- Email: [email protected]
Perinatal and neonatal outcomes of 494 babies delivered from 972 vitrified embryo transfers
Perinatal and neonatal outcomes of 494 babies delivered from 972 vitrified embryo transfers Wenhao Shi, M.D., Xia Xue, M.D., Silin Zhang, M.D., Wanqiu Zhao, M.D., Shan Liu, Ph.D., Hanying Zhou, M.D., Min Wang, M.D., and Juanzi Shi, Ph.D. Assisted Reproduction Center, Maternal and Child Health Care Hospital of Shaanxi Province, Xi'an, People's Republic of China
Objective: To evaluate the safety of vitrification of embryos by comparing the perinatal outcome of vitrified day 3 ETs with fresh cleavage-stage ETs. Design: Retrospective analysis. Setting: Assisted reproduction center. Patient(s): A total of 806 women with 494 infants delivered were included. Intervention(s): Supernumerary embryos and embryos of patients at risk of ovarian hyperstimulation syndrome were vitrified using a Cryotop carrier in an open system and were transferred after warming. Main Outcome Measure(s): Implantation and pregnancy rates, obstetric outcomes, neonatal outcomes, and congenital birth defects. Result(s): A total of 2,543 vitrified embryos were warmed, and 2,375 survived and were transferred. The implantation, pregnancy, miscarriage, and live birth rates were 26.91%, 47.22%, 6.07%, and 38.58%, respectively. In singletons, the mean birth weight was 3,337.44 g, and the mean Apgar scores were 8.91, 9.85, and 9.89 at 1, 5, and 10 minutes, respectively. In multiple gestations, the mean birth weight was 2,556.45 g, and the mean Apgar scores were 8.90, 9.34, and 9.47 at 1, 5, and 10 minutes, respectively. Only seven congenital malformations were observed among the 494 babies. Compared with fresh cleavage-stage ETs, the mean birth weight in the vitrified group was higher in both singleton and multiple gestations. All the other results were similar between the two groups. Conclusion(s): Compared with fresh ETs, vitrified day 3 ET shows no significant differences in obstetrical and neonatal outcomes. The study suggests that vitrification with Cryotop is an effective and safe method for the cryopreservation of human cleavage-stage (day 3) embryos. (Fertil SterilÒ 2012;97: 1338–42. Ó2012 by American Society for Reproductive Medicine.) Key Words: Vitrification, cleavage-stage embryo, Cryotop, neonatal outcome, congenital birth defects
V
itrification is an ultrarapid method of freezing cells in a glass-like state, which avoids ice crystal formation and thereby reduces associated chilling injuries. The benefit of vitrification lies in its simplicity, as the setup time is minimal, and it does not require expensive freezing equipment as used in the slowcooled method (1, 2). Vitrification has become a popular alternative method for embryo cryopreservation. However, there are still some concerns about the impact of toxicity on embryos from using a very high
concentration of cryoprotectants and also about the risks of direct contact with liquid nitrogen (2, 3). In some assisted reproductive technology (ART) clinics, the vitrification method has replaced the traditional slow-cooled method for the cryopreservation of embryos. The current trend of transferring fewer embryos has resulted in more surplus embryos for cryopreservation. Although vitrification has been used widely in ART clinics, the safety of this technique in terms of neonatal outcomes of babies delivered from vitrified embryos is still limited
Received January 1, 2012; revised February 15, 2012; accepted February 29, 2012; published online March 30, 2012. W.S. has nothing to disclose. X.X. has nothing to disclose. S.Z. has nothing to disclose. W.Z. has nothing to disclose. S.L. has nothing to disclose. H.Z. has nothing to disclose. M.W. has nothing to disclose. J.S. has nothing to disclose. The work was supported by funds from the Maternal and Child Health Care Hospital of Shaanxi Province, without receiving any financial support from any third party. Reprint requests: Juanzi Shi, Ph.D., Chair Professor of Center for Assisted Reproduction Technology, Maternal and Child Health Care Hospital of Shaanxi Province, Xi'an, 710003, People's Republic of China (E-mail: [email protected]). Fertility and Sterility® Vol. 97, No. 6, June 2012 0015-0282/$36.00 Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2012.02.051 1338
(4, 5). Rama Raju et al. (5) analyzed 312 cycles from vitrified day 3 embryos and 604 cycles from fresh embryos, and the results supported the safety of vitrification using cryoloop. In our IVF center, we have been using vitrification with Cryotop for embryo cryopreservation for 5 years. Thus, we have a large number of babies delivered from vitrified embryos. In this study, we compared the obstetrical and neonatal outcomes of babies born from vitrified day 3 embryos with those born from fresh embryos from our large data sets.
MATERIALS AND METHODS Patients The Assisted Reproduction Center is a public clinic in Shaanxi Province, China. The policy of the IVF/intracytoplasmic sperm injection (ICSI) program is to perform day 3 ETs. Patients' supernumerary embryos (mostly grade I and VOL. 97 NO. 6 / JUNE 2012
Fertility and Sterility® II embryos) are preserved by vitrification on the day of transfers. The frozen-thawed ET is carried out in those patients who failed to conceive after fresh ET. In patients who were at risk of ovarian hyperstimulation syndrome, no fresh embryos were transferred and all embryos were vitrified. These vitrified embryos were warmed and usually transferred 3 months after oocyte collection. We performed a total of 972 ET cycles with vitrifiedwarmed embryos in 806 patients between January 2008 and December 2010. A total of 2,543 vitrified day 3 embryos were warmed; 2,375 survived (93.39%) and were transferred. During the same period, a total of 1,498 fresh ET cycles were performed in 1,148 patients, which was used as a control group. The main etiology of infertility for the vitrified and fresh embryos groups were male factor (22.58% vs. 25.35%) and female factor (63.52% vs. 59.76%), including tuboperitoneal factors, polycystic ovary syndrome, low ovarian reserve, and endometriosis. This study was approved by the Ethics Review Board of the Maternal and Child Health Care Hospital of Shaanxi Province.
Clinical Procedures, Embryo Culture, and Grading All patients used long and short protocols with GnRH agonist (GnRH-a, Decapeptyl) and recombinant FSH (GONAL-f, Merck Serono; Puregon, Organon) for ovarian hyperstimulation. Thirty-six hours after hCG administration, oocytes were retrieved by transvaginal ultrasound-guided aspiration. Oocytes were fertilized using either conventional IVF or ICSI and incubation in fertilization media (Vitrolife). Normal fertilization was assessed and confirmed by the presence of two pronuclei and second polar body at 16–18 hours after insemination. The embryos were washed and cultured in cleavage media (Vitrolife) for 48 hours before transfer. We used a combination of blastomere number, blastomere size, and fragmentation for embryo scoring (6–8). Based on embryonic cell numbers on day 3, embryos with 8–10 blastomeres were classified as grade I; embryos with 6–7 blastomeres or more than 10 blastomeres were classified as grade II, embryos with 4–5 blastomeres were classified as grade III, and embryos with fewer than 4 blastomeres were classified as grade IV. Based on the uniformity of the blastomeres, the embryos without significant differences in blastomere volume were classified as grade I, embryos with one to two significant differences were classified as grade II, and embryos with two or more significant differences were classified as grade III. Based on an embryo's fragmentation, four grades were classified: grade I, embryos with <10% fragmentation; grade II, embryos with 10%–20% fragmentation; grade III, embryos with 20%–30% fragmentation; and grade IV, embryos with >30% fragmentation. Embryo grades were scored comprehensively according to the above three criteria.
Vitrification The Cryotop method for vitrification was used as described elsewhere by Kuwayama et al. (9). Embryos were equilibrated in equilibration solution (7.5% ethylene glycol [EG] þ 7.5% dimethylsulfoxide [DMSO] in TCM199 medium þ 20% VOL. 97 NO. 6 / JUNE 2012
synthetic serum substitute [SSS]) for 5 minutes at room temperature. They were then placed into vitrification solution (VS; 15% EG þ 15% DMSO þ 0.5 M sucrose). After less than 1 minute in VS, the embryos were placed on the Cryotop strip and plunged into liquid nitrogen immediately. For warming, the Cryotop strip was removed from the liquid nitrogen and plunged directly into thawing solution (1.0 M sucrose in TCM199 þ 20% SSS) for 1 minute at 37 C. After 1 minute, the embryos were placed in dilution solution (0.5 M sucrose in TCM199 þ 20% SSS) at room temperature for 3 minutes. They were then put into washing solution (WS; TCM199 þ 20% SSS) for 5 minutes and 5 minutes again in WS. Finally, the embryos were placed in well-balanced G2.5 (Vitrolife) medium containing 5% human serum albumin (HSA) and cultured in a 37 C, 6% CO2 incubator (America Forma) overnight before transfer. Embryos with R50% survival blastomeres were recorded as survival. Tools and solutions required for vitrification were obtained from Kitazato (Kitazato BioPharma Co.).
Outcome Measures The outcome data were obtained from a postal questionnaire of parents after delivery. The obstetric outcome measures referred to implantation and pregnancy rates, preterm birth rate, mean gestational age, and pregnancy-related complications. The neonatal outcomes evaluated were birth weight, Apgar scores, neonatal intensive care unit admission, neonatal complications, and congenital birth defects.
Statistical Analysis All data management and analyses were performed using SPSS 16.0 software. The differences in outcomes between the two groups were analyzed using c2 analysis, t-test, or Mann-Whitney U-test. Fisher's exact probability test was used in case the expected frequency was less than 5. P< .05 was considered statistically significant.
RESULTS Table 1 shows the clinical parameters between vitrified and fresh ET cycles. In the vitrified embryos group, a total of 972 cycles were performed. The mean maternal age was 29.94 4.08 years. The main diagnoses of infertility were female factor (63.52%), male factor (22.58%), and combined all other factors (13.90%). In this group, 691 (71.09%) were treated by IVF and 248 (25.51%) by ICSI. The mean number of embryos transferred per ET was 2.4, with an implantation rate of 26.91%. A total of 459 clinical pregnancies (47.22%) were achieved: 375 (38.58%) live births, 59 miscarriages, six induced abortions due to fetal anomaly and maternal disease, two (0.44%) fetal deaths, and six pregnancies (1.31%) without completion of the follow-up. In the fresh embryo group, a total of 1,498 cycles were performed in 1,148 patients. The mean maternal age was 30.27 4.11 years. The main diagnoses of infertility were female factor (59.76%), male factor (25.35%), and combined all other factors (14.90%). In this group, 993 (66.29%) were treated by IVF and 370 (24.70%) by ICSI. The mean number of embryos transferred per ET was 2.2, with an implantation rate 30.87%. A total of 733 clinical pregnancies (48.93%) 1339
ORIGINAL ARTICLE: ASSISTED REPRODUCTION
TABLE 1 Comparison of clinical parameters between vitrified and fresh ETs. Parameter
Fresh ETs
No. of patients 1,148 No. of cycles transferred 1,498 Patient age, y 30.27 4.11 Main etiology of infertility, n (%) Female factor 686 (59.76) Male factor 291 (25.35) Combined factor and 171 (14.90) other factors Fertilization procedure, n (%) IVF 993 (66.29) ICSI 370 (24.70) No. of embryos transferred 2.18 0.45 Implantation rate (%) 1,007 (30.87) No. of miscarriages 85 (5.67) (% per ET) No. of ectopic pregnancies 13 (1.77) (% per clinical pregnancy) No. of induced abortions 5c (0.68) (% per clinical pregnancy) No. of fetal deaths 3 (0.41) (% per clinical pregnancy) No. of patients lost to 13 (1.77) follow-up (% per clinical pregnancy) No. of live deliveries 614 (40.99) (% per ET) Singletons (% per live delivery) 421 (68.57) Twins (% per live delivery) 192 (31.27) Triplets (% per live delivery) 1 (0.16)
Vitrified ETs P value 806 972 29.94 4.08
.049a
512 (63.52) 182 (22.58) 112 (13.90)
.117b
691 (71.09) 248 (25.51) 2.42 0.57 639 (26.91) 59 (6.07)
.012 .648 .000 .001 .682
11 (2.40)
.456
d
6 (1.31)
.272
2 (0.44)
1.000
6 (1.31)
.532
375 (38.58)
.233
256 (68.27) 119 (31.73) 0
.922 .879 –
Note: Continuous values are expressed as mean SD. a t-test. b Mann-Whitney U-test. c All for fetal anomaly. d Two for fetal anomaly, four for acute infection. Shi. Perinatal outcome of vitrified embryos. Fertil Steril 2012.
were achieved: 614 live births (40.99%), 85 miscarriages, five induced abortions owing to fetal anomaly and maternal disease, three (0.41%) fetal deaths, and 13 (1.77%) pregnancies without completion of the follow-up. Table 2 shows the obstetrical and neonatal outcomes of singleton and multiple gestations in both groups. For the singleton deliveries of vitrified ETs, mean gestational age and preterm deliveries were not significantly different from the fresh ETs. There were 256 babies (126 males and 130 females) born from vitrified embryos with a mean birth weight of 337.44 505.60 g, which was significantly heavier than the singleton delivery of fresh ETs. The mean Apgar scores were very similar between the two groups, except they were slightly higher in vitrified embryos at 1 minute. Thirty (11.72%) newborns required admission to the neonatal care unit. For the multiple gestations, there were no statistically significant differences in mean gestational age, preterm birth rate, the mean Apgar scores, pregnancy-related complications (antenatal bleeding, gestational diabetes, or pregnancy-induced hypertension), and delivery methods between the two groups. However, again, the mean birth weight of babies delivered from the vitrified embryo group was significantly heavier than that of babies delivered from the fresh embryo group. 1340
Table 3 shows all the congenital birth defects. In the vitrified embryo group, seven (five major and two minor) congenital malformations were observed, including three with congenital heart disease and four with others (spinal bifida, lower limb malformation, inguinal hernia, and polydactyly). In contrast with the fresh embryo group, five (three major and two minor) congenital malformations were observed including two with congenital heart disease, one with trisomy 21, and two with laryngeal cartilage hypoplasia. There were no statistically significant differences between the two groups.
DISCUSSION In this study with a large sample size, we clearly showed that there were no significant differences in perinatal and neonatal outcomes of babies delivered with vitrified versus fresh ETs. This result suggests that vitrification is a safe and alternative method for the cryopreservation of human embryos. Before introduction of vitrification to ART clinics, the traditional slow-cooled embryo freezing technique had been widely used, but inconsistent or low embryo survival rates are a common problem for some ART clinics. Thus vitrification of human embryos has recently become a popular alternative, method that may improve the survival rate of cryopreserved embryos. In the 1930s, B.J. Luyet, who first formulated the theory of vitrification, saw great potential for the successful vitrification of colloids and cells (10). Rall and Fahy successfully vitrified a mouse embryo in 1985 (11), which promoted its widespread use in clinical applications. Although the traditional slowfreezing protocols have been greatly improved in the last two decades, vitrification has provided a more efficient method to preserve oocytes and embryos (12–14). The superiority of vitrification is based on its ability to completely avoid ice formation, as well as its simplicity, rapidity, and economy. Despite its advantages, the main problem is the risk of crosscontamination in liquid nitrogen (3, 15) and the toxic effects (biochemical and osmotic) inherent in the use of high concentrations of cryoprotectants (2). Although vitrification has been used in clinics for the past decade, only a few studies have reported on the safety of this technique in terms of neonatal outcome and child follow-up (4, 5). In 2005, Takahashi et al. first reported that there were no significant differences in the perinatal outcomes of 147 babies born from vitrified blastocysts compared with those born from fresh blastocysts (16). Although more and more clinics have now used vitrification for embryo cryopreservation, few studies have reported on the safety of the technique. In 2009, Rama Raju reported that there were no statistically significant differences in the mean gestational age, incidence of preterm deliveries, mean birth weight, mean Apgar score, and incidence of congenital birth defects between vitrified day 3 embryos and fresh ETs (5). However, only 89 babies were delivered from the vitrified embryos. Thus their sample size was too small to provide sufficient statistical power. In this study, we analyzed the outcomes of 494 babies delivered from vitrified embryos and showed that all prenatal and obstetric parameters were similar between vitrified and fresh embryos. VOL. 97 NO. 6 / JUNE 2012
Fertility and Sterility®
TABLE 2 Comparison of obstetrical and neonatal outcomes between vitrified and fresh ETs. Singleton gestation Parameter No. of vaginal deliveries (%) No. of cesarean sections (%) Mean gestational age, wk No. of preterm deliveries (<37 wk) (%) No. of very preterm deliveries (<32 wk) (%) No. of cases of antenatal bleeding (%) No. of cases of gestational diabetes (%) No. of cases of pregnancyinduced hypertension (%) Live birth Male Female Mean birth weight, g Mean Apgar score 1 min 5 min 10 min Birth weight <1,500 g (%) Birth weight 1,500–2,499 g (%) Birth weight 2,500–4,000 g (%) Birth weight R4,000 g (%) Neonatal intensive care unit admission (%) Neonatal death
Neonatal complications Pathological jaundice Respiratory problems Low birth weight Neonatal septicemia Others
Multiple gestation
Fresh
Vitrification
Fresh
Vitrification
76 (18.05) 345 (81.95) 38.85 1.47 29 (6.89)
34 (13.28) 222 (86.72) 39.02 1.67 17 (6.64)
21 (10.88) 172 (89.12) 36.67 2.01 89 (46.11)
11 (9.24) 108 (90.76) 36.77 1.56 62 (52.10)
1 (0.24)
3 (1.17)
6 (3.11)
1 (0.84)
113 (26.84)
60 (23.44)
62 (32.12)
34 (28.57)
14 (3.33)
6 (2.34)
12 (6.22)
5 (4.20)
16 (3.80)
10 (3.91)
17 (8.81)
19 (15.97)
421 197 (46.79) 224 (53.21) 3,216.87 451.30
256 126 (49.22) 130 (50.78) 3,337.44 505.60a
386 178 (46.11) 208 (53.89) 2,435.23 435.74
238 107 (44.96) 131 (55.04) 2,556.45 432.91a
9.00 0.07 9.91 0.30 9.92 0.27 0 24 (5.70) 371 (88.12) 26 (6.18) 59 (14.01)
8.91 0.45a 9.85 0.47 9.89 0.37 2 (0.78) 10 (3.91) 221 (86.33) 23 (8.98) 30 (11.72)
8.91 0.33 9.36 0.52 9.45 0.57 10 (2.59) 186 (48.19) 190 (49.22) 0 169 (43.78)
8.90 0.39 9.34 0.56 9.47 0.56 0b 99 (41.60)b 138 (57.98)b 1 (0.42)b 71 (30.08)c
0
20 (2 for HDN) 17 18 0 4 (1 for hypoxic-ischemic encephalopathy, 1 for diarrhea, 1 for peptic ulcer, 1 for intracranial haemorrhage)
4 (all for asphyxia)
11 10 5 1 (complicating pneumonia) 0
5 (3 for asphyxia, 1 for congenital heart disease, 1 for neonatal necrotizing enterocolitis)
2 (1 for spinal bifida, 1 for lower limb malformation)
13 21 129 0
11 (1 for HDN) 4 56 0
1 (fungus infection)
1 (fever)
Note: Values are expressed as mean SD. HDN ¼ hemolytic disease of newborn. a P< .05 (t-test). b P< .05 (Mann-Whitney U-test). c P< .05 (c2 analysis). Shi. Perinatal outcome of vitrified embryos. Fertil Steril 2012.
To reduce the toxic effects of the high concentrations of cryoprotectants, it is important to cool the embryos at the fastest possible rate to guarantee vitrification (9). As a result, a physicochemical trade-off exists between the cooling rates and the solute concentrations needed for vitrification (2). An open system was applied in which the embryos are put directly into liquid nitrogen to achieve the fastest possible cooling rate. The carrier is a Cryotop device (Kitazato Supply Co.), which is available to a minimum volume (the smaller the volume of the sample, the higher the cooling rate) (17, 18). To avoid the bias due to multiple pregnancies, perinatal outcomes of singleton and multiple gestations must be compared separately. In this study, the obstetrical and neonatal outcomes were compared between vitrified day 3 ETs and fresh ETs. The VOL. 97 NO. 6 / JUNE 2012
vitrified group was slightly younger than the fresh group, but the difference was not statistically and clinically significant. Given that some of the patients in the frozen ET programs had experienced pregnancy failure after their last fresh or frozen ET cycle, more embryos were intended for transfer in this group. Thus the mean number of embryos transferred was higher and the implantation rate was lower in the vitrified group than in the fresh group. There were no significant differences in terms of mean gestational age, preterm birth rate, or the rate of pregnancy-related complications for both singleton and multiple gestation pregnancies between the two groups. However, the mean birth weight of babies (both singleton and multiple gestation) delivered from vitrified embryos was significantly heavier than that 1341
ORIGINAL ARTICLE: ASSISTED REPRODUCTION 2.
TABLE 3
3.
Incidence of major and minor malformations of babies delivered from vitrified or fresh ETs. Birth defects Major Minor Total (%)
Fresh ETs
Vitrified ETs
3: 1 trisomy 21, 5: 3 congenital heart disease, 2 congenital heart 1 spinal bifida, 1 lower disease limb malformation 2 laryngeal cartilage 2: 1 inguinal hernia, hypoplasia 1 polydactyly 5 (0.62) 7 (1.42)
4.
5. 6.
Shi. Perinatal outcome of vitrified embryos. Fertil Steril 2012.
7.
of fresh embryos. Although babies from multiple gestations had significantly fewer admissions to the neonatal care unit for the vitrified group than the fresh embryo group, there were no significant differences in the singleton babies between the two groups. In conclusion, this study clearly shows that there are no differences in obstetrical and neonatal outcomes of babies delivered between vitrified and fresh ETs. Thus vitrification using Cryotop is a safe and effective method for the cryopreservation of human embryos. However, prospective and randomized control trials in a larger sample size and a long-term follow-up of babies are still needed to further validate the safety of vitrification of human embryos. Acknowledgments: The authors thank all the doctors, scientists, and embryologists in the Maternal and Child Health Care Hospital of Shaanxi Province for their assistance with the data collection, as well the patients for participating in this study. We greatly appreciate and thank Dr. De-Yi Liu from the Melbourne IVF and the University of Melbourne in Australia for his comments and revision of the manuscript. This study was financially supported by the Maternal and Child Health Care Hospital of Shaanxi Province.
REFERENCES 1.
Chian RC, Quinn P. Fertility cryopreservation. New York: Cambridge, UK: Cambridge University Press, 2010.
1342
8. 9.
10. 11. 12.
13.
14.
15. 16.
17.
18.
Roger Gosden. Cryopreservation: a cold look at technology for fertility preservation. Fertil Steril 2011;96:264–8. Bielanski A, Bergeron H, Devenish J. Microbial contamination of embryos and semen during long term banking in liquid nitrogen. Cryobiology 2003;46:146–52. €derstro €m-Anttila V, Bergh C, et al. Children born after Wennerholm UB, So cryopreservation of embryos or oocytes: a systematic review of outcome data. Hum Reprod 2009;24:2158–72. Rama Raju GA, Prakash GJ, Krishna KM, et al. Neonatal outcome after vitrified day 3 embryo transfers: a preliminary study. Fertil Steril 2009;92:143–8. Veeck LL. Preembryo grading and degree of cytoplasmic fragmentation. In: An atlas of human gametes and conceptuses: an illustrated reference for assisted reproductive technology. New York: Parthenon Publishing; 1999:46–51. €gren A, et al. Human embryos with unevenly Hardarson T, Hanson C, Sjo sized blastomeres has lower pregnancy and implantation rates: indications for aneuploidy and multinucleation. Hum Reprod 2001;16:313–8. Van Royen E, Mangelschots K, Vercruyssen M, et al. Multinucleation in cleavage stage embryos. Hum Reprod 2003;18:1062–9. Kuwayama M, Vajta G, Kato O, Leibo SP. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online 2005;11:300–8. Luyet BJ. The vitrification of organic colloids and of protoplasm. Biodynamica 1937;1:1–14. Rall WF, Fahy FM. Ice-free cryopreservation of mouse embryos at 196 C by vitrification. Nature 1985;313:573–5. Cobo A, Diaz C. Clinical application of oocyte vitrification: a systematic review and meta-analysis of randomized controlled trials. Fertil Steril 2011; 96:277–85. Rezazadeh VM, Eftekhari-Yazdi P, Karimian L. Vitrification versus slow freezing gives excellent survival, post warming embryo morphology and pregnancy outcomes for human cleaved embryos. Assist Reprod Genet 2009; 26:347–54. Loutradi KE, Kolibianakis EM, Venetis CA, et al. Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis. Fertil Steril 2008;90:186–93. Bielanski A, Nadin-Davis S, Sapp T, Lutze-Wallace C. Viral contamination of embryos cryopreserved in liquid nitrogen. Cryobiology 2000;40:110–6. Takahashi K, Mukaida T, Goto T, et al. Perinatal outcome of blastocyst transfer with vitrification using cryoloop: a 4-year follow-up study. Fertil Steril 2005;84:88–92. Cobo A, Kuwayama M, Perez S, et al. Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method. Fertil Steril 2008;89:1657–64. Antonella B, Marta C, Francesco T, et al. Ultrastructural evaluation of human metaphase II oocytes after vitrification: closed versus open devices. Fertil Steril 2011;95:928–35.
VOL. 97 NO. 6 / JUNE 2012
Objective: To evaluate the safety of vitrification of embryos by comparing the perinatal outcome of vitrified day 3 ETs with fresh cleavage-stage ETs. Design: Retrospective analysis. Setting: Assisted reproduction center. Patient(s): A total of 806 women with 494 infants delivered were included. Intervention(s): Supernumerary embryos and embryos of patients at risk of ovarian hyperstimulation syndrome were vitrified using a Cryotop carrier in an open system and were transferred after warming. Main Outcome Measure(s): Implantation and pregnancy rates, obstetric outcomes, neonatal outcomes, and congenital birth defects. Result(s): A total of 2,543 vitrified embryos were warmed, and 2,375 survived and were transferred. The implantation, pregnancy, miscarriage, and live birth rates were 26.91%, 47.22%, 6.07%, and 38.58%, respectively. In singletons, the mean birth weight was 3,337.44 g, and the mean Apgar scores were 8.91, 9.85, and 9.89 at 1, 5, and 10 minutes, respectively. In multiple gestations, the mean birth weight was 2,556.45 g, and the mean Apgar scores were 8.90, 9.34, and 9.47 at 1, 5, and 10 minutes, respectively. Only seven congenital malformations were observed among the 494 babies. Compared with fresh cleavage-stage ETs, the mean birth weight in the vitrified group was higher in both singleton and multiple gestations. All the other results were similar between the two groups. Conclusion(s): Compared with fresh ETs, vitrified day 3 ET shows no significant differences in obstetrical and neonatal outcomes. The study suggests that vitrification with Cryotop is an effective and safe method for the cryopreservation of human cleavage-stage (day 3) embryos. (Fertil SterilÒ 2012;97: 1338–42. Ó2012 by American Society for Reproductive Medicine.) Key Words: Vitrification, cleavage-stage embryo, Cryotop, neonatal outcome, congenital birth defects
V
itrification is an ultrarapid method of freezing cells in a glass-like state, which avoids ice crystal formation and thereby reduces associated chilling injuries. The benefit of vitrification lies in its simplicity, as the setup time is minimal, and it does not require expensive freezing equipment as used in the slowcooled method (1, 2). Vitrification has become a popular alternative method for embryo cryopreservation. However, there are still some concerns about the impact of toxicity on embryos from using a very high
concentration of cryoprotectants and also about the risks of direct contact with liquid nitrogen (2, 3). In some assisted reproductive technology (ART) clinics, the vitrification method has replaced the traditional slow-cooled method for the cryopreservation of embryos. The current trend of transferring fewer embryos has resulted in more surplus embryos for cryopreservation. Although vitrification has been used widely in ART clinics, the safety of this technique in terms of neonatal outcomes of babies delivered from vitrified embryos is still limited
Received January 1, 2012; revised February 15, 2012; accepted February 29, 2012; published online March 30, 2012. W.S. has nothing to disclose. X.X. has nothing to disclose. S.Z. has nothing to disclose. W.Z. has nothing to disclose. S.L. has nothing to disclose. H.Z. has nothing to disclose. M.W. has nothing to disclose. J.S. has nothing to disclose. The work was supported by funds from the Maternal and Child Health Care Hospital of Shaanxi Province, without receiving any financial support from any third party. Reprint requests: Juanzi Shi, Ph.D., Chair Professor of Center for Assisted Reproduction Technology, Maternal and Child Health Care Hospital of Shaanxi Province, Xi'an, 710003, People's Republic of China (E-mail: [email protected]). Fertility and Sterility® Vol. 97, No. 6, June 2012 0015-0282/$36.00 Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2012.02.051 1338
(4, 5). Rama Raju et al. (5) analyzed 312 cycles from vitrified day 3 embryos and 604 cycles from fresh embryos, and the results supported the safety of vitrification using cryoloop. In our IVF center, we have been using vitrification with Cryotop for embryo cryopreservation for 5 years. Thus, we have a large number of babies delivered from vitrified embryos. In this study, we compared the obstetrical and neonatal outcomes of babies born from vitrified day 3 embryos with those born from fresh embryos from our large data sets.
MATERIALS AND METHODS Patients The Assisted Reproduction Center is a public clinic in Shaanxi Province, China. The policy of the IVF/intracytoplasmic sperm injection (ICSI) program is to perform day 3 ETs. Patients' supernumerary embryos (mostly grade I and VOL. 97 NO. 6 / JUNE 2012
Fertility and Sterility® II embryos) are preserved by vitrification on the day of transfers. The frozen-thawed ET is carried out in those patients who failed to conceive after fresh ET. In patients who were at risk of ovarian hyperstimulation syndrome, no fresh embryos were transferred and all embryos were vitrified. These vitrified embryos were warmed and usually transferred 3 months after oocyte collection. We performed a total of 972 ET cycles with vitrifiedwarmed embryos in 806 patients between January 2008 and December 2010. A total of 2,543 vitrified day 3 embryos were warmed; 2,375 survived (93.39%) and were transferred. During the same period, a total of 1,498 fresh ET cycles were performed in 1,148 patients, which was used as a control group. The main etiology of infertility for the vitrified and fresh embryos groups were male factor (22.58% vs. 25.35%) and female factor (63.52% vs. 59.76%), including tuboperitoneal factors, polycystic ovary syndrome, low ovarian reserve, and endometriosis. This study was approved by the Ethics Review Board of the Maternal and Child Health Care Hospital of Shaanxi Province.
Clinical Procedures, Embryo Culture, and Grading All patients used long and short protocols with GnRH agonist (GnRH-a, Decapeptyl) and recombinant FSH (GONAL-f, Merck Serono; Puregon, Organon) for ovarian hyperstimulation. Thirty-six hours after hCG administration, oocytes were retrieved by transvaginal ultrasound-guided aspiration. Oocytes were fertilized using either conventional IVF or ICSI and incubation in fertilization media (Vitrolife). Normal fertilization was assessed and confirmed by the presence of two pronuclei and second polar body at 16–18 hours after insemination. The embryos were washed and cultured in cleavage media (Vitrolife) for 48 hours before transfer. We used a combination of blastomere number, blastomere size, and fragmentation for embryo scoring (6–8). Based on embryonic cell numbers on day 3, embryos with 8–10 blastomeres were classified as grade I; embryos with 6–7 blastomeres or more than 10 blastomeres were classified as grade II, embryos with 4–5 blastomeres were classified as grade III, and embryos with fewer than 4 blastomeres were classified as grade IV. Based on the uniformity of the blastomeres, the embryos without significant differences in blastomere volume were classified as grade I, embryos with one to two significant differences were classified as grade II, and embryos with two or more significant differences were classified as grade III. Based on an embryo's fragmentation, four grades were classified: grade I, embryos with <10% fragmentation; grade II, embryos with 10%–20% fragmentation; grade III, embryos with 20%–30% fragmentation; and grade IV, embryos with >30% fragmentation. Embryo grades were scored comprehensively according to the above three criteria.
Vitrification The Cryotop method for vitrification was used as described elsewhere by Kuwayama et al. (9). Embryos were equilibrated in equilibration solution (7.5% ethylene glycol [EG] þ 7.5% dimethylsulfoxide [DMSO] in TCM199 medium þ 20% VOL. 97 NO. 6 / JUNE 2012
synthetic serum substitute [SSS]) for 5 minutes at room temperature. They were then placed into vitrification solution (VS; 15% EG þ 15% DMSO þ 0.5 M sucrose). After less than 1 minute in VS, the embryos were placed on the Cryotop strip and plunged into liquid nitrogen immediately. For warming, the Cryotop strip was removed from the liquid nitrogen and plunged directly into thawing solution (1.0 M sucrose in TCM199 þ 20% SSS) for 1 minute at 37 C. After 1 minute, the embryos were placed in dilution solution (0.5 M sucrose in TCM199 þ 20% SSS) at room temperature for 3 minutes. They were then put into washing solution (WS; TCM199 þ 20% SSS) for 5 minutes and 5 minutes again in WS. Finally, the embryos were placed in well-balanced G2.5 (Vitrolife) medium containing 5% human serum albumin (HSA) and cultured in a 37 C, 6% CO2 incubator (America Forma) overnight before transfer. Embryos with R50% survival blastomeres were recorded as survival. Tools and solutions required for vitrification were obtained from Kitazato (Kitazato BioPharma Co.).
Outcome Measures The outcome data were obtained from a postal questionnaire of parents after delivery. The obstetric outcome measures referred to implantation and pregnancy rates, preterm birth rate, mean gestational age, and pregnancy-related complications. The neonatal outcomes evaluated were birth weight, Apgar scores, neonatal intensive care unit admission, neonatal complications, and congenital birth defects.
Statistical Analysis All data management and analyses were performed using SPSS 16.0 software. The differences in outcomes between the two groups were analyzed using c2 analysis, t-test, or Mann-Whitney U-test. Fisher's exact probability test was used in case the expected frequency was less than 5. P< .05 was considered statistically significant.
RESULTS Table 1 shows the clinical parameters between vitrified and fresh ET cycles. In the vitrified embryos group, a total of 972 cycles were performed. The mean maternal age was 29.94 4.08 years. The main diagnoses of infertility were female factor (63.52%), male factor (22.58%), and combined all other factors (13.90%). In this group, 691 (71.09%) were treated by IVF and 248 (25.51%) by ICSI. The mean number of embryos transferred per ET was 2.4, with an implantation rate of 26.91%. A total of 459 clinical pregnancies (47.22%) were achieved: 375 (38.58%) live births, 59 miscarriages, six induced abortions due to fetal anomaly and maternal disease, two (0.44%) fetal deaths, and six pregnancies (1.31%) without completion of the follow-up. In the fresh embryo group, a total of 1,498 cycles were performed in 1,148 patients. The mean maternal age was 30.27 4.11 years. The main diagnoses of infertility were female factor (59.76%), male factor (25.35%), and combined all other factors (14.90%). In this group, 993 (66.29%) were treated by IVF and 370 (24.70%) by ICSI. The mean number of embryos transferred per ET was 2.2, with an implantation rate 30.87%. A total of 733 clinical pregnancies (48.93%) 1339
ORIGINAL ARTICLE: ASSISTED REPRODUCTION
TABLE 1 Comparison of clinical parameters between vitrified and fresh ETs. Parameter
Fresh ETs
No. of patients 1,148 No. of cycles transferred 1,498 Patient age, y 30.27 4.11 Main etiology of infertility, n (%) Female factor 686 (59.76) Male factor 291 (25.35) Combined factor and 171 (14.90) other factors Fertilization procedure, n (%) IVF 993 (66.29) ICSI 370 (24.70) No. of embryos transferred 2.18 0.45 Implantation rate (%) 1,007 (30.87) No. of miscarriages 85 (5.67) (% per ET) No. of ectopic pregnancies 13 (1.77) (% per clinical pregnancy) No. of induced abortions 5c (0.68) (% per clinical pregnancy) No. of fetal deaths 3 (0.41) (% per clinical pregnancy) No. of patients lost to 13 (1.77) follow-up (% per clinical pregnancy) No. of live deliveries 614 (40.99) (% per ET) Singletons (% per live delivery) 421 (68.57) Twins (% per live delivery) 192 (31.27) Triplets (% per live delivery) 1 (0.16)
Vitrified ETs P value 806 972 29.94 4.08
.049a
512 (63.52) 182 (22.58) 112 (13.90)
.117b
691 (71.09) 248 (25.51) 2.42 0.57 639 (26.91) 59 (6.07)
.012 .648 .000 .001 .682
11 (2.40)
.456
d
6 (1.31)
.272
2 (0.44)
1.000
6 (1.31)
.532
375 (38.58)
.233
256 (68.27) 119 (31.73) 0
.922 .879 –
Note: Continuous values are expressed as mean SD. a t-test. b Mann-Whitney U-test. c All for fetal anomaly. d Two for fetal anomaly, four for acute infection. Shi. Perinatal outcome of vitrified embryos. Fertil Steril 2012.
were achieved: 614 live births (40.99%), 85 miscarriages, five induced abortions owing to fetal anomaly and maternal disease, three (0.41%) fetal deaths, and 13 (1.77%) pregnancies without completion of the follow-up. Table 2 shows the obstetrical and neonatal outcomes of singleton and multiple gestations in both groups. For the singleton deliveries of vitrified ETs, mean gestational age and preterm deliveries were not significantly different from the fresh ETs. There were 256 babies (126 males and 130 females) born from vitrified embryos with a mean birth weight of 337.44 505.60 g, which was significantly heavier than the singleton delivery of fresh ETs. The mean Apgar scores were very similar between the two groups, except they were slightly higher in vitrified embryos at 1 minute. Thirty (11.72%) newborns required admission to the neonatal care unit. For the multiple gestations, there were no statistically significant differences in mean gestational age, preterm birth rate, the mean Apgar scores, pregnancy-related complications (antenatal bleeding, gestational diabetes, or pregnancy-induced hypertension), and delivery methods between the two groups. However, again, the mean birth weight of babies delivered from the vitrified embryo group was significantly heavier than that of babies delivered from the fresh embryo group. 1340
Table 3 shows all the congenital birth defects. In the vitrified embryo group, seven (five major and two minor) congenital malformations were observed, including three with congenital heart disease and four with others (spinal bifida, lower limb malformation, inguinal hernia, and polydactyly). In contrast with the fresh embryo group, five (three major and two minor) congenital malformations were observed including two with congenital heart disease, one with trisomy 21, and two with laryngeal cartilage hypoplasia. There were no statistically significant differences between the two groups.
DISCUSSION In this study with a large sample size, we clearly showed that there were no significant differences in perinatal and neonatal outcomes of babies delivered with vitrified versus fresh ETs. This result suggests that vitrification is a safe and alternative method for the cryopreservation of human embryos. Before introduction of vitrification to ART clinics, the traditional slow-cooled embryo freezing technique had been widely used, but inconsistent or low embryo survival rates are a common problem for some ART clinics. Thus vitrification of human embryos has recently become a popular alternative, method that may improve the survival rate of cryopreserved embryos. In the 1930s, B.J. Luyet, who first formulated the theory of vitrification, saw great potential for the successful vitrification of colloids and cells (10). Rall and Fahy successfully vitrified a mouse embryo in 1985 (11), which promoted its widespread use in clinical applications. Although the traditional slowfreezing protocols have been greatly improved in the last two decades, vitrification has provided a more efficient method to preserve oocytes and embryos (12–14). The superiority of vitrification is based on its ability to completely avoid ice formation, as well as its simplicity, rapidity, and economy. Despite its advantages, the main problem is the risk of crosscontamination in liquid nitrogen (3, 15) and the toxic effects (biochemical and osmotic) inherent in the use of high concentrations of cryoprotectants (2). Although vitrification has been used in clinics for the past decade, only a few studies have reported on the safety of this technique in terms of neonatal outcome and child follow-up (4, 5). In 2005, Takahashi et al. first reported that there were no significant differences in the perinatal outcomes of 147 babies born from vitrified blastocysts compared with those born from fresh blastocysts (16). Although more and more clinics have now used vitrification for embryo cryopreservation, few studies have reported on the safety of the technique. In 2009, Rama Raju reported that there were no statistically significant differences in the mean gestational age, incidence of preterm deliveries, mean birth weight, mean Apgar score, and incidence of congenital birth defects between vitrified day 3 embryos and fresh ETs (5). However, only 89 babies were delivered from the vitrified embryos. Thus their sample size was too small to provide sufficient statistical power. In this study, we analyzed the outcomes of 494 babies delivered from vitrified embryos and showed that all prenatal and obstetric parameters were similar between vitrified and fresh embryos. VOL. 97 NO. 6 / JUNE 2012
Fertility and Sterility®
TABLE 2 Comparison of obstetrical and neonatal outcomes between vitrified and fresh ETs. Singleton gestation Parameter No. of vaginal deliveries (%) No. of cesarean sections (%) Mean gestational age, wk No. of preterm deliveries (<37 wk) (%) No. of very preterm deliveries (<32 wk) (%) No. of cases of antenatal bleeding (%) No. of cases of gestational diabetes (%) No. of cases of pregnancyinduced hypertension (%) Live birth Male Female Mean birth weight, g Mean Apgar score 1 min 5 min 10 min Birth weight <1,500 g (%) Birth weight 1,500–2,499 g (%) Birth weight 2,500–4,000 g (%) Birth weight R4,000 g (%) Neonatal intensive care unit admission (%) Neonatal death
Neonatal complications Pathological jaundice Respiratory problems Low birth weight Neonatal septicemia Others
Multiple gestation
Fresh
Vitrification
Fresh
Vitrification
76 (18.05) 345 (81.95) 38.85 1.47 29 (6.89)
34 (13.28) 222 (86.72) 39.02 1.67 17 (6.64)
21 (10.88) 172 (89.12) 36.67 2.01 89 (46.11)
11 (9.24) 108 (90.76) 36.77 1.56 62 (52.10)
1 (0.24)
3 (1.17)
6 (3.11)
1 (0.84)
113 (26.84)
60 (23.44)
62 (32.12)
34 (28.57)
14 (3.33)
6 (2.34)
12 (6.22)
5 (4.20)
16 (3.80)
10 (3.91)
17 (8.81)
19 (15.97)
421 197 (46.79) 224 (53.21) 3,216.87 451.30
256 126 (49.22) 130 (50.78) 3,337.44 505.60a
386 178 (46.11) 208 (53.89) 2,435.23 435.74
238 107 (44.96) 131 (55.04) 2,556.45 432.91a
9.00 0.07 9.91 0.30 9.92 0.27 0 24 (5.70) 371 (88.12) 26 (6.18) 59 (14.01)
8.91 0.45a 9.85 0.47 9.89 0.37 2 (0.78) 10 (3.91) 221 (86.33) 23 (8.98) 30 (11.72)
8.91 0.33 9.36 0.52 9.45 0.57 10 (2.59) 186 (48.19) 190 (49.22) 0 169 (43.78)
8.90 0.39 9.34 0.56 9.47 0.56 0b 99 (41.60)b 138 (57.98)b 1 (0.42)b 71 (30.08)c
0
20 (2 for HDN) 17 18 0 4 (1 for hypoxic-ischemic encephalopathy, 1 for diarrhea, 1 for peptic ulcer, 1 for intracranial haemorrhage)
4 (all for asphyxia)
11 10 5 1 (complicating pneumonia) 0
5 (3 for asphyxia, 1 for congenital heart disease, 1 for neonatal necrotizing enterocolitis)
2 (1 for spinal bifida, 1 for lower limb malformation)
13 21 129 0
11 (1 for HDN) 4 56 0
1 (fungus infection)
1 (fever)
Note: Values are expressed as mean SD. HDN ¼ hemolytic disease of newborn. a P< .05 (t-test). b P< .05 (Mann-Whitney U-test). c P< .05 (c2 analysis). Shi. Perinatal outcome of vitrified embryos. Fertil Steril 2012.
To reduce the toxic effects of the high concentrations of cryoprotectants, it is important to cool the embryos at the fastest possible rate to guarantee vitrification (9). As a result, a physicochemical trade-off exists between the cooling rates and the solute concentrations needed for vitrification (2). An open system was applied in which the embryos are put directly into liquid nitrogen to achieve the fastest possible cooling rate. The carrier is a Cryotop device (Kitazato Supply Co.), which is available to a minimum volume (the smaller the volume of the sample, the higher the cooling rate) (17, 18). To avoid the bias due to multiple pregnancies, perinatal outcomes of singleton and multiple gestations must be compared separately. In this study, the obstetrical and neonatal outcomes were compared between vitrified day 3 ETs and fresh ETs. The VOL. 97 NO. 6 / JUNE 2012
vitrified group was slightly younger than the fresh group, but the difference was not statistically and clinically significant. Given that some of the patients in the frozen ET programs had experienced pregnancy failure after their last fresh or frozen ET cycle, more embryos were intended for transfer in this group. Thus the mean number of embryos transferred was higher and the implantation rate was lower in the vitrified group than in the fresh group. There were no significant differences in terms of mean gestational age, preterm birth rate, or the rate of pregnancy-related complications for both singleton and multiple gestation pregnancies between the two groups. However, the mean birth weight of babies (both singleton and multiple gestation) delivered from vitrified embryos was significantly heavier than that 1341
ORIGINAL ARTICLE: ASSISTED REPRODUCTION 2.
TABLE 3
3.
Incidence of major and minor malformations of babies delivered from vitrified or fresh ETs. Birth defects Major Minor Total (%)
Fresh ETs
Vitrified ETs
3: 1 trisomy 21, 5: 3 congenital heart disease, 2 congenital heart 1 spinal bifida, 1 lower disease limb malformation 2 laryngeal cartilage 2: 1 inguinal hernia, hypoplasia 1 polydactyly 5 (0.62) 7 (1.42)
4.
5. 6.
Shi. Perinatal outcome of vitrified embryos. Fertil Steril 2012.
7.
of fresh embryos. Although babies from multiple gestations had significantly fewer admissions to the neonatal care unit for the vitrified group than the fresh embryo group, there were no significant differences in the singleton babies between the two groups. In conclusion, this study clearly shows that there are no differences in obstetrical and neonatal outcomes of babies delivered between vitrified and fresh ETs. Thus vitrification using Cryotop is a safe and effective method for the cryopreservation of human embryos. However, prospective and randomized control trials in a larger sample size and a long-term follow-up of babies are still needed to further validate the safety of vitrification of human embryos. Acknowledgments: The authors thank all the doctors, scientists, and embryologists in the Maternal and Child Health Care Hospital of Shaanxi Province for their assistance with the data collection, as well the patients for participating in this study. We greatly appreciate and thank Dr. De-Yi Liu from the Melbourne IVF and the University of Melbourne in Australia for his comments and revision of the manuscript. This study was financially supported by the Maternal and Child Health Care Hospital of Shaanxi Province.
REFERENCES 1.
Chian RC, Quinn P. Fertility cryopreservation. New York: Cambridge, UK: Cambridge University Press, 2010.
1342
8. 9.
10. 11. 12.
13.
14.
15. 16.
17.
18.
Roger Gosden. Cryopreservation: a cold look at technology for fertility preservation. Fertil Steril 2011;96:264–8. Bielanski A, Bergeron H, Devenish J. Microbial contamination of embryos and semen during long term banking in liquid nitrogen. Cryobiology 2003;46:146–52. €derstro €m-Anttila V, Bergh C, et al. Children born after Wennerholm UB, So cryopreservation of embryos or oocytes: a systematic review of outcome data. Hum Reprod 2009;24:2158–72. Rama Raju GA, Prakash GJ, Krishna KM, et al. Neonatal outcome after vitrified day 3 embryo transfers: a preliminary study. Fertil Steril 2009;92:143–8. Veeck LL. Preembryo grading and degree of cytoplasmic fragmentation. In: An atlas of human gametes and conceptuses: an illustrated reference for assisted reproductive technology. New York: Parthenon Publishing; 1999:46–51. €gren A, et al. Human embryos with unevenly Hardarson T, Hanson C, Sjo sized blastomeres has lower pregnancy and implantation rates: indications for aneuploidy and multinucleation. Hum Reprod 2001;16:313–8. Van Royen E, Mangelschots K, Vercruyssen M, et al. Multinucleation in cleavage stage embryos. Hum Reprod 2003;18:1062–9. Kuwayama M, Vajta G, Kato O, Leibo SP. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online 2005;11:300–8. Luyet BJ. The vitrification of organic colloids and of protoplasm. Biodynamica 1937;1:1–14. Rall WF, Fahy FM. Ice-free cryopreservation of mouse embryos at 196 C by vitrification. Nature 1985;313:573–5. Cobo A, Diaz C. Clinical application of oocyte vitrification: a systematic review and meta-analysis of randomized controlled trials. Fertil Steril 2011; 96:277–85. Rezazadeh VM, Eftekhari-Yazdi P, Karimian L. Vitrification versus slow freezing gives excellent survival, post warming embryo morphology and pregnancy outcomes for human cleaved embryos. Assist Reprod Genet 2009; 26:347–54. Loutradi KE, Kolibianakis EM, Venetis CA, et al. Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis. Fertil Steril 2008;90:186–93. Bielanski A, Nadin-Davis S, Sapp T, Lutze-Wallace C. Viral contamination of embryos cryopreserved in liquid nitrogen. Cryobiology 2000;40:110–6. Takahashi K, Mukaida T, Goto T, et al. Perinatal outcome of blastocyst transfer with vitrification using cryoloop: a 4-year follow-up study. Fertil Steril 2005;84:88–92. Cobo A, Kuwayama M, Perez S, et al. Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method. Fertil Steril 2008;89:1657–64. Antonella B, Marta C, Francesco T, et al. Ultrastructural evaluation of human metaphase II oocytes after vitrification: closed versus open devices. Fertil Steril 2011;95:928–35.
VOL. 97 NO. 6 / JUNE 2012