The Developmental Stage at Cryopreservation in Assisted Reproduction: A Systematic Review and Meta-Analysis of Pregnancy Outcomes

Introduction

Since the first pregnancy with frozen-thawed human embryo suc-ceeded in 1983, assisted reproductive technologies (ARTs) have benefited infertile couples who could not conceive without treat-ment [1]. The progress in embryo cryopreservation technologies further advanced the success rate of ART. In addition to patients’ characteristics, the efficacy of embryo transfer is influenced by multiple variables, including hormone supplementation protocols, embryo quality, developmental stages of embryo transferred, cryo-preservation methods, culture media, and synchronization between the embryo and endometrial development.

Among all variables mentioned previously, embryo cryopreser-vation has become a critical and indispensable part of ART. The methods of cryopreservation can be categorized into conventional slow-freezing method and vitrification, the former is known for its lower survival rates for blastocysts, which makes vitrification gradually replaces the dominance of the conventional slow-freez-ing method [2]. Vitrified-thawed embryos have equivalent or even better clinical outcomes as compared with the slow-freezing meth-od in both the cleavage and blastocyst stage embryos [3-8].

In frozen embryo transfer, factors such as extended culture after thawing/warming may further impact pregnancy outcomes. It is suggested that extended culture to the blastocyst stage enables self-selection of viable cells and thus leading to a higher implan-tation rate [9].

Some studies also showed that, although cumu-lative pregnancy rates were similar between cleavage-stage and blastocyst embryos, the transfer frequency before pregnancy was significantly lower in the blastocyst group, indicating shorter time to pregnancy and reduced medical cost [10]. Moreover, for ART complications such as multiple pregnancy, ectopic pregnancy, and miscarriage, it was reported that blastocysts advantage over cleav-age-stage embryos. With a lower number of transferred embryos, blastocyst transfer was logically expected to have a lower multiple pregnancy rate and therefore could be a better strategy to prevent the incidence of multiple pregnancy [10]. Some studies suggested other advantages of replacing fro-zen-thawed blastocysts. Fang et al. have shown that patients re-ceived frozen-thawed Day-5 blastocysts had a lower ectopic preg-nancy rate as compared with frozen-thawed Day-3 embryos and fresh embryos [11]. Nevertheless, prolonged culture results in few-er embryos available for transfer and thus increases the cancella-tion rate of the transfer cycle. On the other hand, blastocyst culture before cryopreservation rather than after thawing may be preferred by clinics for cost-effectiveness issue.

The outcomes of embryo transferred at different developmental stages (the cleavage stage [day 2 or day 3] and the blastocyst stage [day 5 or day 6]) following fresh or fresh and subsequent frozen embryo transfer (cumulative outcomes) have been broadly inves-tigated and well-reviewed [9]. Nevertheless, only limited studies have specifically investigated and compared the outcomes of fro-zen embryo transfer with different developmental stage cryopres-ervation. Although the technology of cryopreservation has greatly advanced in the past decade, there is still no clear consensus on which developmental stage an embryo should be cryopreserved. Additionally, no meta-analyses have specifically investigated whether the developmental stages at which an embryo was frozen would influence the outcomes of embryo transfer.

For the reasons mentioned above, a question was raised: for pa-tients who need frozen-thawed embryo transfer, do embryos fro-zen at different developmental stages affect the outcomes of as-sisted reproduction? To answer this question, we systematically examined literature regarding comparisons of embryo cryopre-served at different developmental stages (2PN [2 pronuclear], cleavage, or blastocyst stages) but transferred at the same stage, and subsequently analyzed the pregnancy outcomes following the frozen-embryo transfer.

Methods

Registration number

The protocol for this systematic review has been registered in PROSPERO (available on https://www.crd.york.ac.uk/prospero/ display_record.asp?ID=CRD42017072828).

Study selection and search strategy

Both prospective and retrospective studies that met eligibility cri-teria and investigated the outcomes of frozen embryo transfer for ART were to be included in this review. The inclusion criteria were as follows: (1) women/couples undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and subsequently receiving frozen embryo transfer (FET); (2) women/couples trans-ferred with embryos cryopreserved using either slow-freezing or vitrification method; (3) studies showed the outcomes of embryos frozen at different stages but transferred at the same stage. The primary outcome was live birth/delivery rate. The secondary outcomes included clinical pregnancy rate, implantation rate, on-going pregnancy rate, miscarriage rate, and multiple pregnancy rate.

In July 2019, the literature search was conducted in PubMed electronic database covering articles published between 2002 and 2017, with keywords including ((vitrify*) OR (cooling) OR (frozen) OR (freez*) OR (cryopreserve*) OR (cryotransfer)) AND (transfer) AND (embryo) AND ((stage) OR (day) OR (de-velopment*) OR (pronuclear) OR (cleavage) OR (blastocy*)). All searches were limited to human studies published in the language of English and excluded reviews, editorials, and case reports. The papers without presenting comparisons between different develop-mental stages at the time of freezing were excluded. Studies with mixed and inseparable results of embryos frozen using vitrification and slow-freezing were excluded as well.

This study was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) state-ment (Table S1 PRISMA Checklist) 

Supporting Information

                        Table: S1 PRISMA checklist

From: Moher D, Liberati A, Tetzlaff J, Altman DG, The PRISMA Group (2009). Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. PLoS Med 6(7): e1000097. doi:10.1371/journal.pmed1000097

Data extraction and quality assessment

The abstracts retrieved from the search were screened by two inde-pendent reviewers (JYPH and ACHL), after which the full articles meeting the inclusion criteria were retrieved. The references cited in retrieved articles were reviewed. Subsequently, the eligibility was assessed, and the data were extracted independently by the two authors (JYPH and ACHL). The risk of bias for eligible cohort studies was evaluated using the Newcastle-Ottawa Scale. Any dis-crepancies were resolved by consensus, and all authors critically analyzed the results.

Data Synthesis and Analysis

In the meta-analysis, the outcomes of blastocyst transfer of em-bryos frozen at the non-blastocyst stage were compared with those frozen at the blastocyst stage. In the case of cleavage stage transfer, the outcomes of embryos cryopreserved at the 2PN stage versus the cleavage stage were analyzed. For insufficient or missing data within the literature, attempts were made to contact the investiga-tors of individual studies via e-mail. Subsequently, dichotomous outcomes of eligible studies for meta-analysis were calculated and expressed in terms of odds ratios (ORs) with 95% confidence intervals (CIs) and combined with Stata (version 13; StataCorp LLC, College Station, TX) using Mantel-Haenszel model (fixed effect) and the DerSimonian and Laird method (random effect). Heterogeneity among studies was assessed by I2 and chi-squared tests. Funnel plots were not generated when there were less than 10 studies, as the plots could be misleading [12].

Results

Study selection and quality assessment

A total of 885 potentially relevant studies was identified via the electronic search on the PubMed. Additional nine articles were identified through reviewing the retrieved articles. Following the screening of abstracts, 856 studies were excluded, and 38 studies were reviewed for eligibility. After reading the manuscripts and assessing the inclusion criteria and methodology, five remaining articles fulfilling the inclusion criteria were included in this review (Figure 1).

Figure 1: PRISMA flow diagram

All five studies were retrospectively conducted as single-center studies. It was found that the methodology varied substantially between studies. While one study vitrified embryos, four studies utilized slow-freezing protocols. Three papers investigated the impact of developmental stages on the outcomes of embryo trans-fer with surplus embryos following fresh cycles, and the rest two studies involved whole embryo freezing (freeze-all). The charac-teristics of these studies are summarized in Table 1, and the risks of bias were assessed and summarized in Table 2.

               Table 1: Characteristics of All Studies Included

OHSS, ovarian hyperstimulation syndrome.

   Table 2: Newcastle-Ottawa Scale Summary Assessment of Risk of Bias

Each study is assessed with eight domains categorized into three groups, Selection, Comparability, and Outcome. A study can be awarded a maximum of one star for each domain except the Com-parability, which can be awarded for a maximum of two stars for important or additional factor [13-17].

Synthesis of Results

To explore the impact of the frozen stage to the clinical outcomes of embryo transfer, the results of studies comparing embryos fro-zen at different stages but transferred at the same stage were syn-thesized for further analysis. Two scenarios were investigated, em-bryos transferred at the cleavage or blastocyst stages.

For blastocyst embryo transfer, the outcomes of blastocyst versus non-blastocyst cryopreservation were analyzed. The forest plots of primary and secondary outcomes are displayed in Figs. 2 and 3, respectively. The results showed that frozen at the non-blastocyst stage was associated with a significant improvement in live birth/ delivery rate as compared with embryos cryopreserved at the blas-tocyst stage (2652 thawing cycles; 3 studies; OR=1.37; 95%CI, 1.13-1.66). Nevertheless, no significant difference was observed in clinical pregnancy rate (2652 thawing cycles; 3 studies; OR=0.92; 95%CI, 0.76-1.12), implantation rate (3821 embryos; 2 studies; OR=0.89; 95%CI, 0.76-1.05), and miscarriage rate (581 clinical pregnancies; 3 studies; OR=0.79; 95%CI, 0.49-1.25). With respect to ongoing pregnancy rate, there was only one study identified for this outcome measure and no statistical significance was observed.

Figure 2: Forest plot of primary outcome for embryos transferred at blastocyst. Meta-analyses of live birth/delivery rate of embryos frozen at blastocyst and non-blastocyst were displayed

Figure 3: Forest plots of secondary outcomes for embryos trans-ferred at blastocyst. Meta-analyses of (A) implantation rate, (B) clinical pregnancy rate, (C) ongoing pregnancy rate, and (D) mis-carriage rate of embryos frozen at blastocyst and non-blastocyst were displayed

For cleavage stage transfer, the outcomes of embryos cryopre-served at the 2PN stage were compared with those frozen at the cleavage stage. As shown in Figure 4, the forest plots of live birth/delivery rate, implantation rate, clinical pregnancy rate, and miscarriage rate are displayed (no data were available for ongo-ing pregnancy rate and multiple pregnancy rate). Only one study meeting the eligibility criteria was identified for the outcomes of live birth/delivery rate, implantation rate, and miscarriage rate. For clinical pregnancy rate, no difference was found between 2PN-and cleavage-stage transfer (805 transfers; 2 studies; OR=1.24; 95% CI, 0.

Figure 4: Forest plots of embryos transferred at cleavage stage. Meta-analyses of (A) live birth/delivery rate, (B) implantation rate, (C) clinical pregnancy rate, and (D) miscarriage rate of embryos frozen at cleavage and 2PN stages were shown

Discussion

The success with frozen embryo transfer has dramatically im-proved in recent years. Embryo freezing allows the surplus good-quality embryos in the fresh cycle can be preserved. In addi-tion, freeze-all of embryos serves as a favorable option for patients at high risk of OHSS during the fresh cycle. Nevertheless, with the increasing need for frozen embryo transfer, there is still no consensus on which developmental stage embryos should be cryo-preserved. For this reason, a systematic review and meta-analysis was conducted to provide insight into the influence of develop-mental stages for embryo freezing. To our knowledge, this is the first systematic review and meta-analysis that specifically investi- gated whether the developmental stages at which an embryo was frozen would impact the outcomes of frozen embryo transfer. The current review analyzed the available results regarding the impact of developmental stage at frozen on the outcomes of ART follow-ing frozen-thawed embryo transfer. Five articles were identified through systematic search. As all studies were carried out and retrospectively reviewed at a single center, different institutional policies thereby directly resulted in different protocols in culture media, freezing protocol, the number of embryos replaced, as well as stimulation protocols. Hence, the design and methodology ap-plied to these studies varied substantially. In addition to the high level of heterogeneity, the definitions and outcomes evaluated also differed between the studies.

For most clinics or hospitals, the number of cleavage embryos re-placed is often greater than that of blastocyst embryos due to dif-ferent implantation potential between cleavage embryos and blas-tocysts. Of the five studies included, significant differences in the number of embryos replaced between the cleavage and blastocyst stages were found in three studies, whereas the rest two had a com-parable number of embryos replaced (Table S2). In the present re-view, we compared outcomes of embryos cryopreserved at differ-ent stages but transferred at the same stage in order to eliminate the impact of implantation potential from different stage of embryos.

Supporting Information

                                         Table: S2 Summary of the number of embryo replaced

NS, not significant; NA, not available: FET, frozen embryo transfer; CSE, transferred at cleavage stage; BL, transferred at blastocyst stage.

In consideration of the fact that freezing methods significantly influenced the survival and pregnancy outcomes of frozen em-bryo transfer, the present review has prospectively designed to exclude studies with mixed results of the different freezing meth-ods [3,4,8,18]. For this reason, three studies have been excluded from the search results, including one randomized controlled trial (RCT) and two population-based studies based on registry data-bases. Although registry-based studies have a large study popula-tion, limitations such as inconsistent data collection, information unavailable, and potential variability in reporting outcomes are hardly avoided. Therefore, these studies are not suitable to be com-bined and analyzed in the systematic review and meta-analysis.

This study aimed to elucidate whether embryos frozen at differ-ent developmental stages would affect the pregnancy outcomes. About a dozen studies were excluded because they focused on the outcomes of the cleavage versus blastocyst stage transfer. Post-thaw culture was only applied in five studies, which were therefore included in this review. Through combining results from different studies, embryos frozen at different stages but transferred at the same stage were allowed to be compared. In our results, no differ- ence was observed in primary and secondary outcomes of cleav-age embryo transfer group. Nevertheless, for blastocyst embryo transfer group, there was a significantly higher delivery/live birth rate with embryos frozen at the non-blastocyst stage as compared with those cryopreserved at the blastocyst stage. In comparison with embryos transferred soon after thawing, extended culture of non-blastocyst embryos after thawing provides a longer recovery time and opportunity to select better blastocyst, and therefore they were relatively competent while being replaced.

Most studies identified in the present review performed frozen em-bryo transfer using supernumerary embryos of the retrieval cycle. For women with a high risk of ovarian hyper stimulation syndrome (OHSS) or adverse endocrinological profile, freezing all embryos provides a valuable alternative when a fresh transfer is not advan-tageous. In addition, because top-quality embryos are generally transferred in fresh cycles, cryopreservation of supernumerary em-bryos may result in lower rates in pregnancy outcomes. Ideally, freeze-all strategies are better to be evaluated as primary therapies. In the current review, only one RCT focused on exploring the out-comes of cryopreserved-embryo transfer exclusively in patients undergoing freeze-all [19]. In the randomized trial conducted by Shapiro et al., embryos frozen at the 2PN or blastocyst stages were both transferred at the blastocyst stage [19]. However, this study was not included in the present review because the results of em-bryo cryopreservation using vitrification or slow-freezing were mixed and inseparable, which makes the study ineligible for inclu-sion. In contrast to Shapiro’s study, the three studies included for meta-analysis in this review were all retrospective, with heteroge-neity in design. Thus, further randomized controlled research is suggested before drawing definitive conclusions.

Blastocyst transfer is suggested to improve embryo-endometrial synchronicity and enable self-selection of viable embryos through the period of extended culture. Thus, culturing of embryos to the blastocyst stage may serve as a suitable strategy without compro-mising the transfer outcomes. However, for blastocyst transfer, freezing at an earlier stage other than blastocyst markedly in-creases the workload imposed on embryologists for the embryo freezing processes. Moreover, if preimplantation genetic test for aneuploidy or embryo biopsies are scheduled, blastocyst extended culture before cryopreservation may be mandatory. However, in the current study, significant higher live birth/delivery rates were noted in a setting of non-blastocyst embryo cryopreservation and post-thawed extended culture to blastocyst and transfer. Thus, whether an extended culture should be performed before or after cryopreservation needs multi-facet evaluations.

There are some limitations in the studies included in the present re-view. No RCTs were included in the final search results so that no results from RCTs were available for the meta-analyses. Studies with extended culture after thawing were limited. Therefore, the analyzable data for data synthesis were restricted. Moreover, some studies only investigated the pregnancy outcome by the number of transfer or thawing cycle without providing information of subject number, which may significantly affect the outcome variable. In addition to the pregnancy outcomes analyzed in the present review, other clinical considerations such as ectopic pregnancy rate, time to live birth, cost-effectiveness, and neonatal outcomes are also of great value. Nevertheless, these outcomes are less frequently investigated and therefore were not included in the present review.

Conclusions

The present review summarizes pregnancy outcomes from five articles evaluating the developmental stages of frozen embryo transfer following IVF or ICSI. Meta-analyses were conducted for the outcomes of embryos frozen at different stages but transferred at the same stage. Although there were no significant differenc-es in various outcomes for frozen-thawed embryo transferred at cleavage stage group, we found that there was a significant dif-ference in blastocyst embryo transfer group. Embryos frozen at non-blastocyst showed a statistically higher live birth/delivery rate as compared with those cryopreserved at blastocyst in the setting of blastocyst frozen embryo transfer cycles. There is currently no sufficient evidence to recommend a specific embryonic stage for cryopreservation in couples undergoing ART. Nevertheless, the fa-vorable live birth/delivery rate of extended culture to blastocyst af-ter non-blastocyst thawing provides a new insight with regards to future clinical practices. Randomized controlled trials are warrant to explore the impact of embryo stage at freezing on pregnancy outcomes.

Data Availability

All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).

Author Contributions

J.Y.P.H. contributed to the study design, acquisition of data, analy-sis, and interpretation of data, as well as article drafting and revis-ing. A.C.H.L. contributed to the acquisition of data, interpretation of data, and revision of the article. All authors read and approved the final manuscript and participated in the decision to submit the manuscript for publication.

Acknowledgments

Medical writing assistance was funded by Merck Ltd., an affiliate of Merck Healthcare KGaA, Darmstadt, Germany. The funder did not have any role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors thank Li-Hsin Chen of Bestat Pharmaservices Corp., Taiwan for providing medical writing support.

Disclosure of Potential Conflicts of Interest

ACHL and JYPH claim no conflicts of interest. There are no addi-tional declarations from the authors relevant to this research relat-ing to employment, consultancy, products in development, patents, or revenues from marketed products to declare.

References

1. Trounson A, Mohr L (1983) Human pregnancy following cryopreservation, thawing and transfer of an eight-cell em-bryo. Nature 305: 707-709.
2. Loutradi KE, Efstratios M Kolibianakis, Christos A Vene-tis, Evangelos G Papanikolaou, George Pados, et al. (2008) Cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis. Fertility and sterility 90: 186-193.
3. Fasano G, Nicolas Fontenelle, Anne-Sophie Vannin, Jamila Biramane, Fabienne Devreker, et al. (2014) A randomized controlled trial comparing two vitrification methods versus slow-freezing for cryopreservation of human cleavage stage embryos. Journal of Assisted Reproduction and Genetics 31: 241-247.
4. Balaban B, B Urman, B Ata, A Isiklar, M G Larman, et al. (2008) A randomized controlled study of human Day 3 em-bryo cryopreservation by slow freezing or vitrification: vit-rification is associated with higher survival, metabolism and blastocyst formation. Human reproduction (Oxford, England) 23: 1976-1982.
5. Summers D, Check JH, Choe JK (2016) A prospective com-parison of outcome following cryopreservation using vitri-fication vs. a modified slow-freeze protocol of 2 pronuclear (2PN) and day 3 multi-cell embryos. Clinical and experimen-tal obstetrics and gynaecology 43: 330-331.
6. Zhu HY, Ya-Mei Xue, Ling-Yun Yang, Ling-Ying Jiang, Chao Ling, et al. (2015) Slow freezing should not be totally substi-tuted by vitrification when applied to day 3 embryo cryopres-ervation: an analysis of 5613 frozen cycles. Journal of Assist-ed Reproduction and Genetics 32: 1371-1377.
7. Levron J, Oshrit Leibovitz, Masha Brengauz, Hila Gitman, Gil M Yerushalmi, et al. (2014) Cryopreservation of day 2-3 embryos by vitrification yields better outcome than slow freezing. Gynecological endocrinology: the official journal of the International Society of Gynecological Endocrinology 30: 202-204.
8. Kuc P, Agnieszka KuczyÅ?ska, Bozena Stankiewicz, Piotr SieczyÅ?ski, Joanna Matysiak, et al. (2010) Vitrification vs. slow cooling protocol using embryos cryopreserved in the 5th or 6th day after oocyte retrieval and IVF outcomes. Folia histochemica et cytobiologica / Polish Academy of Sciences, Polish Histochemical and Cytochemical Society 48: 84-88.
9. Glujovsky D, Farquhar C, Quinteiro Retamar AM, Alvarez Sedo CR, Blake D (2016) Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology. The Cochrane database of systematic reviews Cd002118.
10. Qiao-hua He, Lu Wang, Lin-lin Liang, He-long Zhang, Cui-li-an Zhang, et al. (2016) Clinical outcomes of frozen-thawed single blastocyst transfer in patients requiring whole embryo freezing. Systems biology in reproductive medicine 62: 133-138.
11. Fang C, Huang R, Wei LN, Jia L (2015) Frozen-thawed day 5 blastocyst transfer is associated with a lower risk of ectopic pregnancy than day 3 transfer and fresh transfer. Fertility and sterility 103: 655-661.
12. Anzures-Cabrera J, Higgins J P (2010) Graphical displays for meta-analysis: An overview with suggestions for practice. Re-search synthesis methods 1: 66-80.
13. Veeck LL (2003) Does the developmental stage at freeze im-pact on clinical results post-thaw? Reproductive biomedicine online 6: 367-374.
14. Noyes N, Reh A, McCaffrey C, Tan O, Krey L (2009) Im-pact of developmental stage at cryopreservation and transfer on clinical outcome of frozen embryo cycles. Reproductive biomedicine online 3: 9-15.
15. Vasiliki A Moragianni, Jerry D Cohen, Scott E Smith, Jay S Schinfeld, Stephen G Somkuti, et al. (2010) Outcomes of day-1, day-3, and blastocyst cryopreserved embryo transfers. Fer-tility and sterility 93: 1353-1355.
16. Mesut N, Ciray HN, Mesut A, Aksoy T, Bahceci M (2011) Cryopreservation of blastocysts is the most feasible strategy in good responder patients. Fertility and sterility 96: 1121-1125.
17. Cobo A, María José de los Santos, Damià Castellò, Pilar Gámiz, Pilar Campos, et al. (2012) Outcomes of vitrified early cleavage-stage and blastocyst-stage embryos in a cryopreser-vation program: evaluation of 3,150 warming cycles. Fertility and sterility 98: 1138-1146.
18. Debrock S, K Peeraer, E Fernandez Gallardo, D De Neubourg, C Spiessens, et al. (2015) Vitrification of cleavage stage day 3 embryos results in higher live birth rates than conventional slow freezing: a RCT. Human reproduction (Oxford, England) 30: 1820-1830.
19. Shapiro BS, Daneshmand ST, Garner FC, Aguirre M, Hudson C (2015) Freeze-all at the blastocyst or bipronuclear stage: a randomized clinical trial. Fertility and sterility 104: 1138-1144.