Is vitrification of oocytes useful for fertility preservation for age-related fertility decline and in cancer patients?

Transcription

1 Is vitrification of oocytes useful for fertility preservation for age-related fertility decline and in cancer patients? Ana Cobo, Ph.D., a Juan A. Garcia-Velasco, M.D., b Javier Domingo, M.D., c Jose Remohí, M.D., a and Antonio Pellicer, M.D. a a IVI Valencia, Valencia; b IVI Madrid, Madrid; and c IVI Las Palmas, Las Palmas, Spain The aim of this review is to provide current knowledge on oocyte cryopreservation, with special emphasis on vitrification as a means to preserve fertility in different indications. Major advancements achieved in the past few years in the cryolaboratory have facilitated major changes in our practice. Areas such as fertility preservation for social or oncologic reasons, the possibility to create oocyte banks for egg donation programs, the opportunity to avoid ovarian hyperstimulation syndrome, or to accumulate oocytes in low-yield patients, or even to offer treatment segmentation by stimulating the ovaries, vitrifying, and then transferring in a natural cycle are some of the options that are now available with the development of cryopreservation. We present general experience from our group and others on fertility preservation for age-related fertility decline as well as in oncologic patients, confirming that oocyte vitrification is a standardized, simple, reproducible, and efficient option. (Fertil Steril Ò 2013;99: Ó2013 by American Society for Reproductive Medicine.) Key Words: Fertility preservation, oocyte vitrification, cancer patients, social freezers, fertility decline Discuss: You can discuss this article with its authors and with other ASRM members at fertstertforum.com/coboa-vitrification-oocytes-fertility-preservation-cancer-patients/ Use your smartphone to scan this QR code and connect to the discussion forum for this article now.* * Download a free QR code scanner by searching for QR scanner in your smartphone s app store or app marketplace. Fertility preservation (FP) is an emerging, rapidly evolving branch of reproductive medicine comprising the preservation of gametes (sperm, oocytes) and reproductive tissue (ovarian, testicular), giving individuals at risk of losing their reproductive ability the chance to conceive and have their own genetic offspring. Cancer patients who are to undergo surgery or start chemotherapy or radiotherapy, women with other medical conditions leading to premature menopause, and healthy women wishing to postpone childbearing are the main beneficiaries of this strategy. Options for women to safeguard their fertility include the cryopreservation of ovarian tissue or oocytes. The introduction of vitrification into assisted reproduction (AR) has established efficient female gamete cryopreservation, which provides outcomes similar to those achieved with fresh oocytes and opens up a wide range of applications, including for FP candidates. The present review addresses the clinical use of oocyte vitrification in the FP context for nononcologic and oncologic patients. BACKGROUND History Since its inception, assisted reproduction has accomplished important Received January 17, 2013; revised February 20, 2013; accepted February 25, 2013; published online March 29, A.C. has nothing to disclose. J.A.G.-V. has nothing to disclose. J.D. has nothing to disclose. J.R. has nothing to disclose. A.P. has nothing to disclose. Reprint requests: Ana Cobo, Ph.D., IVI Valencia, Plaza de la Policía Local 3, Valencia, Spain ( ana.cobo@ivi.es). Fertility and Sterility Vol. 99, No. 6, May /$36.00 Copyright 2013 American Society for Reproductive Medicine, Published by Elsevier Inc. advances, true milestones to help increase many couples' means to conceive. Cryopreservation is one of these means and preserves biologic materials at cryogenic temperatures to completely stop biologic reactions. From 1938 to 1945, scientists observed that sperm survived freezing and storage at temperatures as low as 160 C. The first major breakthrough came in 1949 when Polge developed a method using glycerol to protect semen. Embryo cryopreservation has been widely and successfully applied since the very beginning of AR, and the first pregnancy after cryotransfer was published in 1983 (1). In 1985, Lasalle introduced the use of propanediol (2) into a protocol that is still being used with minimal modifications. Conversely, following the first report of a successful pregnancy using a frozen thawed oocyte in 1986 (3), most efforts have been made to develop an ideal oocyte cryopreservation VOL. 99 NO. 6 / MAY

2 VIEWS AND REVIEWS method. Several reasons explain the low success rates traditionally observed, including oocyte size and shape. A large water content probably leads to intracellular ice formation, chilling injury, and osmotic damage, which are the main causes of high oocyte sensitivity to cryopreservation. These effects can be controlled depending on the cryopreservation method applied. There are two main cryobiology strategies: slow freezing and vitrification. During the former, cells are gradually dehydrated in the presence of cryoprotectants (CPAs) and the temperature is lowered at a very slow cooling rate ( 0.3 C) (4). Cells are exposed to low temperatures for a long period, which can lead to chilling injury, defined as irreversible damage after exposing cells to low temperatures (þ15 Cto 5 C) before the nucleation of ice (5, 6). This detrimental event affects mainly the cytoskeleton (7) and cell membranes (8). Ice crystal formation within the cytoplasm must be avoided at all costs to guarantee the survival and integrity of cells when later thawed. The outcome of IVF cycles conducted with slow-frozen/thawed oocytes is limited and has never equalled that achieved with fresh oocytes. Therefore, there is a pressing need to cryopreserve oocytes more effectively. The protocol must reduce the damage to cells caused by ice crystal formation and chilling injury during the freezing process. Vitrification Vitrification is highly effective in avoiding crystallization (9). The first successful vitrification of mammalian (spermatozoa) cells was described more than 60 years ago (10) and was applied to mouse oocytes almost four decades later (11). Initial protocols subjected oocytes to high CPA concentrations for long periods, up to 50 minutes, involved high cytotoxicity, and caused remarkable osmotic stress. Subsequent studies substantially improved these initial experiments. One of the most notable changes was using CPA mixtures to overcome osmotic stress. The ethylene glycol dimethyl sulfoxide 1:1 combination proved to be highly effective (12). Moreover, the probability of achieving vitrification is related directly to the cooling rate and solution viscosity and inversely to sample volume (13). The simplest way of balancing this equation is by reducing the vitrification solution volume when loading samples, followed by direct immersion in liquid nitrogen, which considerably increases the cooling rate. This strategy, in turn, lowers CPA requirements (14). Accordingly, several vitrification systems and protocols using different devices were introduced (15 22). Most of these devices are known as open systems because samples come into direct contact with liquid nitrogen during vitrification. Devices hermetically sealed before vitrification are known as closed systems, which prevent samples from coming into direct contact with liquid nitrogen during vitrification. Although it is true that high cooling rates are required to achieve vitrification, the warming rate is perhaps the best determinant factor for survival, as demonstrated recently (23), leading to the inference that possibly the extremely high cooling rates achieved with open systems are not absolutely necessary. It is more likely that the high viability associated with open systems is due to the extremely high warming rate achieved with these devices (e.g., 40,000 C/min with the Cryotop system [21]). Conversely in closed systems, samples must pass an intermediate stage while they are released from the sealed device. We speculate that this intermediate phase conditions the warming rate and increases the likelihood of ice formation during the process. It probably explains the lower outcomes achieved after oocyte vitrification with the use of closed systems (24, 25). For embryos, this situation differs somewhat, because the results attained with blastocysts or cleavage-stage embryos with the use of closed systems can be most satisfactory (22, 26). Undoubtedly, the great detractor of open systems is the risk of cross-contamination. It is essential to indicate, therefore, that to date there has never been a single cross-contamination case in AR involving a cryotransfer, even with open vitrification systems (27). Nonetheless, certain measures can be taken to avoid this hypothetical risk while applying open vitrification (28, 29). Safety of Vitrification in Relation to Its Effects on Meiotic Spindle: The Chance of Increased Aneuploidy Incidence The extent of clinical oocyte vitrification application can not be reviewed without mentioning the effect on the meiotic spindle (MS), a particularly sensitive structure responsible for chromosome segregation, given the possibility of generating aneuploid embryos. Years ago, it was suggested that the impaired potential of slowly frozen oocytes is related to the MS's high sensitivity to cryopreservation, which may increase the aneuploidy rates in resulting embryos (30, 31). Spindle apparatus disruption caused by low temperature and or cryopreservation procedures is well documented in mice (32 35), cows (36, 37), and humans (38 40). This highly dynamic structure has also been demonstrated to be possibly repolymerized, with a normal appearance in >80% of cases when physiologic conditions return (31, 33 35, 41 45). There is considerable evidence that MS restoration in humans occurs without alterations, with the absence of scatter chromosomes (30, 46). Preliminary studies attributed a stabilizing effect of CPAs on tubulin fibers (31, 47 49), as confirmed by noninvasive studies conducted in living metaphase (metaphase II [MII]) human oocytes, showing complete MS repolymerization in a post-thawing incubation time-dependent manner (50, 51). It was also suggested that suboptimal protocols (44) and suboptimal material, such as aged or spare oocytes (52), compromise their viability and spindles' restoration ability. Several studies using freshly collected oocytes assessed spindle restoration among slow-freezing and vitrification protocols. Most revealed repolymerization, regardless of the cryopreservation method applied (52), although they might depend on temperature (53) and postincubation times (50, 51). Indeed short incubation may be responsible for the poor restoration observed in another study (54). A more recent study confirming MS restoration after crypreservation showed the IVF outcome of 26 cycles conducted with vitrified or slow frozen oocytes (55). Ninety 1486 VOL. 99 NO. 6 / MAY 2013

3 Fertility and Sterility percent of oocytes presented a spindle, with no differences between slow freezing and vitrification (82.0% vs. 83.0%) and a 96% correlation between pre- and postcryopreservation MS visualization. Unlike most studies reporting similarities after vitrification or slow freezing, a recent report evidenced significantly higher spindle visualization in vitrified oocytes (93.5% vs. 72.0%) with a positive correlation (78.3% vs. 50.0%). In addition, terminal deoxynucleotide transferase mediated dutp nick-end labeling assay found no DNA fragmentation in slow-freezing and vitrified oocytes (56). Two studies, which analyzed the chromosomal status of embryos generated from slowly frozen (57) and vitrified oocytes (58), supported adequate MS restoration. The authors of the latter demonstrated that the vitrification of oocytes from infertile patients did not increase aneuploidy incidence in the resulting embryos. This is useful strong evidence to overcome vitrification concerns about aneuploidy generation. The fine study design, including microarray-based DNA fingerprinting, enhanced the validity of the conclusions. Briefly, evidence stemming from recent studies conducted with refined cryopreservation technologies, some including cryopreserved oocytes used in IVF cycles to achieve pregnancy, demonstrates that the MS is able to restore, guarantee proper chromosome segregation, and generate euploid embryos from either slowly frozen or vitrified oocytes. However, the strongest evidence for method safety is that the incidence of congenital anomalies in children born after oocyte cryopreservation is no higher. A study published in 2010 reported the birth of more than 900 babies worldwide resulting from oocyte cryopreservation, with no apparent increase in birth anomalies (59). Yet we think that this figure does not reflect the reality: The number of children born after oocyte vitrification could be significantly higher, because this practice is increasingly applied in routine practice. It is likely that lack of publications on this issue is due to the difficulty of tracking newborns. Nevertheless, it is mandatory to collect increasingly more data on the perinatal outcome of babies born of vitrified oocytes. So it is highly recommended to encourage all groups performing oocyte vitrification to publish such data. CONTRIBUTION OF OOCYTE VITRIFICATION TO CLINICAL PRACTICE IN ASSISTED REPRODUCTION Information in the literature suggests that accurately analyzing oocyte cryopreservation efficiency can prove to be a very difficult task given that techniques and protocols vastly vary. Additionally, a number of patient- and or technique-dependent variables (i.e., age, oocyte quality, cryoprotectant types/concentrations) and various vitrification devices play key roles in final outcomes. Edgar and Gook recently published a comprehensive review on cryopreservation. They concluded that not only is there reproducible evidence that vitrified oocytes retain their normal developmental potential, with survival rates of 90% (60), but that slow-freezing mature oocytes offers low survival rates compared with vitrification, and that resultant embryo development appears to be impaired. These conclusions are in line with those drawn from a meta-analysis of prospective randomized trials comparing the outcomes of slow-freezing MII oocytes versus vitrification, or of fresh versus vitrified oocytes, with outcomes favoring vitrification (25). Nevertheless, heterogeneity was detected when analyzing survival, most probably owing to differential outcomes among the several studies included in the analysis, which involved both open and closed vitrification systems. Interestingly, another review evaluated the oocyte vitrification's clinical efficiency to reveal that the most widespread methodology is that using open systems (61). Although direct contact with liquid nitrogen is controversial, it also seems to be more efficient than the currently available closed systems, at least for oocytes, despite the lack of prospective randomized trials supporting this assumption. When evaluating vitrification's clinical efficiency, to draw conclusions on its usefulness for FP either with or without medical reasons, it is more practical to analyze two different subpopulations currently benefitting by this approach: oocyte recipients and autologous IVF cycles with vitrified oocytes. Oocyte Vitrification and Ovum Donation Egg-banking available for oocyte donation programs is now a reality, thanks to vitrification. The first live birth achieved with vitrified oocytes resulted from a donated egg in 1999 (62). Then reports involved mixed samples (donated and autologous oocytes), with 85% 90% survival rates and a 30% pregnancy rate per transfer (63, 64). In 2008, some publications using exclusively donated oocytes published 85% 95% survival rates and 60% 75% pregnancy rates (65 67), and another, following the protocol of Kuwayama et al. (21) with 30 donors/recipients, demonstrated equivalent development to the blastocyst stage between fresh and vitrified oocytes (48.7% vs. 47.5%) (68). In 2009, Nagy et al. reported their experience with egg banking involving 10 donors and 20 recipients, obtaining a 90% survival rate with a 75% pregnancy rate per transfer (69). In 2011, a joint publication from two centers showed consistent outcomes for ongoing routine clinical use of vitrified donor oocytes after 2 years of consecutively applying egg banking (70). These outcomes mirror a study involving shared vitrified and fresh oocytes, which found similar developmental parameters and clinical outcomes between fresh and cryodonations (71). Other authors evaluated the combined oocyte vitrification and embryo transfer strategy in the blastocyst stage of their ovum donation programs (72). Both blastocyst formation (41.3% vs. 45.3%) and pregnancy rates (61.8% vs. 60%) between vitrified and fresh oocytes were similar. Recently, a prospective observational study involving 14 donors and 20 recipients reported a 90% survival rate and a 43.5% ongoing pregnancy rate (OPR) by considering fresh and cryotransfers of surplus embryos (73). What is most interesting, and unlike most studies published to date, is that this work used a closed vitrification system. The largest published prospective randomized controlled clinical trial assessing egg banking efficiency for ovum donation with 600 recipients (>3,000 oocytes per arm) VOL. 99 NO. 6 / MAY

4 VIEWS AND REVIEWS demonstrated that equivalent outcomes per intention-to-treat populations may be achieved by vitrified or fresh oocytes (74). With overall survival rates of 92.5% and 43.7% OPR, this trial confirmed our previous observations of the capability of undisturbed fertilization and embryo development after oocyte vitrification versus fresh oocytes. In our institution, egg banking is an integral part of our clinical routine, with >30,000 vitrified oocytes donated (unpublished data), positively impacts oocyte donation program management, makes excellent clinical results easier to achieve more efficiently, and equals those obtained with fresh donor oocytes. Oocyte Vitrification and Infertile Patients The potential of oocyte vitrification to resolve the different clinical situations arising in an IVF program have been highlighted. Several studies of the clinical use of autologous vitrified oocytes were published in the first decade of this century using different open carriers and CPAs (65, 75 82). Although these reports comprised small numbers of patients/oocytes, and provided very inconsistent outcomes, they all shaped valuable information on the scope of the strategies applied, and most are still used today. Reported survival rates ranged from 60% to 100%, with a wide range of embryos transferred (1.2 to 4.5) and implantation rates varying from 10% to 60%, with 30% 60% pregnancy rates per embryo transfer. In 2005, Kuwayama et al. reported 90% and 40% survival and pregnancy outcomes, respectively, by applying the widespread Cryotop system. This approach was applied in a prospective randomized study with sibling oocytes from typically infertile patients (mean age 35.5 years) involving 124 vitrified oocytes from 40 patients (83). After achieving a 96.8% survival rate, fertilization (76.6%), embryo development (51.6% excellent quality embryos), 20.4% implantation rate with mean of embryos transferred, and OPRs (30.8%) were similar between vitrified and fresh oocytes. These authors also assessed cumulative outcome in the same infertile population, showing that maternal age negatively affects outcomes for both fresh and vitrified cycles: Implantation rate in patients older than 41 years was 12.2% whereas it was 27.3 for patients younger than 34 years (84). The scope of oocyte vitrification was also evaluated in a selected population of young fertile women, achieving 65% OPR and 20 live births with a mean of embryos transferred and 45.3% implantation rate (85). The authors emphasized the importance of screening candidates for oocyte vitrification to guarantee satisfactory outcomes, with great implications for the objective of this review regarding counseling for FP purposes. Several studies evaluating IVF cycles' efficiency in clinical settings conducted with vitrified versus fresh oocytes (86) or slowly frozen versus vitrified oocytes were published in (55, 87 89). Except for the study by Noyes et al. (55) reporting discretely higher outcomes in the slow freezing group (88% vs. 85% survival rate, 45% vs. 37% blastocyst formation, and 45% vs. 37% pregnancy rate), the reports indicated higher outcomes for vitrified oocytes (80% 90% vs. 60% 85% survival rates, 20% 40% vs. 10% 15% pregnancy rates). Another study by Forman et al. in 2012 concluded a similar aneuploidy incidence between vitrified and fresh oocytes (29.1% vs. 26.4%), and reported lower outcomes in the oocyte vitrification group for fertilization and blastocyst formation (77.9% vs. 90.5% and 34.8% vs. 50.8%) although the OPRs per embryo transferred were similar (53.9% vs. 57.7%) (58). Another study reported high-quality blastocyst development achieved from vitrified oocytes in a typical infertile population, although no comparison with fresh oocytes was made (90). The benefits of oocyte vitrification in two infertile populations include having to avoid the risk of hyperstimulation (91) and low response (LR) (92). For the latter, >1,000 vitrified oocytes obtained from 594 cycles performed with 242 patients (mean 2.5 cycles) were accumulated by vitrification, leading to enhanced cohorts to be inseminated (mean 7.02 MII oocytes) (92). The mean number of embryos transferred was statistically higher in the vitrification group (2.0 [95% confidence interval (CI) ] vs. 1.7 [95% CI ]) although no differences were observed in implantation rates (25.5% vs. 25.0%). Statistically higher cumulative newborn rates per patient initiated, compared with standard-treated LR patients, were observed (42.9% vs. 27.4%). Advancedmaternal-age LR patients (>40 years old with R6 MII oocytes) undergoing PGS analysis also benefited from this strategy: implantation rate 24.4% vs. 19.8% in vitrification versus fresh oocytes (93). The results obtained in this population may prove to be useful for practitioners treating women desiring FP. Finally, a multicenter prospective longitudinal cohort study with 3,000 vitrified oocytes evaluated the efficacy and reproducibility of oocyte vitrification and achieved an 85% overall survival rate, mean 1.91 (95% CI ) embryos replaced to the uterus, and 26.3% delivery with a 15.8% live birth rate per transfer (94). No significant differences between centers were found for survival, fertilization, and embryo development, which reflects the method's reproducibility. Three variables were detected as determinants of success: patient age (<38 years), number of vitrified MII oocytes (R8), and blastocyst stage on embryo transfer. Each year of maternal age decreased the delivery rate by 7%. The positive correlation between survival and age merely reflects oocyte quality, which declines with age. Additionally, when the number of vitrified oocytes was higher than eight, delivery rates increased from 22.6% to 46.4%. This finding may be comparable with Cobo et al.'s in which the mean number of vitrified oocytes was 7.2 (92). This is perhaps the most interesting evidence of these studies given its implication for FP counseling. It also suggests that it is most advisable to inform patients that they should ensure a reasonable number of cryopreserved oocytes, for which more than one stimulation cycle is likely required. Basically, evidence to date indicates that oocyte vitrification has significantly improved the clinical outcome of cryopreserved oocytes by achieving results similar to those with fresh oocytes, which makes its implementation as an efficient FP method feasible VOL. 99 NO. 6 / MAY 2013

5 Fertility and Sterility OOCYTE VITRIFICATION FOR FERTILITY PRESERVATION Age-Related Fertility Decline (Social Reasons) and Nononcologic Patients In today's society, many women push pregnancy further away from the right childbearing age. The change in motherhood trends stems from women being able to choose their profession, financial situation, partner, among other social conditions. The affects childbearing, given the well known decline in female fertility beyond the age of 30 years (95 97). AR is unable to fully overcome the effect of age on fertility loss after the age of 35 years. Additionally, a noteworthy increase in adverse reproductive outcomes and a higher proportion of maternal and/or fetal morbidity and mortality are associated with advanced maternal age. Therefore, medical organizations' recommend promoting educational programs that encourage procreation at a maternal age of years (98). Delaying motherhood iaffects modern society with political, ethical, and even religious implications. A study analyzed the effect of delayed maternity on fertility dynamics in Europe. It found that childbearing age is higher in countries with high child care provision and part-time employment opportunities than in countries with limited childcare availability and less flexible labor market (99). A novel indication for FP is known as social or FP because of age-related fertility decline. The term social has been widely used to describe elective fertility preservation for women who decide to collect their oocytes to not use them immediately but to preserve them for future use. This term is controversial, because many have attributed a pejorative connotation that denigrates social freezers' motivations (100). Undoubtedly, the social impact and ethical connotations need to be profoundly analyzed. Despite the focus of the present review being oocyte vitrification efficiency for FP rather than related ethical challenges, we must mention interesting publications on this matter. Lockwood (100) discussed the extremely difficult demarcation between medical and social needs. Dondorp and de Wert's contribution offers a wide-ranging analysis of ethical aspects of FP for healthy women (101). Challenges such as gender inequality and fertility, biologic limits, late pregnancy, and motherhood and its implications, and the invasive nature of procedures required to harvest oocytes or ovarian tissue from healthy women have been addressed. The problem of public health system coverage of a social problem, i.e., delaying motherhood, has been raised. From an ethical/moral standpoint, the authors concluded that there was no reason for healthy women to not benefit from FP. However, based on a combined opinion on cryopreservation of ovarian tissue and oocytes, the authors suggested offering FP only under experimental conditions based on uncertainty as to the efficacy and safety of available techniques, which was in accordance with the experimental label conferred by major regulatory bodies ( ). An emerging mindset change motivated by a growing body of clinical evidence regarding the technique is perceived is some publications ( ). Indeed the strategy is increasingly accepted in the USA, where >50% of IVF settings offer FP options (112). Eight years after their first guideline publications, the European Society for Human Reproduction and Embryology task force considered that oocyte cryopreservation should also be available for nonmedical reasons (98). Similarly, a very recent publication by the practice committees of the American Society for Reproductive Medicine and Society for Assisted Reproductive Technology published their guideline document. It concluded that oocyte vitrification should no longer be considered to be experimental, thanks to evidence that the outcomes achieved with vitrified oocytes are similar to those obtained with fresh oocytes (113). Despite some centers increasingly applying FP as a routine strategy, the needs of those patients who wish to use FP options are not always met adequately, mostly because of concerns regarding lack of clinical evidence for method safety, lack of knowledge, and appropriate training for the most successful techniques available (114). A survey study that addressed AR professionals, bioethicists, medical students, and the general population regarding their acceptance of oocyte cryopreservation to preserve fertility for personal reasons showed that nonexperts indicated a greater level of ambivalence than experts, who were mostly in favor (115). Another similar study assessed the willingness of women at reproductive ages to undergo social FP and concluded that a significant proportion of young women would consider it (116). Widespread FP application among healthy women will undoubtedly affect IVF practice and patients' economic resources. Remarkable divergences among two cost-effective analyses of nonmedical FP in a European study led to positive conclusions (117) but disagreed with a USA-based analysis (118), although several differences in study design contributed to this discrepancy (119). Clinical Outcome of Oocyte Vitrification for FP for Nononcologic Reasons Specific reports in the literature on FP outcomes for nononcologic reasons are lacking, owing to its recent incorporation into the clinical routine. The intrinsic notion of delaying motherhood for the future implies that a several years' wait is needed to build a large case set. Nonetheless, we are starting to collect information from women who return to use their oocytes after deciding to delay childbearing. A retrospective observational study comprising 560 nononcologic patients (mean age years) showed that 90.6% decided to delay motherhood for nonmedical reasons, and the rest reported other medical conditions apart from cancer, such as endometriosis, imminent adnexectomy, etc. (120). In all, 5,498 MII oocytes ( per patient) harvested from 726 controlled ovarian stimulation (COS) cycles were vitrified. Twenty-six patients had returned to attempt pregnancy (191 warmed oocytes of the 253 available). Mean oocyte storage time, number of warmed oocytes per patient, and survival rate were months; oocytes, and 84.8%, respectively. The OPR per warming cycle was 30.7% and 33.3% after cryotransferring supernumerary embryos, leading to a cumulative OPR per patient of 70.9%. VOL. 99 NO. 6 / MAY

6 VIEWS AND REVIEWS Five healthy babies have been born (four from fresh embryo transfers and one from a cryotransfer). There is another FP branch in nononcologic patients other than that motivated by social reasons (age-related fertility decline), i.e., related to other medical conditions, such as some autoimmune or genetic diseases that can lead to premature menopause. Two examples are provided in the case reports on the vitrification of 8 and 14 oocytes after COS in 22- and 14-year-old patients affected with mosaic Turner syndrome (121, 122). Oncologic Patients Progress in cancer treatment using radiotherapy or chemotherapy has improved survival rates among malignant diseases. This is particularly evident in children and breast cancer patients (104, ). Unfortunately, most suffer chronic adverse effects of radiation or cytotoxic chemotherapy, including gonadal failure and infertility, which often cause distress, low self-esteem, and undermined quality of life (126, 127). Thus the need is evident for an effective FP strategy that provides the chance to conceive a child with one's own gametes. In contrast to FP for nonmedical reasons, the possibility of safeguarding fertility in cancer patients has always been accepted, albeit initially under experimental conditions (105). The literature contains numerous reports assessing different FP options for oncologic patients: embryo, ovarian tissue, or oocyte cryopreservation (124, ). Until recently, practically all guideline documents agreed that the best option was embryo cryopreservation, given the lack of strong evidence for the success of the two other options (104, 108). As discussed above, this situation has drastically changed thanks to evidence for similar outcomes between vitrified and fresh oocytes, which changes the positioning of main scientific societies (98, 113). Here, we do not assess the embryo or ovarian tissue cryopreservation option, but we wish to mention that they have long since been applied and have led to several live births (138, 139). Offering oocyte vitrification to cancer patients involves the disadvantage of needing COS to harvest oocytes, which delays chemotherapy initiation and may allow high E 2 levels (140). The time interval between cancer diagnoses and treatment initiation may vary among malignancies; breast cancer patients usually wait 4 6 weeks between surgery and chemotherapy, which is sufficient to obtain oocytes for vitrification (135). The potential risk associated with high E 2 has led to much controversy among clinicians, especially oncologists, which may be overcome by different approaches: oocyte retrieval in unstimulated cycles, i.e., natural cycles, where a single oocyte is aspirated on natural ovulation; or immature oocyte retrieval with further in vitro maturation (IVM) and vitrification of MII oocytes. Immature oocytes can be retrieved after oophorectomy in women with ovarian malignancy (141), by aspiration from unstimulated ovaries (142, 143), or by a strategy combining ovarian tissue cryobanking and immature oocyte retrieval (144, 145). The mean maturation rate among these studies varied from 50% to 80% with a mean number of 7 10 vitrified MII oocytes. Some authors also inseminated IVM oocytes to store embryos (143, 146). In unstimulated cycles, immature oocyte retrieval was performed throughout the menstrual cycle in the midfollicular or the luteal phase, with no differences in the number of retrieved oocytes, maturation and fertilization rates, or total number of cryopreserved oocytes and embryos (146). Although IVM followed by vitrification of MII oocytes has been implemented, no live birth has been reported. A different strategy to collect MII oocytes for vitrification, given the need for FP, consists of ovarian stimulation before oophorectomy to retrieve mature oocytes ex vivo (147, 148). In a recent report, immature oocytes were retrieved with the use of COS in a 13-year-old premenarcheal female diagnosed with myelodysplastic syndrome (149). Despite this encouraging report, the undefined response to COS in children in earlier puberty stages and the quality of the oocytes retrieved at these ages should be considered (150). Another study provided a different option of using immature oocytes, which involved collecting them with mature ones after ovarian stimulation to improve cryopreserved reserves (151). Third, the use of aromatase inhibitors (letrozole) with COS as a safe approach to obtain a reasonable number of mature oocytes with serum E 2 levels similar to natural ovulation cycles was described by Oktay et al. (152, 153). Recently, letrozole proved to be an effective ovulation induction agent in humans by inhibiting the negative feedback that E 2 exerts on the hypothalamus and pituitary and by increasing sensitivity of the FSH receptor in ovarian granulosa cells (154, 155). Preliminary experience reveals that letrozole can induce ovulation without raising estrogen levels which remain at physiological levels when used alone or in combination with FSH. Interestingly, statistical analysis found no difference between women with cancer undergoing ovarian stimulation and control subjects (152, 153, 156). Another study, with 223 cancer patients, assessed the response to COS in hormone-dependent and nonhormone-dependent patients. It showed that after letrozole stimulation, the mean E 2 level remained low (381 pg/ml), although a poorer response was observed in breast cancer versus nonhormone-dependent patients: mean (95% CI) difference in E 2 levels: 1,362 (1,102 1,623); mean difference in number of MII oocytes: 1.9 ( ) (157). Clinical Outcome of Oocyte Vitrification for FP for Oncologic Patients Information about outcomes achieved in cancer patients who preserved their fertility through oocyte vitrification is still lacking, mainly because their gametes have not yet been used. First, this is because this option has been available only until recently. Second, following their diagnosis, oncologic patients have a difficult time: They decide to preserve their fertility, complete healing must be established, and some need to find the right partner before deciding to become mothers. However, there is strong evidence in oocyte recipients, and especially in infertile patients, as previously discussed, for the potential of vitrification technology to efficiently preserve cancer patients' fertility VOL. 99 NO. 6 / MAY 2013

7 Fertility and Sterility TABLE 1 Clinical outcomes and live births reported in cancer patients who preserved fertility through oocyte cryopreservation (slow freezing and vitrification). Yang et al., 2007 (158) Porcu et al., 2008 (159) Sanchez Serrano et al., 2009 (160) Kim et al., 2011 (161) García-Velasco, 2013 (120) Type of malignancy Hodgkin lymphoma Borderline ovarian tumor Breast cancer Chronic myeloid leukemia Non-Hodgkin lymphoma Cryopreservation technique Slow freezing Slow freezing Combined OTC-SF þ OV (Cryotop) Vitrification (EMG) Vitrification (Cryotop) Age at FP, y No. of cryopreserved oocytes Storage time (y) Twin or single pregnancy Single a Twin Twin Single Single No. of live births Weeks of gestation þ 3 d 39 Weight of baby, g 3,062 2,100 and 2,400 1,650 and 1,830 2,410 3,440 Sex of baby Male Females Males Male Male Note: EMG ¼ electron microscope grids; FP ¼ fertility preservation; OTC-SF ¼ ovarian tissue cryopreservation; OV ¼ oocyte vitrification. a Gestational carrier. Cobo. Oocyte vitrification for fertility preservation. Fertil Steril There are two reports (Table 1) of babies born to cancer patients after the slow freezing of oocytes (158, 159). Despite the present review's focus on vitrification, we consider that these outcomes are noteworthy given their relevance and the scarcity of data on live births. Another study reported the birth of 20 babies after slow freezing of embryos for FP in breast cancer patients (139). Very few studies report clinical outcomes with vitrified oocytes for FP in cancer patients (Table 1). The birth of twins is described in one study that combined ovarian tissue cryopreservation/grafting and oocyte vitrification in a breast cancer patient who returned 20 months after cancer treatment (160). Following the first IVF failure after grafting, the decision of accumulate oocytes from successive COS cycles through vitrification was taken. Two day-3 embryos were transferred, leading to the birth of two healthy babies. This study highlights the chance of increasing IVF success in poor-prognosis patients after grafting ovarian tissue, thus avoiding the short lifespan consequences of transplanted tissues (160). In 2011, a study by Kim et al. reported the birth of the first baby after oocyte vitrification in a patient with chronic myeloid leukemia, whose oocytes were vitrified for FP before chemo- and radiotherapy (161). Seven oocytes were vitrified with the use of electron microscope grids and were stored for 9 years, when the patient returned to attempt pregnancy. A recent report provides clinical outcomes from an FP program for cancer patients who opted for oocyte vitrification after their oncologists agreed (120). In this large report, 97.4% of patients chose oocyte vitrification over ovarian tissue cryopreservation. It included 340 patients (mean age years) diagnosed with breast cancer (67%), Hodgkin lymphoma (11%), non-hodgkin lymphoma (5%), and gastrointestinal tumors (3.5%). The mean E 2 levels on the day of triggering were statistically lower in patients receiving letrozole stimulation (404 vs. 1,369 pg/ml), and 939 MII oocytes were vitrified ( per patient). Two pregnancies were achieved in two of the four patients who returned to use their vitrified oocytes. Unfortunately, one miscarried at gestation week 6. A second pregnancy was achieved in a 33-year-old patient with non-hodgkin lymphoma who attempted pregnancy 2 years after finishing oncologic treatment. Four oocytes were warmed, all developed into viable embryos, of which two were transferred. A boy weighing 3,440 g was born in gestation week 39 (Table 1). As far as we know, the reports by Kim et al. (161) and Garcia-Velasco et al. (120) are the first publications on the birth of babies born to cancer patients who chose oocyte vitrification for FP before being treated for malignant diseases. CONCLUSION Oocyte vitrification currently offers advantageous clinical results in diverse populations, such as oocyte recipients and typical infertile patients, thus making it a very effective FP option for medical and nonmedical reasons. Based on current evidence, we can state that oocyte vitrification is a standardized technique. However, a degree of variability due to multiple factors, including specific VOL. 99 NO. 6 / MAY

8 VIEWS AND REVIEWS populations, methodologies applied, particular protocols, and types of device and cryoprotectants, is seen. Despite oocyte vitrification protocols being simple to perform, experienced hands are needed to guarantee success. Although the current evidence on the outcome of oocyte vitrification for FP is very limited, experience available in infertile patients may be useful for counseling purposes. FP should be tailored specifically to each patient to achieve optimal results. Special care must be paid to any condition and to the decision on the number of oocytes to be stored. Patients must be counseled objectively according to their possibilities and current evidence to avoid false hopes. Interdisciplinary collaboration is required, especially in cancer patients, with oncologists etc. The collection of clinical outcome data from IVF cycles conducted with vitrified oocytes stored by women affected or not by cancer disease is emerging. 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