Impact of cell loss after warming of human vitrified day 3 embryos on obstetric outcome in single frozen embryo transfers.

Purpose Does cell loss (CL) after vitrification and warming (V/W) of day 3 embryos have an impact on live birth rate (LBR) and neonatal outcomes?

This retrospective analysis includes cleavage stage day 3 embryos vitrified/warmed between 2011 and 2018. Only single vitrified/warmed embryo transfers were included. Pre-implantation genetic screening, oocyte donation, and age banking were excluded from the analysis. The sample was divided into two groups: group A (intact embryo after warming) and group B (≤ 50% blastomere loss after warming).

On the total embryos (n = 2327), 1953 were fully intact (83.9%, group A) and 374 presented cell damage (16.1%, group B). In group B, 62% (232/374) of the embryos had lost only one cell. Age at cryopreservation, cause of infertility, insemination procedure, and semen origin were comparable between the two groups. The positive hCG rate (30% and 24.3%, respectively, for intact vs CL group, p = 0.028) and LBR (13.7% and 9.4%, respectively, for intact vs CL group, p = 0.023) per warming cycle were significantly higher for intact embryos. However, LBR per positive hCG was equivalent between intact and damaged embryos (45.6% vs 38.5%, respectively, p = 0.2). Newborn measurements (length, weight, and head circumference at birth) were comparable between the two groups. Multivariate logistic regression showed that the presence of CL is not predictive for LB when adjusting for patients’ age.

LBR is significantly higher after transfer of an intact embryo compared to an embryo with CL after warming; however, neonatal outcomes are comparable between the two groups.

Vitrification of embryos, Cell damage, Blastomere loss, Neonatal outcome, Live birth rate

The application of frozen embryo transfer (FET) has progressively increased during the last two decades due to advances in the efficacy and safety of cryopreservation strategies [1–3]. Indeed, vitrification has replaced the slow-freezing technique as a result of improved survival rates and higher implantation rates. Although today FET is widely used in assisted reproductive technology, several concerns have recently emerged regarding its safety in terms of pregnancy, obstetric, and perinatal outcomes. Following slow freezing, blastomere loss impairs embryo post-thawing in vitro development [4–6]. Moreover, compared to fully intact embryos, day 3 vitrified embryos with cell loss after warming also show lower overnight cleavage [7]. However, the latter study also reported that when a damaged embryo underwent overnight cleavage, similar implantation rates were found between intact and damaged embryos. Likewise, Edgar et al. emphasized the importance of further cleavage after warming for implantation rate, irrespective of blastomere survival [4]. Early reports initially based on slow freezing concluded that transfers of embryos with cell loss negatively affect implantation and conception rate [8–10]. Other studies showed that transfers of embryos with blastomere loss are not associated with lower implantation rates compared to those with intact blastomeres [11–13]. In addition, a recent retrospective study compared obstetrical outcomes of neonates born after a transfer of an intact embryo with those deriving from an embryo with cell loss, highlighting an increased risk to deliver small for gestational age babies or with transient tachypnea at birth [14]. In contrast, recent evidence concluded that blastomere loss is not associated with an increased risk of any adverse neonatal outcome in the singletons, describing comparable neonatal conditions between embryos derived from blastomere loss embryos and intact embryos [15]. In clinical practice, embryos with blastomere loss are generally transferred because they can implant and further develop; nevertheless, the possibility of increased adverse neonatal outcomes remains a concern for clinicians. In this scenario, the present study aims at investigating whether transfers of single vitrified day 3 embryo with blastomere loss impact on pregnancy, live birth rate, and obstetric outcome.

Study design This is a retrospective, single-center cohort study including day 3 vitrified–warmed embryos that were transferred between 2011 and 2018 at the Centre for Reproductive Medicine, Universitair Ziekenhuis Brussel, Belgium. The study was approved by the Ethics Committee of the Universitair Ziekenhuis Brussel (B.U.N.143201940782). Study population The analysis included all vitrified/warmed day 3 single embryo transfers obtained from consecutive treatments with standard in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Each woman was included only once in our analysis. Patients undergoing in vitro maturation (IVM) procedures, who were oocyte donors or recipients or had undergone embryo biopsy for pre-implantation genetic diagnosis, were not included. Embryos were divided into two groups, namely group A (intact embryo after warming) and group B (embryo with blastomere loss after warming). An intact embryo was defined as an embryo that remained fully intact after warming, not showing any blastomere loss. Conversely, an embryo with blastomere loss was defined as an embryo that lost one or more blastomeres after thawing, referred to as partially damaged, but with at least 50% of the blastomeres intact. Stimulation protocol Ovarian stimulation was started on day 2 or 3 of the menstrual cycle with daily injections of gonadotrophins, followed by a daily dose of 0.25 mg of GnRH antagonist in a fixed protocol, starting 6 days after the gonadotrophin intake. Cycle monitoring was performed through serum estradiol (E2), progesterone (P), and luteinizing hormone (LH) assessments, and serial trans-vaginal ultrasound examinations [16]. Ovulation triggering was performed with the administration of hCG or GnRH agonist in case of risk for OHSS [17], as soon as three follicles reached 17 mm of diameter. Oocyte retrieval was performed 36 h later. Collected oocytes were inseminated via either conventional IVF or ICSI. FET preparation protocol FET preparation methods used included hormonal replacement therapy (HRT) and natural cycle (NC) protocol with spontaneous or triggered ovulation. In the HRT protocol, estradiol valerate 6 mg daily (Progynova, Bayer HealthCare Pharmaceuticals) was administered orally from day 1 or 2 of the menstrual cycle onwards. Embryo transfers were planned on the fifth day of progesterone intake (Utrogestan, Vifor Pharma, 400 mg twice daily). Exogenous hormonal supplementation was continued for 14 days until a blood B-hCG test was performed. Patients with a positive test continued with hormone supplementation until 12 weeks of gestation. Conversely, the use of NC with spontaneous ovulation did not require any pharmacological intervention [18]. Serial blood test for hormonal workup and ultrasound monitoring during the proliferative phase were exclusively performed to identify the presence of a dominant follicle as well as the LH surge, in order to schedule the transfer when the endometrium was synchronized to the developmental stage of the embryo. Embryo transfers were planned on day 5 from the LH peak. In case of NC with triggered ovulation protocol, hCG 5000 IU was administered as soon as a dominant follicle of > 16 mm was observed. Embryos were warmed 1 day before embryo transfer, cultured overnight, and transferred on the sixth day from the hCG administration. Embryo selection before vitrification On day 3 in the morning, embryos with at least 6 cells and ≤ 20% fragmentation were selected for vitrification. Vitrification method The cryopreservation method used for all the embryos analyzed in the study was closed vitrification using CBS-VIT High-Security straws (Cryo Bio System, L'Aigle, France) using DMSO–ethylene glycol (EG)–sucrose(S) as the cryoprotectants (Irvine ScientificR Freeze kit, Newtownmountkennedy, County Wicklow, Ireland) [7]. The device and vitrification media did not change during the study period; neither did the protocol for day 3 vitrification change over time. Cell loss assessment after warming and quality assessment at transfer Day 3 embryos were warmed 1 day prior to transfer and transferred as day 4 embryos after overnight culture. The number of surviving cells was indicated on the total number of cells that were visible at evaluation immediately after warming (e.g., 5/8 or 8/8). If a single ET was planned, one embryo (the best according to quality at freezing) was put in culture for overnight cleavage when at least 50% of the cells were surviving. If more than 2 cells were damaged and other embryos were available, a second embryo was thawed, since we know that further cleavage is more likely in embryos without or with minimal damage [7]. On day 4, further cleavage was assessed and could be taken into consideration to determine the final embryo quality at transfer. Embryo quality at transfer was categorized into 4 qualities depending on the degree of further cleavage and final cell stage. Quality 1 embryos were embryos that were already compacting after overnight cleavage or even reached the blastocyst stage; embryo quality 2 included embryos with > 8 cells and with at least further cleavage of 2 blastomeres. Embryo quality 3 included embryos with at least 8 cells and cleavage of 1 blastomere. Embryo quality 4 was defined as embryos which had < 8 blastomeres and/or no signs of further cleavage. An extra embryo thawing on day 4 was never realized to avoid embryo–endometrial asynchrony. Assisted hatching was not performed after warming. Main outcome measures The primary aim of this retrospective cohort study was to evaluate the possible association between cell loss after warming and live birth rate (LBR). The secondary endpoint was to evaluate the potential association between embryo cell loss after warming and neonatal measurements (length, weight, and head circumference at birth). Statistical analysis Continuous data were presented as mean ± standard deviation (SD), and categorical data were described as number and percentages. Continuous variables were analyzed using the independent t test or Mann–Whitney U test depending on the normality of the distribution. Normality was examined using the Shapiro–Wilk test. Categorical variables were analyzed by Pearson’s chi-squared test or Fisher’s exact test, as appropriate. To study the association between blastomere loss and pregnancy as well as neonatal outcomes (length, weight, and head circumference at birth), odds ratios were calculated for each outcome, after adjusting for potential confounders, using multivariate logistic regression. All covariates (age of the patient at the moment of embryo cryopreservation, and intact or no embryos) were simultaneously entered into the multivariable logistic regression model. The assumptions for the final model were successfully tested. All statistical tests used a two-tailed α of 0.05. A p value < 0.05 was considered as statistically significant. The analyses were exploratory. No formal sample size calculation was performed. Analyses were performed using STATA 15.0.

Demographic characteristics Demographic characteristics—such as maternal age at cryopreservation, maternal age at the embryo transfer, cause of infertility, insemination procedure (ICSI\IVF), and semen origin—were comparable between the two groups (Table 1). Table 1. | Grouping | ||| |---|---|---|---| | Group A (n = 1953) | Group B (n = 374) | p values | | | Age at the time of embryo cryopreservation | 34 ± 4.8 | 34 ± 4.9 | 0.98 | | Age at ET | 34.8 ± 4.8 | 34.6 ± 4.9 | 0.557 | | Cause of infertility | 0.583 | || | 1. Male factor | 782 (45.2) | 150 (43) | | | 2. Endometriosis | 109 (6.3) | 29 (8.3) | | | 3. Idiopathic | 451 (26.1) | 94 (26.9) | | | 4. Ovarian insufficiency | 82 (4.7) | 16 (4.6) | | | 5. PCO | 118 (6.8) | 18 (5.1) | | | 6. Tubal factor | 188 (10.9) | 42 (12) | | | Procedure for injection | 0.899 | || | ICSI | 1888 (94.7) | 366 (94.6) | | | IVF | 105 (5.3) | 21 (5.4) | | | Procedure semen extraction | 0.441 | || | Ejaculate | 1950 (97.8) | 381 (98.5) | | | Testicular biopsy | 43 (2.2) | 6 (1.5) | Data are expressed as mean ± SD, number (n) and percentage (%) Group A intact embryo after warming, Group B blastomere loss after warming, ET embryo transfer, PCO polycystic ovarian syndrome, ICSI intracytoplasmic sperm injection, IVF in vitro fertilization, n number Embryo characteristics A total of 2327 vitrified/warmed day 3 transferred embryos were included in the analysis, of which 1953 (83.9%) embryos were fully intact after warming (group A), and 374 (16.1%) presented with cell loss (group B). Characterization of all frozen–thawed embryos according to cell stage at freezing and cell loss after thawing is displayed in Table 2. The majority of embryos presented 8 cells and/or more than 8 cells at the time of vitrification (897, 38.4%, and 1027, 44.3%, respectively). With regard to embryo quality at transfer, defined by the number of cells that further cleaved after warming and final cell stage at transfer, a total of 2071 (89.0%) embryos were of quality 1 and 2 (1558 and 513, respectively) (Table 3). Considering embryo quality at ET, the percentage of embryos represented in each quality class (1–4) was not significantly different between the two groups (class 1 = 67.7% vs 64.2, class 2 = 21.8% vs 23.2%, class 3 = 3.7% vs 7.6%, class 4 = 6.8% vs 5%, respectively; p = 0.198) (Table 3). In only a minor number of transfers, embryos without further cleavage were transferred. Table 2. | Cell stage at vitrification | Number thawed | Number of damaged cells | |||| |---|---|---|---|---|---|---| | 0 | 1 | 2 | 3 | 4 | || | 6 cells | 120 (5.1) | 107 | 9 | 4 | – | – | | 7 cells | 283 (12.2) | 242 | 32 | 7 | 2 | – | | 8 cells | 897 (38.4) | 780 | 83 | 25 | 8 | 1 | | > 8 cells | 1027 (44.3) | 824 | 108 | 56 | 29 | 10 | | Total | 2327 | 1953 (83.7) | 232 (9.9) | 92 (3.9) | 39 (1.6) | 11 (0.5) | Data are expressed as number (n) and percentage (%) Table 3. | Grouping | |||| |---|---|---|---|---| | n | Group A (n = 1953) | Group B (n = 374) | p values | | | Embryo quality at ET | 1.5 ± 0.9 | 1.5 ± 0.8 | 0.198* | | | 1 | 1558 | 1320 (67.7) | 238 (64.2) | | | 2 | 513 | 426 (21.8) | 87 (23.2) | | | 3 | 104 | 74 (3.7) | 30 (7.6) | | | 4 | 152 | 133 (6.8) | 19 (5) | Data are expressed as mean ± SD, number (n), and percentage (%) Group A intact embryo after warming, Group B blastomere loss after warming, ET embryo transfer, n number *Wilcoxon sum rank Obstetrical outcome The chemical pregnancy rate and LBR per warming cycle were significantly higher in the intact embryo group versus the cell loss group (585/1953, 30%, versus 91/374, 24.3%, p = 0.028, and 267/1953, 13.7%, versus 35/374, 9.4%, p = 0.023). However, LBR per positive hCG was equivalent between group A and B (45.6% vs 38.5%, p = 0.2). Biochemical pregnancy rate per frozen embryo transfer (4.2% vs 2.7%, p = 0.115) and miscarriage rate (5.3% vs 6.9%, p = 0.252) were similar between groups with and without blastomere loss, respectively (Table 5). Newborn measurements (length, weight, and head circumference at birth) showed no statistical difference between the two groups (50.1 ± 2.7 cm vs 50.1 ± 2.8 cm, p = 0.918; 3334.7 ± 643.7 g vs 3362.4 ± 517.7 g, p = 0.774; 38.2 ± 37.2 cm vs 34.5 ± 35.2 cm, p = 0.169, respectively, for intact and damaged embryos) (Table 4). Table 5. | OR | p value | 95% C.I | || |---|---|---|---|---| | Intact vs CL | 1.4 | 0.18 | 0.86 | 2.2 | | Age at cryo | 0.98 | 0.25 | 0.95 | 1.01 | LB live birth, OR odds ratio, C.I. confidence interval, Intact vs CL intact embryos versus cell loss embryos after thawing, Age at cryo maternal age at cryopreservation Table 4. | Grouping | ||| |---|---|---|---| | Group A (n = 1953) | Group B (n = 374) | p values | | | Chemical pregnancy, no. (%) | 585 (30) | 91(24.3) | 0.028 | | Biochemical pregnancy (per frozen embryo transfer) | 53 (2.7) | 16 (4.2) | 0.115 | | Miscarriage, no. (%) | 134 (6.9) | 20 (5.3) | 0.252 | | Live birth (per ET cycle), no. (%) | 267 (13.7) | 35 (9.4) | 0.023 | | Live birth rates (per positive hCG) (%) | 45.6 | 38.5 | 0.2 | | Gestational age (including all positive hCG) | 35.9 ± 23.5 | 34.8 ± 10.7 | 0.619 | | Neonatal length (cm) | 50.1 ± 2.7 | 50.1 ± 2.8 | 0.918 | | Neonatal weight (g) | 3334.7 ± 643.7 | 3362.4 ± 517.7 | 0.774 | | Neonatal head circumference (cm) | 38.2 ± 37.2 | 34.5 ± 35.2 | 0.169 | Data are expressed as mean ± SD or number (no.) and percentage (%) ET embryo transfer, n number, hCG human chorionic gonadotropin Multivariable logistic regression analysis Multivariable logistic regression analysis showed no association between transfer of intact or damaged embryos and LB (adjusted OR = 1.4, 95% CI = 0.86–2.2, p = 0.18), when adjusting for the potential confounder such as patient age at cryopreservation (Table 5).

The results of this large retrospective cohort study indicated that transfer of embryos with blastomere loss derived from V/W was associated with lower live birth and chemical pregnancy rates when compared with those of fully intact embryos. However, when adjusting for patient age at cryopreservation, the negative effect of cell damage on LB was not observed. Moreover, we noted that LBR per positive hCG was equivalent between the intact and cell loss groups indicating that if implantation occurs, LB is not affected by cell loss. Furthermore, cell loss after warming of cleavage-stage embryos had no impact on newborn measurements (neonatal length, weight, and head circumference at birth). These findings confirm the results of our previous study [7] in which an association between cell loss and diminished developmental potential was demonstrated, even in case such a loss was limited. However, the latter study showed that if the embryo resumed cleavage after warming, there was no effect of the number of cells lost on its implantation potential (although analyzed on small numbers). Our findings are in line with other studies [10, 19, 20] which report that damaged embryos with blastomere loss after cryopreservation may diminish embryo developmental potential and pregnancy rate, when only considering blastomere loss per se. FET cycles performed with completely intact embryos after warming achieved superior reproductive outcomes than those performed with partially damaged embryos. Indeed, a fully intact embryo represents a marker of better embryonic developmental potential, irrespective of its morphological quality [10, 21]. In this context, several studies [8, 22] reported that the capacity of the frozen–thawed embryo to further cleave in vitro is impaired in embryos showing blastomere loss after warming compared with intact embryos. Consistent with this notion, blastomere loss after thawing accounts for 30% of implantation potential reduction and approximately for 40% of clinical pregnancy rate decrement [4]. In addition, necrotic blastomeres may produce a toxic effect on the remaining cells and affect embryo viability [23]. Considering the proportion of cell loss after cryopreservation, embryos with less than 50% of the original number of blastomeres are considered unsuitable for transfer [10], while scientific evidence suggested that embryo competency is not impacted below the limit of 25% of blastomere loss [12, 14]. According to this, a double embryo transfer should not be performed, even to compensate for those with blastomere loss, to avoid the risk for multiple pregnancy. However, after vitrification, extensive cell loss is less frequent than after slow freezing [7]. Our results are partly in agreement with those reported by Wu et al. [14], who found that the transfer of embryos with blastomere loss following vitrification is associated with lower rates of implantation, clinical pregnancy, and live birth, compared to transfers of fully intact embryos. However, the same authors reported several adverse outcomes in newborns resulting from the implantation of embryos with blastomere loss, such as a higher rate of small for gestational age babies or transient tachypnea at birth. Nevertheless, the study conducted by Wu et al. [14] included both single and double embryo transfers. Moreover, although it aimed to collect data about major birth defects, a long-term follow-up on neonatal growth and development was not performed. In addition, our results are in line with those recently described by Jiang et al. [15] who showed that transfer of vitrified/warmed day 3 embryos with blastomere loss is related to impaired embryo developmental potentials in terms of live birth rates when compared to transfer of fully intact embryos. However, the neonatal outcomes of embryos derived from blastomere loss embryos and intact embryos were similar, concluding that blastomere loss was not associated with increased risk of any adverse neonatal condition. Finally, it has to be mentioned that HRT protocol for FET, per se, seems to be associated with increased risk of adverse maternal and neonatal outcomes such as preeclampsia [24, 25], preterm birth, and low birth weight when compared to NC-FET [26]. The strength of the present study relies on its design, including a large sample size of cleavage-stage vitrified day 3 embryos. Moreover, only single embryo transfers were included in the analysis. Study limitations, however, exist and should be taken into consideration when interpreting the results. The retrospective nature of our study is inherent to risk of bias. Therefore, although a significant effort was made to eliminate all known sources of systematic error through multivariate analysis, unknown sources of bias may exist and have an impact on measured outcomes. Finally, neonatal outcomes might only be analyzed for a specific subgroup of patients (those who delivered); however, demographic characteristics were comparable between the two groups included in the initial analysis [27]. In conclusion, the transfer of embryos with blastomere loss caused by V/W is associated with lower chemical pregnancy and live birth rates when compared with transfer of fully intact embryos. However, the negative effect of blastomere loss is not observed when adjusting for patient age at cryopreservation. Moreover, if implantation occurs, LBR is not impacted by cell loss. Finally, blastomere loss is not associated with neonatal length, weight, and head circumference at birth. Larger cohort studies are warranted to validate these findings.

The authors would like to thank Walter Meul for the contribution to the data management of the study. Author contribution F.D.G. was responsible for the concept and the initial draft of the article. A.R. was responsible for the concept and conducted the statistical analysis. G.C., A.B., P.D., S.M., H.T., G.V., and C.B. contributed to the interpretation and editing of the article. L.V.L. contributed to the conception and draft of the article. All of the authors critically reviewed the content and approved the final version of the work. Declarations Conflict of interest The authors declare no conflict of interest. Footnotes Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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