Frozen versus fresh single blastocyst transfer in ovulatory women: a multicentre, randomised controlled trial

doi:10.1016/S0140-6736(18)32843-5

The Lancet

Volume 393, Issue 10178, 30 March–5 April 2019, Pages 1310-1318

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https://doi.org/10.1016/S0140-6736(18)32843-5 Get rights and content

Summary

Background

Elective single embryo transfer (eSET) has been increasingly advocated, but concerns about the lower pregnancy rate after reducing the number of embryos transferred have encouraged transfer of multiple embryos. Extended embryo culture combined with electively freezing all embryos and undertaking a deferred frozen embryo transfer might increase pregnancy rate after eSET. We aimed to establish whether elective frozen single blastocyst transfer improved singleton livebirth rate compared with fresh single blastocyst transfer.

Methods

This multicentre, non-blinded, randomised controlled trial was undertaken in 21 academic fertility centres in China. 1650 women with regular menstrual cycles undergoing their first cycle of in-vitro fertilisation were enrolled from Aug 1, 2016, to June 3, 2017. Eligible women were randomly assigned to either fresh or frozen single blastocyst transfer. The randomisation sequence was computer generated, with block sizes of two, four, or six, stratified by study site. For those assigned to frozen blastocyst transfer, all blastocysts were cryopreserved and a delayed frozen-thawed single blastocyst transfer was done. The primary outcome was singleton livebirth rate. Analysis was by intention to treat. This trial is registered at the Chinese Clinical Trial Registry, number ChiCTR-IOR-14005405.

Findings

825 women were assigned to each group and included in analyses. Frozen single blastocyst transfer resulted in higher rates of singleton livebirth than did fresh single blastocyst transfer (416 [50%] vs 329 [40%]; relative risk [RR] 1·26, 95% CI 1·14–1·41, p<0·0001). The risks of moderate or severe ovarian hyperstimulation syndrome (four of 825 [0·5%] in frozen single blastocyst transfer vs nine of 825 [1·1%] in fresh single blastocyst transfer; p=0·16), pregnancy loss (134 of 583 [23·0%] vs 124 of 481 [25·8%]; p=0·29), other obstetric complications, and neonatal morbidity were similar between the two groups. Frozen single blastocyst transfer was associated with a higher risk of pre-eclampsia (16 of 512 [3·1%] vs four of 401 [1·0%]; RR 3·13, 95% CI 1·06–9·30, p=0·029).

Interpretation

Frozen single blastocyst transfer resulted in a higher singleton livebirth rate than did fresh single blastocyst transfer in ovulatory women with good prognosis. The increased risk of pre-eclampsia after frozen blastocyst transfer warrants further studies.

Funding

The National Key Research and Development Program of China.

Introduction

Studies have shown that elective single embryo transfer (eSET) is the most efficient approach to reduce the risk of multiple gestations and their associated risks to mothers and children after in-vitro fertilisation (IVF).¹ Despite strong advocacy for its universal adoption,² its widespread uptake is slow because of concerns about the lower pregnancy rate after reducing the number of embryos transferred.³ Many efforts had been made to improve the selection of a single embryo that is likely to implant and thus to increase pregnancy rate after eSET.

Extending embryo culture to blastocyst from cleavage stage allows for better evaluation of the implantation potential of the embryo.⁴ The implantation rate is higher after blastocyst transfer than after cleavage-stage embryo transfer during fresh embryo transfer cycles.⁵ However, there are drawbacks to blastocyst culture. The failure to reach an embryo transfer because of poor or arrested embryo development increases in women seeking a blastocyst-stage embryo transfer compared with women with a cleavage-stage embryo transfer,⁶ especially in those with few day-3 cleavage-stage embryos and those with poor prognosis. Some cleavage-stage embryos that do not survive prolonged in-vitro culture, however, may continue to develop into viable pregnancies if they are transferred into the uterus on day 3,⁷ because in-vitro culture condition differs from the in-vivo environment. As a result, single blastocyst transfer strategy is recommended primarily to women with a good prognosis,⁸ who will probably have more cleavage-stage embryos available for extended culture.

In addition to embryo selection, improving the peri-implantation uterine environment could also contribute to better success rates after single embryo transfer. The supra-physiological level of administered gonadotropins or resultant increase in steroid hormones after ovarian stimulation might adversely affect the endometrial development.⁹ The endometrium after ovarian stimulation has been shown to exhibit histological advancement, alteration in gene expression, and structural abnormalities.¹⁰ With the development in cryopreservation technology, embryos could be more safely frozen and preserved for later use.¹¹ Elective frozen embryo transfer avoids the exposure of the endometrium to the adverse sequelae of ovarian stimulation and has been shown to result in a higher rate of livebirth than has fresh embryo transfer in women with polycystic ovary syndrome (PCOS).¹² However, in ovulatory women, frozen embryo transfer seemed to be as efficient and as safe as fresh embryo transfer to achieve a livebirth.13, 14 In previous trials, embryo transfer was done at cleavage stage and up to two embryos were transferred; the proportion of multiple pregnancies was high at approximately 30%, leading to increased maternal and fetal morbidity.13, 14 In the past 5 years, with the refinement of techniques for blastocyst culture and in compliance with the guidelines for reducing the risk of multiple pregnancies,¹⁵ the application of single blastocyst transfer has become increasingly popular. Frozen single blastocyst transfer could optimise pregnancy rates and maintain perinatal safety compared with fresh single blastocyst transfer.

Research in context

Evidence before this study

We searched Pubmed and Cochrane Library from database inception to May 1, 2018, with the keywords “frozen embryo” OR “frozen-thawed cycle” OR “cryopreservation” OR “vitrification” OR “freeze all” AND “fresh embryo”. We identified one Cochrane systematic review published in 2017, and five additional randomised trials that found conflicting results. The Cochrane review reported four randomised trials comparing fresh embryo transfer versus elective frozen embryo transfer. The authors concluded that frozen embryo transfer resulted in lower rates of miscarriage and ovarian hyperstimulation syndrome (OHSS), but a higher rate of pregnancy complications. No difference in the cumulative livebirth rate (based on subsequent embryo transfers of embryos cryopreserved from the study cycle of ovarian stimulation) was found. There was great heterogeneity among the trials included in the Cochrane review in terms of study populations, developmental stages of the transferred embryos, freezing methods, and the number of embryos transferred. The result was dominated by the trial undertaken in women with polycystic ovary syndrome (PCOS). Subsequently, two large randomised trials were undertaken in ovulatory women with cleavage-stage embryo transfer with consistent results showing that elective frozen embryo transfer led to similar rates of pregnancy, pregnancy loss, and livebirth compared with fresh embryo transfer. Another randomised trial compared frozen versus fresh euploid blastocyst transfer after preimplantation genetic screening and found higher rates of pregnancy and livebirth after frozen embryo transfer. The risk of obstetric complications was not reported. There were two other trials that were respectively undertaken in women with gonadotropin releasing hormone (GnRH) antagonist regimen and GnRH agonist trigger for ovarian stimulation and in women with elevated progesterone on the day of triggering; results showed no significant difference in the rates of pregnancy and livebirth. In all these trials, up to two embryos were transferred in both the fresh and frozen embryo transfer groups, leading to higher rates of multiple pregnancies and their associated perinatal morbidity. Whether frozen single blastocyst transfer could improve singleton livebirth rate compared with fresh single blastocyst transfer remained to be determined.

Added value of this study

In this multicentre randomised trial, 1650 ovulatory women with good prognosis from 21 fertility centres in China were randomly assigned to undergo either a frozen single blastocyst transfer or a fresh single blastocyst transfer. Frozen single blastocyst transfer resulted in a higher rate of singleton livebirth attributed to a higher rate of implantation than did fresh single blastocyst transfer. Frozen single blastocyst transfer also led to a higher singleton birthweight, which was accompanied by a higher risk of pre-eclampsia. The risks of OHSS, pregnancy loss, and other obstetric complications including preterm delivery and congenital anomalies were similar after frozen and fresh single blastocyst transfer.

Implications of all the available evidence

A strategy to transfer a single frozen blastocyst versus two cleavage-stage embryos results in a marked decrease in twin livebirth rates with a comparable overall livebirth rate. The available evidence on so-called freeze-all strategy suggested the risk–benefit ratio of elective frozen embryo transfer was influenced by several factors, including the patient diagnosis and the stage of embryo transferred. Elective frozen embryo transfer seems a better choice to achieve livebirth for women with PCOS, women with a higher risk of OHSS, and women with good prognosis who are planning to undergo single blastocyst transfer. However, its potential for increased maternal pre-eclampisa, as well as the long-term effects on offspring, warrant further studies.

In this randomised trial, we compared pregnancy outcomes and obstetric and perinatal complications after frozen versus fresh single blastocyst transfer.

Section snippets

Study design and participants

This study was a non-blinded, multicentre, randomised controlled trial undertaken in 21 academic fertility centres in China. The trial was approved by the ethics committees of all study sites. All the couples including female and male partners gave written informed consent. A data and safety monitoring board was established to oversee the study. We have previously published the protocol.¹⁶

This trial included women with regular menses who were undergoing the first cycle of IVF with or without

Results

Recruitment was done between Aug 1, 2016, and June 3, 2017. 1650 women were included and randomly assigned to either fresh or frozen single blastocyst transfer groups (figure). The fresh and frozen groups were comparable in baseline demographics and clinical characteristics (table 1) and the outcomes of ovarian stimulation (table 2).

98 (12%) women who were assigned to the fresh embryo transfer group actually underwent a frozen embryo transfer, while 37 (5%) women assigned to the frozen embryo

Discussion

In ovulatory women with a good prognosis, a frozen single blastocyst transfer resulted in a higher rate of singleton livebirth, which was mainly mediated by a higher rate of implantation, than did fresh single blastocyst transfer. The rate of pregnancy loss after frozen and fresh single blastocyst transfer was similar. Frozen blastocyst transfer led to a higher singleton birthweight but also a higher risk of LGA than did fresh single blastocyst transfer. The incidence of pre-eclampsia was

Data sharing

The study protocol and statistical analysis plan will be available online with publication. Data collected for the study, including specified dataset and a data dictionary defining each field in the set, will be made available to others with publication. Investigators can request data sharing by emailing the corresponding author. Our publication committee established for this trial will review and approve the request. An agreement on how to collaborate will be reached based on the overlaps and

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External bending of cryodevice during vitrification leads to cryoprotectant cracks and damage to embryo blastomeres
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Embryo blastomeres and the zona pellucida are occasionally damaged during vitrification; is this a result of crack-induced mechanical damage in the glass state, caused by external bending of the device?
A stereomicroscope was used to observe external bending-induced cracks in a cryoprotectant. Thereafter, 309 human cleavage-stage embryos derived from abnormally fertilized eggs were used to assess embryo damage under two external bending conditions: forward bending and backward bending, with three bending degrees applied. Three distinct embryo positions were used to examine the correlation between bending and embryo damage. Damage was assessed by looking at blastomere lysis rates, and overall rates of damaged and surviving embryos.
A series of parallel cracks were identified in the cryoprotectant used for external bending, which led to damage to the embryo blastomeres. Compared with forward bending and control, the embryos were found to be more easily damaged by backward bending, indicated by significantly higher blastomere lysis and embryo damage rates, and lower embryo survival rate of backward bending than forward bending (P < 0.001). The degree of embryo damage also increased as the degree of external forces increased. Embryo position correlated with degree of embryo damage.
Cryoprotectant crack-induced damage was identified as the cause of embryo damage. Mechanical damage to the glass state occurs because of improper external bending of the cryodevice strip in liquid nitrogen during vitrification. To prevent damage, bending of the strip should be avoided and the embryos should be placed near the tip of the strip.
Does embryo biopsy, independent of vitrification, impact perinatal outcomes? An analysis of perinatal outcomes following preimplantation genetic testing biopsy in fresh and frozen embryo transfer cycles
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To compare neonatal outcomes in pregnancies resulting from embryos that have undergone preimplantation genetic testing (PGT) biopsy compared with no biopsy in both fresh and frozen embryo transfers (ETs) and determine whether findings are mediated by multiple births.
Retrospective cohort study.
Society of Assisted Reproductive Technologies-Clinical Outcomes Reporting System data, 2014–2015.
Autologous in vitro fertilization treatment cycles using fresh or frozen blastocyst ET, with or without PGT biopsy.
Not applicable.
Large for gestational age (LGA), small for gestational age, and preterm delivery. Secondary outcomes included high birthweight, low birthweight, and clinical pregnancy measures. Outcomes were evaluated using log-binomial regression models with repeated measures. Models were used to estimate the controlled direct effects of biopsy on birth outcomes that were not mediated by multiple gestations.
In fresh ET, biopsy was associated with an increase in LGA (relative risk [RR] 1.45, confidence interval [CI] 1.04–2.02) that persisted in the model mediated for multiple gestation (RR 1.36, 95% CI 1.01–1.83) but was not present in an analysis restricted to elective single ET (RR 0.99, 95% CI 0.91–1.09). In frozen ET, there were no differences in any of the primary outcomes after accounting for multiple gestations.
In a large multicenter database, there were no differences in neonatal outcomes after PGT biopsy in frozen ET cycles, and an increase in LGA was noted in fresh transfers that persisted even after accounting for multiple gestations but was not present in analysis restricted to elective single ET.
Frozen autologous and donor oocytes are associated with differences in clinical and neonatal outcomes compared with fresh oocytes: a Society for Assisted Reproductive Technology Clinic Outcome Reporting System Analysis
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To study the clinical and neonatal outcomes of embryos derived from frozen oocytes relative to fresh oocytes in both autologous and donor oocyte cycles after fresh embryo transfer (ET).
This is a retrospective cohort study using the Society for Assisted Reproductive Technology Clinic Outcome Reporting System database between 2014 and 2015.
The Society for Assisted Reproductive Technology Clinic Outcome Reporting System database was used to identify autologous and donor oocyte cycles that resulted in a fresh ET during 2014 and 2015.
There were 154,706 total cycles identified that used embryos derived from fresh or frozen oocytes and resulted in a fresh ET, including 139,734 autologous oocyte cycles and 14,972 donor oocyte cycles.
Generalized linear regression models were used to compare the clinical and neonatal outcomes of frozen oocytes relative to fresh oocytes. Models were adjusted for maternal age, body mass index, smoking status, parity, infertility diagnosis, number of embryos transferred, and preimplantation genetic testing. An additional sensitivity analysis was performed to examine singleton pregnancies separately.
The live birth (LB) rate was the primary outcome. Secondary outcomes include pregnancy and birthweight outcomes.
Differences in clinical and neonatal outcomes between fresh and frozen-thawed oocytes after fresh ET were observed. Specifically, our study found a higher incidence of high-birthweight infants after the use of frozen oocytes relative to fresh oocytes in both autologous oocytes (12.5% [frozen] vs. 4.5% [fresh], adjusted risk ratio [aRR] 2.67, 95% confidence interval [CI] 1.65–4.3) and donor oocyte cycles (6.2% [frozen] vs. 4.6% [fresh], aRR 1.42, 95% CI 1.1–1.83). This finding remained true when the analysis was restricted to singleton gestations only for both groups: autologous (17.3% [frozen] vs. 7.1% [fresh], aRR 2.77, 95% CI 1.74–4.42) and donor oocytes (9.4% [frozen] vs. 7.8% [fresh], aRR 1.38, 95% CI 1.07–1.77). Additionally, we observed a decrease in LB (aRR 0.81, 95% CI 0.77–0.85); clinical pregnancy (aRR 0.83, 95% CI 0.8–0.87); and an increase in biochemical pregnancy loss (aRR 1.22, 95% CI 1.05–1.43) after the use of frozen oocytes in donors, but not autologous cycles.
Our findings of an increased incidence of high-birthweight infants after the transfer of embryos derived from frozen oocytes in both autologous and donor oocyte cycles raise questions about oocyte vitrification and deserve further study. Additionally, the finding of a decreased likelihood of LB with frozen-donor oocytes compared with fresh donor oocytes is an important finding, especially because more patients are seeking to use frozen oocytes in their donor egg cycles. Future research should be directed toward these findings to optimize the use of frozen oocytes in clinical practice.
Impact of trophectoderm biopsy for preimplantation genetic testing on obstetric and neonatal outcomes: a meta-analysis
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This study aimed to investigate whether trophectoderm biopsy for preimplantation genetic testing is associated with an increased risk of adverse obstetrical and neonatal outcomes compared with conventional in vitro fertilization or intracytoplasmic sperm injection without preimplantation genetic testing.
Entries between January 1990 and August 2022 were searched using MEDLINE, Embase, Web of Science, the Cochrane Library, and Google Scholar.
Publications comparing the outcomes of pregnancies after preimplantation genetic testing using trophectoderm biopsy and in vitro fertilization or intracytoplasmic sperm injection were included. Only human studies with a cohort or case-control design or randomized controlled trials were eligible for inclusion.
The study selection process was performed independently by 2 investigators. The quality of the observational studies was assessed using the Newcastle-Ottawa Scale, and the Cochrane risk-of-bias tool version 2 was used to grade the level of bias in randomized controlled trials. The pooled odds ratio and 95% confidence interval were calculated using a random-effects model when substantial heterogeneity occurred (indicated by I² of >50% and P<.1). Otherwise, a fixed-effects model was used.
This meta-analysis included 13 studies involving 11,469 live births after preimplantation genetic testing treatment with trophectoderm biopsy before embryo transfer and 20,438 live births after in vitro fertilization or intracytoplasmic sperm injection only. The odds ratio of preterm delivery was higher in the trophectoderm-biopsied group than in the routine in vitro fertilization or intracytoplasmic sperm injection group (pooled odds ratio, 1.12; 95% confidence interval, 1.03–1.21); however, the difference did not exist after sensitivity analysis (odds ratio, 0.97; 95% confidence interval, 0.84–1.11). The risk of low birthweight did not increase among the biopsied pregnancies (pooled odds ratio, 1.01; 95% confidence interval, 0.85–1.20). No marked difference was observed in the risk of other obstetrical or neonatal outcomes between the biopsy and control groups. Furthermore, no difference was noted in the perinatal outcomes between trophectoderm-biopsied and nonbiopsied groups in the subgroup analyses by intracytoplasmic sperm injection, frozen-thawed transfer, or single embryo transfer.
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Ambient ozone and ovarian reserve in Chinese women of reproductive age: Identifying susceptible exposure windows
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Little is known about the association of ambient ozone with ovarian reserve. Based on a retrospective cohort study of 6008 women who attended a fertility center in Hubei, China, during 2018–2021, we estimated ozone exposure levels by calculating averages during the development of follicles (2-month [W1], 4-month [W2], 6-month [W3]) and 1-year before measurement (W4) according to Tracking Air Pollution in China database. We used multivariate logistic regression and linear regression models to investigate association of ozone exposure with anti-müllerian hormone (AMH), the preferred indicator of ovarian reserve. Each 10 μg/m³ increases in ozone were associated with 2.34% (0.68%, 3.97%), 2.08% (0.10%, 4.01%), 4.20% (1.67%, 6.67%), and 8.91% (5.79%, 11.93%) decreased AMH levels during W1-W4; AMH levels decreased by 15.85%, 11.90%, 16.92% in the fourth quartile during W1, W3, and W4 when comparing the extreme quartile, with significant exposure-response relationships during W4 (P < 0.05). Ozone exposure during W1 was positively associated with low AMH. Additionally, we detected significant effect modification by age, body mass index, and temperature in ozone-associated decreased AMH levels. Our findings highlight the potential adverse impact of ozone pollution on female ovarian reserve, especially during the secondary to small antral follicle stage and 1-year before measurement.
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^†: These authors contributed equally to this Article

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ArticlesFrozen versus fresh single blastocyst transfer in ovulatory women: a multicentre, randomised controlled trial

Summary

Background

Methods

Findings

Interpretation

Funding

Introduction

Section snippets

Study design and participants

Results

Discussion

Data sharing

Best Pract Res Clin Obstet Gynaecol

Fertil Steril

Fertil Steril

Fertil Steril

Am J Obstet Gynecol

Fertil Steril

Elective single-embryo transfer

Fertil Steril

Elective single embryo transfer: an update to UK Best Practice Guidelines

Hum Fertil (Camb)

In vitro fertilization with single blastocyst-stage versus single cleavage-stage embryos

N Engl J Med

Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology

Cochrane Database Syst Rev

Is it time for a paradigm shift in understanding embryo selection?

Reprod Biol Endocrinol

Blastocyst culture and transfer in clinical-assisted reproduction: a committee opinion

Fertil Steril

The endometrium in stimulated cycles for IVF

Hum Reprod Update

Gene expression profiles and structural/functional features of the peri-implantation endometrium in natural and gonadotropin-stimulated cycles

J Clin Endocrinol Metab

Oocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance

Hum Reprod Update

Articles
Frozen versus fresh single blastocyst transfer in ovulatory women: a multicentre, randomised controlled trial