Oocyte slow freezing using a M sucrose concentration protocol: is it really the time to trash the cryopreservation machine?

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1 Oocyte slow freezing using a M sucrose concentration protocol: is it really the time to trash the cryopreservation machine? Veronica Bianchi, Ph.D., Michela Lappi, B.Sc., Maria Antonietta Bonu, B.Sc., and Andrea Borini, M.D. Tecnobios Procreazione, Centre for Reproductive Health, Bologna, Italy Objective: To update results on outcomes with frozen/thawed oocytes using a differential sucrose concentration during dehydration (0.2 M) and rehydration (0.3 M), combined with a one-step propanediol exposure. Design: Retrospective cohort study. Setting: Private IVF centers. Patient(s): Infertile couples undergoing IVF treatment. Intervention(s): Oocyte thawing cycles between May 2004 and December Main Outcome Measure(s): Survival, fertilization, and cleavage rates were reported to evaluate biological outcomes. Clinical pregnancy and implantation rates were analyzed as markers of efficiency. Result(s): Three hundred forty-two patients and 443 cycles were monitored; the survival was 71.8%, fertilization 77.9%, and of the embryos obtained 83.8% were classified as grade 1 and 2. Three hundred ninety-four transfers were performed, resulting in 90 pregnancies. The pregnancy rate per transfer was 22.8% and per patient was 26.3%, with 122 gestational sacs. The implantation rate per embryo was 13.5%. Patients were divided into three groups according to their age: %34 years (group A), years (group B), and R39 years (group C). Biological outcomes were comparable in all three groups, whereas the pregnancy rate per transfer was higher in the first group (27.7% vs. 21.4% and 17.6%). The implantation rates per injected egg were 11.8%, 8.0%, and 7.5% for the three groups, respectively. Conclusion(s): The biological and clinical data obtained on 443 cycles are consistent with our previous results showing that slow freezing of oocytes can be a valid tool in IVF practice when performed with a suitable protocol. (Fertil Steril Ò 2012;97: Ó2012 by American Society for Reproductive Medicine.) Key Words: Oocyte cryopreservation, slow freezing, ICSI, implantation/oocyte, clinical pregnancy Inthe last 10 years there have been significant improvements in assisted reproductive technologies (ART), primarily concerning the field of oocyte cryopreservation. Since the first pregnancy in 1986 (1), several groups have published more consistent results, giving rise to a new wave of interest in this field (2 4). To date, oocyte freezing has been recommended primarily to preserve fertility in women diagnosed with cancer or other pelvic diseases that may compromise their chances to conceive. Presently early detection programs, combined with improved treatment regimens, have allowed women with cancer to live longer. Therefore, quality of life issues, such as maintaining fertility and future parenthood, have become extremely important. The optimization of oocyte freezing represents a major advancement for these women because embryo cryopreservation was previously the only option available. In cases in which female cancer patients did not have a partner, the only option to preserve fertility after remission was by embryo freezing, requiring them to choose a sperm Received August 19, 2011; revised and accepted January 27, 2012; published online February 22, V.B. has nothing to disclose. M.L. has nothing to disclose. M.A.B. has nothing to disclose. A.B. has nothing to disclose. Reprint requests: Andrea Borini, M.D., Tecnobios Procreazione, Via Dante 15, Bologna 40125, Italy ( borini@tecnobiosprocreazione.it). Fertility and Sterility Vol. 97, No. 5, May /$36.00 Copyright 2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert donor, often resulting in moral and psychological distress. Currently the remarkable improvements in oocyte freezing techniques make it possible to guarantee higher chances of success for these patients to plan a family. Additionally, some countries have legal restrictions that require IVF clinics to adopt egg freezing as a standard practice to avoid repeated ovarian stimulations. Another benefit of oocyte cryopreservation is the possibility to circumvent moral and legal issues associated with embryo freezing, such as legal status and ownership of cryopreserved embryos in the event of divorce. Finally, for egg donation programs oocyte freezing avoids [1] synchronization issues between donor and recipient, and [2] the risk for transmission of infectious pathogens due to quarantine of the donor eggs. VOL. 97 NO. 5 / MAY

2 ORIGINAL ARTICLE: ASSISTED REPRODUCTION Nevertheless, the technique of oocyte cryopreservation remains under experimental status in terms of outcomes and biological variables involved in the process. Little is known about the intrinsic quality of oocytes, and much of the limited information available usually requires invasive procedures that compromise the oocyte; several reports in the literature to date have shown that the successful recovery and ongoing development of oocytes after thawing (5) is highly dependent upon the selection of good-quality oocytes before freezing. Several studies on ultrastructural damages (6, 7), meiotic spindle repolymerization (8), and permeability evaluations (9) after thawing have been published to validate new protocols. The unique feature of the human egg as one of the largest cells in the body (approximately 130-mm diameter) is the main aspect to overcome during oocyte freezing. The low surface area to volume ratio reduces the efficacy of the exchange between cryoprotectant agents and water. In slow-cooling protocols the concentration of permeating cryoprotectants is approximately 1.5 M (usually propanediol [PROH]), whereas the nonpermeating agents (usually sucrose) are M in concentration. The evaluation of cell volume dynamics is important to target improvements in freezing protocols; optimal exposure time combined with the minimizing of osmotic stress should allow sufficient dehydration to achieve protection from freezing injury. The approaches to address these issues have been varied. Yang et al. (10) increased cryoprotectant exposure temperature to achieve faster dehydration rates. Quintans et al. (11) adopted a stepwise addition of the permeating cryoprotectant (PROH) to reduce volume excursions, whereas Boldt et al. (12) tried to use a sodium-depleted freezing medium combined with a lower seeding temperature to improve postthaw recovery. The success of these methods seemed to be confined to sporadic cases and overall was not reproducible. The approach conducted by our group, in collaboration with Paynter et al. (9), was based on measurements of oocyte osmotic response to cryoprotectants, and consequently a new protocol for slow freezing was designed in The objective of this article is to present data from an infertile cohort of patients who decided to cryopreserve their oocytes using a slowfreezing approach with differential sucrose concentration. The hypothesis was to confirm our preliminary data reported in 2007 (13). MATERIALS AND METHODS Study Design This is a retrospective cohort study of patients who decided to freeze their supernumerary oocytes. Institutional approval for the present study was obtained by the Institutional Review Board of Tecnobios Procreazione, Centre for Reproductive Health, Bologna, Italy. With the introduction in 2004 of a very restrictive law (40/ 2004) in Italy, a maximum of three oocytes could be inseminated because all the embryos produced must be transferred. As a consequence, egg freezing became a very important tool in IVF practice to avoid repeated ovarian stimulations. However, on the May 8, 2009 the Italian Constitutional Court declared that the law (40/2004) was unconstitutional, affirming the need to empower the attending physician with the means to carry out a full evaluation regarding the number of eggs to inseminate, and eventually to freeze resulting embryos (14). Setting This study was carried out on 342 patients undergoing at least one thawing cycle from May 2004 to December Included among these subjects were 78 patients whose data had previously been published in 2007 (13) and 264 newly enrolled patients. The biological parameters recorded were survival, fertilization, and cleavage rates. Oocytes with an intact membrane, clear cytoplasm, and no sign of degeneration were considered to have survived after thawing. All eggs were checked before culture and before injection. The clinical variables considered were pregnancy rates (per patient, per thawing cycle, and per transfer) and implantation rates (per transferred embryo and per thawed and injected oocyte). Clinical pregnancy was defined as the presence of a gestational sac at ultrasound examination. Patients who achieved a pregnancy were monitored during the entire period, and follow-up of the babies born was recorded. Neonatal outcomes have also been reported. Participants and Variables The study was carried out on 342 infertile patients (male, tubal, ovarian, or idiopathic infertility) with at least three supernumerary eggs to freeze; all oocytes were cryopreserved using a slow-freezing protocol previously described (13). All the subjects included in the cryopreservation program were informed about the procedure and provided written, informed consent. Induction of multiple follicular growth was obtained by administering exogenous gonadotropins after pituitary desensitization with GnRH analogue, as previously described (15). Ovulation was induced with 10,000 IU hcg when at least two follicles of 22 mm in diameter were observed, with corresponding E 2 values detected in the bloodstream. Egg retrievals were performed transvaginally via ultrasound guidance approximately 35 to 36 hours after hcg injection; oocytes were cultured in fertilization media (SAGE Cooper Surgical) supplemented with 5% human serum albumin (HSA; SAGE) at 37 C in a 5% CO 2 humidified incubator. Endometrial preparation for the thawing cycles was performed in all women as previously described (16). In case of pregnancy, hormone replacement therapy was continued for 60 days after transfer. Oocyte Culture and Preparation Before Cryopreservation The oocyte cumulus complexes were isolated from their follicular fluid, washed in human tubal fluid medium supplemented with 10% HSA (Irvine Scientific), and cultured in 5% CO 2 at 37 C. Complete removal of the cumulus cells was performed enzymatically using hyaluronidase (80 IU/mL; 1102 VOL. 97 NO. 5 / MAY 2012

3 Fertility and Sterility Sigma) and mechanically with 170-mm and 140-mm micropipettes (Denuding Flexipet, Cook Australia). Only oocytes in metaphase II with no sign of morphologic alterations were cryopreserved, and the others were discarded. Oocyte Freezing Oocytes were cryopreserved using a slow-freezing protocol adapted from the one originally described for embryos (17). Eggs were equilibrated in one-step homemade solution containing 1.5 M PROH supplemented with 20% HSA (Irvine Scientific) for 10 minutes, then transferred for 5 minutes into a loading solution containing 1.5 M PROH þ 0.2 M sucrose þ 20% HSA. Afterward the oocytes were loaded into plastic straws (Paillettes Crystal 133 mm; Cryo Bio System) and placed into an automated Kryo 10 series III biological freezer (Planer Kryo 10/1,7 GB). All the procedures were carried out at room temperature (approximately 25 C). Once the loaded straws were placed in the machine the temperature was gradually lowered from 20 Cto 7 C at a rate of 2 C/min. Manual seeding was induced during a 10-minute holding ramp at 7 C. The temperature was then decreased to 30 Cat a rate of 0.3 C/min and finally rapidly to 150 C at a rate of 50 C/min. The straws were then plunged into liquid nitrogen and stored for later use. Oocyte Thawing The straws were air-warmed for 30 seconds and then placed in a30 C water bath for 40 seconds. The cryoprotectant was removed by stepwise dilution of PROH at room temperature. The thawing solutions were homemade and contained [1] 1.0 M PROH þ 0.3 M sucrose þ 20% HSA (5-minute equilibration); [2] 0.5 M PROH þ 0.3 M sucrose þ 20% HSA (5-minute equilibration); or [3] 0.3 M sucrose þ 20% HSA (10-minute exposure before the final dilution in PBS solution for another 10 minutes). Surviving oocytes were finally placed in culture at 37 C and 5% CO 2 and rechecked 1 hour later to confirm their good status. In all cases insemination was performed within 2 hours after thawing by intracytoplasmic sperm injection, and oocytes were incubated in cleavage medium (SAGE Cooper Surgical) supplemented with 5% HSA (SAGE). Fertilization was checked hours later, and normal zygotes were transferred into fresh cleavage medium (SAGE). Embryos were checked the following day, and those that had cleaved were transferred. Bias To avoid possible bias due to patient age the population was divided into three groups (%34 years, years, and R39 years), and outcome measures were evaluated separately for each group. Statistics Data were analyzed and compared using the c 2 test. Significance was accepted when the c 2 value was >3.84 and P<.05. The Student t test was used to evaluate whether the three groups were comparable for number of patients. RESULTS Three hundred forty-two patients with a mean (SD) age of years underwent 443 thawing cycles; 2,458 oocytes were thawed, and 71.8% survived; of these, 1,296 were microinjected and 1,010 (77.9%) fertilized normally, whereas the rate of abnormal fertilization was 7.4%. The embryos were graded on day 2 from 1 to 4 (best to worst) according to fragmentation rate, cytoplasm quality, and the presence of multinucleated blastomeres (18). Nine hundred fifty-five zygotes cleaved, and of these, 83.8% were grade 1 and 2 embryos with regular blastomeres and a maximum of 10% fragmentation. The rate of four-cell embryo development was higher than the rate of two-cell development (48.1% vs. 25.9%, respectively) at the same observation time, indicating a good cleavage time frame (Table 1). Nine hundred seven embryos were replaced in 394 transfers, resulting in 90 pregnancies. The pregnancy rate per transfer was 22.8%, and per patient was 26.3%. The number of gestational sacs confirmed by ultrasound assessment was 122, with 93 fetal heartbeats (FHBs). The implantation rate per embryo was 13.5%, and the implantation per injected oocyte was 9.4% (Table 2). To avoid possible bias, patients were subsequently divided into three distinct groups and evaluated according to their age: %34 years (group A), years (group B), and R39 years (group C). Complete data are shown in Tables 3 and 4. The results from this age-dependant analysis show comparable (no significant difference) outcomes for biological parameters (survival, fertilization, and cleavage rates), regardless of age. The respective survival rates for the three groups were 74.2%, 70.0%, and 70.6%, the fertilization rates were 79.6%, 79.8%, and 73.1%, and the cleavage rates were TABLE 1 Overall survival, fertilization, and cleavage rates. Patients undergoing at least one 342 thawing cycle Age (y), mean SD Thawing cycles 443 Oocytes Thawed 2,458 Survived 1,766 (71.8) Microinjected 1,296 Fertilization 2PN 1,010 (77.9) 1PN/3PN 96 (7.4) Degenerated 48 (3.7) Embryos Cleaved 955 (94.5) G1 339 (35.5) G2 461 (48.3) G3 104 (10.9) G4 51 (5.3) 2 cells 247 (25.9) 3 cells 129 (13.5) 4 cells 460 (48.1) >4 cells 119 (12.5) Note: Values are number (percentage) unless otherwise noted. PN ¼ pronuclei; G ¼ grade. VOL. 97 NO. 5 / MAY

4 ORIGINAL ARTICLE: ASSISTED REPRODUCTION TABLE 2 Overall clinical outcome. Patients undergoing at least one 342 thawing cycle Thawing cycles 443 Embryo transfers 394 Embryos transferred 907 Pregnancies 90 Pregnancy rate/et 90/394 (22.8) Pregnancy rate/thawing cycle 90/443 (20.8) Pregnancy rate/patient 90/342 (26.3) Multiple pregnancy rate 26/90 (28.9) Gestational sacs 122 FHB 93 Implantation rate (%) 13.5 Implantation/injected oocyte 9.4 rate (%) Abortions 32 (35.5) Babies born at the time of 77 publication Note: Values are number (percentage) unless otherwise noted. 93.2%, 95.4%, and 95.6%, with no significant differences among the groups. Conversely, the clinical outcomes after ET demonstrate the expected age-related decrease. The pregnancy rates per transfer were higher for younger patients, even though no statistical significance was shown (27.7% in group A, 21.4% in group B, and 17.6% in group C). The implantation rate was higher in the A group (16.7%) compared with groups B and C (11.6% and 10.8%, respectively), but the difference was not statistically significant. The implantation per injected egg showed a similar trend, with a high rate TABLE 3 Survival, fertilization, and cleavage of frozen oocytes according to age. Parameter 34 y y R39 y Patients undergoing at least one thawing cycle Age (y), mean SD Thawing cycles Oocytes Thawed Survived 737 (74.2) 570 (70.0) 459 (70.6) Microinjected Fertilization 2PN 410 (79.6) 347 (79.8) 253 (73.1) 1PN/3PN 31 (6.0) 30 (6.9) 35 (10.1) Degenerated 10 (1.9) 19 (4.4) 19 (5.5) Embryos Cleaved 382 (93.2) 331 (95.4) 242 (95.6) G1 133 (34.8) 105 (31.8) 102 (42.2) G2 186 (48.7) 169 (51.0) 106 (43.8) G3 37 (9.7) 41 (12.4) 26 (10.7) G4 26 (6.8) 16 (4.8) 8 (3.3) 2 cells 97 (25.4) 89 (26.9) 60 (24.8) 3 cells 53 (13.9) 47 (14.2) 29 (12.0) 4 cells 180 (47.1) 162 (48.9) 119 (49.1) >4 cells 52 (13.6) 33 (10.0) 34 (14.0) Note: Values are number (percentage) unless otherwise noted. Abbreviations as in Table 1. TABLE 4 Clinical outcome of patients divided according to the age. Parameter 34 y y R39 y Patients undergoing at least one thawing cycle Thawing cycle Embryo transfers Embryos transferred Pregnancies Pregnancy rate/et (%) Pregnancy rate/thawing cycle (%) Pregnancy rate/patient (%) Multiple pregnancies 16 (37.2) 4 (14.3) 6 (31.6) Gestational sacs FHB 49 a 24 a 20 Implantation rate (%) Implantation/injected oocyte (%) Abortions 10 (23.3) 12 (42.8) 10 (52.6) Babies born at the time of publication Note: Values are number (percentage) unless otherwise noted. a P<.05. of 11.8% in group A vs. 8.0% and 7.5% in groups B and C, respectively (nonsignificant). The miscarriage rate was approximately the same in the three groups (10%, 12%, and 10%, respectively; nonsignificant). The number of FHBs was significantly higher (P<.05) in group A (49 FHBs) compared with B (24 FHBs) and C (20 FHBs), as reported in Table 4. At the time of publication 77 babies have been born, and one pregnancy is still ongoing. Overall 32 miscarriages were reported, and they all occurred within 12 weeks of gestation. Neonatal anomalies were reported in one baby, which had choanal atresia. Thirty-five newborns were male, and 42 were female. DISCUSSION To date oocyte freezing has led to the birth of more than 900 babies, using mostly a slow freeze rapid thaw approach. Fortunately the offspring arising from frozen eggs have no significant difference regarding the incidence of abnormalities compared with those found in naturally conceived children (19). Therefore, it has been proposed that the experimental label be removed from oocyte cryopreservation (20). In the last decade, data published by Italian groups (2, 3, 21, 22) have been replicated worldwide (23, 24). In the beginning the slow-freezing process was considered the only approach to cryopreserve eggs and embryos, because of the numerous studies published in the literature. However, after the work published by Kuwayama et al. in 2005 (4) regarding the technique of vitrification, this ultra-rapid freezing method has been evaluated as an alternative for human oocyte cryopreservation. One of the issues that must be resolved when freezing eggs is the preservation of the structural integrity of this unique cell. To accomplish this, various modifications to cryopreservation methods have been made to avoid 1104 VOL. 97 NO. 5 / MAY 2012

5 Fertility and Sterility intracellular ice crystal formation, osmotic shock, or other types of freezing damage that might compromise the developmental competence (9). The results from these studies have revealed that survival is not a guarantee of viability. This was clearly demonstrated when, despite good biological outcomes (25), clinical data were not satisfying (2, 3, 21). Collectively, the survival rate in all the studies was almost 80%, but the implantation rate was approximately 8%, significantly low when compared with embryo cryopreservation outcomes. The modification to slow-freezing protocols described by some authors (26, 27) showed improved results in a limited group of patients. In 2007 we published (13) results using a modification in sucrose concentration that allowed a more gradual dehydration of the oocyte during the freezing process. The survival rate was 75.9%, which was comparable to previously published data (2) from the use of a higher sucrose concentration. Similar rates were reported for fertilization and cleavage rates, showing that this modification was not leading to poor biological outcomes. During the thawing phase a higher sucrose concentration was used, to prevent the osmotic shock that was previously observed when the sucrose concentration was the same during freezing and thawing (24). The different sucrose concentration served to reduce the rate of dehydration of the oocyte during freezing and did not compromise recovery after thawing (13). The excessive shrinkage of the oocyte achieved using 0.3 M sucrose does not compromise the survival rate but significantly lowered the potential for a pregnancy (2, 3, 21). During this procedure, eggs were exposed to a single-step PROH solution with a 1.5-M concentration for 10 minutes. A longer exposure to the cryoprotective agent has shown lower pregnancy and implantation rates (28). After 6 years of experience with this protocol, we are reporting an increased number of cases with consistently reproducible results. After an overall data evaluation, patients were divided in three groups according to their age: group A, %34 years; group B, years; and group C, R39 years. This grouping provides information regarding the age-related potential for a patient to achieve a pregnancy (29). The survival, fertilization, and cleavage rates for good-quality embryos were comparable among the three groups, with no significant differences, indicating that the biological outcomes are not strictly correlated to the average age. However, clinical outcomes were higher in group A (younger patients) compared with groups B and C. The pregnancy rate per transfer was 27.7% in group A and decreased to 21.4% in group B and 17.6% in group C. Because several patients had multiple transfers with the same cohort of oocytes, we also reported the pregnancy rate per patient, which was, respectively, 31.2%, 24.6%, and 21.1% for the three groups. Focusing on the implantation rate, it was observed that the rate was higher in group A compared with groups B and C (16.7%, 11.6%, and 10.8%, respectively). Recently the concept of calculating the implantation rate per oocyte has been introduced (30) to describe the biological loss in IVF. This parameter allows for a comparison of the outcomes using different protocols, provides a more direct correlation of the results based on the number of eggs used, and can give an idea of the approximate number of pregnancies (or live births) that can result from 100 frozen oocytes. Survival, fertilization, cleavage, and embryo quality are no longer considered separately but are effectively consolidated in one measurement. It is possible to compare the outcome of the previously published data using different slow cryopreservation protocols and freezing mixtures. The first report by our group using 0.1 M sucrose (31) was clearly inadequate, with an overall implantation rate per thawed oocyte of 2.3%; this was mostly due to the low survival and fertilization rates. In the following years several studies were reported using an increased (0.3 M) sucrose concentration, and despite the high survival and fertilization results the rate of implantation per thawed oocyte remained low: 2.6% in our group (2), 1.9% in the study by Levi Setti et al. (3), and 2.6% reported by De Santis et al. (21). By using a sodium-depleted protocol, Boldt et al. (26) reported a remarkable implantation rate of 5.3% per thawed egg. However, this result was derived from the treatment of only 23 patients and has yet to be confirmed in a larger patient population. In our previous work (13) a similar value of implantation rate (5.9%) was reported in 78 patients, following the application of a protocol based on different concentrations of sucrose in the freezing (0.2 M) and thawing (0.3 M) solutions and a one-step PROH exposure. Using the same protocol the rate of implantation per thawed egg is consistent with our previous results in the overall group (5.0%) and becomes higher in the younger patients group (6.1%). Nevertheless, it is important to point out that in Italy, because of legal restrictions, not all surviving eggs were injected, so it would be more appropriate to consider the implantation rate per injected oocyte. According to this calculation our implantation rate per injected oocyte was 9.4% in the overall group, whereas when patients were divided by age the rates were 11.8% in group A, 8.0% in group B, and 7.5% in group C. With the introduction of the vitrification technique improved results have been published, primarily by two groups worldwide (32, 33, 34). However, these groups are using open devices that are not approved by the US Food and Drug administration to store biological material, and thus the chosen carrier device for vitrification remains a controversial matter. In 2009 Fadini et al. (35) compared slow-freezing with ultra-rapid cooling methods in programs run in different time periods. A total of 1,348 slow-frozen oocytes were thawed in 286 cycles, whereas 285 vitrified eggs were warmed in 59 cycles. The protocol for slow cooling was based on a 0.3-M sucrose concentration. The author used an open device (Cryoleaf) for vitrification cases. The survival rate obtained with the slowfreezing method was 59.7%, considerably lower compared with the literature (2, 3, 25), whereas the recovery was higher with vitrification (78.8%). Fertilization and implantation rates were also better in the vitrified/warming cycles. Nevertheless, the implantation rate per thawed oocyte was 1.3% for slow freezing and 3.5% for vitrification. In 2009 Cao et al. (36) reported a higher survival rate using vitrification rather than slow freezing (91.8% vs. 61%, respectively) but provided no data on pregnancy and implantation rates. VOL. 97 NO. 5 / MAY

6 ORIGINAL ARTICLE: ASSISTED REPRODUCTION More recently, in 2010 Kim et al. (37) reported a 6.4% implantation rate per warmed oocyte in a group of fertile patients with a mean age of years, using vitrification. Here we report an implantation rate per thawed oocyte of 5.0% in the overall study (all groups combined) and 6.1% in group A (younger patients). These results obtained with slow freezing are comparable to those reported in the literature using vitrification. Few comparative randomized studies are available in the literature; in 2010 Smith et al. (38) published a randomized study comparing the two methods and also reported that oocyte survival and pregnancy rates were significantly higher after vitrification compared with slow cooling (67% vs. 81% for survival rate and 13% vs. 38% for pregnancy rate, respectively). Although these data suggest that vitrification may be a more efficient technique for successful cryopreservation of oocytes, the protocol used in these studies for slow freezing was not the method previously demonstrated (13) to give better outcomes. In contrast, a closed vitrification system has been used by Grifo and Noyes (24) to cryopreserve oocytes in cancer patients, with results comparable to the slow-freezing method (using a 0.3-M sucrose concentration). The survival rates were not different between the two protocols (88% for slow cooling and 95% for vitrification), and comparable results were also observed for fertilization rates and blastocyst formation. These results show that either method can be successful. Conversely, the majority of randomized studies currently available in the literature compare vitrification (using open devices) with slow freezing using 0.3 M sucrose. Although the open system is the best option for vitrification outcomes, the high sucrose concentration in the slow-freezing protocols is suboptimal and showed poor outcomes worldwide (2, 3, 21). The present study aimed to determine the survival and pregnancy rates after slow freezing of oocytes using the modified protocol with M sucrose and confirms that these rates are comparable to our previously published (2007) report, which involved a smaller number of patients (13). Our results were also compared with those reported in the literature using slow-freezing and vitrification methods (2, 3, 21, 28, 38). Another aspect considered in this study concerns the health of babies born. It has been assumed that cryodamage induced by freezing could somehow influence the risk of developmental anomalies in babies born after oocyte cryopreservation, but lately this concern has been partially overcome because of the growing number of births reported worldwide (19). In this study 77 births were reported, with one ongoing pregnancy at the time of publication. No abnormal karyotype was reported, and one baby showed choanal atresia. Currently available evidence does not show an increase in the incidence of anomalies at birth using oocyte freezing. Nevertheless further clinical studies, including long-term follow-up, should be considered with either slow-freezing or vitrification techniques. REFERENCES 1. Chen C. Pregnancies after human oocyte cryopreservation. Lancet 1986;1: Borini A, Sciajno R, Bianchi V, Sereni E, Flamini C, Coticchio G. Clinical outcome of oocyte cryopreservation after slow cooling with a protocol utilizing a high sucrose concentration. Hum Reprod 2006;21: Levi Setti PE, Albani E, Novara PV, Cesana A, Morreale G. Cryopreservation of supernumerary oocytes in IVF/ICSI cycles. Hum Reprod 2006;21: Kuwayama M, Vajta G, Kato O, Leibo SP. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online 2005;11: De Santis L, Cino I, Coticchio G, Fusi FM, Papaleo E, Rabellotti E, et al. Objective evaluation of the viability of cryopreserved oocytes. Reprod Biomed Online 2007;15: Nottola SA, Macchiarelli G, Coticchio G, Bianchi S, Cecconi S, De Santis L, et al. Ultrastructural of human mature oocytes after slow cooling cryopreservation using different sucrose concentrations. Hum Reprod 2007;22: Coticchio G, De Santis L, Rossi G, Borini A, Albertini D, Scaravelli G, et al. Sucrose concentration influences the rate of human oocytes with normal spindle and chromosome configuration after slow cooling cryopreservation. Hum Reprod 2006;21: Bianchi V, Coticchio G, Fava L, Flamigni C, Borini A. Meiotic spindle imaging in human oocytes frozen with a slow freezing procedure involving high sucrose concentration. Hum Reprod 2005;20: Paynter SJ, Borini A, Bianchi V, De Santis L, Flamigni C, Coticchio G. Volume changes of mature human oocytes on exposure to cryoprotectant solutions used in slow cooling procedures. Hum Reprod 2005;20: Yang DS, Blohm PL, Winslow KL, Cramer L. A twin pregnancy after microinjection of human cryopreserved oocyte with a specially developed oocyte cryopreservation regime. Fertil Steril 1998;70(Suppl 1):S Quintans CJ, Donaldson MJ, Bertolino MV, Pasquallini RS. Birth of two babies using oocytes that were cryopreserved in a choline-based freezing medium. Hum Reprod 2002;17: Boldt J, Cline D, McLaughlin D. Human oocyte cryopreservation as an adjunct to IVF-embryo transfer cycles. Hum Reprod 2003;18: Bianchi V, Coticchio G, Distratis V, Di Giusto N, Flamigni C, Borini A. Differential sucrose concentration during dehydration (0.2 mol/l) and rehydration (0.3 mol/l) increases the implantation rate of frozen human oocytes. Reprod Biomed Online 2007;14: Benagiano G, Gianaroli L. The Italian Constitutional Court modifies Italian legislation on assisted reproduction technology. Reprod Biomed Online 2010;20: Dal Prato L, Borini A, Coticchio G, Cattoli M, Flamigni C. Half-dose depot triptorelin in pituitary suppression for multiple ovarian stimulation in assisted reproduction technology: a randomized study. Hum Reprod 2004;19: Dal Prato L, Borini A, Cattoli M, Bonu MA, Sciajno R, Flamigni C. Endometrial preparation for frozen-thawed embryo transfer with or without pretreatment with gonadotropin-releasing hormone agonist. Fertil Steril 2002;77: Lassalle B, Testart J, Renard JP. Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol. Fertil Steril 1985;44: Cummins JM, Breen TM, Harrison KL, Shaw JM, Wilson LM, Hennessey JF. A formula for scoring human embryo growth rates in in vitro fertilization: its value in predicting pregnancy and in comparison with visual estimates of embryo quality. J In Vitro Fert Embryo Transf 1986;3: Noyes N, Porcu E, Borini A. Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies. Reprod Biomed Online 2009;18: Noyes N, Boldt J, Nagy P. Oocyte cryopreservation: is it time to remove its experimental label? J Assist Reprod Genet 2010;27: De Santis L, Cino I, Rabellotti E, Papaleo E, Calzi F, Fusi FM, et al. Oocyte cryopreservation: clinical outcome of slow-cooling protocols differing in sucrose concentration. Reprod Biomed Online 2007;14: Parmegiani L, Bertocci F, Garello C, Salvarani MC, Tambuscio G, Fabbri R. Efficiency of human oocyte slow freezing: results from five assisted reproduction centers. Reprod Biomed Online 2009;18: VOL. 97 NO. 5 / MAY 2012

7 Fertility and Sterility 23. Gook DA, Edgar DH. Human oocyte cryopreservation. Hum Reprod Update 2007;13: Grifo J, Noyes N. Delivery rate using cryopreserved oocyte is comparable to conventional in vitro fertilization using fresh oocytes: potential fertility preservation for female cancer patients. Fert Steril 2010;93: Fabbri R, Porcu E, Marsella T, Rocchetta G, Venturoli S, Flamigni C. Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod 2001;16: Boldt J, Tidswell N, Sayers A, Kilani R, Cline D. Human oocyte cryopreservation: 5-year experience with a sodium-depleted slow freezing method. Reprod Biomed Online 2006;13: Azambuja R, Petracco A, Okada L, Michelon J, Badalotti F, Badalotti M. Experience of freezing human oocytes using sodium-depleted media. Reprod BioMed Online 2011;22: Borini A, Levi Setti PE, Anserini P, De Luca R, De Santis L, Porcu E, et al. Multicentric observational study on slow-cooling oocyte cryopreservation: clinical outcome. Fertil and Steril 2010;94: Dor J, Seidman DS, Ben-Shlomo I, Levran D, Ben-Rafael Z, Mashiach S. Cumulative pregnancy rate following in-vitro fertilization: the significance of age and infertility aetiology. Hum Reprod 1996;11: Patrizio P, Sakkas D. From oocyte to baby: a clinical evaluation of the biological efficiency of in vitro fertilization. Fertil Steril 2009;91: Borini A, Bonu MA, Coticchio G, Bianchi V, Cattoli M, Flamigni C. Pregnancies and births after oocyte cryopreservation. Fertil Steril 2004;82: Cobo A, Kuwayama M, Perez S, Ruiz A, Pellicer A, Remohí J. Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method. Fertil Steril 2008;89: Ubaldi F, Anniballo R, Romano S, Baroni E, Albicci L, Colamaria S, et al. Cumulative ongoing pregnancy rate achieved with oocyte vitrification and cleavage stage transfer without embryo selection in a standard infertility program. Hum Reprod 2010;25: Cobo A, Vajta G, Remohí J. Vitrification of human mature oocytes in clinical practice. Reprod Biomed Online 2009;19(Suppl 4): Fadini R, Brambillasca M, Mignini Renzini M, Merola M, Comi R, De Ponti E, et al. Human oocyte cryopreservation: comparison between slow and ultra rapid methods. Reprod Biomed Online 2009;19: Cao YX, Xing Q, Li L, Cong L, Zhang Z, Wei Z, et al. Comparison of survival and embryonic development in human oocytes cryopreserved by slow freezing and vitrification. Fertil Steril 2009;92: Kim TJ, Laufer LR, Hong SW. Vitrification of oocytes produces high pregnancy rates when carried out in fertile women. Fertil Steril 2010;93: Smith GD, Serafini PC, Fioravanti J, Yadid I, Coslovsky M, Hassun P, et al. Prospective randomized comparison of human oocyte cryopreservation with slow-rate freezing or vitrification. Fertil Steril 2010;94: VOL. 97 NO. 5 / MAY