Keywords

Introduction

Embryo cryopreservation is today the most established method of fertility preservation [1]. Oocyte cryopreservation now offers a new option for single women in the reproductive-age group in need of delaying childbearing for any reason. Due to practical problems related to the structure of the oocyte and optimization of freezing methods, it has taken more than 20 years for oocyte cryopreservation to evolve into a technique with acceptable clinical pregnancy rates.

This transition was made possible by three important achievements: utilization of intracytoplasmic sperm injection (ICSI), improvements in cryoprotectants, and introduction of vitrification [2–5]. The improvements in the technique and the recent removal of the “experimental” label on oocyte cryopreservation by the American Society of Reproductive Medicine (ASRM) Practice Guideline Committee [6] have ushered in a new era.

Oocyte cryopreservation is expected to take the lead in fertility preservation. It is also likely to become a useful adjunct to routine IVF in various clinical scenarios such as the unavailability of sperm at the time of egg retrival [7, 8], in cases of ovarian hyperstimulation syndrome [9], in poor responders [10, 11], in patients at risk of losing their fertility potential due to genetic abnormalities such as BRCA mutation carrier status [12], Turner syndrome [13], fragile X syndrome, and deletions of the X chromosome [6] and for couples who do not wish to cryopreserve supernumerary embryos for ethical, legal, or religious concerns [14].

Another indication for oocyte vitrification that has now become a reality [15, 16] is the establishment of donor oocyte banks. In future, IVF cycles using frozen-thawed donor oocytes may outnumber those using fresh donor oocytes. However, elective oocyte cryopreservation (EOC) for deferring childbearing remains the most controversial but, surprisingly, the most common indication for oocyte cryopreservation. Most centers currently performing oocyte vitrification in the United States do so for elective indications [17].

There are many factors that affect the efficiency of success with oocyte cryopreservation such as factors related to host (age, donor/nondonor oocyte, infertility factor), stimulation protocols, cryopreservation methods (slow freezing and vitrification), protocols, and devices (cryotop, cryoleaf, cryotip). Hence, it is difficult to reliably estimate the success of oocyte cryopreservation from various studies. The majority of the studies on oocyte cryopreservation are observational, and only six RCTs with clinical outcomes have been published [15, 18–22].

History

The first live birth with oocyte cryopreservation was reported in 1986 with slow freezing [23]; but due to very low success rates, there were only five live births reported initially [24]. In 1997, intracytoplasmic sperm injection (ICSI) was first used to fertilize frozen-thawed oocytes, circumventing zona hardening caused by the cryopreservation process [25]. Further optimization of oocyte cryopreservation required another decade. In 1999, the first live birth with oocyte cryopreservation after vitrification was reported [26] followed by a few case reports until 2005 [24].

There were only 100 reported live births from oocyte cryopreservation. A meta-analysis [24] of these concluded that success rates with oocyte cryopreservation using slow freezing were lower than that of IVF with fresh oocytes. Comparisons of vitrification with either slow freezing or fresh oocyte cycles could not be performed because of the limited number of reports with vitrification at the time of publication. However, the success rates with vitrification showed encouraging results compared with slow freezing.

Following the first RCT comparing slow freezing and vitrification, which showed that vitrification was more successful in terms of both embryological and clinical outcomes [18], more workers reported improved clinical outcomes using vitrification [14–16, 19, 27–31]. Efficiency of slow-freezing protocols has since improved [32–40]. However, success rates remain lower for slow freezing compared with vitrification.

With the improvements in oocyte cryopreservation technology and associated clinical outcomes, its clinical applications widened, with more than a thousand live births reported to date [17, 41]. Over the past 5 years, oocyte cryopreservation, especially with vitrification, has proven to be an efficient technique, resulting in pregnancy outcomes similar to that of IVF with fresh oocytes [15].

A recent RCT has shown that aneuploidy rates in embryos derived from vitrified oocytes were similar to those from fresh oocytes in young infertile women undergoing IVF with their own eggs [21]. This suggests that oocyte cryopreservation does not have an adverse effect on chromosome segregation during meiotic division.

Clinical Application

Research into IVF outcome parameters using oocyte cryopreservation falls into one of two main categories: (1) studies assessing donor oocyte cryopreservation/thaw cycles representing young fertile women and (2) studies assessing infertile women with failed IVF attempts, who have supernumerary oocytes for cryopreservation. Studies on oocyte cryopreservation for poor responders [11] and for IVF cycles with failed sperm retrieval [8] constitute the exceptions.

Nondonor or Autologous Oocyte Cryopreservation

Randomized Controlled Trials

Most reports on cryopreservation of nondonor oocytes are observational studies, with only a few RCTs performed in infertile women undergoing IVF who prefer cryopreservation of their surplus oocytes. These women declined embryo cryopreservation due to ethical or legal concerns. Also, there are studies that compare the efficacy of oocyte cryopreservation by temporarily cryopreserving oocytes under an institutional review board approval [21]. There is only one single study to date assessing the efficiency of nondonor oocyte cryopreservation when applied to young fertile women [42].

There are four published RCTs on nondonor oocyte crypreservation; all four report the outcomes of IVF using vitrified/warmed nondonor oocytes from infertile patients [18–21]. At present, there are no RCTs evaluating IVF outcomes of slow frozen oocytes compared with fresh oocytes. Only one study compared slow freezing and vitrification, reaching the conclusion that vitrification is superior to slow freezing in terms of oocyte survival, fertilization, implantation, and clinical pregnancy rates. This is the only RCT comparing the two techniques [18].

Two RCTs were conducted in infertile couples with supernumerary oocytes available to vitrify and warm only if pregnancy was not achieved in the fresh cycle [19, 20]. Fresh sibling oocytes were transferred in the first cycle. If pregnancy failed to occur, then the cryopreserved sibling oocytes were thawed, fertilized, and transferred to the same patient in a subsequent cycle. Using this design, the authors were able to compare the fertilization and embryo developmental rates of vitrified and fresh sibling oocytes. Both studies concluded that similar fertilization and embryo development rates were achieved with fresh and vitrified oocytes [19, 20].

In the most recently published RCT, Forman et al. [21] adopted a unique design, which allowed the comparison of clinical outcomes with nondonor vitrified and fresh oocytes. In this study, the authors divided retrieved oocytes from infertile patients less than 35 years of age. One group of oocytes underwent temporary vitrification while their others remained in culture. Later, vitrified oocytes were thawed; vitrified and nonvitrified oocytes were fertilized with ICSI, and resulting embryos were cultured to the blastocyst stage.

Embryos of sufficient quality to transfer or cryopreserve underwent trophectoderm biopsy for genotyping and a karyotype was assigned to each embryo. Blastocysts obtained from vitrified and fresh oocytes were then transferred in pairs, and embryonic aneuploidy was assessed in each one. To determine the identity of the implanted embryos, DNA fingerprinting was performed on cell-free fetal DNA enriched from maternal serum specimens drawn at 9 weeks of gestation or on newborn DNA taken from a buccal swab.

The authors detected no differences between the two groups regarding aneuploidy. In addition, the ongoing pregnancy rate per transferred embryo was similar for vitrified and fresh oocytes. However, the fertilization and embryo development rates were lower in vitrified compared with fresh oocytes. This finding is in contrast with previous trials reporting similar fertilization and embryo development rates for both nondonor [19, 20] and donor cryopreserved oocytes [15, 22] compared with fresh oocytes.

Importantly, oocyte vitrification does not seem to increase the rate of aneuploidy or diminish the implantation potential of viable blastocysts. The authors demonstrate that clinical success rates with nondonor vitrified oocytes from young infertile women are similar to their sibling fresh oocytes.

Overall, RCTs investigating the use of cryopreserved nondonor oocytes from infertile patients suggest that vitrification is more successful compared with slow freezing [18]; fertilization and embryo development rates of vitrified oocytes are comparable to fresh oocytes [19, 20], and for women less than 35 years, pregnancy rates and embryo aneuploidy rates of vitrified oocytes are similar to fresh oocytes [21].

Observational Studies

Many observational studies on the efficacy of oocyte cryopreservation have been reported, and most of these studies (almost 90 % of slow freezing and 50 % of vitrification studies to date) were conducted in centers located in Italy. This is because Italian law prohibits insemination of more than three oocytes and has banned embryo cryopreservation, which has forced oocyte cryopreservation into routine clinical practice.

In the largest of these studies, supernumerary oocytes from infertile women were cryopreserved using slow freezing, and 940 thaw cycles were performed in eight centers [38]. The overall survival rate of thawed oocytes was 55.8 %. The fertilization rate (72.5 vs. 78.3 %), implantation rate (10.1 vs. 15.4 %), pregnancy rate per transfer (17 vs. 27.9 %), and delivery rate per transfer (11.6 vs. 21.6 %) were all significantly lower for cryopreserved oocyte cycles compared with fresh cycles.

Despite the reported lower success rates, the protocol is still evolving [39]. Recently, Azambuja et al. [40] using a sodium-depleted media and Bianchi et al. [39] using a modified slow-freezing protocol reported higher encouraging success rates with slow freezing.

Novel Indications

Although oocyte cryopreservation is proposed for preserving fertility in cancer patients, the data on clinical success of oocyte cryopreservation in such patients are limited. For the purposes of counseling, success rates might be extrapolated from other populations.

Cancer patients are treated with the assumption that their reproductive potential is similar to that of age-matched healthy individuals.

Some studies suggest comparable results with nondonor patients [43–46], whereas others show diminished oocyte yield [47–50]. If further studies with larger sample size confirm that women with cancer have diminished ovarian reserve, appropriate counselling of these women is crucial as women with diminished ovarian reserve are expected to be more susceptible to gonadotoxic agents.

Oocyte cryopreservation has been used in poor responders [11] and in situations when sperm cannot be obtained for IVF [7, 8]. Cobo et al. [11] have reported a new strategy with vitrification for managing poor responder patients. They have proposed that for poor responders, accumulation of oocytes by vitrification and simultaneous insemination yields live birth rates comparable to those in normoresponders.

Donor Oocyte Cryopreservation

There are two RCTs using vitrified donor oocytes [15, 22]. The largest RCT including 600 recipients of donor oocytes demonstrated similar ongoing pregnancy rates with vitrified donor oocytes when compared with fresh donor oocytes [15]. This study reported implantation and clinical pregnancy per embryo transfer rates of 39.9 vs. 40.9 % and 55.4 vs. 55.6 % for vitrified donor and fresh donor oocytes, respectively.

Oocyte donors are women under the age of 35; therefore, the results of these studies may be extrapolated to young patients seeking fertility preservation. With the current excellent reported success with cryopreserved donor oocytes, it is now possible to justify and establish oocyte cryobanking. This strategy will also allow quarantine of oocytes.

Trends in Nondonor Oocyte Cryopreservation

There are numerous nonrandomized studies investigating IVF outcome parameters associated with oocyte cryopreservation. As there are differences between these studies in design, cryopreservation protocols, indications for cryopreservation, age of the patients, number of oocytes thawed and embryos transferred, it is not appropriate to compare the success between different studies. However, these studies show that while the clinical success rates with slow freezing have an increasing trend with time, vitrification has been more successful than slow freezing.

Since 2006, implantation and live birth rates increased from 2 to 14 % and 2 to 27 % [39, 40, 51] for slow freezing, while they ranged from 13 to 20 % and 23 to 35 %, respectively, following a more closer trend for vitrification [19, 20, 29, 52].

Age Trends

Most of the studies published to date reported results according to the mean ages of the patients, which range from 29.9 ± 2.3 [21] and 35.7 ± 5.7 [36]. However, it is not appropriate to use the reported success rates when counselling patients individually, as the success of IVF using cryopreserved oocytes is likely to be affected by the patient’s age.

According to an individual patient data meta-analysis, live birth success rates with cryopreserved oocytes show an age-related decline regardless of the freezing technique used, and an aged-based probability of live birth may be calculated for cryopreserved oocytes [53].

Estimated age-based success rates may also change according to the indication for oocyte cryopreservation, such as elective oocyte cryopreservation or oocyte cryopreservation in poor responders. For example, in poor responders, accumulating cryopreserved oocytes in consecutive cycles followed by thaw, ICSI, and embryo transfer is reported to yield comparable success rates to those observed in normal responders [10, 11].

When this strategy was applied to poor responders over 40, live birth/patient success rates were higher (15.8 %) for the vitrified oocyte group compared with the fresh oocyte group (7.1 %) [11]. Recently Melzer et al. [54] reported a similar approach for patients undergoing EOC. In that study of 132 patients undergoing multiple cycles of EOC with an average age of 38.4 at first and 39 at subsequent cycles, when more than one cycle was applied, subsequent cycles resulted in greater oocyte yield, albeit with the implementation of a higher dose.

Elective Oocyte Cryopreservation

Among the programs in the USA offering EOC, half accepted women aged 38–40 years, and about one-third accepted women above 40 years [17]. A recent study analyzing 491 women reported that mean age of the patients undergoing EOC was 38 [55] in accordance with two other studies [56, 57]. Importantly, more than 80 % of women undergoing EOC were over 35 years old (range: 36–41) [56].

Despite the reported interest of older reproductive-age women toward oocyte cryopeservation [55–57], one of these studies found that the mean age of women inquiring about this procedure was 35.2 ± 5.4 years [57]. The same study reported that the age for the application of EOC decreased from 39 + 1.4 years in 2005 to 37.4 + 2.3 years in 2011.

This shows that EOC is primarily utilized by older reproductive-age women, although women inquire about the procedure earlier. However, to achieve higher success rates with IVF, both the age of inquiry and application of EOC should be at an age younger than 35 years [58].

Conclusion

Following the first live birth with cryopreserved oocytes in 1986 and a very slow progress for 20 years, clinical outcomes using cryopreserved oocytes have made great strides during the past decade. Recent RCTs show that fertilization, embryo development, and pregnancy rates with vitrified nondonor and donor oocytes are similar to fresh oocytes. Vitrification remains the protocol of choice as the overall success rates with slow freezing remain lower compared with vitrification.

These improvements in the cryopreservation technique and clinical outcomes may result in an increased utilization of oocyte vitrification in clinical practice. In order to provide appropriate counselling to women considering oocyte cryopreservation for fertility preservation or as an elective procedure for deferring child bearing, it is necessary to arrive at age-specific and indication-specific success rates so that we may better inform our patients.