a live birth after transplantation was 39 years [ 3 ]. Thus, the current technology may not be clinically useful to cryopreserve ovarian tissue in women older than 40 years of age to preserve fertility and extend ovarian endocrine function [ 59 , 60 ] (see Outstanding Questions ). Therefore, we propose guidelines for selecting patients who are most likely to benefit from elective ovarian tissue cryopreservation (see Clinician’s Corner ).