Abnormal cleavage up to Day 3 does not compromise live birth and neonatal outcomes of embryos that have achieved full blastulation: a retrospective cohort study.

STUDY QUESTION: Do embryos displaying abnormal cleavage (ABNCL) up to Day 3 have compromised live birth rates and neonatal outcomes if full blastulation has been achieved prior to transfer? SUMMARY ANSWER: ABNCL is associated with reduced full blastulation rates but does not impact live birth rates and neonatal outcomes once full blastulation has been achieved. WHAT IS KNOWN ALREADY?: It is widely accepted that ABNCL is associated with reduced implantation rates of embryos when transferred at the cleavage stage. However, evidence is scarce in the literature reporting birth outcomes from blastocysts arising from ABNCL embryos, likely because they are ranked low priority for transfer. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study included 1562 consecutive autologous in vitro fertilization cycles (maternal age 35.1 ± 4.7 years) performed at Fertility North, Australia between January 2017 and June 2022. Fresh transfers were performed on Day 3 or 5, with remaining embryos cultured up to Day 6 before vitrification. A total of 6019 embryos were subject to blastocyst culture, and a subset of 664 resulting frozen blastocysts was included for live birth and neonatal outcome analyses following single transfers. PARTICIPANTS/MATERIALS, SETTING, METHODS: ABNCL events were annotated from the first mitotic division up to Day 3, including direct cleavage (DC), reverse cleavage (RC) and <6 intercellular contact points at the 4-cell stage (<6ICCP). For DC and RC in combination, the ratios of affected blastomeres over the total number of all blastomeres up to Day 3 were also recorded. All pregnancies were followed up until birth with gestational age, birthweight, and sex of the baby being recorded. MAIN RESULTS AND THE ROLE OF CHANCE: Full blastulation rates for embryos showing DC (19.5%), RC (41.7%), <6ICCP (58.8%), and mixed (≥2) ABNCL types (26.4%) were lower than the rates for those without ABNCL (67.2%, P < 0.01 respectively). Subgroup analysis showed declining full blastulation rates with increasing ratios of combined DC/RC affected blastomeres over all blastomeres up to the 8-cell stage (66.2% when 0 affected, 47.0% when 0.25 affected, 27.4% when 0.5 affected, 14.5% when 0.75 affected, and 7.7% when all affected, P < 0.01). However, once full blastulation had been achieved, no difference was detected between DC, RC, <6ICCP, and no ABNCL blastocysts following single frozen transfers in subsequent live birth rates (25.9%, 33.0%, 36.0% versus 30.8%, P > 0.05, respectively), gestational age (38.7 ± 1.6, 38.5 ± 1.2, 38.3 ± 3.5 versus 38.5 ± 1.8 weeks, P > 0.05, respectively) and birthweight (3343.0 ± 649.1, 3378.2 ± 538.4, 3352.6 ± 841.3 versus 3313.9 ± 509.6 g, P > 0.05, respectively). Multiple regression (logistic or linear as appropriate) confirmed no differences in all of the above measures after accounting for potential confounders. LIMITATIONS, REASONS FOR CAUTION: Our study is limited by its retrospective nature, making it impossible to control every known or unknown confounder. Embryos in our dataset, being surplus after selection for fresh transfer, may not represent the general embryo population. WIDER IMPLICATIONS OF THE FINDINGS: Our findings highlight the incremental impact of ABNCL, depending on the ratio of affected blastomeres up to Day 3, on subsequent full blastulation. The reassuring live birth and neonatal outcomes of ABNCL blastocysts imply a potential self-correction mechanism among those embryos reaching the blastocyst stage, which provides valuable guidance for clinical practice and patient counseling. STUDY FUNDING/COMPETTING INTEREST(S): This research is supported by an Australian Government Research Training Program (RTP) Scholarship. All authors report no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Comparison of single euploid blastocyst transfer cycle outcome derived from embryos with normal or abnormal cleavage patterns.

RESEARCH QUESTION: To assess incidence of abnormal cleavage among biopsied blastocysts; to compare euploidy rates of the blastocysts with abnormal and normal cleavage; and to compare single euploid blastocyst transfer (SEBT) outcome derived from embryos with normal or abnormal cleavage. DESIGN: Retrospective analysis of prospectively collected data in a private IVF clinic. Consecutive 554 patients (749 cycles) undergoing preimplantation genetic testing for aneuploidy (n = 497; 671 cycles) or monogenic diseases (n = 57; 78 cycles) were included. All assessments for abnormal cleavage were carried out retrospectively; presence of abnormal cleavage was not a factor in deciding which euploid embryo to transfer. A total of 1015 blastocysts were biopsied and 295 SEBT procedures were carried out. Main outcome measure was live birth rate (LBR). RESULTS: Incidence of reverse cleavage, direct cleavage, and reverse plus direct cleavage, were 7.7%, 6.4% and 2.3%, respectively. Of the 1015 biopsied blastocysts, 35.0% were euploid. Blastocysts with abnormal cleavage, in total, had a significantly higher euploidy rate compared with blastocysts with normal cleavage (44.6% [74/166] versus 33.1% [281/849]; P = 0.017). The LBR after SEBT with normal, reverse and direct cleavage, and direct cleavage plus reverse cleavage, was 133/238 (55.9%), 6/26 (23.1%), 8/24 (33.3%) and 0/3 (0.0%) (P < 0.001). Generalized estimating equation analysis showed that the presence of abnormal cleavage pattern was the only independent predictor of LBR (OR 0.316; 95% CI 0.115 to 0.867; P = 0.013). CONCLUSIONS: Blastocysts with direct or reverse cleavage should be biopsied in preimplantation genetic testing cycles if they are morphologically eligible. Euploid blastocysts with abnormal cleavage, however, have approximately half the LBR of those euploid blastocyst with normal cleavage, hence, blastocysts with abnormal cleavage should have lower priority for transfer.

The effect of early irregular cell division of human embryos on blastocyst euploidy: considerations from the subsequent development of the blastomeres by direct or reverse cleavage.

OBJECTIVE: To investigate whether blastocysts that divide irregularly reduce subsequent blastocyst euploidy. DESIGN: Retrospective study. SETTING: Private clinic. PATIENT(S): A total of 122 blastocysts for which consent for disposal and research use was obtained. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Results of next-generation sequencing analysis of the blastocysts and whether blastomeres by normal or irregular divisions subsequently participated in blastocyst formation or not. RESULT(S): The embryos were classified according to their dynamics until the second cleavage. The blastocyst euploidy rates were 33.3% (19/57) in the normal cleavage (NC) group, 38.3% (18/47) in the direct cleavage (embryos with one cell dividing into 3 cells) (DC) group, and 72.2% (13/18) in the reverse cleavage (RC) (embryos with fused cells once divided) group. The rate of the RC group was significantly higher than that of the NC group. The blastocyst participation rate of the blastomeres were 95.6% in the NC group and 56.5% in that derived from DC of the first cleavage, and 91.7% in that of blastomeres derived from normal division of the second cleavage and 53.6% in that derived from DC of the second cleavage, both of which were significantly lower in the latter. In the RC group, the rates of fused and nonfused blastomeres were 62.1% and 87.5%, respectively, with no significant difference. CONCLUSION(S): The blastomeres generated by DC were often excluded from blastocyst formation, and we speculate that this is one reason why their division does not reduce blastocyst euploidy. The association between RC and euploidy of blastocysts merits further study.

Incidence, dynamics and recurrences of reverse cleavage in aneuploid, mosaic and euploid blastocysts, and its relationship with embryo quality.

BACKGROUND: During embryonic development, the normality of cleavage and the ploidy state are closely related to the final clinical outcome. At present, many research teams are focusing on the combined application of timelapse (TL) technology and preimplantation genetic testing (PGT) technology, hoping to find a connection between the two aspects of morphodynamics and genes. In the process of embryonic cleavage, there is a common abnormal cleavage pattern called reverse cleavage (RC). RC refers to blastomere fusion and failed cytokinesis. There are very few reports about it. Whether the occurrence of RC affects blastocyst euploidy is even less clear. Whether the RC phenomenon affects the embryonic developmental potential and whether it is related to the embryo ploidy. This is important for clinicians and embryologists. In this study, we used TL to observe whether there was a phenomenon of RC in each biopsy embryo and then combined it with the ploidy state to give an answer, which provided support for the selection strategy of RC embryos. METHODS: A total of 405 TL-PGT cycles and 1,467 blastocysts were included in the study. All TL data were collected from the Reproductive Medicine Center, Huazhong University of Science and Technology Hospital. Embryos images throughout embryonic development, from post-insemination to day 5 or 6 until biopsy and cryopreservation, were acquired by the Embryoscope Plus TL microscopy system from January 2019 to December 2020. This study investigated the overall incidence of RC during cleavage; the relationship between RC phenomenon and the number of occurrences and ploidy results; the relationship between RC occurrence and blastocyst developmental quality, as well as the dynamics of RC embryos. RESULTS: Among the 1,453 blastocysts biopsied, 400 blastocysts showed RC phenomenon at the cleavage stage, and the incidence rate was 25.9%. In euploid, mosaic and aneuploid embryos, the incidence of RC was 27.2%, 26.6%, and 25.0%, respectively. The incidence of RC was similar among these three groups with no significant difference (P > 0.05). The number of RC occurrences was not associated with embryo ploidy status (P > 0.05). In general, the blastocyst quality of the RC + group was lower than that of the RC- group. In the ICM score, the proportion of A score in the RC + group was significantly lower than that in RC- group (P < 0.05). In the TE score, there was no significant difference between the two groups of A-grade blastocysts, but the proportion of B-grade blastocysts in the RC + group was significantly lower than that in the RC- group (P < 0.01). In terms of developmental kinetic parameters, the cleavage synchrony parameters s2 and s3 were significantly longer in RC + embryos than in RC- embryos (P < 0.05). However, these changes in kinetic parameters were not significantly different between the euploid, mosaic and aneuploid groups. CONCLUSIONS: The chromosomal euploidy of cleavage-stage embryos with RC phenomenon developed to the blastocyst stage was not significantly different from that of cleavage normal blastocysts. Therefore, RC embryos should not be discarded. It is recommended to select and utilize blastocyst culture, which has similar clinical value to normal cleavage embryos.

Prevalence, consequence, and significance of reverse cleavage by human embryos viewed with the use of the Embryoscope time-lapse video system.

OBJECTIVE: To investigate the prevalence and potential causes of reverse cleavage (RC) by human early-cleavage embryos and its associations with embryonic development and implantation after transfer. DESIGN: Clinical retrospective cohort study. SETTING: Private fertility treatment center. PATIENT(S): A total of 126 consecutive in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment cycles, with 353 IVF and 436 ICSI embryos cultured in the Embryoscope until day 3. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Embryo assessment on day 3, incidence of abnormal division, embryo morphokinetic parameters, and fetal heart beat. RESULT(S): RC, referring to either blastomere fusion or failed cytokinesis, occurred up to three times per individual embryo in 27.4% of embryos during the first three cleavage cycles. A higher incidence was associated with GnRH antagonist cycles compared with agonist cycles (odds ratio [OR] 1.683), or with ICSI compared with IVF (OR 1.600). After ICSI, sperm progressive motility was associated with RC (area under the receiver operating characteristic curve: 0.573). Compared with RC-negative embryos, a lower proportion of RC-positive embryos reached 6-cell stage or beyond by day 3 (47.7% vs. 71.7%), and were more likely to have multinucleation at the 4-cell stage (10.1% vs. 5.0%). Embryos showing RC had significantly poorer performance in both conventional grading and morphokinetic parameters, and they implanted less (0/22 vs. 29/131) than those not showing RC. CONCLUSION(S): RC significantly compromised embryo development, culminating in poor implantation potential. For each embryo, it can occur on more than one occasion at any stage during the first 3 days of culture. It is associated with factors affecting both oocyte and sperm.