Improving Fertility Preservation for Girls and Women by Coupling Oocyte in vitro Maturation with Existing Strategies

“Oocytes from large preovulatory follicles inherently have the highest capacity to generate a pregnancy…”

Background

Advances in the treatment of cancer have resulted in a significant increase in cancer survival rates over the past two decades. Therefore, there is an increasing demand to prevent or decrease the loss of fertility in young female cancer patients who are facing life-preserving, but fertility-destroying, chemo or radiation therapy. Fertility preservation covers a range of clinical approaches and laboratory technologies, many of which are still experimental. Ovarian hyperstimulation and multiple oocyte collection, as per a full IVF cycle, offers cancer patients the best chance of preserving their fertility. Therefore, this is often the first line of treatment for cancer patients, providing good pregnancy outcomes. However, a full IVF stimulation cycle is not suitable for certain cohorts of patients undergoing cancer treatment, such as prepubertal girls, patients who have estrogen-sensitive cancers or those who have insufficient time for a full IVF cycle (∼2 weeks).

Ovarian tissue is increasingly being collected from cancer patients and cryopreserved for fertility preservation purposes. Ovarian tissue preservation has been practiced for many decades despite the fact that, until recently, the prospects of generating a pregnancy from this material have been very limited. Recently, there has been encouraging successes using tissue-transplantation approaches with now >60 healthy children born. Oocyte in vitro maturation (IVM) is a procedure that involves the collection of immature oocytes from minimally unstimulated ovaries followed by their maturation in the laboratory before being fertilized or stored. IVM is used to treat infertility. There are significant opportunities to improve fertility preservation by coupling differing variants of IVM to existing fertility preservation strategies.

Placing IVM in the spectrum of fertility preservation strategies

It is important that we attempt to capture the reproductive potential of all oocytes within an ovary of a cancer patient who is at risk of loss of fertility. Oocytes from large preovulatory follicles inherently have the highest capacity to generate a pregnancy and their fertility potential can be adequately captured by IVF, however, the numbers of such oocytes are limited. At the other end of the spectrum, oocytes from the primordial follicles are extremely numerous in the ovary, but currently it is still technically very challenging to achieve a pregnancy using this material, and just a handful of clinics worldwide have this capability (e.g., using tissue transplantation). Between the primordial and preovulatory follicles lie a large range of numerous preantral and small antral follicles, the reproductive potential of which are typically not captured in the majority of fertility preservation clinics. However, this can be readily achieved by using IVM. IVM is a specialized reproductive technology that generates mature oocytes from growing antral follicles of unstimulated or mildly stimulated ovaries [1]. A great challenge in fertility preservation is generating preovulatory-sized follicles, required to obtain developmentally competent oocytes, irrespective of which approach is taken; whether via ovarian hyperstimulation, tissue transplantation or in vitro follicle growth. In all these approaches, advanced stages of follicle development are difficult to achieve or come at a cost to the patient. This technical hurdle can be overcome by aiming to collect oocytes from smaller follicles using these approaches, but using them in combination with IVM, in particular, IVM technologies specifically tailored for oocytes from small antral follicles [2,3]. This is the basic philosophy of integrating IVM together with a number of existing strategies to preserve fertility in cancer patients.

Current clinical applications of IVM to fertility preservation

Routine IVM

It may not be possible or advisable to offer a stimulated IVF cycle to all young women who are to undergo fertility threatening treatment. Clinical IVM offers the next best chance of achieving a pregnancy, using immature oocytes aspirated in vivo from unstimulated or mildly stimulated ovaries. Treatment of infertility using IVM is a routine clinical procedure in some centers, and with significant recent advances, live birth rates of up to 40% per egg collection procedure have been achieved [4]. In human IVM, mild ovarian stimulation prior to oocyte collection is usual, but in domestic animal breeding, where IVM is in widespread use (∼400,000 offspring/year), immature oocytes are nearly always collected from unstimulated ovaries [5].

The major advantages of IVM to cancer patients are first, that it reduces or potentially avoids elevation in circulating concentrations of estradiol, which is important for patients with estrogen-sensitive cancers to mitigate the risk of inadvertent stimulation of their cancer. Second, it can be offered to the cohort of patients who cannot delay the start of their cancer treatments, as an IVM oocyte collection can proceed at very short notice and at any stage of the menstrual cycle. As there are numerous small–medium antral follicles present in ovaries at all stages of the menstrual/ovarian cycle, it is feasible to collect oocytes for IVM at any stage of the cycle and indeed this is standard IVM practice in the veterinary sector [5]. In reproductive medicine, this has been termed random start IVM or emergency IVM. Recent evidence shows that random start IVM is a viable approach for oocyte retrieval in cancer patients [6,7], although to date few pregnancies have been reported. Routine IVM, as it is currently practiced, is not suitable for prepubertal girls, as oocytes are collected via transvaginal aspiration. Oocytes could potentially be collected laparoscopically as girls have ovaries containing antral follicles, although to our knowledge this has not been attempted in a fertility preservation context.

An IVM collection procedure yields immature oocyte–cumulus complexes, which are more difficult to cryopreserve than mature (metaphase II) oocytes, most likely due to adverse effects of cryopreservation on cumulus cell transzonal processes, which are important for oocyte maturation. Hence, for cancer patients, these immature oocytes are best matured in vitro (∼30 h) prior to cryopreservation of mature oocytes or of embryos following fertilization if the patient has a partner.

Ex vivo IVM

IVM is also currently used clinically in women and girls by collecting immature oocytes ex vivo from small antral follicles as ovarian tissue is processed for cryopreservation (ex vivo IVM). It is common for patients to have ovarian tissue cryopreserved before chemo/radiotherapy, with the objective of preserving the reproductive potential in the large number of primordial follicles in the ovarian cortex. By contrast, the majority of small antral follicles reside in the medulla of the ovary, which at present is not cryopreserved. These numerous small antral follicles contain oocytes that have the potential to yield a pregnancy using IVM, particularly if an IVM system is used that is designed for oocytes from small antral follicles [2,3]. This approach generates additional mature oocytes for cancer patients that are otherwise wasted in the standard ovarian tissue cryopreservation process.

Collecting oocytes from small antral follicles from ovaries ex vivo has long been used and indeed is standard practice in animal IVM research laboratories. This approach has only recently become more widespread in fertility preservation practices with numerous publications of successful oocyte maturation, including from prepubertal girls [8]. Recently, there have been three reports of pregnancies and live births from cancer survivors using this approach [9–11]. A downside of this approach is that the number of oocytes available for use is usually low (<15). However, ex vivo IVM is never used alone as it goes hand-in-hand with ovarian tissue cryopreservation. Moreover, combining tissue preservation and ex vivo IVM with a routine IVM oocyte retrieval prior to ovarian resection maximizes oocyte yield [12]. The additional oocytes obtained from ex vivo IVM are particularly important for certain hematological cancer types where retransplantation of cryopreserved ovarian tissue is contraindicated because of risk of reimplanting malignant cells back to the host [13], and given that other options such as in vitro follicle growth (see below) are still at the experimental stage.

Potential future clinical applications of IVM to fertility preservation

IVM & ovarian tissue transplantation

Another viable fertility restoring approach for young women with cancer is the freezing and autologous transplantation of ovarian tissue after cancer treatment [14]. Frozen–thawed ovarian tissues are commonly transplanted to orthotopic sites such as the remaining ovary if present, on the broad ligament or into ovarian fossa enabling either natural conception or a stimulated IVF cycle. Either approach can be used to achieve pregnancy after the restoration of endocrine function, which is usually seen a few months after transplantation. Orthotopic tissue transplantation has proved to be a clinically viable approach to fertility preservation, with over 60 reported live births and a success rate of approximately 25% [15]. There are, however, concerns of reintroducing neoplastic cells back to the patients. Heterotopic transplantation sites such as the abdominal wall or forearm have also been attempted but far fewer pregnancies have been reported [16].

The ovarian tissue-transplantation approach may also benefit from IVM, as an alternative to hyperstimulating the transplanted tissue and attempting IVF. As the ovarian tissue is transplanted in small pieces and the resultant growing follicles do not have the normal stromal and vascular support of an intact ovary, these pieces do not respond to exogenous gonadotrophin stimulation in the same manner as does a normal whole ovary. Hence few follicles grow to ovulatory size (∼20 mm) and it is difficult to trigger oocyte maturation using LH-analogs in a controlled manner in the transplant, particularly in heterotopic grafts. Conversely growth of small- to mid-antral-sized follicles (2–14 mm) is more readily achieved in a tissue graft, and oocytes from these follicles could then be used for IVM. In this scenario, cryopreserved ovarian tissue containing primordial follicles would be thawed, transplanted (orthotopic or heterotopic), ovarian endocrine activity monitored, follicles would be stimulated to grow with low-dose follicle stimulating hormone, monitored by ultrasound until they reach 2–10 mm, immature oocytes would be collected and matured in vitro and then either cyropreserved or proceed with IVF. This approach has not yet been attempted in human but would involve a simple combination of existing procedures. Further research in this area is warranted in order to test this hypothesis for the benefit of cancer patients in the future.

IVM & in vitro follicle culture

In an analogous manner, if in vitro follicle culture is ever to offer hope to cancer patients, it will need to be used in conjunction with a specialized IVM system adapted for this technology. Strategies to grow follicles in vitro have been attempted for many years, in order to fully capitalize on the use of frozen–thawed ovarian tissue in fertility preservation. The great advantage of this approach would be that it would eliminate the risk of reimplanting neoplastic cells back to the cancer patient. However, complete folliculogenesis from primordial to preovulatory stage, with subsequent oocyte retrieval, IVF, embryo transfer and production of live offspring, has only ever been achieved in mice. Exhaustive efforts over decades to adapt this technology to higher mammals have so far failed to achieve a pregnancy.

It is estimated that in women it takes approximately 90 days for a preantral follicle to grow to the preovulatory stage (15–20 mm) in vivo [17]. To date, the largest human follicles grown in vitro (from secondary follicles) are approximately 0.6–1.5 mm, with considerably smaller sizes achieved when starting from primordial follicles [18]. There have been recent important advances in this technology, such as the use of 3D alginate hydrogels and/or multistep culture systems [18,19]; however, this approach requires further development before it will be possible to produce a healthy human pregnancy from such oocytes. It may be impossible, at least for the foreseeable future, to culture follicles to large antral size in vitro and to stimulate these follicles with LH-analogs to yield a mature oocyte. Therefore, the logical route is to combine advanced follicle culture technologies with IVM [18]. Proof of principle has been established recently, using this combination of technologies, with the production of a small number of mature human oocytes [19], however, the developmental normality of these is yet to be determined. It is unlikely that standard IVM culture systems (as used clinically) are optimal for these oocytes from very small follicles [20]. There have been significant recent advances in specialized IVM systems designed for oocytes from the smallest antral follicles (e.g., <3 mm), for example, by using pre-IVM systems containing cAMP modulators and/or IVM systems containing GDF9, BMP15 and/or cumulin [2,3].

Conclusion

There is an ever increasing demand for fertility preservation as increasing numbers of young women and girls survive cancer and seek to have children. Oocyte IVM is an established procedure for the treatment of infertility. IVM has the capacity to capture the reproductive potential of oocytes in growing follicles that is commonly lost in most fertility preservation clinics. As IVM can be easily coupled with, and can augment, existing fertility preservation strategies, it can be anticipated that in the near future most modern fertility preservation clinics will have IVM capabilities as part of their treatment repertoire.