Obstetric and neonatal outcomes after transfer of vitrified early cleavage embryos

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1 Human Reproduction, Vol.28, No.8 pp , 2013 Advanced Access publication on April 7, 2013 doi: /humrep/det104 ORIGINAL ARTICLE Embryology Obstetric and neonatal outcomes after transfer of vitrified early cleavage embryos S.Y. Liu 1, B. Teng 1,J.Fu 1,X.Li 1, Y. Zheng 1, and X.X. Sun 1,2, * 1 Shanghai Ji Ai Genetics & IVF Institute, Obstetrics & Gynecology Hospital, Fudan University, Shanghai , China 2 Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai , China *Correspondence address. xiaoxi_sun@yahoo.cn Submitted on October 28, 2012; resubmitted on March 1, 2013; accepted on March 13, 2013 study question: Does vitrification and warming of Day 3 embryos have an impact on neonatal outcome when compared with Day 3 embryos that are slow cooled and thawed, or with embryos from a fresh cycle? summary answer: The median birthweight was higher in the vitrified group versus the slow cooled or fresh embryo transfer, and the rate of low birthweight in twin babies was lower in the vitrified group. what is known already: Vitrification has been successfully used for cryopreserving human oocytes and blastocyst-stage embryos. Most published studies looking at the neonatal outcomes after transfer of vitrified embryos refer to blastocyst-stage embryos. Information on children born after transfer of Day 3 vitrified embryos is relatively rare. study design, size, duration: A retrospective, single-centre study of children born after Day 3 embryo transfer from fresh, slow frozen or vitrified embryos during the period January 2006 to May 2011 was conducted. Each patient contributed only one cycle per group. Children born were followed-up at 7 30 days after delivery. Outcome measures include obstetric and neonatal outcomes, which were evaluated by medical records and questionnaires sent to parents. participants/materials, setting, methods: Patients underwent transfer of vitrified Day 3 embryos (n ¼ 2617 transfers, Cryotop method), slow frozen Day 3 embryos (n ¼ 4681) and fresh Day 3 embryos (n ¼ 9194). All cycles were performed at the Shanghai Ji Ai Genetics & IVF Institute. mainresultsandtheroleofchance:frequencies of hypertensive disorder, gestational diabetes, placenta previa and abruptio placenta were similar in all groups. Five hundred and forty five, 986 and 1914 singleton babies were born from vitrified, slow freezing and fresh transfers, and the median gestational ages were 38.7, 38.7 and 38.7 weeks, respectively. Preterm birth (32 37 weeks) occurred in 7.5, 9.2 and 7.8% of the vitrified, slow freeze and fresh groups, respectively. The median birthweight from vitrified embryos ( g) was higher than that from slow freezing ( g) and fresh ( g) transfers (P, for both). The rate of perinatal mortality was 0.4% for all transfer groups. Three hundred and eighty two, 734 and 1322 twin babies were born from vitrified, slow freezing and fresh transfers, respectively. There were no differences among groups in mean gestational age and in the rate of preterm birth. The median birthweight for babies born from vitrified embryos ( g) was higher than that from the slow freezing ( g) or fresh ( g) transfer groups (vitrified versus fresh: P ¼ ; vitrified versus slow freeze: P ¼ 0.049). The rate of low birthweight ( g) from vitrified (30.4%) was lower than that from fresh (36.2%) transfer (P ¼ 0.034). limitations, reasons for caution: The main limitation of this study is that the obstetric and neonatal data were obtained by questionnaires sent to the parents without checking medical records. This is, especially, problematic for reporting on congenital malformations, so birth defects were excluded from the data. wider implications of the findings: Transfer of vitrified and warmed Day 3 embryos does not seem to have an adverse effect on neonatal outcome. Children born following the transfer of vitrified embryos seem to have a higher birthweight when compared with those of fresh or slow frozen embryos. study funding/competing interest(s): This study received no outside funding and none of the authors has any conflict of interest. Key words: vitrification / cryopreservation / cleavage-stage embryo / obstetric outcome / neonatal outcome & The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oup.com

2 2094 Liu et al. Introduction Embryo cryopreservation has become a routine component of clinical IVF programs. Cryopreservation offers the opportunity to reduce the number of embryos transferred per procedure, helping to reduce the occurrence of higher-order multiple gestations, while optimizing the clinical use of excess embryos. The trend of transferring fewer embryos has resulted in more embryos being available for freezing. For many years, slow cooling was the dominant method for cryopreservation of human embryos, but vitrification has generally become the preferred method in recent years. Vitrification has been successfully used in freezing human oocytes (Nagy et al., 2009; Ubaldi et al., 2010; Garcia et al., 2011; Trokoudes et al., 2011) as well as cleavage-stage human embryos and human blastocyst-stage embryos (Stehlik et al., 2005; Balaban et al., 2008; Rezazadeh Valojerdi et al., 2009; Van Landuyt et al., 2011). One of the limitations of traditional slow freezing is the formation of intracellular ice, which can lead to cell damage (Shaw and Jones, 2003). Vitrification is an ultrarapid method of cooling cells into a glass-like state, which avoids ice crystal formation and thereby reduces associated cell injuries. However, in order to achieve these results, higher concentrations of cryoprotectants must be used. Many published reports have shown better post-thawing survival rates and encouraging pregnancy rates when compared with slow freezing (Kuleshova and Lopata, 2002; Kuwayama, 2007), especially when used to cryopreserve oocytes and blastocyst-stage embryos. Although vitrification is now used widely in assisted reproduction treatment (ART) clinics, there are still concerns regarding the safety of vitrification. Most published reports looking at the neonatal outcomes after transfer of vitrified embryos have used blastocyst-stage embryos (Takahashi et al., 2005; Mukaida et al., 2009; Wikland et al., 2010). Reported data on children born after transfer of Day 3 vitrified embryos are relatively rare (Rama Raju et al., 2009; Kato et al., 2012; Shi et al., 2012). In this study, our slow freezing protocol was used prior to January 2010, then vitrification was utilized from January 2010 onwards. Except for the switch in the type of cryopreservation, no other major changes were made in laboratory or clinical protocols, In addition, similar patient populations were treated during these time periods. The aim of the present study was to compare the obstetric and neonatal outcomes of babies born following transfer of from vitrified Day 3 embryos with those born after slow frozen or fresh embryo transfer during the same time period based on our large data sets in the same clinic. Materials and Methods Patients Nine thousand one hundred and ninety four fresh transfer cycles were performed from January 2006 to May Between January 2006 and December 2009, 4681 embryo transfers were performed using slow freezing, and between January 2010 and May 2011, 2617 embryo transfers were performed using vitrification. Each patient contributed only one cycle per group. All of the transferred embryos were at the Day 3 stage. Mixed fresh/thawed embryo transfer cycles and mixed cleavage/ blastocyst embryo transfer cycles were excluded from this data set. Children born after transfer of vitrified, slow freeze and fresh Day 3 embryos during the period January 2006 to May 2011 at the Shanghai Ji Ai Genetics & IVF institute were followed up at 7 30 days after delivery. Ovarian stimulation In the fresh cycles, ovarian stimulation was performed using long or short protocols with GnRH agonists (Ferring Pharmaceuticals, Switzerland) and recombinant FSH (Gonal F, Merck-Serono, Geneva, Switzerland). HCG (Profasi, Merck-Serono) was injected when, at least, three follicles of 17 mm diameter were seen on ultrasound. Oocyte retrieval was performed by transvaginal ultrasound h after HCG injection. Oocytes were fertilized using either conventional IVF or ICSI and incubated in fertilization media (Vitrolife, Sweden). Normal fertilization was assessed and confirmed by the presence of two pronuclei and second polar body at h after insemination. The embryos were washed and cultured in cleavage medium (Vitrolife) for 48 h before being transferred or frozen. Routine examination of embryo quality included the number of blastomeres, the degree of fragmentation and the uniformity of blastomeres. Embryo morphology was scored according to a grading system that was a modification of a previously published scheme (Hardarson et al., 2001). The new scheme comprised four grades: Grade 1, no fragments and equal blastomeres; Grade 2,,20% fragments, equal or unequal blastomeres; Grade 3, equal or unequal blastomeres, 20 50% fragments; Grade 4, equal or unequal blastomeres,.50% fragments. An embryo with at least seven blastomeres and Grades 1 and 2 was defined as good quality. Embryos with at least six blastomeres and fragments,50% were frozen. Slow freezing/thawing of Day 3 embryos Cryopreservation was performed using a slow freezing protocol (Tesart et al., 1986). Briefly, embryos were equilibrated in 1.5 M propanediol (PROH) at room temperature for 10 min, then transferred to 1.0 M PROH/0.1 M sucrose and loaded into plastic straws. Straws were put in a controlled-rate freezer (Kryo-360, Planer Product Ltd., UK) and cooled at a rate of 228C/min to 278C, at which point seeding was performed manually with liquid nitrogen-cooled forceps. Cooling was then continued at a rate of 20.38C/min to 2308C and then rapidly cooled at a rate of 2508C/min to 21408C before plunging into and storing in liquid nitrogen. Embryos were thawed rapidly by moving straws from liquid nitrogen to air for 30 s. Then, the embryos were sequentially incubated in each of the following solutions for 5 min: 1.0 M PROH/0.2 M sucrose, 0.2 M sucrose and sucrose-free HEPES. Embryos were assessed for the number of intact blastomeres. Post-thaw survival of cryopreserved embryos was defined as having 50% blastomeres intact. Embryo transfer was performed 1 h after thawing. Vitrification and warming protocols The Cryotop method for vitrification was used as described by Kuwayama (2007). Day 3 embryos were equilibrated in equilibration solution [7.5% ethylene glycol (EG) + 7.5% dimethylsulfoxide (DMSO) in HEPES-based medium plus 20% synthetic serum substitute (SSS)] for 5 min at room temperature. The embryos were then placed into vitrification solution (VS; 15% EG + 15% DMSO M sucrose). After,1 min 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 HEPES-based medium plus 20% SSS) for 1 min at 378C, then were sequentially incubated in each of the following solutions for 3 min: 0.5 M sucrose, 0.25 M sucrose and sucrose-free HEPES. Then, the embryos were placed in G2 (Vitrolife) medium containing 20% SSS and cultured in an incubator at 378C with 6% CO 2 until transfer.

3 Outcomes after transfer of Day 3 vitrified embryos 2095 Transfer of frozen/thawed Day 3 embryos Natural cycles were performed only in those women who had spontaneous ovulation. In women with amenorrhea or irregular menstrual cycles, hormone replacement treatment (HRT) was performed. On Day 3 of the replacement cycle, estradiol valerate (E 2, Progynova, Schering AG, Berlin, Germany) was commenced 2 mg daily, with increasing doses up to 4 mg per day. When a thickness of the triple endometrial layers of at least 8 mm was observed, vaginal progesterone 90 mg per day (Crinone, Merck-Serono, Switzerland) was administered 3 days, whereupon embryos were thawed and transferred. For natural cycles, ovarian scanning was performed on cycle Days If there was a leading follicle of mm diameter, plasma E 2, progesterone and LH were measured every day. Embryo transfer was performed 4 days after an LH surge. Clinical pregnancy was diagnosed by the presence of a gestational sac on vaginal ultrasound 4 weeks after the embryo transfer. A maximum of three embryos were transferred. All pregnant women continued to have E 2 and progesterone administered daily until 10 weeks of gestation. Follow-up of pregnancy, delivery and perinatal outcome In fresh transfer cycles, gestational age was determined by subtraction of the oocyte retrieval date from the date of birth plus an additional 14 days. In women with regular menstrual periods with frozen transfers, gestational age was calculated from the date of last menstrual period to the date of birth. In HRT cycles, gestational age was determined by subtracting the date of embryo transfer from the date of birth plus an additional 17 days. Patients were routinely followed up by ultrasound scans performed at our centre until the end of the first trimester. After establishing an ongoing pregnancy at 12 weeks following embryo transfer, they were given a questionnaire (with prepaid envelopes) that they had to fill out and send back after delivery. Those patients who did not respond to this questionnaire were reminded by a telephone call 1 month after expected date of childbirth. This survey contained questions about the duration of pregnancy, pregnancy-related complications, mode of delivery, birthweight and perinatal mortality. When a patient delivered in our hospital, data were collected from the hospital s medical record database. Statistics All statistical analyses were performed using STATA (version 11). Descriptive statistics were calculated on all measures to determine the characteristics of the sample, to check normality assumptions and to ensure adequate variability. Univariate analyses were performed using the x 2 test for dichotomous variables, analysis of variance for continuous variables and the Student Newman Keuls test for pairwise comparison. Single linear regression analysis was applied to quantify the effect of the studied factors (grouped by vitrification or slow freeze or fresh transfer) on the outcomes, and multivariate linear stepwise regression to assess the relationship between grouping variable and all of the variables that were significant at the univariate level to obtain point and interval estimates of association parameters after controlling for potential confounding factors. A P-value of,0.05 was considered to be significant. For neonatal outcome, logistic regression was used to adjust for potential maternal confounding factors (parity and BMI). Smoking was excluded as a confounding factor owing to the small number of smokers. Outcome measures The obstetric outcome measures refer to the implantation and pregnancy rates, mean gestational age and pregnancy-related complications. The neonatal outcomes evaluated were birthweight, preterm birth (delivery between 32 and 37 weeks), very preterm birth (delivery before 32 weeks), low birthweight (birthweight between 1500 and 2500 g) and very low birthweight (birthweight,1500 g). Deliveries included all live born babies and stillborns after 28 weeks of gestation. Perinatal mortality included mortality in live borns within 7 days of age and stillborns after 28 weeks of gestation (Wikland et al., 2010). Results The number of ongoing pregnancies, lost to follow-up, was two in the vitrified group, three in the slow-freeze group and seven in the fresh group. Table I summarizes the comparisons of clinical parameters among vitrified, slow freezing and fresh transfer cycles. Maternal characteristics were comparable in all the three groups. Pregnancy complications were also similar in the three groups. Both the overall embryo survival rate and the proportion of embryos with 100% intact blastomeres were significantly higher with vitrification than that with slow freezing (P, for both). The mean numbers of embryos thawed (per embryo transfer) were 2.6 in the vitrified group and 3.3 in the slow freezing group (P, 0.001), respectively. The implantation and clinical pregnancy rates were similar in the three groups. The frequency of ectopic pregnancy in the fresh group was significantly higher when compared with that in the vitrified (P ¼ 0.002) or slow freezing group (P, 0.001). Obstetric and neonatal outcomes in singletons Obstetric and neonatal outcomes in singletons are presented in Table II. The frequencies of hypertensive disorder, gestational diabetes, placenta previa and abruptio placenta were similar in all groups. Five hundred and forty five, 986 and 1914 singleton babies were born from vitrified, slow freezing and fresh transfers, respectively, and the median gestational ages were 38.7, 38.7 and 38.7 weeks, respectively. Preterm birth (32 37 weeks) occurred in 7.5, 9.2 and 7.8% of the vitrified, slow freeze and fresh groups, respectively (P. 0.05). The median birthweight of babies born after transfer of vitrtified embryos was significantly higher than that of slow frozen or fresh embryos (both P, ). When adjustment was made for maternal BMI and parity, the differences remained significant (adjusted P-value, vitrified versus fresh: P ¼ 0.001; vitrified versus slow freeze: P, ). No significant difference was found for the rate of perinatal mortality. Obstetric and neonatal outcomes in twin pregnancies Obstetric and neonatal outcomes of twins are presented in Table III. Three hundred and eighty two, 734 and 1322 babies were born from vitrified, slow freezing and fresh transfers, respectively. There were no differences in mean gestational age, rate of preterm birth or perinatal mortality among groups. The median birthweight of babies born after transfer from vitrified embryos was significantly

4 2096 Liu et al. Table I Comparison of clinical parameters among vitrified, slow freeze and fresh Day 3 embryo transfers, January 2006 to May Vitrified Slow freeze Fresh... Embryo transfer cycles Natural cycles (NC) 811 (31.0%) 1545 (33.0%) NA HRT cycles 1806 (69.0%) 3136 (67.0%) NA Cycles of follow-up 2587 (98.9%) 4613 (98.5%) 9010 (98.0%) NC HRT Pregnancies (% per cycles of follow-up) 938 (36.3%) 1693 (36.7%) 3343 (37.1%) NC 296 (36.9%) 556 (36.5%) HRT 642 (36.0%) 1137 (36.8%) Deliveries (% per cycles of follow-up) 736(28.4%) 1353 (29.3%) 2575 (28.6%) NC 231 (28.8%) 440 (28.9%) HRT 505 (28.3%) 913 (29.5%) Maternal age (years) (22 46) (22 46) (21 44) Years (of infertility) 4.7 (1 10) 4.6 (1 9) 4.4 (1 9) Cause of infertility Male factor 881(33.7%) 1638 (35.0%) 3006 (32.7%) Tubal 856 (32.7%) 1545 (33.0%) 3089 (33.6%) Combined 751 (28.7) 1231 (26.3%) 2639 (28.7%) Unexplained 129 (4.9%) 267 (5.7%) 460 (5.0%) Insemination methods IVF 1514 (57.9%) 2668 (57.0%) 5443 (59.2%) ICSI 1103 (42.1%) 2013 (43.0%) 3751 (40.8%) Embryo survival rates 97.6% (6696/6861) 87.5% (13 352/15 260) NA Intact blastomere rates 89.2% (6122/6861) 57.1% (8713/15 260) NA Mean embryos thawed (% per embryo transfer) NA Mean embryos left NA Mean thawing cycles might be repeated NA Mean embryos transferred Ectopic pregnancies 36 (3.8%) 58 (3.4%) 221 (6.6%) Spontaneous miscarriages (% per pregnancy, before 12 weeks) 118 (12.6%) 205 (12.1%) 368 (11.0%) Lost to follow-up after 12 weeks Pregnancies follow-up after 12 weeks Spontaneous miscarriages (% per pregnancy, after 12 weeks) 46 (5.9%) 74 (5.2%) 172 (6.3%) Embryo survival rates: vitrified versus slow freeze, P, Intact rates: vitrified versus slow freeze, P, The mean number of embryos thawed: vitrified versus slow freeze, P, The mean number of embryos transferred: compared with both vitrified and slow freeze, P, The mean number of embryos left: vitrified versus slow freeze, P, The mean number of thawing cycles might be repeated: vitrified versus slow freeze, P, Ectopic pregnancies: compared with vitrified, P ¼ 0.002; compared with slow freeze, P, higher than those from the slow freezing or fresh transfer groups (vitrified versus fresh: P ¼ and vitrified versus slow freeze: P ¼ 0.049). After adjustments for maternal BMI and parity, this difference was still significant (adjusted P-value, vitrified versus fresh: P, and vitrified versus slow frozen: P ¼ 0.063). The rate of low birthweight babies ( g) from vitrified embryos (30.4%) was significantly lower than that of fresh (36.2%) embryos (P ¼ 0.034). Discussion In this study, a total of transfer cycles were evaluated for several outcome measures. Perinatal outcomes from vitrified Day 3 embryo transfers were compared with those of slow freezing or fresh Day 3 embryo transfers, and no adverse outcome was observed regarding mean gestational age, frequency of preterm birth and perinatal mortality. The mean birthweight of both singleton and twins

5 Outcomes after transfer of Day 3 vitrified embryos 2097 Table II Obstetric and neonatal outcomes in singleton pregnancies after transfer of vitrified, slow freeze and fresh Day 3 embryos. Vitrified (n 5 545) Slow freeze (n 5 986) Fresh (n ) Test between groups, P-value... Vitrified Vitrified versus slow versus freeze fresh... Maternal age, (years) (22 45) (22 44) (21 43) BMI (kg/m 2 ) 21.4 ( ) 21.6 ( ) 21.6 ( ) Primiparous 295 (54.1%) 523 (53.0%) 1072 (56.0%) Educational level, university 218 (40.0%) 366 (37.1%) 861 (45.0%) Babies born Live borns Gender Male 276 (50.6%) 491 (49.8%) 959 (50.1%) Female 269 (49.4%) 495 (50.2%) 955 (49.9%) Spontaneous vaginal delivery 60 (11.0%) 107 (10.9%) 189 (9.9%) Vacuum or forceps extraction 2 (0.4%) 3 (0.3%) 7 (0.4%) Caesarean delivery 483 (88.6%) 876 (88.8/%) 1718 (89.8%) Hypertensive disorder 27 (5.0%) 41 (4.2%) 74 (3.9%) Hypertension 6 (1.1%) 8 (0.8%) 13 (0.7%) Pre-eclampsia 21 (3.9%) 33 (3.3%) 61 (3.2%) Gestational diabetes 18 (3.3%) 29 (2.9%) 55 (2.9%) Placenta previa 2 (0.4%) 5 (0.5%) 6 (0.3%) Abruptio placenta 1 (0.2%) 3 (0.3%) 5 (0.3%) Gestational age (weeks) ( ) ( ) ( ) (7.5%) 91 (9.2%) 149 (7.8%) (0.7%) 8 (0.8%) 6 (0.3%) ,28 1 (0.2%) 1 (0.1%) 4 (0.2%) Birthweight (g) ( ) ( ) ( ) (1.8%) 34 (3.5%) 49 (2.6%) , (0.4%) 6 (0.6%) 8 (0.4%) SGA 8 (1.5%) 20 (2.0%) 35 (1.8%) Apgar score, 7 at 5 min 15 (2.8%) 45 (4.6%) 56 (2.9%) Transferred to NICU 44 (8.1%) 71 (7.2%) 153 (8.0%) Perinatal mortality 2 (0.4%) 4 (0.4%) 7 (0.4%) Stillborns Early neonatal mortality SGA: small for gestational age; NICU: neonatal intensive care unit. born after vitrified embryo transfer was significantly higher than that after slow freezing or fresh embryo transfer. Significantly, fewer newborn twins in the vitrified group were low birthweight, compared with the fresh group. The reasons for better outcomes after vitrified embryo transfer compared with slow freezing or fresh transfers are not known. Recent studies have shown that the surrounding environment can alter the epigenome which can, in turn, impact on embryo metabolism and developmental competence (Chason et al., 2011). Numerous genes are associated with birthweight (Zhao et al., 2009; Adkins et al., 2010). For example, the growth-promoting function of insulinlike growth factor (IGF)2 is important during mouse embryogenesis and is mediated in part by signaling through the insulin receptor (Louvi et al., 1997). H19 is growth inhibitory and functionally antagonistic to IGF2 (Gabory et al., 2009), both of these genes are imprinted. However, there are very limited data regarding possible effects of cryopreservation on the epigenome of embryos. A recent study in a murine model reported that the process of embryo vitrification significantly augmented the loss of methylation in the H19 differentially methylated domain, especially in the placenta. IGF2 expression in the fetus of the IVF and vitrified groups decreased significantly compared with the control group, but increased significantly in the

6 2098 Liu et al. Table III Obstetric and neonatal outcomes in twin pregnancies after transfer of vitrified, slow freeze and fresh Day 3 embryos. Vitrified (n 5 191) Slow freeze (n 5 367) Fresh (n 5 661) Test between groups, P-value... Vitrified Vitrified versus slow versus freeze fresh... Maternal age, (years) (22 41) (22 40) (21 41) BMI (kg/m 2 ) 21.5 ( ) 21.7 ( ) 21.6 ( ) Primiparous 118 (61.8%) 217 (59.1%) 379 (57.3%) Educational level, university 87 (45.5%) 144 (39.2%) 311 (47.0%) Babies born Live borns Gender Male 194 (50.8%) 365 (49.7%) 665 (50.3%) Female 188 (49.2%) 369 (50.3%) 657 (49.7%) Mode of delivery Spontaneous vaginal delivery 5 (2.6%) 9 (2.5%) 19 (2.9%) Vacuum or forceps extraction 1 (0.5%) 1 (0.3%) 2 (0.3%) Caesarean delivery 185 (96.9%) 357 (97.3%) 640 (96.8%) Hypertensive disorder 17 (8.9%) 28 (7.6%) 63 (9.5%) Hypertension 1 (0.5%) 2 (0.5%) Pre-eclampsia 16 (8.4%) 26 (7.1%) 63 (9.5%) Gestational diabetes 10 (5.2%) 17 (4.6%) 23 (3.5%) Placenta previa 2 (1.0%) 5 (1.4%) 8 (1.2%) Abruptio placenta (0.2%) 1.0 Gestational age (weeks) ( ) ( ) ( ) (49.2%) 194 (52.9%) 330 (49.9%) (4.7%) 17 (4.6%) 18 (2.7%) ,28 1 (0.5%) 1 (0.3%) 5 (0.8%) Birthweight (g) ( ) ( ) ( ) (30.4%) 250 (34.1%) 479 (36.2%) , (1.8%) 20 (2.7%) 32 (2.4%) SGA 16 (4.2%) 29 (4.0%) 57 (4.3%) Apgar score, 7 at 5 min 14 (3.7%) 31 (4.2%) 53 (4.0%) Tranferred to NICU 153 (40.1%) 325 (44.3%) 568 (43.0%) Perinatal mortality 1 (0.3%) 11 (1.5%) 15 (1.1%) Stillborns Early neonatal mortality placenta. Compared with the IVF group, the IGF2 expression in the fetuses of the vitrified group increased (Wang et al., 2010). As the genome-wide demethylation and remethylation processes occur in embryos during the preimplantation period, embryo cryopreservation may cause modifications to imprinted regions of the genome and affect development of the fetus. In the present study, a higher birthweight after vitrified transfer may be associated with modifications of the epigenome of embryos. However, additional research is needed to test the impact of vitrification on the epigenetic marks and embryonic developmental competence. Another explanation put forward for better results in pregnancies resulting from vitrified transfer is that the physical effects of freezing and thawing embryos may filter out weaker embryos and allow only good quality ones to survive, resulting in improved fetal growth (Shih et al., 2008). Conventional slow freezing has been widely used for nearly 30 years as an adjunct to fresh ART cycles. However, embryo survival rate, especially intact blastomere rate, has remained low, in part, because of ice crystal formation. Vitrification has significantly improved the situation in our hands. This is based partly on the avoidance of ice crystal

7 Outcomes after transfer of Day 3 vitrified embryos 2099 formation with these rapid cooling methods. In several studies, as well as at our own institute, a higher intact blastomere rate was obtained by using vitrification (Stehlik et al., 2005; Rezazadeh Valojerdi et al., 2009) rather than classical slow cooling methods. In the present study, the higher rates of embryo survival and intact blastomere rate using vitrification versus slow cooling (97.6 and 89.2% versus 87.5 and 57.1%, respectively) led to a decrease in the number of thawed embryos required for successful embryo transfer. It is, thus, feasible to increase the number of frozen transfer cycles from a single egg collection, enhancing the cumulative probability of pregnancy. When appraising the relative efficacy of vitrification versus slow cooling, it must be emphasized that embryo survival is an important end-point, but not sufficient to determine which method is actually superior in clinical practice (Edgar and Gook, 2012). Ultimately, the success of cryopreservation must be defined by clinical efficacy, and also by comparing outcomes with those from appropriate fresh controls. Consistent clinical outcomes similar to those from fresh transfers have been reported following vitrification. Rama Raju et al. (2009) reported survival of 90.4% in a larger series of 907 warmed embryos, with an implantation rate of 18.1% which was comparable with that of fresh embryos (23.5%). A survival rate of 85% and an implantation rate of 20% were also reported using a vitrification method based on DMSO, EG and sucrose (Desai et al., 2007). The literature on vitrification has mainly focused on cryopreservation of blastocyst-stage embryos. Blastocyst transfer is thought to result in higher implantation and delivery rates than that of cleavagestage embryos (Papanikolaou et al., 2008; Guerif et al., 2009). However, one drawback of blastocyst culture is the risk of transfer cancelation if no blastocyst has developed by Day 6, despite adequate embryo development at Day 3. Although vitrification has been used clinically in recent years, there is still a limited amount of published information on neonatal outcome following transfer of vitrified Day 3 embryos. Shi et al. (2012) compared the outcomes of 494 babies delivered from vitrified Day 3 embryos and 807 babies from fresh Day 3 embryo transfer and found that there were no significant differences in mean gestational age, preterm birth rate or the rate of pregnancy-related complications for both singleton and multiple gestation pregnancies. However, they observed that the mean birthweight of babies (both singleton and multiple gestation) delivered from vitrified embryos was significantly higher than that of fresh embryos. A large retrospective study found that singletons born after embryo/ blastocyst vitrification had a higher birthweight and no increase in adverse perinatal outcomes or birth defects when compared with fresh embryo/blastocyst transfer cycles (Kato et al., 2012). A systematic review published in 2012 found that the children born after cryopreservation had better perinatal outcomes compared with those born after fresh embryo transfer (Maheshwari et al., 2012). In this study, vitrification, slow freezing and fresh Day 3 embryo transfers have been employed in the same centre. The comparisons here were useful because many other confounding variables were kept constant and the large study population size provides significant statistical power. One limitation of this study was the fact that some of the data were obtained from patient questionnaires, rather than the medical record. However, we were able to obtain a compliance rate of over 99% thanks to our diligent nursing staff. In conclusion, this study demonstrates that transfer of vitrified and warmed Day 3 embryos does not seem to have any adverse effect on neonatal outcome. Vitrified embryo transfer seems to be associated with a higher birthweight when compared with pregnancies conceived after fresh or slow freezing embryo transfer, implying an improved perinatal outcome. However, long-term follow-up of babies is still needed to further evaluate the safety of vitrified embryos. Acknowledgement The authors gratefully acknowledge Dr Andrew Dorfmann, Genetics & IVF Institute in Fairfax, USA, for reviewing the paper and giving suggestions. The authors also thank Yan Zhang for help with the statistical analysis. Authors roles S.Y.L. and X.X.S.: conception and design, acquisition of data, analysis and interpretation of data, and writing of the article. B.T., J.F., X.L. and Y.Z.: collection and assembly of data, and data analysis. All authors critically reviewed and approved the final version of the manuscript. Funding This study received no external funding. Conflict of interest None declared. References Adkins RM, Somes G, Morrison JC, Hill JB, Watson EM, Maqann EF, Krushkal J. Association of birth weight with polymorphisms in the IGF2, H19, and IGF2R genes. Pediatr Res 2010;68: Balaban B, Urman B, Ata B, Isiklar A, Larman MG, Hamilton R, Gardner DK. A randomized controlled study of human Day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation. Hum Reprod 2008;23: Chason RJ, Csokmay J, Segars JH, DeCherney AH, Armant DR. 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