The Role of Oocyte Cryopreservation in Assisted Reproduction

Abstract

Oocyte cryopreservation (OC) has progressed rapidly from an experimental procedure with limited success to a clinically accepted procedure, in large part due to significant improvements in the techniques and widespread laboratory adaptation of vitrification. With significant improvements in clinical outcome, elective oocyte cryopreservation has gained in popularity as a means of overcoming diminishing ovarian reserve associated with aging. With clinical pregnancy rates equal to utilizing retrieved oocytes, oocyte cryopreservation is being increasingly utilized as an adjunct to standard IVF and now plays a significant role in egg donation with the establishment of egg banks analogous to sperm banks. Continuing research and clinical experience will be instrumental in defining the role of OC going forward.

1. Introduction

The Nobel Prize winning work of RG Edwards lead to the first successful pregnancy resulting from in vitro fertilization (IVF) and the birth of Louise Brown in England in 1978 [1]. Numerous improvements in the technique of controlled ovarian hyperstimulation (COH), as well as laboratory fertilization and culture technique, subsequently resulted in rapid improvement in pregnancy success and worldwide clinical acceptance. Although the cryopreservation of mammalian embryos was originally described as early as 1947 [2], the first successful live births from cryopreserved/thawed embryos following IVF were reported in 1983 by Alan Trounson’s group [3]. Cryopreservation of embryos following IVF quickly gained clinical acceptance by increasing overall pregnancy success and decreasing the need for additional procedures.

Cryopreservation of unfertilized human oocytes for the purpose of fertility preservation has always been an attractive alternative to cryopreservation and storage of human embryos, posing fewer ethical, moral, religious and legal problems. However, initial attempts at oocyte cryopreservation based on the laboratory success of cryopreservation of fertilized embryos was disappointing, resulting in only a handful of successful live births by 1987 [4].

Unfertilized oocyte cryopreservation had been vexed with the technical problems of potential meiotic spindle disruption, possibly resulting in aneuploidy when traditional slow freezing utilizing cryoprotectants was used [5]. In addition, although acceptable rates of freeze–thaw survival were observed, poor fertilization with IVF was common, as was polyspermic fertilization [6]. These problems were found to be a result of changes in the zona pellucida associated with cryopreservation [7]. As such, the procedure did not attain initial widespread clinical acceptance and was relegated to being considered an experimental procedure for many years.

The hurdle of cryopreservation of mature unfertilized human oocytes was overcome by the use of ultra-rapid freezing or “vitrification”. Significant improvement in fertilization of cryopreserved-thawed oocytes was the result of the widespread clinical application of intracytoplamsic sperm injection (ICSI), where a single sperm is injected through the zona pelucida into the ooplasm, overcoming the problems of poor fertilization and potential polyspermic fertilization. As a result, many live-births have been achieved over the last 25 years, and the safety and efficacy of the procedure was confirmed [8, 9]. Of interest, following thousands of live-births world wide, the procedure was deemed no longer “experimental” in 2012 by the American Society for Reproductive Medicine (ASRM), leading to widespread clinical acceptance of the procedure [10].

Oocyte cryopreservation has long been suggested as a means of fertility preservation in young women wishing to delay childbearing into their fourth and fifth decades. Likewise, fertility preservation is paramount in young women faced with potentially sterilizing procedures such as chemotherapy or radiation treatment, as well as surgery associated with modern oncology treatments. Cryopreservation of mature unfertilized oocytes can also be a valuable adjunct in IVF, particularly when sperm for fertilization is not available on the day of oocyte retrieval. Finally, oocyte cryopreservation has the potential to play a significant role in oocyte donation: adding convenience as well as screening and expanded donor selection choices, analogous to sperm donation using commercial sperm banks.

2. Vitrification

Fertilized cleavage or blastocyst stage embryos contain a diploid chromosomal complement encased within the nucleus of each cell. Cryopreservation of embryos was done originally via slow freezing using cryoprotectants that were required to avoid damage associated with cooling to sub-zero temperatures. Cryoprotectants, either permeating such as propanediol (PROH), glycerol, or dimethysulfoxide (DMSO) or non-permeating such as sucrose, were utilized to displace intracellular water and avoid cellular damage from intracellular ice formation. Seeding, to induce ice formation outside the specimen, was followed by more rapid cooling to −196 degrees Celsius and storage in liquid nitrogen [11].

In 1985, Rall and Fahey introduced the process of vitrification for the cryopreservation of mammalian embryos [12]. Using higher concentrations of cryoprotectant and rapid cooling results in vitrification (glass formation), thereby avoiding intracellular ice formation. Trounson subsequently described ultrarapid freezing techniques in human embryos [5]. The potential toxicity of cryoprotectants at higher concentrations at room temperature requires their addition at lower temperatures, but the rapid exposure to low temperatures obviates the need for costly programmed biological freezing equipment. Significant advances in the techniques of vitrification have resulted in widespread clinical acceptance [13].

Unlike embryos, immature human oocytes, which are arrested in the diplotene stage of the first meiotic prophase, are recruited by gonadotropin stimulation at the beginning of the follicular phase at which time they experience significant antral growth or atresia. Developing antral follicles morphologically exhibit the germinal vesicle (GV) by light microscopy which contains the chromosomal complement. The oocyte resumes meiosis in response to the midcycle luetenizing hormone (LH) surge, as evidenced morphologically by extrusion of the first polar body which can be seen beneath the zona pellucida (ZP). The mature oocyte therefore demonstrates evidence of germinal vesicle breakdown (GVBD) which is associated with the appearance of the spindle apparatus and the presence of the first polar body, but remains arrested in the second meiotic metaphase (MII). With fertilization, meiosis is completed and the second polar body is extruded. The polar body contains the discarded haploid chromosomal complement of the oocyte, which is visible beneath the zona pelucida followed by formation of 2 pronuclei (2pn), visible morphologically on light microscopy.

Because MII oocytes contain chromosomes which are still attached to microtubular spindle, there were concerns regarding disassembly of the meiotic spindle and dispersal of the polar pericentriolar material upon freezing and thawing that could result in an increase in chromosomal aneuploidy. Early studies of murine oocyte ultrastructure using transmission electron microscopy (TEM) have shown little adverse effect on the meiotic spindle structure following freezing/thawing either with slow cooling or ultrarapid cooling [14]. Likewise, reports of cytogenetic evaluation following oocyte freezing/thawing followed by IVF have been reassuring in both mouse and in human [8, 9], suggesting no significant increase in meiotic nondisjunction resulting in aneuploidy.

Although early reports by Chen [15] suggested excellent survival following cryopreservation of mature oocytes, with the first successful pregnancy in humans reported in 1986, poor fertilization following freezing and thawing limited the clinical success of the procedure. Poor fertilization of previously cryopreserved mature oocytes as well as problems with polyspermic fertilization have suggested changes to the ZP, such as hardening of the zona, possibly resulting from premature cortical granule release associated with changes to the oolema as a consequence of freezing and thawing. TEM evaluations of cryopreserved/thawed oocytes have shown no evidence of premature cortical granule release [6], however using TEM studies in mammalian oocyte have revealed cracks in the ZP following freezing and thawing. This suggests that physical changes in the glycoprotein architecture of the ZP may be the cause of “hardening,” preventing normal fertilization or conversely for polyspermic fertilization.

Intracytoplasmic Sperm Injection (ICSI), in which a single viable spermatozoa is injected through the ZP into the oolema, was developed as treatment for severe male factor infertility and resulted in the first live births in 1992 [16]. The development of the technique followed earlier attempts at zona drilling and sub-zonal insertion of sperm to enhance fertilization, which were also plagued by polyspermic fertilization. Extensive experience with ICSI has demonstrated excellent fertilization rates, often higher than seen with standard IVF [17] and obviates the problem of polyspermic fertilization through the injection of a single sperm. Based on these results, ICSI was subsequently found to provide high rates of normal fertilization in previously cryopreserved oocytes [18], resulting in the first live birth in 1997 [19].

2.1 Fertility preservation prior to cancer treatment

Over the past 50 years; significant advances in cancer therapies, and in particular chemotherapy, has resulted in major improvement in long-term patient survival [20]. Chemotherapy and radiation therapy as well as gonadectomy can decrease the reserve of viable oocytes, resulting in immediate or premature ovarian failure (POF). The type of chemo/radiotherapy, duration, cumulative dose and patient age, have all been shown to predict POF [21]. As survival for cancer patients continues to improve, counseling prior to potential iatrogenic infertility due to planned oncology therapy in reproductive age patients has become a clinical necessity regarding the available options for fertility preservation prior to treatment [20].

For long term cancer survivors who have experienced POF due to gonadotoxic therapies or even gonadectomy, options for having children include adoption or oocyte donation. For reproductive age women in a stable committed relationship, standard IVF utilizing COH followed by fertilization using her partner’s sperm and cryopreservation and storage of resulting embryos is a reasonable option for women wishing to preserve the chance of having their own biologic offspring. COH and egg retrieval can be accomplished in a relatively short time period (14–21 days), allowing time to schedule prior to starting chemotherapy, radiation therapy, or surgery, and can even be accomplished successfully after early potential gonadotoxic therapy has begun [22]. Cryopreservation of embryos offers a predictable likelihood of pregnancy success based on the age of the patient as well as the number and the quality of embryos stored. Although data on live birth rates from stored embryos prior to cancer therapy are limited, patients can be counseled based on live birth rates following use of cryopreserved embryos from the general infertility population [20].

Oocyte cryopreservation has the advantage of not requiring a partner for single women facing cancer therapy and has fewer ethical, moral, religious and legal problems than the current widespread cryopreservation of embryos. Mature oocyte cryopreservation also requires COH using gonadotropin therapy, followed by out-patient surgical oocyte retrieval and cryopreservation. Like cryopreservation of embryos, information on the pregnancy success rates following fertilization of mature oocytes from cancer patients is limited. It is clear that the age of the patient at vitrification and the number of oocytes stored are predictors of pregnancy success [23]. Live birth rates from donor and infertile patients can be a guide to counseling cancer patients however, with live birth rates as high as 46.8% reported for women less than 35 years of age [20].

For prepubertal and adolescent women facing cancer treatment, gonadotropin therapy and egg retrieval are not reasonable options for obtaining multiple mature oocytes for cryopreservation. However, ovarian biopsy, in which ovarian cortical tissue containing several hundreds or thousands of immature oocytes can be obtained for cryopreservation, is an option. Laparoscopic Ovarian Biopsy (LOB) has been shown to be a safe and effective method for obtaining ovarian tissue for cryopreservation [24]. It can be performed at the time of general anesthesia for lymph node biopsy or central line placement, immediately prior to planned cancer therapies. Histologic evaluation from ovarian biopsies performed in pre-pubertal or adolescent females have shown viable immature oocytes, even after initiation of conservative chemotherapy [24]. This allows for obtaining ovarian tissue for cryopreservation prior to more complete myeloablative therapies such as total body irradiation used in preparation for bone marrow transplantation (BMT) [24].

Due to the complexity and limited success of in vitro maturation, frozen–thawed immature human oocytes, like those found in cryopreserved ovarian tissue, require in vivo maturation [25]. This involves reimplantation of autologous cryo-thawed ovarian tissue back into the patient, survival of the autologous graft, and normal maturation of oocytes which can be harvested and used in conjunction with standard IVF to achieve pregnancy and live-births [26].

Two competing strategies have been championed. Oktay and others have pursued reimplantation of the previously cryopreserved-thawed ovarian tissue into the forearm [27].

This is analogous to the procedure used for preserving parathyroid gland function following total thyroidectomy. Technical problems related to monitoring the tissue, poor graft survival, and difficulty retrieving mature eggs, have limited the widespread acceptance of this procedure over the years.

An alternative option involves re-implantation of the cryo-thawed tissue into the remaining ovary or the ovarian bed as originally described by Gosden et al. in 1994 [25]. This approach has the advantage of superior graft survival, likely due to the excellent blood supply and high oxygen tension, ease of monitoring, and ease of oocyte retrieval which is unchanged from standard IVF and widely accessible to clinicians performing egg retrieval.

Concerns have been raised regarding the theoretic risk of reintroducing malignant cells or tissue, resulting in relapse of the original cancer or disease which prompted the cryopreservation of the ovarian tissue [28]. It is important to evaluate thawed ovarian tissue prior to autotransplantation and to involve pathology and oncology specialists in the consideration of its use. With proper screening, the risk appears small, with no reported recurrences [29]. With continued experience, overall the data on safety and efficacy, as well as reproductive outcomes, has by 2019 lead to ovarian tissue cryopreservation to be considered an established medical procedure [30, 31].

3. Planned oocyte cryopreservation

With improvements in vitrification of mature oocytes, as well as significantly improved fertilization using ICSI, the pregnancy rates using cryopreserved oocytes were found to be comparable to those found using fresh oocytes with IVF [32]. More importantly, studies of the health of babies born following the use of oocyte cryopreservation have shown no increase in congenital abnormalities [8, 9]. With these reassuring clinical results, OC was no longer considered experimental by the ASRM in 2012 [10].

The success of oocyte cryopreservation has led to increased interest in cryopreserving oocytes to extend the reproductive capacity in otherwise healthy women wishing to delay childbearing. Although the ASRM initially declined to recommend OC for the “sole purpose of circumventing reproductive aging in healthy women,” intense interest and an undeniable increase in efficacy lead the organization to publish a risk/benefit “fact sheet” regarding OC by 2014, followed by a stronger supportive endorsement for the procedure by 2018 [33].

The rate of first birth to women age 35–39, as well as age 40–44, continues to increase in the U.S. [34]. Increasing emphasis on education, later age at marriage, access to effective contraception and opportunity for career advancement are among many of the reasons for this trend. However, with increasing maternal age, fertility decreases dramatically beginning at 35, due to decreasing oocyte quantity and quality, resulting in increasing chromosomal abnormalities seen in failure to conceive, miscarriage and birth defects [35].

Egg donation, using higher quality eggs from young healthy donors, has historically been the treatment of choice for women wishing to conceive in their fourth and fifth decades of life. OC performed at a younger age, prior to decreasing ovarian reserve, allows for having a child using a woman’s own genetic material later in life. Because a woman’s age, number and quality of oocytes strongly determine the chance of pregnancy, OC cryopreservation is likely to be most successful for younger women [23]. By the age of 38, research suggests that 25–30 oocytes may be required to provide a reasonable chance of pregnancy success [36]. The cost of the procedure must be considered, as well as the cost of long term storage, particularly in young women. In addition, the possibility of achieving pregnancy naturally or with standard fertility treatments, should be taken into consideration when considering planned OC [37]. Due to the fact that at age 20–30, the time of maximal career advancement also corresponds a woman’s to optimal fertility, the available option for OC has prompted several large corporations to cover the costs associated with the procedure [38], providing additional incentive for career advancement and delayed childbirth.

3.1 In vitro fertilization and oocyte cryopreservation

The success and ready availability of OC has also resulted in increasing use of the technique as an adjunct to standard IVF procedures. On the day of egg retrieval, typically a fresh semen sample is required to prepare viable spermatozoa for either standard insemination or for ICSI to accomplish fertilization of multiple mature oocytes retrieved following COH. In cases of severe oligo-asthenospermia, several samples may be cryopreserved and “banked” to ensure adequate numbers of viable spermatozoa on the day of oocyte retrieval. In cases of obstructive azoospermia, surgical extraction of spermatozoa from the epididymis or testis is typically performed prior to the planned oocyte retrieval and cryopreserved, or the planned procedure for sperm retrieval can be scheduled on the same day to obtain a fresh specimen.

In clinical practice, there are cases when fresh sperm cannot be obtained on the day of egg retrieval, either because the male partner is unexpectedly unavailable or unable to provide a sample. Also, thawing of severely oligo-asthenic semen samples may yield insufficient viable spermatozoa to fertilize any or all of the mature oocytes retrieved, particularly in cases when several dozen oocytes are obtained following COH. Likewise, planned surgical extraction procedures can be unexpectedly delayed due to surgical scheduling requirements or fail to yield viable spermatozoa sufficient for fertilization. In these cases, cryopreservation of the unfertilized mature oocytes can be performed until such time adequate viable spermatozoa are available to accomplish IVF without compromising the success of the procedure.

Other common clinical situations can arise when insufficient viable mature oocytes are obtained at the time of egg retrieval, particularly in older patients or those with significantly decreased ovarian reserve. In these cases, additional cycles of COH and egg retrieval can be performed to increase the overall number of oocytes used in IVF and in particular when genetic screening using preimplantation genetic screening (PGS) is used [39], increasing the overall pregnancy success. In addition, for patients wishing to limit or avoid freezing embryos, supernumary mature oocytes retrieved following COH can be cryopreserved [40]. As seen for other indications for oocyte cryopreservation, pregnancy rates following transfer of fresh embryos in such cycles and embryos from previously cryopreserved “sister oocytes” from the same cycle are similar [41], suggesting no significant decrease in oocyte or embryo quality.

4. Oocyte donation

The clinical problem of infertility due to inadequate number or quality of oocytes available to produce a healthy pregnancy was overcome by the introduction of oocyte donation in 1984 [42]. Patients with diminished ovarian reserve due to advanced age, POF, or gonadectomy for cancer or benign disease could conceive a pregnancy with her partner, carry the pregnancy to term and deliver a healthy child using donated oocytes from a young, healthy woman, fertilized by the patient’s husband’s sperm, and the resulting embryos transferred to her uterus.

Options for obtaining oocytes include “known donors,” such as family members including younger sisters, cousins or same-sex partners. Alternatively, anonymous donors, analogous to sperm donors, which are chosen by matching physical characteristics such as height, weight, hair color, eye color, ethnic background, etc., can be used. Anonymous donors must be carefully screened for the absence of infectious or heritable disease which could adversely affect the health of the offspring.

Sperm donors have the advantage of providing semen samples, which can be easily cryopreserved and banked, allowing for quarantine against potential infectious agents with long incubation periods and rapid availability of a wide selection of potential donors. Unlike sperm donors, oocyte donors initially require selection of a potential donor who would undergo COH using gonadotropin treatment and ultrasound monitoring followed by oocyte retrieval and insemination of the fresh mature oocytes using the patient’s partner’s fresh or previously banked semen sample.

Although thorough screening of a potential oocyte donor for health, infectious disease risk factors and family history of potential genetic disease is similar to semen donors, actual screening for infectious disease is required within 3 days of oocyte retrieval by the U.S. Food and Drug Administration (FDA), compared with a 6 week quarantine period required for banked semen samples. Cryopreservation of oocytes from healthy young donors allows for similar quarantining and “banking”, with ready availability of healthy, screened oocytes which can be chosen by matching the donor’s physical traits, as has been in use for sperm donors for many decades. However, concerns with respect to commercialization and marketing as well as cost effectiveness and accurate reporting of pregnancy outcomes remain.

Because commercial “egg banks” are not required to report clinical outcomes per cycle start, including pregnancy, miscarriage and live birth rates, as required by law under the auspices of the Society for Assisted Reproductive Technology (SART) to the centers of disease control (CDC), caution should be exercised when interpreting the pregnancy success rates advertised by such commercial enterprises as they compete for patients seeking donor eggs [43]. Known factors influencing pregnancy success using previously cryopreserved mature oocytes include younger age of the donor [44]. Also, donors who have had previous pregnancy success in fresh cycles are associated with a higher live birth rate using cryopreserved oocytes [45].

It is also clear that as the number of donor oocytes thawed increases, there is an associated increase in the cumulative live birth rate [45]. This raises the question of cost-effectiveness of utilizing commercial egg banks and their pricing structure in terms of the cost per oocyte. Considering the rate of fertilization, embryo cleavage, blastocyst formation, implantation and miscarriage, the chance of live birth has been estimated at 8% per thawed oocyte [46]. This must be compared with the multiple oocytes and embryos obtained through conventional egg donation, which may yield multiple embryos for transfer, as well as for cryopreservation of supernumary embryos for additional attempts at pregnancy, which can increase the overall cumulative probability of successful live birth.

5. Summary

The clinical success of IVF has resulted in the rapid development and adoption of innovations including COH, embryo cryopreservation, ICSI and oocyte donation, which have been successful in overcoming infertility from multiple etiologies resulting in the birth of more than 1 million children as of 2012 [47]. By 2018, 40 years after the birth of Louise Brown, more than 8 million children have been born, worldwide [48]. Due to significant technical challenges, largely overcome by rapid improvements in vitrification and fertilization, the innovation of clinically successful oocyte cryopreservation has been much more recent, having been approved for widespread use only since 2012 [10].

The principal application of the technology remains preserving fertility potential, both for medically necessary and elective indications. Cryopreservation of unfertilized eggs has the advantage of significantly fewer ethical, moral, religious and potentially legal problems when compared with the cryopreservation and potential long-term storage of embryos. The clinical utility of OC is also clear as an adjunct to fertility treatment using IVF as well as having a significant potential role in oocyte donation. As with all important emerging innovations in the field of assisted reproductive technology, continuing research and clinical experience will be instrumental in defining the role of OC going forward.