Five years' experience using oocyte vitrification to preserve fertility for medical and nonmedical indications

ORIGINAL ARTICLE: FERTILITY PRESERVATION Five years' experience using oocyte vitrification to preserve fertility for medical and nonmedical indications Juan A. Garcia-Velasco, M.D., a,d Javier Domingo, M.D., b Ana Cobo, Ph.D., c Maria Martínez, M.D., a Luis Carmona, M.D., b and Antonio Pellicer, M.D. c a IVI-Madrid, Madrid; b IVI-Las Palmas, Las Palmas; c IVI-Valencia, Valencia; and d Rey Juan Carlos University, Madrid, Spain Objective: To evaluate the results of controlled ovarian hyperstimulation (COH) for oocyte vitrification to preserve fertility for medical and nonmedical indications. Design: A retrospective, multicenter, observational study performed between March 2007 and June 2012. Setting: University-affiliated infertility clinics. Patient(s): Of 560 nononcological patients and 475 oncological patients, we performed 1,080 oocyte vitrification cycles, 725 for nonmedical reasons and 355 in patients affected with cancer. Cycle outcome is presented, including 30 women who returned to use their frozen eggs with, 20 pregnancies obtained, 6 newborns, and 8 ongoing pregnancies. Intervention(s): Controlled ovarian hyperstimulation, oocyte retrieval, warming of oocytes, and ET in those who already came back. Main Outcome Measure(s): Days of stimulation, total dose of gonadotropins, estrogen (E) and P levels, number of oocytes retrieved and vitrified, pregnancy rate (PR). Result(s): Comparable results were obtained in both groups of patients, with lower total dose of gonadotropins used and lower serum E 2 levels in patients affected with cancer. Frozen/thawed oocytes performed similarly in both groups. Conclusion(s): Patients who vitrify eggs for medical or nonmedical reasons perform similarly, as observed in this large series. This technique offers realistic expectations to both groups of patients to have a child with their own eggs. These data could be used to adequately counsel our patients. (Fertil Steril Ò 2013;99:1994 9. Ó2013 by American Society for Reproductive Medicine.) Key Words: Fertility preservation, oocyte vitrification, cancer, social freezing, newborns Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/garcia-velascoj-oocyte-vitrification-fertility-preservation-cancer/ 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. After more than two decades of continuous efforts made to improve cryopreservation techniques, evolution and perfection of different vitrification strategies have achieved efficient and reproducible protocols to successfully cryopreserve the female gamete. Embryo development parameters, as well as clinical outcomes, have proven to be unaltered after oocyte vitrification compared with fresh oocytes in ovum donation programs (1 4). In addition, vitrification has shown to be effective in autologous IVF cycles (5, 6). The advent of vitrification as a means of fertility preservation (FP) has opened a window of opportunity for many female cancer patients to be a mother with her own gametes after having overcome the disease (7, 8). The first reported case in Europe of a pregnancy after FP using vitrification of oocytes was from our group Received December 27, 2012; revised February 5, 2013; accepted February 6, 2013; published online March 1, 2013. J.A.G.-V. reports consultancy, payment for lectures, and grants from Merck Serono, MSD, and Ferring. J.D. has nothing to disclose. A.C. has nothing to disclose. M.M. has nothing to disclose. L.C. has nothing to disclose. A.P. has nothing to disclose. Supported by FIS PI11/02747 from Ministerio de Ciencia e Innovacion, Madrid, Spain. Reprint requests: Juan A. Garcia-Velasco, M.D., IVI Madrid, Av del Talgo 68, 28023 Madrid, Spain (E-mail: juan.garcia.velasco@ivi.es). Fertility and Sterility Vol. 99, No. 7, June 2013 0015-0282/$36.00 Copyright 2013 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2013.02.004 (9), in a patient whose ovarian cortex was cryopreserved first, and after grafting, four stimulation cycles were performed to accumulate and vitrify mature oocytes; a later IVF cycle successfully ended in a twin pregnancy. Although FP may initially concern cancer patients, there are many other medical conditions that may compromise fertility, such as endometriosis or high risk for early ovarian failure, where an intervention as safeguarding gametes for future use is required to uphold fertility potential. In addition, age-related decline of fertility is a very common condition in assisted reproductive technology (ART). Elective oocyte vitrification for nonmedical conditions is increasingly accepted as an option to postpone motherhood. 1994 VOL. 99 NO. 7 / JUNE 2013

Fertility and Sterility Fertility preservation has being used routinely for relatively few years (7, 8). There is still a gap in knowledge about the performance of the technique in the future when patients come back to use their oocytes. At present we have been predicting success based on the available knowledge of oocytes from ovum donation programs and also on the autologous IVF cycles carried out with vitrified oocytes. Needless to say, the accuracy of such predictions will be substantially enhanced when we have available data from FP women, who for medical or nonmedical reasons, decide to attempt pregnancy. The aim of the current study is to describe the outcome achieved with oncological and nononcological patients who decided to preserve their fertility by means of oocyte vitrification in the past 5 years and of those who already came back to use their oocytes. MATERIALS AND METHODS A total of 1,035 patients were enrolled in this observational, multicenter study since we started the Fertility Preservation Program at our institution. The sample included 560 nononcological patients (n ¼ 725 cycles) and 475 oncological patients (n ¼ 355 cycles) who vitrified oocytes for FP purposes from March 2007 to June 2012. All FP cycles in cancer patients were carried out after obtaining the approval of the oncologist. Our Institutional Review Board approved this study and all patients gave written informed consent. Twenty-six nononcological patients have already returned to use their oocytes to attempt pregnancy, whereas only four cancer patients have tried to become pregnant by using their vitrified oocytes after overcoming their disease. Controlled Ovarian Hyperstimulation Protocol for Nononcological Patients Controlled ovarian hyperstimulation (COH) was initiated on day 2 or 3 of a spontaneous cycle. An initial dose of 225 300 IU recombinant FSH (Gonal-F, Merck-Serono; Puregon, MSD) and/or highly purified hmg (Menopur, Ferring Pharmaceuticals) were administered. From day 6 onward, the gonadotropin dosage was estimated according to serum E 2 levels and a transvaginal ultrasound scan. When a leading follicle reached 14 mm, GnRH antagonist (0.25 mg/d; Cetrotide, Merck-Serono; Orgalutran, MSD) was administered. Final oocyte maturation was triggered with 250 mg of recombinant hcg (Ovitrelle, Merck-Serono) as soon as two follicles were R18 mm in mean diameter. Oocyte retrieval was scheduled 36 hours after hcg injection. COH Protocol for Oncological Patients Our COH protocol did not change throughout the duration of the program and has been described elsewhere (10). In nonhormone-dependent cancers, COH was performed using a similar protocol: starting recombinant FSH on day 2 or 3 of a spontaneous cycle under a GnRH antagonist protocol with letrozole not being administered. In hormonedependent cancer patients, aromatase inhibitors were used for ovarian stimulation. Letrozole (5 mg/d) (Femara 2.5 mg; Novartis) was administered orally starting the second or third day of a spontaneous cycle until the day of triggering and then until the first day of menstruation after oocyte retrieval. After 2 days of letrozole administration, 150 225 IU/d of recombinant FSH (Gonal F; Merck-Serono) was added. GnRH antagonist (0.25 mg/d) (Cetrotide; Merck-Serono) was administered when the leading follicle reached 14 mm, and final oocyte maturation was triggered with 0.2 mg of GnRH agonist triptorelin (Decapeptyl 0.1, Ipsen-Pharma) as soon as two follicles were R20 mm. Because time was a limiting factor for those patients who came in the ovulatory phase or the early luteal phase at the time of their first visit we used a similar protocol but with minor modifications. Patients received GnRH antagonist (0.25 mg/d) for 3 days, and their serum E 2 levels were measured. If the E 2 level was <60 pg/ml, COH was started regardless of bleeding, if the level was higher, one extra administration of GnRH antagonist was given, and COH was started. No patient required more than four doses of GnRH antagonist to reduce E 2 levels to less than 60 pg/ml. Oocyte Vitrification/Warming The Cryotop method used for oocyte vitrification was the one described by Kuwayama et al. (11), with minimal modifications. Oocytes were denuded by enzymatic means 2 hours after ovum pick-up. They were then equilibrated at room temperature for 15 minutes in 7.5% (vol/vol) ethylene glycol þ 7.5% dimethyl sulfoxide (DMSO) in TCM199 medium þ 20% synthetic serum substitute (SSS) (Kitazato). After 12 minutes the oocytes were checked for rehydration, if complete they were subjected to the vitrification step. Oocytes were then placed in vitrification solution containing 15% ethylene glycol þ 15% DMSO þ 0.5 M sucrose. After 1 minute in this solution, oocytes were placed on the Cryotop strip (1) and immediately submerged in liquid nitrogen. No more than four oocytes per Cryotop were loaded. For warming, the Cryotop was removed from the liquid nitrogen and warmed in 1.0 M sucrose in TCM199 þ 20% SSS at 37 C. Any manipulation of oocytes was avoided at this point. After 1 minute, oocytes were placed in 0.5 M sucrose in TCM199 þ 20% SSS at room temperature for 3 minutes with no extra manipulations. Finally, one 5-minute wash followed by one 1-minute wash was performed with TCM199 þ 20% SSS at room temperature before incubating the oocytes in routine culture media for 2 hours before intracytoplasmic sperm injection (ICSI). Embryo Transfer Embryos were selected for transfer strictly according to their morphological appearance. Surplus embryos suitable for additional cryopreservation were vitrified following the Cryotop protocol for embryos (12). Hormonal therapy (HT) protocol was used for endometrial preparation. Women with functioning ovaries were first down-regulated in the luteal phase with a single dose of GnRH-agonist depot (Decapeptyl, 3.75 mg; Ipsen Pharma; Procrin depot 3.75, Abbott; Gonapeptyl 3.75 mg, Ferring Pharmaceuticals) and received 6 mg of oral E 2 valerate (Progynova, Schering) after menses. VOL. 99 NO. 7 / JUNE 2013 1995

ORIGINAL ARTICLE: FERTILITY PRESERVATION Approximately 10 15 days after initiation of E 2 valerate, serum E 2 levels and endometrial thickness were determined. If the E 2 levels were >150 pg/ml, endometrial thickness R7 mm, and a triple layer endometrial pattern was confirmed, administration of micronized P (800 mg/d, vaginally) (Progeffik, Effik Laboratories) was initiated. Embryo transfer was scheduled according to the embryo developmental stage. Vaginal P was administered starting on the day of fertilization until 12 weeks of gestation or was discontinued if pregnancy was not achieved. For natural cycles, follicular growth was monitored. When the leading follicle reached a mean diameter of 18 mm, 250 mg of recombinant hcg (Ovitrelle, Merck-Serono) was administered to trigger ovulation. Micronized vaginal P was initiated on the day of ET (400 mg/d, vaginally). All ETs were routinely performed under transabdominal ultrasound guidance. Clinical pregnancy was defined as an intrauterine sac with heartbeat at 6 weeks' gestation. Ongoing pregnancies were defined as the presence of at least one developing embryo of >12 weeks' gestation. Statistical Analysis Continuous data are presented as mean SD, whereas categorical data were expressed as number and percentage. Kolmogorov-Smirnov-Wilk criteria for normality of distribution were used. The c 2 test, Fisher test, and double-sided Student's t test were used as appropriate. Significance was set at.05. The analyses were performed using SPSS 17.0 software. RESULTS Nononcological Patients Seven hundred twenty-five COH cycles were performed to 560 nononcological patients (mean age 36.7 4.2 years). Among them, 505 patients (90.6%) decided to cryopreserve gametes due to their wish to delay motherhood: endometriosis was the reason for FP in 38 patients (6.8%); 8 patients (1.4%) vitrified their oocytes before adnexectomy; 3 women (0.5%) presented with genetic disease (X fragile and Rend u-osler disease); 2 women (0.4%) had fibroids; and 1 patient (0.2%) hadrheumatological disease (Fig. 1). Mean length of stimulation was 10.1 2.1 days. Total doses of gonadotropin used during COH were 1,923 336 UI/mL of recombinant FSH and 1,115 106 UI/ml of hmg (Table 1). Mean E 2 levels on the day of triggering were 2,214 566 pg/ml and mean P levels were 0.8 0.7 ng/ml. A total of 7,225 oocytes were obtained after oocyte retrieval (13.0 24.0 per patient) out of which 5,498 metaphase II oocytes were vitrified (9.9 22.6 per patient). Twenty-six patients have returned trying to attempt pregnancy (Table 2). Among them 20 patients (76.9%) vitrified their oocytes to delay childbearing, 5 (19.2%) vitrified due to the presence of endometriosis, and 1 patient (3.8%) underwent oophorectomy after the FP cycle. Six patients underwent two vitrification cycles, whereas 20 patients required just one COH cycle. Mean oocytes storage time was 20.5 0.8 months. Of the 253 vitrified oocytes, 191 have been warmed (7.3 3.9) with a survival rate of 84.8%. All patients, except for one, had available embryos for transfer. Ongoing pregnancy rate (PR) per warming cycle was 30.7% and 33.3% after cryotransfer. Cumulative ongoing PR per patient was 70.9% considering both fresh and cryotransfers. A total of five healthy babies have been born (4 from fresh ETs and 1 from a cryotransfer). Oncological Patients Since March 2007, we attended to 475 patients diagnosed with cancer who asked about FP options (mean age 31.9 5.1 years). Breast cancer was the most frequent diagnosis in our series (67%), followed by Hodgkin lymphoma (11%), non- Hodgkin lymphoma (5%), and gastrointestinal tumors (3.5%). A total of 340 patients (71.6%) underwent COH for oocyte vitrification, and 24 patients opted for other FP alternatives. One hundred eleven patients (22.6%) opted not to undergo any treatment for different reasons: lack of time to complete COH, nonconformity with the concept and uncertainty of the expectations, lack of oncologist's agreement, unwillingness to undergo laparoscopy with general anesthesia, or ineligibility for medical reasons. Most of the patients could be stimulated only once, although this treatment may be the only opportunity for them to preserve some oocytes, and therefore the cancellation rate was low (6.6%). Fifty-six patients (11.3%) decided to preserve their fertility, even if they had previous had children (Table 1). A total of 4,104 oocytes were obtained (11.8 8 per patient) after oocyte retrieval from which 2,939 metaphase oocytes were vitrified (71.6%) (8.5 6.4 per patient). Total doses of gonadotropin used during COH for hormonaldependent tumors were 1,493 940 IU recombinant FSH and 1,851 979 IU recombinant FSH for nonhormonaldependent tumors (P¼.001). Mean E 2 levels on the day of triggering were 404 676 pg/ml for hormonal-dependent tumors and 1,369 1,371 pg/ml for the remaining cancer cases (P<.001). Mean time required to start ovarian stimulation was 9.4 12.5 days from the first consultation, with a mean length of stimulation of 9.5 5.9 days. We have warmed oocytes for four oncological patients who used their frozen oocytes and two pregnancies were obtained (Table 2). A miscarriage occurred at 6 weeks after a second ET with vitrified embryos in a patient affected with endometrioid adenocarcinoma. The second pregnancy was in a 33-year-old patient who had non-hodgkin lymphoma. She was a very low responder patient and after two COH cycles only two oocytes were obtained per cycle. Two years later, after having finished her treatment, she attempted IVF. The four oocytes were from four warmed embryos who developed two were transferred and two were vitrified. A 3,440-g boy was born by a vaginal delivery, induced after spontaneous rupture of membranes on the 39th week of pregnancy (Table 2). DISCUSSION Using vitrification oocyte freezing has been shown to be an effective technique that may allow women to preserve their 1996 VOL. 99 NO. 7 / JUNE 2013

Fertility and Sterility FIGURE 1 (A) Fertility preservation (FP) options distribution of the oncological patients. (B) Distribution of nononcological patients according to their indication to cryopreserve (cryo) oocytes. (C) Indications of all the FP cases from our program. Endomet ¼ endometriosis; Ov ¼ ovarian; repet ¼ repeat. Garcia-Velasco. 5-year experience with oocyte vitrification. Fertil Steril 2013. gametes for future fertility, which can be damaged by oncological treatments or by age-related quality decline. In our series, we have accumulated extensive experience with a simple, safe, and efficient protocol, providing women a possibility to postpone childbearing until therapy is over or until they find the right time for motherhood. The fact that many societies consider egg freezing as nonexperimental (13 15), along with the accumulating evidence from different centers around the world confirming that it is a reproducible, safe, and effective technique, makes vitrification a tremendously attractive option for women who need to consider FP. Interestingly, when the indication for FP is oncological or medical it seems to be out of discussion, and all efforts are not enough to freeze eggs or even ovarian tissue, with the approval of most, if not all, of the medical community. However, if FP is decided for social reasons, frequently it is questioned, as these women may reproduce but decide against it at present. In fact, there are some considerations to be made regarding the different options. First, FP for oncological reasons seems out of question, but needs to be clearly specified which women may benefit from oocyte freezing and from ovarian tissue freezing. Recommendation and counseling should be individualized based on multiple factors such as the age of the patient, the urgency of the treatment, and the type of cancer treatment and associated risk of gonadal failure (15). Ovarian response to COH in these patients may be lower than expected (10, 16, 17), but this is still a controversial issue. In fact, in our series a wide range of ovarian response was observed, but the pregnancy described in a patient with lymphoma came from cycles with a very poor response. There a growing trend to freeze ovarian cortex, as there is an increasing number of newborns as a result of ovarian transplantation (7, 8, 18). In our series, only 2.6% women opted for ovarian cortex freezing as a means of FP. The legal situation in our country, in which ovarian transplantation is not allowed in most private hospitals, may partially explain these numbers, making oocyte vitrification a much more popular option. Second, the demand for FP for social reasons is rapidly increasing. Just looking at the increase in the demand of infertility treatment from 2003 2009 according to the American Society for Reproductive Medicine show us the reality. It is alarming to confirm that the 9% increase in the demand jumps up to 41% if we focus on women >40 years old (19). Not only there is a well-known delay in the age of first pregnancy, but also this delay translates in fewer children born to women less than 35 years and a higher risk of being VOL. 99 NO. 7 / JUNE 2013 1997

ORIGINAL ARTICLE: FERTILITY PRESERVATION TABLE 1 Patient demographics and characteristics of the stimulation cycles for both oncological and nononcological FP patients. Nononcological FP (n [ 560) Oncological FP (n [ 355) Age (y) 36.7 4.2 31.9 5.1 a Previous children 11.3% Days from 1st visit to COH 9.4 Length of stimulation (d) 10.1 2.1 9.5 5.9 a Cancellation rate (%) 2.7 6.7 a Total no. of oocytes 7,225 4,104 Total MII (%) 5,498 (76) 2,939 (71.6) a MII/patient 9.9 8.5 Total FSH/hMG with 1,493 940 letrozole (IU) Total FSH/hMG (IU) 3,038 337 1,851 979 a Peak serum E 2 at triggering 404 676 with letrozole (pg/ml) Peak serum E 2 at triggering (pg/ml) 2,214 566 1,369 1,371 a Note: FP ¼ fertility preservation; MII ¼ metaphase II. a P<.001. Garcia-Velasco. 5-year experience with oocyte vitrification. Fertil Steril 2013. childless with increasing maternal age. Also, at present the PR is quite low for the infertility treatments available due to advanced maternal age, not only because women at this age respond poorly to the drugs, but also because most of these oocytes will be aneuploid (20). Thus, the potential benefits of oocyte freezing at an earlier age, when the best oocytes are produced, become clearer. Women could have children with their own eggs at the age at which they decide providing women reproductive autonomy (21, 22) reducing the need for oocyte donors that still has relevant ethical connotations (23), diminishing the pressure on these women to have a child in a certain time-frame just TABLE 2 Clinical outcome of nononcological FP patients. Nononcological Oncological No. of patients/warming cycles 26 4 Fresh ETs (%) 24 (92.3) 4 No. of embryos transferred 37 (1.5 0.6) 8 (2) CPR/patient (%) 11 (42.3) 1 (25) OPR/patient (%) 8 (30.7) 1 (25) No. of patients with surplus 17 (65.3) 2 (50) embryos No. of surplus embryos vitrified 49 (2.8 4.2) 4 (2) No. of cryotransfers 15 (88.2) 1 No. of embryos transferred per 2.3 0.7 2 cryotransfer CPR/patient (%) 7 (46.6) 1 (100) OPR/patient (%) 5 (33.3) 0 Total live birth 5 1 Mean birth weight (g) 3,150 0.3 3,440 Sex of the baby Female (%) 3 (60) 0 Male (%) 2 (40) 1 Note: Unless otherwise indicated, values are mean SD. CPR ¼ clinical pregnancy rate; FP ¼ fertility preservation; OPR ¼ ongoing pregnancy rate. Garcia-Velasco. 5-year experience with oocyte vitrification. Fertil Steril 2013. because the biological clock is ticking, as well as the number of inefficient infertility treatments being performed today at advanced ages (13, 14). In addition, labels are important and age-related oocyte freezing seems more appropriate than social freezing. The term social has connotations that these women may reproduce but voluntarily decided not to. It may be because they decided to wait for financial, professional, or personal maturity. It could be because they do not have a partner, or the right partner who they want to father their child. In fact, most women who freeze their eggs not for medical reasons choose to do it based on the lack of a partner and being aware that time is against their fertility options. But still, if we look at our own data, these women are coming late fertility declines after age 35 years (24), and mean age of these women is almost 37 years. Thus, our responsibility as physicians is to increase social awareness about the impact of age on fertility and offer younger women who voluntarily postpone their first pregnancy the information about FP. Third, who should cover these expenses? If young women do not have the financial stability to raise a family, they may not be ready to support the cost of egg freezing (22). Still at present, women who opt to freeze their egg for nonmedical indications have an average reported age of 38 years, and are usually highly educated (25, 26). However, it is well know that their chances of establishing a successful pregnancy would increase if they froze their oocytes at an earlier age. But most likely, they do not come in earlier, not only are they not aware of the technique, but they may have different priorities at that time such as completing their education, establishing an stable relationship, and finding financial security (27). Informing women and raising awareness campaigns about the decline in fertility with age may make them feel helped, and may lower the mean age of the so-called social freezers. Fourth, how many women will be coming back for the frozen eggs? It is difficult to estimate as this is a new social trend and not enough time has elapsed to evaluate hard data. We can speculate from two points of view: [1] based on modeling, if 61% of women who froze their eggs at younger ages come back for treatment, oocyte vitrification would be more cost effective than current practice of waiting until an older age and undergoing underperforming or ineffective infertility treatments (28) and [2] for cancer patients, the simple fact of undergoing FP improves the patient's experience with oncological treatments (29). To conclude, oocyte vitrification is a simple, safe, and efficient option to preserve gametes. This can be done for different indications, from oncological therapies to age-related fertility decline. In any case, we should inform women about their individual chances of oocyte survival, which depends heavily on their age, and the possibilities of having a live birth according to the number of frozen eggs and avoid unrealistic expectations or overpromises. Acknowledgments: The authors thank the IVI Foundation and all embryologists and gynecologists working for our Fertility Preservation Program at the IVI clinics. We also 1998 VOL. 99 NO. 7 / JUNE 2013

Fertility and Sterility express our gratitude to Merck Serono-Spain for providing the medication for our oncological patients. REFERENCES 1. Cobo A, Kuwayama M, Perez S, Ruiz A, Pellicer A, Remohi J. Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method. Fertil Steril 2008;89:1657 64. 2. Cobo A, Meseguer M, Remohi J, Pellicer A. Use of cryo-banked oocytes in an ovum donation programme: a prospective, randomized, controlled, clinical trial. Hum Reprod 2010;25:2239 46. 3. Herrero L, Martínez M, Garcia-Velasco JA. Current status of human oocyte and embryo cryopreservation. Curr Opin Obstet Gynecol 2011;23:245 50. 4. Martinez-Burgos M, Herrero L, Megias D, Salvanes R, Montoya MC, Cobo A, et al. Vitrification versus slow freezing of oocytes: effects on morphologic appearance, meiotic spindle configuration, and DNA damage. Fertil Steril 2011;95:374 7. 5. Rienzi L, Cobo A, Paffoni A, Scarduelli C, Capalbo A, Vajta G, et al. 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