Introduction
Since it was first reported in 1984, frozen-thawed embryo transfer (FET) has been increasingly used in assisted reproductive technology (ART). Reference Zeilmaker, Alberda and van Gent1 As an essential part of ART, embryo cryopreservation has been used to store surplus embryos after oocyte retrieval and in vitro fertilization (IVF). With the development of cryobiology and refinement of cryopreservation over the last few decades, vitrification has been adopted as a preferred method by most reproductive medicine centers. Reference Fasano, Fontenelle and Vannin2,Reference Debrock, Peeraer and Fernandez3 However, due to the use of cryoprotectants in vitrification, concerns regarding the safety of transferring vitrified embryos have been raised, Reference Zaat, Zagers and Mol4–Reference Bosch, De Vos and Humaidan6 particularly in terms of pregnancy and perinatal outcomes.
The effect of cryopreservation on the human embryo and health of the offspring is still a matter of debate. Many studies have demonstrated that the incidences of macrosomia and large-for-gestational-age (LGA) babies after FET are significantly higher than those following fresh embryo transfer or spontaneous conception. Reference Wennerholm, Henningsen and Romundstad7,Reference Pelkonen, Koivunen and Gissler8 Notably, there is no report in the literature regarding the adverse effects of FET in terms of birth defects. Thus, embryo cryopreservation offers many infertile patients the opportunity to undergo FET under ideal conditions, such as after an appropriate endometrial lining is prepared, and to avoid severe ovarian hyperstimulation syndrome (OHSS). Reference Tiitinen, Halttunen and Harkki9,Reference Thurin, Hausken and Hillensjo10 With the recent abolition of the one-child policy in China, some infertile patients with pregnancy intentions have resorted to transferring surplus embryos that had been cryopreserved during their previous IVF cycles. Although there are several previous reports of cases undergoing FET of embryos that had been cryopreserved for up to 16 years, Reference Yuan, Mai and Ma11–Reference Revel, Safran and Laufer13 there has been no systematic study on long-term cryopreservation duration and perinatal outcomes.
Several recent reports have indicated that embryo cryopreservation duration has no effect on the survival rate of the embryo after thawing and neonatal birthweight in singletons. Reference Riggs, Mayer and Dowling-Lacey14–Reference Li, Yin and Wang17 Nevertheless, no study has focused on the effect of embryo cryopreservation duration on maternal safety in terms of pregnancy-related complications. To fill this knowledge gap, this retrospective cohort study was designed to explore whether embryo cryopreservation duration has adverse effects on maternal health and neonatal outcomes after FET and to provide more data on the safety of the vitrification technique.
Method
Study design and participants
This retrospective cohort study included all infertile women undergoing FET and had live births in the International Peace Maternity and Child Health Hospital (IPMCH) from May 2010 to September 2017. Women who received donated oocytes or sperm, or women who underwent preimplantation genetic testing (PGT) were excluded. Mixed transfers with two embryos retrieved from different oocyte retrieval cycles were also excluded as they were cryopreserved at different times. Eligible FET cycles which resulted in live birth were eventually recruited in the analysis, and categorized into four groups according to the duration of embryo cryopreservation (Group I: ≤3 months; Group II: 4–6 months; Group III: 7–12 months; and Group IV: >12 months). Written informed consent about follow-up until delivery was routinely obtained from all women when they initiated their ART cycles in IPMCH. Ethical approval was obtained from the Institutional Review Board of the International Peace Maternity and Child Health Hospital (GKLW 2016-21).
ART procedures
The ART procedures, including ovarian stimulation, oocyte retrieval, and insemination, by either conventional IVF or intracytoplasmic sperm injection (ICSI), were conducted according to our standard protocols. Fertilization assessment was carried out 16–20 h after insemination. Embryo cryopreservation was performed by vitrification due to one of the following indications: 1) there was a maternal condition that was unsuitable for fresh embryo transfer, such as a high estrogen level, OHSS, or a desynchronized endometrium; 2) embryos had been harvested in a previous, unsuccessful IVF cycle, or there were surplus embryos after a fresh embryo transfer; or 3) the infertile couple had chosen to delay the transfer for personal reasons. FET was performed following endometrial preparation by natural monitoring, an ovarian stimulation cycle, or hormone replacement therapy (HRT).
Embryo vitrification and warming procedures
All embryos were vitrified and warmed with the open device. Before November 9, 2015, the embryo vitrification and thawing kit of JieYing Laboratory Inc (Longueuil, Quebec, Canada) was applied, Reference Cai, Qian and Wang18,Reference Wang, Okitsu and Zhao19 and after that, the Cryotop® of Kitazato BioPharma Co. Ltd (Fuji, Japan) was used. Reference Parmegiani, Beilby and Arnone20 The operation procedures were in accordance with the manufacturer’s instructions. When vitrification: 1) Transfer embryos to Equilibration Solution (ES) for 5 min (JieYing Kit)/7–8 min (Cryotop® Kit) at room temperature; 2) Transfer embryos to Vitrification Solution (VS) with minimal volume of ES and equilibrate for 1 mi (JieYing Kit)/30–60 s (Cryotop® Kit); 3) Quickly place the embryos on the JY straw/Cryotop straw with minimal volume of VS (each straw contains 1–2 embryos); 4) Plunge the straw into sterile liquid nitrogen and fit the straw cap; 5) Transfer the straw to storage dewar (MVE XC47/11-6SQ, Chart Industries, GA, USA). When thawing: 1) JieYing Kit: warm Thawing Solution (TS) 1, 2, 3, and 4 to room temperature; Cryotop® Kit: warm TS to 37°C, Diluent Solution (DS) and Washing Solution (WS) to room temperature; 2) Remove the straw cap from the straw in liquid nitrogen; 3) Quickly immerse the straw into TS1 (JieYing Kit)/TS (Cryotop® Kit) and gently wash for 1 min; 4) Transfer the embryos to TS2 (JieYing Kit)/DS (Cryotop® Kit) for 3 min; 5) Transfer the embryos to TS3 (JieYing Kit) for 5 min/WS (Cryotop® Kit) for 3 min; 6) Transfer the embryos to TS4 (JieYing Kit) for 5 min/another WS (Cryotop® Kit) for 3 min; 7) Transfer and incubate the embryos to culture medium at a 37°C incubator to complete recovery. The liquid nitrogen dewars were only opened when the embryos need to be taken out, and closed immediately after taking out. Sterile liquid nitrogen was refilled regularly every week. Only embryologists who have achieved a recovery rate of more than 98% on discarded embryos were allowed to take up the job. The quality control assessment was carried out every year, and if the embryologist failed, he/she would be retrained.
Data collection and variable definition
Sociodemographic characteristics (including maternal age at oocyte retrieval and embryo transfer, residence, educational attainment, occupation, smoking status during pregnancy), reproductive history (including parity, number of previous abortions, previous ectopic pregnancy, primary infertility, cause of infertility [tubal infertility, anovulation, endometriosis, male-factor infertility, unexplained infertility, or combined cause], and duration of infertility [1–2, 3–4 or ≥5 years]) were extracted from the ART files, which were recorded at the first visit. The maternal height and weight were measured, and her body mass index (BMI) was calculated.
The patient’s clinical data regarding the ART procedure, including oocyte retrieval and embryo transfer, were collected from the patient’s hospital records as previously described. Reference Wu, Li and Zhu21 ART procedures were conducted per routine protocols, and patient information during the ART procedure, including controlled ovarian hyperstimulation (COH) protocol (gonadotropin-releasing hormone [GnRH] agonist protocol, GnRH antagonist protocol, microflare protocol or other protocol), type of insemination (IVF or ICSI), number of oocytes retrieved (≤10, 11–20 or >20), type of endometrial preparation (natural cycle, HRT cycle, or ovarian stimulation cycle), day of embryo transfer (day 3, day 4, or day 5), and number of embryos transferred (1 or 2), was documented. Endometrial thickness before embryo transfer was measured by highly trained sonographers via transvaginal ultrasound (Acuson X300, Siemens, Germany).
The follow-up interview on the pregnancy-related complications and neonatal outcomes were preformed after their deliveries. Data on pregnancy-related complications (including gestational age, gestational hypertensive disorder, gestational diabetes mellitus (GDM), intrahepatic cholestasis of pregnancy (ICP), meconium staining of the amniotic fluid, preterm birth, and mode of delivery) and neonatal outcomes (including birthweight and sex of the neonates) were extracted from hospital records provided by participants. Small-for-gestational-age (SGA) or LGA was defined according to a global reference for birthweight for a given gestational age and sex. Reference Mikolajczyk, Zhang and Betran22
Statistical analysis
Continuous variables with a normal distribution are represented as the means ± standard deviations, and differences among groups were tested by one-way analysis of variance. Categorical variables are represented as frequencies with proportions, and differences in trends were detected by the Cochran–Mantel–Haenszel χ2 test.
To investigate the associations between the embryo cryopreservation duration and pregnancy-related complications, odds ratios (ORs) and 95% confidence intervals (CIs) were calculated and adjusted for potential confounding factors for each outcome using multinomial logistic regression. Neonatal outcomes were stratified according to the delivery of a singleton or multiples. To analyze the neonatal outcomes of singletons, multinomial logistic regression analyses were performed to adjust ORs for potential confounding factors. When we analyzed the neonatal outcomes of multiples, ORs and 95% CIs were obtained using multilevel logistic regression, and the analysis was adjusted for the same confounding factors as those used for the singletons, according to Carlin et al. Reference Carlin, Gurrin and Sterne23
SAS software version 9.3 (SAS Institute, Inc., Cary, NC) was used to perform all statistical analyses. All p values were calculated using two-sided tests. Differences were considered statistically significant at a p value of less than 0.05.
Results
The flow chart of the study is shown in Fig. 1. A total of 3987 women with live birth deliveries from 12,158 FET cycles were included in the analysis. Among them, 123 women who could not provide delivery medical records were defined as lost to follow-up. With the increasing of embryo cryopreservation duration among groups, the number of participants was decreasing, and the longest cryopreservation duration was 6.7 years. The frequency distribution graph with number of live birth deliveries from FET per year is shown in Fig. 2.
Fig. 1. Patient inclusion flow chart. a. PGT, preimplantation genetic testing. b. Mixed transfer cycle was defined as the transfer of two embryos from different oocyte retrieval cycles.
Fig. 2. Frequency distribution graph with number of live birth deliveries from frozen-thawed embryo transfer (FET) per year. Blue bars represent the number of FET cycles in this year. Red bars represent the number of live birth deliveries from the FET in this year.
The distributions of the maternal sociodemographic characteristics and reproductive history data among groups are shown in Table 1. Although maternal age at embryo transfer was comparable among groups (p trend = 0.318), women who underwent transfers of embryos with longer cryopreservation times were younger at oocyte retrieval (p trend = 0.007). Additionally, all groups showed comparable proportions in terms of residence, educational attainment, occupation, and smoking status during pregnancy. The proportions of women who experienced previous abortions (p trend < 0.001) were much lower in the groups of women who underwent transfers of embryos with longer cryopreservation times. The proportions of parous women were higher in the 7–12-month and over-12-month cryopreservation groups than in the ≤3-month and 4–6-month cryopreservation groups (p trend < 0.001). No significant differences were found among groups in terms of previous ectopic pregnancy, duration of infertility, primary infertility, or cause of infertility.
Table 1. Maternal characteristics of all FET groups with different embryo cryopreservation durations
FET, frozen-thawed embryo transfer; BMI, body mass index; SD, standard deviations.
a Combined is defined as two or more infertility causes mentioned above.
The results of the differences in the procedures for oocyte retrieval and frozen-thawed embryo transfer in each group are provided in Table 2. The distributions of COH protocol, type of insemination, number of oocytes retrieved, day of embryo transfer and number of embryos transferred were not different between any groups. Endometrial thickness was comparable among groups before embryo transfer. However, the endometrial preparation protocol was found to be significantly different among the groups. Women who underwent FET within 3 months of cryopreservation were much more likely to undergo a natural cycle protocol and less likely to undergo a HRT cycle (58.62% in the natural cycle and 30.54% in the HRT cycle, p trend < 0.001).
Table 2. ART procedures in all FET groups with different embryo cryopreservation
ART, assisted reproductive technology; FET, frozen-thawed embryo transfer; COH, controlled ovarian stimulation; GnRH, Gonadotropin-releasing hormone; IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; OS, ovarian stimulation; HRT, hormonal replacement therapy.
Table 3 shows the associations between embryo cryopreservation duration and pregnancy-related complications after adjusting for confounding factors, including age at oocyte retrieval, age at embryo transfer, parity, number of previous abortions, type of fertilization, type of endometrial preparation, and number of fetus. Compared to cryopreservation for up to 3 months, long-term cryopreservation duration did not increase the risk of any pregnancy-related complication, including GDM, gestational hypertension, preeclampsia, ICP, meconium staining of the amniotic fluid, preterm birth, and cesarean section deliveries. The associations between embryo cryopreservation duration and neonatal outcomes are presented in Table 4. FETs after different embryo cryopreservation durations had similar proportions in terms of neonatal sex for both singletons and multiples. No significant trends in the association between birthweight and increased embryo cryopreservation duration were found for singletons (Group I: 3323.76 ± 522.00, Group II: 3334.63 ± 525.53, Group III: 3300.23 ± 512.75, and Group IV: 3292.02 ± 565.46, p trend = 0.447) or multiples (Group I: 2503.91 ± 441.64, Group II: 2499.27 ± 438.26, Group III: 2532.34 ± 450.39, and Group IV: 2495.78 ± 501.33, p trend = 0.761). The rates of low birthweight and macrosomia were also comparable among groups in singletons, and no association was found between the risk of LBW or macrosomia and embryo cryopreservation duration. A similar null effect was also observed in multiples with respect to LBW, and no case of macrosomia was found among multiples in the four groups. Additionally, there was no evidence of an association between SGA or LGA and embryo cryopreservation duration among either singletons or multiples.
Table 3. Complications of pregnancies following the transfer of frozen-thawed embryos with different cryopreservation durations
aOR, adjusted odds ratio; CI, confidence interval.
a aOR was adjusted for age at oocyte retrieval, age at embryo transfer, parity, number of previous abortions, type of insemination, type of endometrial preparation, and number of fetus.
Table 4. Outcomes of neonates born after FET with different embryo cryopreservation durations
FET, frozen-thawed embryo transfer; aOR, adjusted odds ratio; CI, confidence interval; NA, not accessible; AGA, appropriate for gestational age; SGA, small for gestational age; LGA, large for gestational age; SD, standard deviations.
a aOR was adjusted for age at oocyte retrieval, age at embryo transfer, parity, number of previous abortions, type of insemination, and type of endometrial preparation.
Considering the possible impact of the embryo vitrification/thawing kits replacement, a stratified analysis according to the date of embryo frozen and thawed was conducted. And it was shown that regardless of whether embryos were frozen before or after November 9, 2015, different cryopreservation duration has no effect on pregnancy complications and neonatal outcomes (Supplementary Table S1-S2). The same results were also found in the stratified analysis of embryo thawed date (Supplementary Table S3-S4).
Discussion
In this retrospective cohort study, we found no association between embryo cryopreservation duration before FET and pregnancy-related complications, including GDM, gestational hypertension, preeclampsia, meconium staining of the amniotic fluid, and preterm birth. In addition, embryo cryopreservation duration seemed to have no adverse effect on abnormal birthweight, including LBW, SGA, macrosomia, and LGA. The findings of our study suggest that it is safe to cryopreserve human embryos for a longer time period and long-term cryopreservation will not result in adverse effects on maternal health or neonatal birthweight.
Since FET was first introduced, the safety of the procedures has been a concern. Although FET has been regarded as having a higher live birth rate than fresh embryo transfer and a comparable rate of birth defects, Reference Zhu, Zhang and Cao24,Reference Davies, Moore and Willson25 there are still problems resulting from embryo cryopreservation technology. Our previous study indicated that blastomere loss after embryo thawing could lead to a decreased pregnancy rate after embryo transfer. Reference Wu, Li and Zhu21 In addition to blastomere loss, other factors, namely, embryo vitrification, open vitrification systems, and vitrification duration, have raised concern regarding their impacts on pregnancy outcomes and neonatal safety. Reference Wirleitner, Vanderzwalmen and Bach15,Reference Rienzi, Gracia and Maggiulli26,Reference Cai, Niringiyumukiza and Li27 Although the long duration of cryopreservation makes it difficult to study the impact of duration on the safety of vitrification, some studies have indicated comparable pregnancy rates for FETs after short- and long-term cryopreservation and reported that birthweight in singletons is not influenced by vitrification duration. Reference Riggs, Mayer and Dowling-Lacey14–Reference Li, Yin and Wang17 Our findings were consistent with these documented findings. In addition, our study was the first to compare the relationship between embryo cryopreservation duration and birthweight in multiples, which also reached the same conclusion as singletons.
Besides, these studies failed to evaluate the impacts of cryopreservation duration on adverse maternal health conditions during pregnancy. Reference Riggs, Mayer and Dowling-Lacey14 Pregnancy-related complications, especially GDM and gestational hypertensive disorder, have been regarded as risk factors for chronic noninfectious diseases of the offspring in adulthood, Reference Kajantie, Osmond and Eriksson28,Reference Miranda, Cerqueira and Barros29 which cannot be detected in the short-term postpartum follow-up in these studies. Thus, it is critical to assess maternal health during pregnancy after transferring long-term cryopreserved embryos. From our findings, transferring embryos cryopreserved within or more than 12 months did not have any effect on the risk of GDM, gestational hypertensive disorder, ICP, or other pregnancy-related complications.
Some studies have found that the embryo cryopreservation does not increase the incidence of chromosomal abnormalities Reference Li, Zhang and Sun30,Reference Forman, Li and Ferry31 and DNA damage, Reference Kopeika, Thornhill and Khalaf32 while increasing evidence suggests that cryopreservation may be associated with deviations from the physiological epigenetic marks, such as DNA methylation, Reference Wang, Xu and He33 histone modifications, Reference Maldonado, Penteado and Faccio34 and noncoding RNA. Reference Hiura, Hattori and Kobayashi35 Although owing to the ethical issues, most of these studies were on animals. A multi-omics study found that FET seemed to introduce more disturbance into infant epigenomes than fresh embryo transfer did, and the epigenetic alterations highly enriched in the processes related to nervous system, cardiovascular system, and glycolipid metabolism. Reference Chen, Peng and Ma36 These epigenetic alterations remind us that the cryopreservation may have long-term effects on the offspring of FET. Hiura et al. have observed that ART offspring has increased incidences of normally rare imprinting disorders such as Beckwith-Wiedemann syndrome (BWS), Angelman syndrome (AS), Prader-Willi syndrome (PWS), and Silver-Russell syndrome (SRS). Reference Hiura, Okae and Miyauchi37,Reference Hattori, Hiura and Kitamura38 Due to the long follow-up period and difficulty in obtaining human biological samples, the long-term effects of embryo cryopreservation on FET offspring still need more research to confirm.
Although embryo cryopreservation and FET have been widely applied, to the best of our knowledge, this is the first broad study to evaluate whether vitrified embryos that are cryopreserved for a longer time are associated with adverse maternal health or neonatal outcomes. Our results indicated that cryopreservation duration did not have a negative impact on these outcomes. However, it should be noted that the longest cryopreservation duration in our study was up to 6.7 years, and the mean duration of cryopreservation in Group IV was approximately 2.7 years. Therefore, it is apparently safe to transfer embryos that have been cryopreserved for approximately 3 years. Additionally, the transfer of these embryos could reduce the economic burden and physical pain of these women by allowing them to avoid undergoing a new ovarian stimulation cycle.
Due to the patients’ concern on the adverse effects of extremely long-term embryo cryopreservation on both mothers and babies, they refused to have these embryos transferred; thus, the study population with extremely long-term cryopreservation duration is lacking in this study. This is one of the limitations of our study. Yuan et al. reported an analysis of long-term embryo cryopreservation (≥12 years) in 20 patients; 4 successfully conceived. Among them, one patient developed GDM, while one developed GDM and had a preterm delivery. Reference Yuan, Mai and Ma11 It is worth noting that the sample size of their study was quite small, that the embryos were cryopreserved by means of slow-freezing methods, and that the patients were at an advanced age when the embryos were transferred (38–51 years old). Reference Yuan, Mai and Ma11 On the other hand, we must also be aware of the influence of iatrogenic damage during long-term cryopreservation, such as human errors of freezing, preservation, the daily use of liquid nitrogen tanks and even the equipment failures rather than the increasing storage time itself. Reference Tomlinson39 Due to concerns from both patients and clinical doctors, more robust evidence on the safety of transferring long-term cryopreserved embryos is urgent and necessary.
During the 7 years in this study, embryo vitrification and thawing kits have been replaced. In order to study the impact of kit replacement, we added a stratified analysis, and the result showed that it was comparable with the overall result. Parmegiani et al. conducted a randomized controlled trial to study the efficacy and efficiency of a universal warming protocol on vitrified embryos with two different embryo vitrification/thawing kits, including Cryotop® Kit (Kitazato Japan) and Sage Kit (Origio, Denmark), and indicated that the survival rates and implantation rates among the combination of kits of different manufacturers were comparable, and a thawing kit of a given manufacturer could be used to warm embryos vitrified with another kit. Reference Parmegiani, Beilby and Arnone20 Similarly, in our study, although the embryo vitrification/thawing kits was replaced, it did not affect the results.
In summary, this retrospective cohort study proves the safety of transferring long-term cryopreserved embryos in terms of pregnancy-related complications. Further studies with long-term follow-up are still required to assess the possible effects of long-term cryopreservation on child growth and development.
Acknowledgments
The authors wish to thank the staff at the reproductive medicine center and obstetrics department of International Peace Maternity and Child Health Hospital for their work in assembling the data for this study. The authors wish to thank American Journal Expert to improve the readability of the text and the authors are entirely responsible for the scientific content of the paper.
Financial Support
This study was supported by the National Key Research and Development Program of China (WYT, grant numbers 2018YFC1002804, 2016YFC1000203), (HFH, grant number 2017YFC1001300); National Natural Science Foundation of China (WYT, grant number 81671412); Shanghai Shen Kang Hospital Development Center (WYT, grant number SHDC12018X17), (LC, grant number SHDC12018622); Shanghai Municipal Health Commission (WYT, grant number 201840210); the Interdisciplinary Key Program of Shanghai Jiao Tong University (WYT, grant number YG2019GD04); and International Peace Maternity and Child Health Hospital (LC, grant number CR2018SY02).
Conflicts of Interest
The authors declare no conflict of interest.
Ethical Standards
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national guidelines and with the Helsinki Declaration of 1975, as revised in 2008, and has been approved by the Institutional Review Board of the International Peace Maternity and Child Health Hospital (GKLW 2016-21). Written informed consent was obtained from all participants before inclusion.
Supplementary material
To view supplementary material for this article, please visit https://doi.org/10.1017/S2040174421000192
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