Behind the scenes: Cleavage patterns of 9600 embryos

Behind the scenes: Cleavage patterns of 9600 embryos | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Behind the scenes: Cleavage patterns of 9600 embryos Maya Shavit, Daniel Gonen, Yuval Atzmon, Nardin Aslih, Asaf Bilgory, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2746951/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study investigates the incidence of irregular cleavage (IRC) among human embryos and their influence on IVF treatment outcomes. This study was designed as a prospective observational study in a single-centre IVF clinic including 1,001 women who underwent 1,976 assisted reproduction treatments during 2016–2021. Morphokinetics of embryos was analysed and evaluated for the association between IRC and women’s characteristics, treatment characteristics, and pregnancy outcome. We found IRC incidence to be 17.5% (1,689/9,632 embryos). Of these, 85% embryos had one IRC, and 15% had multiple IRC. 35% of IRC events occurred during the embryo's first cell cycle. IRC embryos were found to correlate with male factor (p = 0.01) and higher ICSI rate (p = 0.01). Age, BMI, parity, basal FSH level, stimulation protocol and number of retrieved oocytes did not differ between groups. Embryos with early IRC or more than one IRC event had lower blastulation rate (p = 0.01 and p = 0.01, respectively). Fresh cycles with IRC embryos had a lower clinical pregnancy rate (p = 0.01), and early IRC embryos had a lower live birth rate (p = 0.04) compared to embryos without IRC. Frozen embryo transfer (FET) cycles of blastocyst embryos, with and without IRC, had comparable results. In conclusion, number of abnormal cleavage events and their timing are of great importance for the prognosis of the developing human embryo. Irregular cleavage direct cleavage clinical pregnancy rate live birth rate time-lapse imaging morphokinetics Introduction Selecting embryos with the greatest implantation potential is critical for achieving higher live birth rates and reducing both unsuccessful transfers and multiple birth rates in IVF cycles. There are two accepted methods for performing embryo selection. The traditional method based on morphology is known to be subjective and is associated with high intra- and inter-personal disagreement 1 . The novel morphokinetic assessment and analyses of preimplantation human embryo development with time-lapse monitoring and use of artificial intelligence can provide indicative and objective data on embryo quality and implementation potential 2 – 5 . As the use of time lapse monitoring systems becomes more common, we gain qualitative data regarding embryo cleavage patterns, and its relation to embryonic viability and implantation rate. Abnormal cell cleavage in the form of direct cleavage of a mother cell to more than two daughter cells is defined as irregular cleavage (IRC). This phenomenon is generally associated with lower developmental potential 6 – 10 . Only a few studies about embryo cleavage patterns have been published 6 , 11 – 14 . All of those were retrospective and with a relatively small sample size. Furthermore, results published so far from different studies are contradictory regarding euploidy, implantation and live birth rates. The purpose of this study was to contribute to the current literature regarding IRC embryos, their incidence, correlation to patient and treatment characteristics, and to compare IVF treatment outcomes derived from embryos with regular and irregular cleavage patterns. Materials And Methods Study design This prospective observational cohort study was conducted at the IVF Unit of Hillel Yaffe Medical Center from January 2016 to December 2021. Patients All patients whose embryos were assessed with time-lapse technology for morphological and morphokinetic developmental patterns during fresh embryo development were included in the study. Clinical outcomes were evaluated for 1,001 patients undergoing 1,976 IVF cycles, with 23,605 retrieved oocytes, and 9,632 developed embryos. Demographic characteristics of the women enrolled (including age, parity, BMI, smoking, basal FSH level, and infertility cause) were collected from medical records. Ovarian stimulation protocol (long GNRH agonist or GNRH antagonist), ovulation trigger (GNRH agonist or hCG), number of retrieved oocytes, and ICSI rate were analysed. Outcome for fresh and frozen transfer cycles was analysed according to embryo age at transfer (day 3 or day 5) and number of embryos per transfer (one to three). IRC IRC defined as an abnormal cell cleavage in the form of a direct cleavage of a mother cell to more than two daughter cells regardless of the cell size. Only cells containing visible nuclei were considered blastomeres; otherwise, they were annotated as fragment. Reverse cleavage was not evaluated in this study. We distinguished between early IRC (occurred in the first 24 hours from the presence of 2PN) and late IRC (up to the third cleavage). We also distinguished between single and multiple events of IRC. The analysis was based on these IRC characteristics. Outcome Measures Outcomes included morphokinetics parameters (multinucleation, fragmentation, time to blastulation, uneven blastomere rate), blastulation rate, high quality embryos remaining for use (frozen or transferred) rate, clinical pregnancy rate (CPR) which was defined as ultrasound confirmation of an intrauterine gestational sac with a positive heartbeat, live birth rate (LBR), miscarriage rate (spontaneous termination before 24 weeks of gestation), preterm labour rate (live deliveries occurring earlier than 37 weeks of pregnancy), and placental complication rate during gestation, including gestational hypertension, pre-eclampsia, abruption, intrauterine growth restriction (IUGR), or intra-uterine foetal demise (IUFD). Ethical Issues The Institutional Review Board (Helsinki Committee) at Hillel-Yaffe Medical Center approved the study protocol on 27/04/2020. Approval number is 0026-20-HYMC. As the protocol involved only the observational review of patient charts, the need for individual patient informed consent was waived by the Institutional Review Board (Helsinki Committee) at Hillel-Yaffe Medical Center. All methods in the present study were strictly performed in accordance with the Declaration of Helsinki for Medical Research. Statistical analysis Statistical analysis was performed using SPSS,-22.0 package for windows (IBM Corp., USA). Categorical variables were analysed with chi-squared test or Fisher’s exact test. Continuous variables were analysed using a t-test. For results that were significant or showed a statistical trend in univariate analysis, multivariable analysis was performed with a multiple logistic regression model to assess the potential impact of those parameters and to further investigate and find the morphokinetic parameters and patient characteristics predictive of a successful or unsuccessful pregnancy outcome, CPR and LBR. A p-value < 0.05 was considered statistically significant. All statistical tests were two-tailed. We also conducted a logistic regression for nominal parameters. Results The incidence of IRC embryos was 17.5% (1,689/9,632 embryos). Of these, 35% had early IRC (occurring during the embryo's first cell cycle) and 65% had late IRC (occurring after the embryo's first cell cycle ended). In addition, 7,943 (82.5%) embryos had no IRC, 1,447 (15%) embryos had one IRC, and 242 (2.5%) embryos had two or more IRC. Patient characteristics Age, BMI, parity, and basal FSH level were not correlated with the number of IRC events or with their timing. IRC embryos were found to correlate with male factor infertility (35.7% vs. 31.2%, p = 0.01; Tables 1 and 2 ). Table 1 Basic patient and treatment characteristics by number of irregular cleavage events during embryo development Characteristic No IRC (n = 7,943) 1 IRC (n = 1,447) 2 + IRC (n = 242) P value Maternal age (years ± SD) 34.2 ± 6.5 33.9 ± 6.3 34.1 ± 6.2 0.336 Parity (mean ± SD) 0.4 ± 0.7 0.4 ± 0.6 0.4 ± 0.6 0.207 BMI (kg/m ²± SD) 26.3 ± 6.2 26.5 ± 6.0 25.5 ± 5.9 0.214 Basal FSH level (mean ± SD) 7.7 ± 4.0 7.6 ± 3.6 7.9 ± 3.9 0.673 Infertility cause (n (%)) Anovulation Endometriosis Male factor Tubal factor Unexplained 824 (10.4%) 213 (2.7%) 2,480 (31.2%) 980 (12.3%) 1,552 (19.5%) 127 (8.8%) 38 (2.6%) 520 (35.9%) 188 (13%) 278 (19.2%) 21 (8.7%) 2 (0.8%) 84 (34.7%) 25 (10.3%) 59 (24.4%) 0.136 0.206 0.001 0.480 0.160 Ovarian stimulation protocol, n (%)) Long GNRH agonist GNRH antagonist Other 786 (9.8%) 5,922 (74.6%) 1235 (15.5%) 168 (11.6%) 1,043 (72.1%) 236 (16.3%) 29 (12%) 178 (73.6%) 35 (14.4%) 0.199 GNRH agonist ovulation trigger, n (%) 802 (10.1%) 162 (11.2%) 22 (9.1%) 0.375 Number retrieved oocytes (mean ± SD) 14.5 ± 9.3 14.9 ± 8.7 14.3 ± 8.2 0.315 ICSI (n (%) 6,910 (87%) 1,301 (90%) 208 (86%) 0.007 IRC, irregular cleavage; SD, standard deviation; BMI, Body mass index; ICSI, Intracytoplasmic sperm injection Table 2 Basic patient and treatment characteristics by timing of first Irregular cleavage event during embryo development Characteristic No IRC (n = 7,943) Early IRC (n = 588) Late IRC (n = 1,101) P value Maternal age (years ± SD) 34.2 ± 6.5 33.7 ± 6.3 34.1 ± 6.3 0.169 Parity (mean ± SD) 0.4 ± 0.7 0.4 ± 0.7 0.4 ± 0.6 0.175 BMI (kg/m² ± SD) 26.3 ± 6.2 25.8 ± 6.0 26.3 ± 6.0 0.178 Basal FSH level (mean ± SD) 7.7 ± 4.0 7.8 ± 4.0 7.5 ± 3.1 0.534 Infertility cause (n (%)) Anovulation Endometriosis Male factor Tubal factor Unexplained 824 (10.4%) 213 (2.7%) 2,480 (31.2%) 980 (12.3%) 1,552 (19.5%) 58 (9.9%) 18 (3.1%) 208 (35.4%) 72 (12.2%) 114 (19.4%) 90 (8.2%) 22 (2.0%) 396 (36%) 141 (12.8%) 223 (20.3%) 0.074 0.328 0.001 0.901 0.846 Ovarian stimulation protocol, n (%) Long GNRH agonist GNRH antagonist Other 786 (9.8%) 5,922 (74.6%) 1235 (15.5%) 70 (11.9%) 421 (71.6%) 97 (16.4%) 127 (11.5%) 800 (72.7%) 174 (15.8%) 0.224 GNRH agonist ovulation trigger (n (%)) (10.1%)802 (13.1%)77 (9.7%)107 0.057 Retrieved oocytes number (mean ± SD) 14.5 ± 9.3 15.0 ± 9.0 14.7 ± 8.5 0.383 ICSI (n (%)) 6,910 (87%) 518 (88%) 991 (90%) 0.011 IRC, irregular cleavage; SD, standard deviation; BMI, Body mass index; ICSI, Intracytoplasmic sperm injection Treatment characteristics Neither the number of IRC events nor their timing was influenced by the stimulation protocol, trigger medication or number of retrieved oocytes. IRC embryos had higher ICSI rate (89% vs. 87%, p-value 0.01) (Tables 1 and 2 ). Other morphokinetic parameters Both higher number of IRC events along the embryo’s development course and earlier time to first IRC event were found to correlate with a higher multi-nucleation rate, and a higher rate of uneven blastomere size. An early IRC event also correlated with a higher fragmentation rate (14.9%, 18% and 12% for none, early and late IRC events, respectively, p < 0.01) (Tables 3 and 4 ). Table 3 IVF outcome for embryos by number of IRC events during embryo development Characteristic No IRC (n = 7,943) 1 IRC (n = 1,447) 2 + IRC (n = 242) P value Multi-nucleation (mean ± SD) 0.98 ± 0.86 1.3 ± 0.9 1.7 ± 0.8 < .001 Fragmentation (%) 14.9% 14.3% 13.2% .128 Uneven blastomere, n (%) 2,224 (28%) 651 (45%) 118 (49%) < .001 Blastocyst formation, n (%) 1,581 (19.9%) (20.8%) 301 (11.4%) 26 .004 Time to blastulation (mean ± SD) 108.5 ± 10.1 112.4 ± 9.5 111.1 ± 8.9 < .001 Usable embryos, n (%) 4,280 (53.9%) (14.0%) 202 (9.5%) 23 < .001 Fresh cycles outcome Fresh cycles with ET, n (%) Clinical pregnancy, n (%) Live birth, n (%) Miscarriage, n (%) Preterm birth, n (%) Placental complications, n (%) 1,808 (22.7%) (30.8%) 562/1,808 (20.5%) 356/1,808 (22%) 127/562 57/562 (10%) 33/562 (6%) 113 (7.8%) (23.4%) 26/113 (14.1%) 16/113 6/26 (23%) 6/26 (23%) 0/26 (0%) 20 (8.2%) (5.0%)1 (5.0%) 1 (0%) 0/1 (0%) 0/1 (0%) 0/1 < .001 .009 .095 .999 .037 .091 IRC, irregular cleavage; SD, standard deviation; ET, embryo transfer Table 4 IVF outcome for embryos by timing of first IRC event during embryo development Characteristic No IRC (n = 7,943) Early IRC (n = 588) Late IRC (n = 1,101) P value Multi-nucleation (mean ± SD) 0.98 ± 0.86 1.61 ± 1.0 1.3 ± 0.8 < .001 Fragmentation (%) 14.9% 18% 12% < .001 Uneven blastomere, n (%) 2,224 (28%) 411 (70%) 385 (35%) < .001 Blastocyst formation, n (%) 1,581 (19.9%) 92 (15.6%) 239 (21.7%) .011 Time to blastulation, minutes (mean ± SD) 108.5 ± 10.1 112.1 ± 10.6 112.4 ± 9.0 < .001 Usable embryos, n (%) 4,280 (53.9%) 53 (9.0%) 175 (15.9%) < .001 Fresh cycle outcomes Fresh cycles with ET, n (%) Clinical pregnancy, n (%) Live birth, n (%) Miscarriage, n (%) Preterm birth, n (%) Placental complication, n (%) 1,808 (22.7%) (30.8%) 562/1,808 (20.5%) 56/1,808 (22%) 127/562 57/562 (10%) 33/562 (6%) 32 (5.4%) 3/32 (9.4%) 1/32 (3.1%) 2/3 (66%) (0%) 0/3 (0%) 0/3 101 (9.2%) 24 (23.7%) 16 (15.8%) 3/24 (12.5%) 6/24 (25%) 0/24 (0%) < .001 .010 .042 .093 .021 .105 IRC, irregular cleavage; SD, standard deviation; ET, embryo transfer Ivf Outcomes Blastulation Blastulation rate was lower in the two or more IRC group (11.4%) compared with none (19.9%) or one (20.8%) IRC only (p = 0.01). Blastulation rate was also lower when comparing early IRC (15.6%) to no (19.9%) or late IRC (21.7%) (p = 0.01). Also of importance, is that time to blastulation was longer for IRC embryos in comparison to those without (Tables 3 and 4 ). Rate of usable embryos The usable embryo rate was defined as embryos chosen for either transfer or freeze. The rate was significantly lower for embryos with IRC and even more so for embryos with two or more IRC (53.9%, 14%, and 9.5% respectively, p < 0.01). The rate was also lower for embryos with an early IRC vs. late IRC (9.0% vs. 15.9%, p < 0.01) (Tables 3 and 4 ). Clinical pregnancy, live birth, and miscarriage rate in fresh cycles There were 133 fresh cycles with embryo transfer (ET) of an IRC embryo. All fresh transfer cycles (cleavage stage and blastocyst) were combined for outcome analysis due to the small sample size. Among these, 23 were single embryo transfers (SET), and the remaining 110 cycles were of two or three embryos. In most cycles with multiple embryos, at least one embryo did not have an IRC event. The CPR per transfer was lower with increasing number of IRC events (30.8%, 23.4%, and 5.0% for none, one, or two or more IRC events, respectively; p = 0.01). ET with an early IRC embryo had both lower CPR and lower LBR compared with late and no IRC (9.4%, 23.7%, 30.8%, p = 0.01 and 3.1%, 15.8%. 20.5%, p = 0.04). Miscarriage rate did not differ significantly between the study groups (Tables 3 and 4 ). Preterm labour and placental complications in fresh cycles Preterm labour was significantly more prevalent in the IRC groups (23% in the single IRC event group and 25% in the late IRC groups vs. 10% in the no IRC group, p = 0.04 and 0.02, respectively). There were no preterm deliveries in the group of early or more than one IRC event group. No significant difference was found in the rate of placental complications between the study groups (Tables 3 and 4 ). IRC frozen embryo transfer outcomes A total of 95 embryos with IRC were frozen, all at blastocyst stage. Of these embryos, 51 were transferred in a frozen cycle, of which 32 were SET and 19 dual embryo transfers (DET). In the DET cycles, the transferred embryo included one embryo with an IRC event and the other had normal cleavage. The CPR of these cycles was 25% (8/32) in the SET cycles and 36% (7/19) in the DET cycles (p = 0.369). In comparison, CPR for SET of a blastocyst without IRC in a frozen cycle in our centre was 37% (p = 0.17) and LBR was 22% (p = 0.37). We cannot compare DET for blastocysts without IRC as our centre’s policy is to transfer only one blastocyst when there was no IRC. The LBR was 15.6% (5/32) in the SET cycles and 10.5% (2/19) in the DET cycles (p = 0.61; Table 5 ). Table 5 Irregular cleavage (IRC) frozen embryo transfer outcomes Characteristic No IRC (n = 261) IRC (n = 95) P value Single embryo transfer Clinical pregnancy rate, n (%) Live birth rate, n (%) 95/261 (37%) 59/261 (22%) 8/32 (25%) 5/32 (15.6%) 0.175 0.367 Dual embryo transfer Clinical pregnancy rate, n (%) Live birth rate, n (%) Not applicable Not applicable 7/19 (36%) 2/19 (10.5%) N/A N/A Logistic regression When logistic regression analysis was used to determine the variables that contributed to the LBR, only younger age and higher parity were found to be significant parameters for achieving both CPR and LBR. BMI, duration of infertility, aetiology of infertility, number of oocytes retrieved, time to cleavage stage embryo, degree of embryo fragmentation, embryo degree of multi-nucleation, number of IRC events and their timing were not related to achieving clinical pregnancy or to live delivery (Table 6 ). Table 6 Multivariable logistic regression analysis for clinical pregnancy rate Variable Odds ratio 95% CI for Odds ratio P value Lower Upper Age 0.928 0.907 0.949 < .001 Parity 1.274 1.053 1.542 0.013 CI, confidence interval Discussion The present study evaluated the impact of IRC on IVF treatment and pregnancy outcomes and contributes to the existing, yet meagre literature regarding the reproductive potential of embryos with no, single or multiple IRC events and their timing 6 , 8 – 14 . We demonstrated that male factor and ICSI correlate with IRC, that a single early IRC event or multiple occasions of IRC have deleterious effects on embryo development and IVF outcomes in term of blastulation rate. However, the blastulation rate was not affected by a single late IRC event. Unlike blastulation rate, the CPR was found to be significantly lower in cases with single late IRC and even more so in cases with an early IRC or multiple instances of IRC. We found that frozen blastocysts with IRC could lead to pregnancies, although the CPR was lower in comparison to FET with normal cleavage embryos. In addition, to the best of our knowledge, this is the first study to follow these pregnancies until delivery and investigate preterm labour and placental complications in pregnancies resulting from an IRC ET. Male Factor, ICSI and IRC Analysis of IRC events in relation to the cause of infertility revealed a connection between sperm defects and IRC. ICSI was used more frequently with the single and late IRC embryo groups. Our findings contradict a previous study 14 that found higher IVF compared to ICSI in the single IRC group, and no significant difference between the other groups. They also reported that sperm origin (testicular/epididymal as opposed to ejaculated) influences embryo cleavage patterns, while paternal age did not. The impact of paternal age is debated in the literature, Ozbek et al. 6 found a correlation between advanced paternal age and IRC. One of the possible hypotheses underlying IRC is the formation of multipolar spindles through the introduction of either incomplete, defective, or supernumerary centrioles by defective sperm 15 . Our hypothesis regarding the higher ICSI rate is that the procedure might damage the ovum miotic spindle, which in turn will manifest as IRC. It will be interesting re-examine our findings and those of Zhan et al. 14 as well, as to further investigate whether certain semen parameters, such as DNA fragmentation or teratospermia, are predictive of IRC. The impact of IRC and blastulation In agreement with previous studies 13 , 14 , we found a lower blastulation rate for embryos with early or multiple IRC events. However, lower blastulation rate was not demonstrated for embryos with single and/or late IRC. This is most likely due to a less deleterious effect of the IRC, as well as our centre policy of growing those embryos to day 5, while good quality embryos without IRC are more commonly transferred at cleavage stage. Early vs. late IRC As previously mentioned, early as compared to late IRC, has a detrimental effect on embryonic development, as manifested by lower rates of blastulation, fewer usable embryos, lower clinical pregnancy, and live birth rates. The molecular mechanisms that underlie IRC are not entirely clear, but it is widely accepted that mitotic errors play a vital role in enabling irregular division. When IRC occurs late, there is at least one blastomere that cleaves normally and contributes to the chromosomal balance of the embryo, thereby bolstering its viability and eventually leading to higher implantation and live birth rates. Lagalla et al. 11 explored the possibility of a potential ‘aneuploidy rescue’ mechanism, as they observed that IRC embryos that excreted some cells during the compaction process developed into euploid blastocysts. Further genetic analysis could reveal new information about the differences in the self-correction mechanisms between early and late IRC and about the molecular underpinnings of both abnormal cleavage patterns. A few recent studies included pregestational genetic analysis of the IRC embryos. These studies included mainly patients with poor prognosis: advanced maternal age, severe male factor, recurrent unexplained pregnancy loss and repeated implantation failure. Most of embryo biopsies reported were done at blastocyst stage and only some of the studies mentioned the timing of IRC event during embryonic development. Lagalla et al. 11 and Zhan et al. 14 reported that the euploidy rate gradually increased when the IRC event occurred later. Surprisingly, IRC embryos were found to have euploid rates comparable to those of embryos without IRC. In our study, genetic analysis of the embryos was not available. One vs. several IRC Higher number of IRC events during embryonic development represents more mitotic errors, and fewer blastomeres that cleave normally. Only one earlier study 14 referred to this issue. In agreement with Zhan et al. 14 , we also found lower blastulation rates and lower CPR. Moreover, we found fewer usable embryos and a higher preterm birth rate. Zhan et al. 14 also performed genetic analyses and did not find significant differences between one vs. multiple IRC events. The performance of frozen embryos with IRC Based on the accumulated genetic data regarding the euploidy status of IRC embryos who reached blastocyst stage 6 , 11 , we also found that IRC embryos that reached blastocyst stage achieved reasonable CPR and LBR in FET and fresh cycles. We found a higher rate of preterm labour in the late IRC group. There were no preterm deliveries in the early or more than one IRC event group, but this is most likely due to chance given the small size of the groups. This finding of higher preterm birth rates should be confirmed by further studies. Strengths and limitations The strengths of the present study include a relatively large sample size of human embryos incubated in EmbryoScope®, in a single IVF centre with a limited number of embryologists; all with high level of expertise and homogeneous guidelines regarding embryo estimation and management. All IRC annotations were confirmed by the embryologists. This was a prospective observational study; hence, the data were gathered prospectively and therefore, are highly reliability. Only the statistical analysis was done retrospectively. This study had few limitations. The embryos transferred did not undergo genetic analysis. Another drawback was the number of embryos transferred per cycle. In many of the cycles with an IRC embryo, a parallel normal cleaved embryo was transferred. This makes it more difficult to interpret the CPR and LBR. In conclusion, given the results of this study and previous studies, embryos with IRC pattern should have lower priority for transfer, mainly in the cleavage stage. Since live birth rate after ET of an IRC embryo is significantly lower compared with normal cleavage embryos. We recommend that blastocysts with irregular cleavage patterns should be considered a second-choice candidate for embryo transfer if they are morphologically eligible. Declarations Acknowledgements: We would like to thank all the participants and co-workers in Hillel-Yaffe Medical Centre IVF Unit for their assistance in this study. Author contributions: Shavit M: Data analysis, manuscript writing. Gonen D: Data collection, manuscript writing. Atzmon Y, Aslih N, Bilgory A, Shibli Y, Sharqawi M, Estrada Garcia D: Manuscript editing. Michaeli M, Poltov D: Data collection, embryo morphokinetics annotation. Shalom-Paz E: Project development, supervision, manuscript editing. Data availability statement: All datasets used and analyzed in this study are available from the corresponding author on request. Competing interests statement: None of the authors have any relationships with industry or financial associations which may pose a conflict of interest. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References Storr, A., Venetis, C. A., Cooke, S., Kilani, S. & Ledger, W. Inter-observer and intra-observer agreement between embryologists during selection of a single Day 5 embryo for transfer: a multicenter study. Hum. Reprod. 32 , 307–314 (2017). Motato, Y. et al. Morphokinetic analysis and embryonic prediction for blastocyst formation through an integrated time-lapse system. Fertil. Steril. 105 , 376–84.e9 (2016). Chen, A. A., Tan, L., Suraj, V., Reijo Pera, R. & Shen, S. Biomarkers identified with time-lapse imaging: discovery, validation, and practical application. Fertil. Steril. 99 , 1035–1043 (2013). Herrero, J. & Meseguer, M. Selection of high potential embryos using time-lapse imaging: the era of morphokinetics. Fertil. Steril. 99 , 1030–1034 (2013). Kirkegaard, K., Agerholm, I. E. & Ingerslev, H. J. Time-lapse monitoring as a tool for clinical embryo assessment. Hum. Reprod. 27 , 1277–1285 (2012). Ozbek, I. Y. et al. Comparison of single euploid blastocyst transfer cycle outcome derived from embryos with normal or abnormal cleavage patterns. Reprod. Biomed. Online 42 , 892–900 (2021). Liu, Y. et al. Between-laboratory reproducibility of time-lapse embryo selection using qualitative and quantitative parameters: a systematic review and meta-analysis. J. Assist. Reprod. Genet. 37 , 1295–1302 (2020). Barrie, A. et al. Preliminary investigation of the prevalence and implantation potential of abnormal embryonic phenotypes assessed using time-lapse imaging. Reprod. Biomed. Online 34 , 455–462 (2017). Athayde Wirka, K. et al. Atypical embryo phenotypes identified by time-lapse microscopy: high prevalence and association with embryo development. Fertil. Steril. 101 , 1637–48.e1 (2014). Rubio, I. et al. Limited implantation success of direct-cleaved human zygotes: a time-lapse study. Fertil. Steril. 98 , 1458–1463 (2012). Lagalla, C. et al. Embryos with morphokinetic abnormalities may develop into euploid blastocysts. Reprod. Biomed. Online 34 , 137–146 (2017). Almagor, M., Or, Y., Fieldust, S. & Shoham, Z. Irregular cleavage of early preimplantation human embryos: characteristics of patients and pregnancy outcomes. J. Assist. Reprod. Genet. 32 , 1811–1815 (2015). Desai, N., Goldberg, J. M., Austin, C. & Falcone, T. Are cleavage anomalies, multinucleation, or specific cell cycle kinetics observed with time-lapse imaging predictive of embryo developmental capacity or ploidy? Fertil. Steril. 109 , 665–674 (2018). Zhan, Q., Ye, Z., Clarke, R., Rosenwaks, Z. & Zaninovic, N. Direct unequal cleavages: embryo developmental competence, genetic constitution and clinical outcome. PLoS ONE 11 , e0166398 (2016). Somfai, T. et al. Relationship between the length of cell cycles, cleavage pattern and developmental competence in bovine embryos generated by in vitro fertilization or parthenogenesis. J. Reprod. Dev. 56 , 200–207 (2010). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2746951","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":191689326,"identity":"9290040a-8429-4632-bf1d-2615cc05f4a4","order_by":0,"name":"Maya Shavit","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYFADduYDQFJChgQtzGwJIC08pGjhMQBRhLXotp8x/MDwyyaPn5nn86sbNRY8DOyHj27Ap8XsTI6xBGNfWrFkM+8265xjQIfxpKXdwKvlQI6BBGPP4cQNh3m3GeewAbVI8Jjh13L+jfEPxp7/ifsP8zwzzvlHjJYbOWYSDD8OJG5g5mF+nNtGlJZnZRaJDcmJMw6zmTHn9knwsBH0y/nkzTc+/LFL7G9vfvw551udHD/74WN4tTAwcBgwJLaBWWwSYBK/chBgf8DA8AfMYv5AWPUoGAWjYBSMRAAAD1RHqxCzIYAAAAAASUVORK5CYII=","orcid":"","institution":"Hillel-Yaffe Medical Center","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Maya","middleName":"","lastName":"Shavit","suffix":""},{"id":191689327,"identity":"f4ce8591-6f02-4a5e-b9ab-99a11704dfbb","order_by":1,"name":"Daniel Gonen","email":"","orcid":"","institution":"The Technion – Israel Institute of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Gonen","suffix":""},{"id":191689328,"identity":"60b3ef34-964b-4594-945f-c3465bf3226a","order_by":2,"name":"Yuval Atzmon","email":"","orcid":"","institution":"Hillel-Yaffe Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuval","middleName":"","lastName":"Atzmon","suffix":""},{"id":191689329,"identity":"ebc965a6-2052-4c07-8f5b-f79aad47cd96","order_by":3,"name":"Nardin Aslih","email":"","orcid":"","institution":"Hillel-Yaffe Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nardin","middleName":"","lastName":"Aslih","suffix":""},{"id":191689331,"identity":"5748390f-ee22-4329-84a3-02b2033d2501","order_by":4,"name":"Asaf Bilgory","email":"","orcid":"","institution":"Hillel-Yaffe Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Asaf","middleName":"","lastName":"Bilgory","suffix":""},{"id":191689333,"identity":"9d90b5d0-8993-4af1-8b6f-393fd90d7cf6","order_by":5,"name":"Yasmin Shibli","email":"","orcid":"","institution":"Hillel-Yaffe Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yasmin","middleName":"","lastName":"Shibli","suffix":""},{"id":191689335,"identity":"95dd3ab1-b829-4296-bb15-51c268b92bd4","order_by":6,"name":"Moamina Sharqawi","email":"","orcid":"","institution":"Hillel-Yaffe Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Moamina","middleName":"","lastName":"Sharqawi","suffix":""},{"id":191689338,"identity":"e850f76d-6961-45d4-8c48-01c04fae243d","order_by":7,"name":"Daniela Estrada Garcia","email":"","orcid":"","institution":"Hillel-Yaffe Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Daniela","middleName":"Estrada","lastName":"Garcia","suffix":""},{"id":191689341,"identity":"9850117c-8bf6-4812-8640-80fd43b48c1b","order_by":8,"name":"Mediea Michaeli","email":"","orcid":"","institution":"Hillel-Yaffe Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mediea","middleName":"","lastName":"Michaeli","suffix":""},{"id":191689343,"identity":"eada8040-de98-4b68-8a0d-e622661fafbe","order_by":9,"name":"Diana Poltov","email":"","orcid":"","institution":"Hillel-Yaffe Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Diana","middleName":"","lastName":"Poltov","suffix":""},{"id":191689346,"identity":"3684fb6b-e7f4-421f-a87c-098018f3993e","order_by":10,"name":"Einat Shalom-Paz","email":"","orcid":"","institution":"Hillel-Yaffe Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Einat","middleName":"","lastName":"Shalom-Paz","suffix":""}],"badges":[],"createdAt":"2023-03-28 12:14:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2746951/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2746951/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":36982781,"identity":"225ccaef-7f24-4ed6-98ab-e574482c5475","added_by":"auto","created_at":"2023-05-13 07:14:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":453432,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2746951/v1/3b04801f-07fa-4495-8bba-f3182cfe9f1a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Behind the scenes: Cleavage patterns of 9600 embryos","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSelecting embryos with the greatest implantation potential is critical for achieving higher live birth rates and reducing both unsuccessful transfers and multiple birth rates in IVF cycles. There are two accepted methods for performing embryo selection. The traditional method based on morphology is known to be subjective and is associated with high intra- and inter-personal disagreement \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The novel morphokinetic assessment and analyses of preimplantation human embryo development with time-lapse monitoring and use of artificial intelligence can provide indicative and objective data on embryo quality and implementation potential \u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs the use of time lapse monitoring systems becomes more common, we gain qualitative data regarding embryo cleavage patterns, and its relation to embryonic viability and implantation rate. Abnormal cell cleavage in the form of direct cleavage of a mother cell to more than two daughter cells is defined as irregular cleavage (IRC). This phenomenon is generally associated with lower developmental potential \u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOnly a few studies about embryo cleavage patterns have been published \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. All of those were retrospective and with a relatively small sample size. Furthermore, results published so far from different studies are contradictory regarding euploidy, implantation and live birth rates.\u003c/p\u003e \u003cp\u003eThe purpose of this study was to contribute to the current literature regarding IRC embryos, their incidence, correlation to patient and treatment characteristics, and to compare IVF treatment outcomes derived from embryos with regular and irregular cleavage patterns.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eThis prospective observational cohort study was conducted at the IVF Unit of Hillel Yaffe Medical Center from January 2016 to December 2021.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003eAll patients whose embryos were assessed with time-lapse technology for morphological and morphokinetic developmental patterns during fresh embryo development were included in the study. Clinical outcomes were evaluated for 1,001 patients undergoing 1,976 IVF cycles, with 23,605 retrieved oocytes, and 9,632 developed embryos.\u003c/p\u003e \u003cp\u003eDemographic characteristics of the women enrolled (including age, parity, BMI, smoking, basal FSH level, and infertility cause) were collected from medical records. Ovarian stimulation protocol (long GNRH agonist or GNRH antagonist), ovulation trigger (GNRH agonist or hCG), number of retrieved oocytes, and ICSI rate were analysed. Outcome for fresh and frozen transfer cycles was analysed according to embryo age at transfer (day 3 or day 5) and number of embryos per transfer (one to three).\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eIRC\u003c/h2\u003e \u003cp\u003eIRC defined as an abnormal cell cleavage in the form of a direct cleavage of a mother cell to more than two daughter cells regardless of the cell size. Only cells containing visible nuclei were considered blastomeres; otherwise, they were annotated as fragment.\u003c/p\u003e \u003cp\u003eReverse cleavage was not evaluated in this study. We distinguished between early IRC (occurred in the first 24 hours from the presence of 2PN) and late IRC (up to the third cleavage). We also distinguished between single and multiple events of IRC. The analysis was based on these IRC characteristics.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eOutcome Measures\u003c/h3\u003e\n\u003cp\u003eOutcomes included morphokinetics parameters (multinucleation, fragmentation, time to blastulation, uneven blastomere rate), blastulation rate, high quality embryos remaining for use (frozen or transferred) rate, clinical pregnancy rate (CPR) which was defined as ultrasound confirmation of an intrauterine gestational sac with a positive heartbeat, live birth rate (LBR), miscarriage rate (spontaneous termination before 24 weeks of gestation), preterm labour rate (live deliveries occurring earlier than 37 weeks of pregnancy), and placental complication rate during gestation, including gestational hypertension, pre-eclampsia, abruption, intrauterine growth restriction (IUGR), or intra-uterine foetal demise (IUFD).\u003c/p\u003e\n\u003ch3\u003eEthical Issues\u003c/h3\u003e\n\u003cp\u003e The Institutional Review Board (Helsinki Committee) at Hillel-Yaffe Medical Center approved the study protocol on 27/04/2020. Approval number is 0026-20-HYMC. As the protocol involved only the observational review of patient charts, the need for individual patient informed consent was waived by the Institutional Review Board (Helsinki Committee) at Hillel-Yaffe Medical Center. All methods in the present study were strictly performed in accordance with the Declaration of Helsinki for Medical Research.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using SPSS,-22.0 package for windows (IBM Corp., USA). Categorical variables were analysed with chi-squared test or Fisher\u0026rsquo;s exact test. Continuous variables were analysed using a t-test. For results that were significant or showed a statistical trend in univariate analysis, multivariable analysis was performed with a multiple logistic regression model to assess the potential impact of those parameters and to further investigate and find the morphokinetic parameters and patient characteristics predictive of a successful or unsuccessful pregnancy outcome, CPR and LBR. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. All statistical tests were two-tailed. We also conducted a logistic regression for nominal parameters.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe incidence of IRC embryos was 17.5% (1,689/9,632 embryos). Of these, 35% had early IRC (occurring during the embryo's first cell cycle) and 65% had late IRC (occurring after the embryo's first cell cycle ended). In addition, 7,943 (82.5%) embryos had no IRC, 1,447 (15%) embryos had one IRC, and 242 (2.5%) embryos had two or more IRC.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePatient characteristics\u003c/h2\u003e \u003cp\u003eAge, BMI, parity, and basal FSH level were not correlated with the number of IRC events or with their timing. IRC embryos were found to correlate with male factor infertility (35.7% vs. 31.2%, p\u0026thinsp;=\u0026thinsp;0.01; Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBasic patient and treatment characteristics by number of irregular cleavage events during embryo development\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo IRC\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7,943)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 IRC\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;1,447)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u0026thinsp;+\u0026thinsp;IRC\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;242)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaternal age (years\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.9\u0026thinsp;\u0026plusmn;\u0026thinsp;6.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.336\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParity (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.207\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI (kg/m \u0026sup2;\u0026plusmn; SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.214\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasal FSH level (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.673\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInfertility cause (n (%))\u003c/p\u003e \u003cp\u003eAnovulation\u003c/p\u003e \u003cp\u003eEndometriosis\u003c/p\u003e \u003cp\u003eMale factor\u003c/p\u003e \u003cp\u003eTubal factor\u003c/p\u003e \u003cp\u003eUnexplained\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e824 (10.4%)\u003c/p\u003e \u003cp\u003e213 (2.7%)\u003c/p\u003e \u003cp\u003e2,480 (31.2%)\u003c/p\u003e \u003cp\u003e980 (12.3%)\u003c/p\u003e \u003cp\u003e1,552 (19.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e127 (8.8%)\u003c/p\u003e \u003cp\u003e38 (2.6%)\u003c/p\u003e \u003cp\u003e520 (35.9%)\u003c/p\u003e \u003cp\u003e188 (13%)\u003c/p\u003e \u003cp\u003e278 (19.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21 (8.7%)\u003c/p\u003e \u003cp\u003e2 (0.8%)\u003c/p\u003e \u003cp\u003e84 (34.7%)\u003c/p\u003e \u003cp\u003e25 (10.3%)\u003c/p\u003e \u003cp\u003e59 (24.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.136\u003c/p\u003e \u003cp\u003e0.206\u003c/p\u003e \u003cp\u003e0.001\u003c/p\u003e \u003cp\u003e0.480\u003c/p\u003e \u003cp\u003e0.160\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOvarian stimulation protocol, n (%))\u003c/p\u003e \u003cp\u003eLong GNRH agonist\u003c/p\u003e \u003cp\u003eGNRH antagonist\u003c/p\u003e \u003cp\u003eOther\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e786 (9.8%)\u003c/p\u003e \u003cp\u003e5,922 (74.6%)\u003c/p\u003e \u003cp\u003e1235 (15.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e168 (11.6%)\u003c/p\u003e \u003cp\u003e1,043 (72.1%)\u003c/p\u003e \u003cp\u003e236 (16.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29 (12%)\u003c/p\u003e \u003cp\u003e178 (73.6%)\u003c/p\u003e \u003cp\u003e35 (14.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.199\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGNRH agonist ovulation trigger, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e802 (10.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e162 (11.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22 (9.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.375\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber retrieved oocytes (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.5\u0026thinsp;\u0026plusmn;\u0026thinsp;9.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.3\u0026thinsp;\u0026plusmn;\u0026thinsp;8.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.315\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eICSI (n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6,910 (87%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,301 (90%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e208 (86%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eIRC, irregular cleavage; SD, standard deviation; BMI, Body mass index; ICSI, Intracytoplasmic sperm injection\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBasic patient and treatment characteristics by timing of first Irregular cleavage event during embryo development\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo IRC\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7,943)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEarly IRC (n\u0026thinsp;=\u0026thinsp;588)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLate IRC (n\u0026thinsp;=\u0026thinsp;1,101)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaternal age (years\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.7\u0026thinsp;\u0026plusmn;\u0026thinsp;6.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.169\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParity (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.175\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI (kg/m\u0026sup2; \u0026plusmn; SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.178\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasal FSH level (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.534\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInfertility cause (n (%))\u003c/p\u003e \u003cp\u003eAnovulation\u003c/p\u003e \u003cp\u003eEndometriosis\u003c/p\u003e \u003cp\u003eMale factor\u003c/p\u003e \u003cp\u003eTubal factor\u003c/p\u003e \u003cp\u003eUnexplained\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e824 (10.4%)\u003c/p\u003e \u003cp\u003e213 (2.7%)\u003c/p\u003e \u003cp\u003e2,480 (31.2%)\u003c/p\u003e \u003cp\u003e980 (12.3%)\u003c/p\u003e \u003cp\u003e1,552 (19.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58 (9.9%)\u003c/p\u003e \u003cp\u003e18 (3.1%)\u003c/p\u003e \u003cp\u003e208 (35.4%)\u003c/p\u003e \u003cp\u003e72 (12.2%)\u003c/p\u003e \u003cp\u003e114 (19.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90 (8.2%)\u003c/p\u003e \u003cp\u003e22 (2.0%)\u003c/p\u003e \u003cp\u003e396 (36%)\u003c/p\u003e \u003cp\u003e141 (12.8%)\u003c/p\u003e \u003cp\u003e223 (20.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.074\u003c/p\u003e \u003cp\u003e0.328\u003c/p\u003e \u003cp\u003e0.001\u003c/p\u003e \u003cp\u003e0.901\u003c/p\u003e \u003cp\u003e0.846\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOvarian stimulation protocol, n (%)\u003c/p\u003e \u003cp\u003eLong GNRH agonist\u003c/p\u003e \u003cp\u003eGNRH antagonist\u003c/p\u003e \u003cp\u003eOther\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e786 (9.8%)\u003c/p\u003e \u003cp\u003e5,922 (74.6%)\u003c/p\u003e \u003cp\u003e1235 (15.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70 (11.9%)\u003c/p\u003e \u003cp\u003e421 (71.6%)\u003c/p\u003e \u003cp\u003e97 (16.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e127 (11.5%)\u003c/p\u003e \u003cp\u003e800 (72.7%)\u003c/p\u003e \u003cp\u003e174 (15.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.224\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGNRH agonist ovulation trigger (n (%))\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(10.1%)802\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(13.1%)77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(9.7%)107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.057\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRetrieved oocytes number (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.5\u0026thinsp;\u0026plusmn;\u0026thinsp;9.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.0\u0026thinsp;\u0026plusmn;\u0026thinsp;9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.7\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.383\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eICSI (n (%))\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6,910 (87%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e518 (88%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e991 (90%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.011\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eIRC, irregular cleavage; SD, standard deviation; BMI, Body mass index; ICSI, Intracytoplasmic sperm injection\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTreatment characteristics\u003c/h2\u003e \u003cp\u003eNeither the number of IRC events nor their timing was influenced by the stimulation protocol, trigger medication or number of retrieved oocytes. IRC embryos had higher ICSI rate (89% vs. 87%, p-value 0.01) (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eOther morphokinetic parameters\u003c/h2\u003e \u003cp\u003eBoth higher number of IRC events along the embryo\u0026rsquo;s development course and earlier time to first IRC event were found to correlate with a higher multi-nucleation rate, and a higher rate of uneven blastomere size. An early IRC event also correlated with a higher fragmentation rate (14.9%, 18% and 12% for none, early and late IRC events, respectively, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIVF outcome for embryos by number of IRC events during embryo development\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo IRC\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7,943)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 IRC\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;1,447)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u0026thinsp;+\u0026thinsp;IRC\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;242)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMulti-nucleation (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFragmentation (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.9%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.128\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUneven blastomere, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,224 (28%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e651 (45%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e118 (49%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlastocyst formation, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,581 (19.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(20.8%) 301\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(11.4%) 26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime to blastulation (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e108.5\u0026thinsp;\u0026plusmn;\u0026thinsp;10.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e112.4\u0026thinsp;\u0026plusmn;\u0026thinsp;9.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e111.1\u0026thinsp;\u0026plusmn;\u0026thinsp;8.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUsable embryos, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4,280 (53.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(14.0%) 202\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(9.5%) 23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFresh cycles outcome\u003c/p\u003e \u003cp\u003eFresh cycles with ET, n (%)\u003c/p\u003e \u003cp\u003eClinical pregnancy, n (%)\u003c/p\u003e \u003cp\u003eLive birth, n (%)\u003c/p\u003e \u003cp\u003eMiscarriage, n (%)\u003c/p\u003e \u003cp\u003ePreterm birth, n (%)\u003c/p\u003e \u003cp\u003ePlacental complications, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,808 (22.7%)\u003c/p\u003e \u003cp\u003e(30.8%) 562/1,808\u003c/p\u003e \u003cp\u003e(20.5%) 356/1,808\u003c/p\u003e \u003cp\u003e(22%) 127/562\u003c/p\u003e \u003cp\u003e57/562 (10%)\u003c/p\u003e \u003cp\u003e33/562 (6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e113 (7.8%)\u003c/p\u003e \u003cp\u003e(23.4%) 26/113\u003c/p\u003e \u003cp\u003e(14.1%) 16/113\u003c/p\u003e \u003cp\u003e6/26 (23%)\u003c/p\u003e \u003cp\u003e6/26 (23%)\u003c/p\u003e \u003cp\u003e0/26 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20 (8.2%)\u003c/p\u003e \u003cp\u003e(5.0%)1\u003c/p\u003e \u003cp\u003e(5.0%) 1\u003c/p\u003e \u003cp\u003e(0%) 0/1\u003c/p\u003e \u003cp\u003e(0%) 0/1\u003c/p\u003e \u003cp\u003e(0%) 0/1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.001\u003c/p\u003e \u003cp\u003e.009\u003c/p\u003e \u003cp\u003e.095\u003c/p\u003e \u003cp\u003e.999\u003c/p\u003e \u003cp\u003e.037\u003c/p\u003e \u003cp\u003e.091\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eIRC, irregular cleavage; SD, standard deviation; ET, embryo transfer\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIVF outcome for embryos by timing of first IRC event during embryo development\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo IRC\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7,943)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEarly IRC (n\u0026thinsp;=\u0026thinsp;588)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLate IRC (n\u0026thinsp;=\u0026thinsp;1,101)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMulti-nucleation (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFragmentation (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.9%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUneven blastomere, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,224 (28%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e411 (70%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e385 (35%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlastocyst formation, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,581 (19.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e92 (15.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e239 (21.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.011\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime to blastulation, minutes (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e108.5\u0026thinsp;\u0026plusmn;\u0026thinsp;10.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e112.1\u0026thinsp;\u0026plusmn;\u0026thinsp;10.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e112.4\u0026thinsp;\u0026plusmn;\u0026thinsp;9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUsable embryos, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4,280 (53.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53 (9.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e175 (15.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFresh cycle outcomes\u003c/p\u003e \u003cp\u003eFresh cycles with ET, n (%)\u003c/p\u003e \u003cp\u003eClinical pregnancy, n (%)\u003c/p\u003e \u003cp\u003eLive birth, n (%)\u003c/p\u003e \u003cp\u003eMiscarriage, n (%)\u003c/p\u003e \u003cp\u003ePreterm birth, n (%)\u003c/p\u003e \u003cp\u003ePlacental complication, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,808 (22.7%)\u003c/p\u003e \u003cp\u003e(30.8%) 562/1,808\u003c/p\u003e \u003cp\u003e(20.5%) 56/1,808\u003c/p\u003e \u003cp\u003e(22%) 127/562\u003c/p\u003e \u003cp\u003e57/562 (10%)\u003c/p\u003e \u003cp\u003e33/562 (6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32 (5.4%)\u003c/p\u003e \u003cp\u003e3/32 (9.4%)\u003c/p\u003e \u003cp\u003e1/32 (3.1%)\u003c/p\u003e \u003cp\u003e2/3 (66%)\u003c/p\u003e \u003cp\u003e(0%) 0/3\u003c/p\u003e \u003cp\u003e(0%) 0/3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e101 (9.2%)\u003c/p\u003e \u003cp\u003e24 (23.7%)\u003c/p\u003e \u003cp\u003e16 (15.8%)\u003c/p\u003e \u003cp\u003e3/24 (12.5%)\u003c/p\u003e \u003cp\u003e6/24 (25%)\u003c/p\u003e \u003cp\u003e0/24 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.001\u003c/p\u003e \u003cp\u003e.010\u003c/p\u003e \u003cp\u003e.042\u003c/p\u003e \u003cp\u003e.093\u003c/p\u003e \u003cp\u003e.021\u003c/p\u003e \u003cp\u003e.105\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eIRC, irregular cleavage; SD, standard deviation; ET, embryo transfer\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIvf Outcomes\u003c/h3\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eBlastulation\u003c/h2\u003e \u003cp\u003eBlastulation rate was lower in the two or more IRC group (11.4%) compared with none (19.9%) or one (20.8%) IRC only (p\u0026thinsp;=\u0026thinsp;0.01). Blastulation rate was also lower when comparing early IRC (15.6%) to no (19.9%) or late IRC (21.7%) (p\u0026thinsp;=\u0026thinsp;0.01). Also of importance, is that time to blastulation was longer for IRC embryos in comparison to those without (Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRate of usable embryos\u003c/h2\u003e \u003cp\u003eThe usable embryo rate was defined as embryos chosen for either transfer or freeze. The rate was significantly lower for embryos with IRC and even more so for embryos with two or more IRC (53.9%, 14%, and 9.5% respectively, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The rate was also lower for embryos with an early IRC vs. late IRC (9.0% vs. 15.9%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eClinical pregnancy, live birth, and miscarriage rate in fresh cycles\u003c/h2\u003e \u003cp\u003eThere were 133 fresh cycles with embryo transfer (ET) of an IRC embryo. All fresh transfer cycles (cleavage stage and blastocyst) were combined for outcome analysis due to the small sample size. Among these, 23 were single embryo transfers (SET), and the remaining 110 cycles were of two or three embryos. In most cycles with multiple embryos, at least one embryo did not have an IRC event. The CPR per transfer was lower with increasing number of IRC events (30.8%, 23.4%, and 5.0% for none, one, or two or more IRC events, respectively; p\u0026thinsp;=\u0026thinsp;0.01). ET with an early IRC embryo had both lower CPR and lower LBR compared with late and no IRC (9.4%, 23.7%, 30.8%, p\u0026thinsp;=\u0026thinsp;0.01 and 3.1%, 15.8%. 20.5%, p\u0026thinsp;=\u0026thinsp;0.04). Miscarriage rate did not differ significantly between the study groups (Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003ePreterm labour and placental complications in fresh cycles\u003c/b\u003e\u003c/h2\u003e \u003cp\u003ePreterm labour was significantly more prevalent in the IRC groups (23% in the single IRC event group and 25% in the late IRC groups vs. 10% in the no IRC group, p\u0026thinsp;=\u0026thinsp;0.04 and 0.02, respectively). There were no preterm deliveries in the group of early or more than one IRC event group. No significant difference was found in the rate of placental complications between the study groups (Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eIRC frozen embryo transfer outcomes\u003c/h2\u003e \u003cp\u003eA total of 95 embryos with IRC were frozen, all at blastocyst stage. Of these embryos, 51 were transferred in a frozen cycle, of which 32 were SET and 19 dual embryo transfers (DET). In the DET cycles, the transferred embryo included one embryo with an IRC event and the other had normal cleavage. The CPR of these cycles was 25% (8/32) in the SET cycles and 36% (7/19) in the DET cycles (p\u0026thinsp;=\u0026thinsp;0.369). In comparison, CPR for SET of a blastocyst without IRC in a frozen cycle in our centre was 37% (p\u0026thinsp;=\u0026thinsp;0.17) and LBR was 22% (p\u0026thinsp;=\u0026thinsp;0.37). We cannot compare DET for blastocysts without IRC as our centre\u0026rsquo;s policy is to transfer only one blastocyst when there was no IRC. The LBR was 15.6% (5/32) in the SET cycles and 10.5% (2/19) in the DET cycles (p\u0026thinsp;=\u0026thinsp;0.61; Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIrregular cleavage (IRC) frozen embryo transfer outcomes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo IRC\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;261)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIRC\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;95)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSingle embryo transfer\u003c/p\u003e \u003cp\u003eClinical pregnancy rate, n (%)\u003c/p\u003e \u003cp\u003eLive birth rate, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e95/261 (37%)\u003c/p\u003e \u003cp\u003e59/261 (22%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8/32 (25%)\u003c/p\u003e \u003cp\u003e5/32 (15.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.175\u003c/p\u003e \u003cp\u003e0.367\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDual embryo transfer\u003c/p\u003e \u003cp\u003eClinical pregnancy rate, n (%)\u003c/p\u003e \u003cp\u003eLive birth rate, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7/19 (36%)\u003c/p\u003e \u003cp\u003e2/19 (10.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eLogistic regression\u003c/h2\u003e \u003cp\u003eWhen logistic regression analysis was used to determine the variables that contributed to the LBR, only younger age and higher parity were found to be significant parameters for achieving both CPR and LBR. BMI, duration of infertility, aetiology of infertility, number of oocytes retrieved, time to cleavage stage embryo, degree of embryo fragmentation, embryo degree of multi-nucleation, number of IRC events and their timing were not related to achieving clinical pregnancy or to live delivery (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMultivariable logistic regression analysis for clinical pregnancy rate\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eOdds ratio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e95% CI for Odds ratio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLower\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUpper\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.928\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.907\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.053\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.542\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.013\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eCI, confidence interval\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study evaluated the impact of IRC on IVF treatment and pregnancy outcomes and contributes to the existing, yet meagre literature regarding the reproductive potential of embryos with no, single or multiple IRC events and their timing \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12 CR13\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. We demonstrated that male factor and ICSI correlate with IRC, that a single early IRC event or multiple occasions of IRC have deleterious effects on embryo development and IVF outcomes in term of blastulation rate. However, the blastulation rate was not affected by a single late IRC event. Unlike blastulation rate, the CPR was found to be significantly lower in cases with single late IRC and even more so in cases with an early IRC or multiple instances of IRC. We found that frozen blastocysts with IRC could lead to pregnancies, although the CPR was lower in comparison to FET with normal cleavage embryos. In addition, to the best of our knowledge, this is the first study to follow these pregnancies until delivery and investigate preterm labour and placental complications in pregnancies resulting from an IRC ET.\u003c/p\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMale Factor, ICSI and IRC\u003c/h2\u003e \u003cp\u003eAnalysis of IRC events in relation to the cause of infertility revealed a connection between sperm defects and IRC. ICSI was used more frequently with the single and late IRC embryo groups. Our findings contradict a previous study \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e that found higher IVF compared to ICSI in the single IRC group, and no significant difference between the other groups. They also reported that sperm origin (testicular/epididymal as opposed to ejaculated) influences embryo cleavage patterns, while paternal age did not. The impact of paternal age is debated in the literature, Ozbek et al. \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e found a correlation between advanced paternal age and IRC. One of the possible hypotheses underlying IRC is the formation of multipolar spindles through the introduction of either incomplete, defective, or supernumerary centrioles by defective sperm \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Our hypothesis regarding the higher ICSI rate is that the procedure might damage the ovum miotic spindle, which in turn will manifest as IRC.\u003c/p\u003e \u003cp\u003eIt will be interesting re-examine our findings and those of Zhan et al. \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e as well, as to further investigate whether certain semen parameters, such as DNA fragmentation or teratospermia, are predictive of IRC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eThe impact of IRC and blastulation\u003c/h2\u003e \u003cp\u003eIn agreement with previous studies \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, we found a lower blastulation rate for embryos with early or multiple IRC events. However, lower blastulation rate was not demonstrated for embryos with single and/or late IRC. This is most likely due to a less deleterious effect of the IRC, as well as our centre policy of growing those embryos to day 5, while good quality embryos without IRC are more commonly transferred at cleavage stage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eEarly vs. late IRC\u003c/h2\u003e \u003cp\u003eAs previously mentioned, early as compared to late IRC, has a detrimental effect on embryonic development, as manifested by lower rates of blastulation, fewer usable embryos, lower clinical pregnancy, and live birth rates.\u003c/p\u003e \u003cp\u003eThe molecular mechanisms that underlie IRC are not entirely clear, but it is widely accepted that mitotic errors play a vital role in enabling irregular division. When IRC occurs late, there is at least one blastomere that cleaves normally and contributes to the chromosomal balance of the embryo, thereby bolstering its viability and eventually leading to higher implantation and live birth rates. Lagalla et al. \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e explored the possibility of a potential \u0026lsquo;aneuploidy rescue\u0026rsquo; mechanism, as they observed that IRC embryos that excreted some cells during the compaction process developed into euploid blastocysts. Further genetic analysis could reveal new information about the differences in the self-correction mechanisms between early and late IRC and about the molecular underpinnings of both abnormal cleavage patterns.\u003c/p\u003e \u003cp\u003eA few recent studies included pregestational genetic analysis of the IRC embryos. These studies included mainly patients with poor prognosis: advanced maternal age, severe male factor, recurrent unexplained pregnancy loss and repeated implantation failure. Most of embryo biopsies reported were done at blastocyst stage and only some of the studies mentioned the timing of IRC event during embryonic development. Lagalla et al. \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and Zhan et al. \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e reported that the euploidy rate gradually increased when the IRC event occurred later. Surprisingly, IRC embryos were found to have euploid rates comparable to those of embryos without IRC. In our study, genetic analysis of the embryos was not available.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eOne vs. several IRC\u003c/h2\u003e \u003cp\u003eHigher number of IRC events during embryonic development represents more mitotic errors, and fewer blastomeres that cleave normally. Only one earlier study \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e referred to this issue. In agreement with Zhan et al. \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, we also found lower blastulation rates and lower CPR. Moreover, we found fewer usable embryos and a higher preterm birth rate. Zhan et al. \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e also performed genetic analyses and did not find significant differences between one vs. multiple IRC events.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eThe performance of frozen embryos with IRC\u003c/h2\u003e \u003cp\u003eBased on the accumulated genetic data regarding the euploidy status of IRC embryos who reached blastocyst stage \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, we also found that IRC embryos that reached blastocyst stage achieved reasonable CPR and LBR in FET and fresh cycles.\u003c/p\u003e \u003cp\u003eWe found a higher rate of preterm labour in the late IRC group. There were no preterm deliveries in the early or more than one IRC event group, but this is most likely due to chance given the small size of the groups. This finding of higher preterm birth rates should be confirmed by further studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003eStrengths and limitations\u003c/h2\u003e \u003cp\u003e The strengths of the present study include a relatively large sample size of human embryos incubated in EmbryoScope\u0026reg;, in a single IVF centre with a limited number of embryologists; all with high level of expertise and homogeneous guidelines regarding embryo estimation and management. All IRC annotations were confirmed by the embryologists. This was a prospective observational study; hence, the data were gathered prospectively and therefore, are highly reliability. Only the statistical analysis was done retrospectively.\u003c/p\u003e \u003cp\u003eThis study had few limitations. The embryos transferred did not undergo genetic analysis. Another drawback was the number of embryos transferred per cycle. In many of the cycles with an IRC embryo, a parallel normal cleaved embryo was transferred. This makes it more difficult to interpret the CPR and LBR.\u003c/p\u003e \u003cp\u003eIn conclusion, given the results of this study and previous studies, embryos with IRC pattern should have lower priority for transfer, mainly in the cleavage stage. Since live birth rate after ET of an IRC embryo is significantly lower compared with normal cleavage embryos. We recommend that blastocysts with irregular cleavage patterns should be considered a second-choice candidate for embryo transfer if they are morphologically eligible.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e We would like to thank all the participants and co-workers in Hillel-Yaffe Medical Centre IVF Unit for their assistance in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e Shavit M: Data analysis, manuscript writing. Gonen D: Data collection, manuscript writing. \u0026nbsp;Atzmon Y, Aslih N, Bilgory A, Shibli Y, Sharqawi M, Estrada Garcia D: Manuscript editing. Michaeli M, Poltov\u003csup\u003e\u0026nbsp;\u003c/sup\u003eD: Data collection, embryo morphokinetics annotation. Shalom-Paz\u0026nbsp;E: Project development, supervision, manuscript editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eData availability statement:\u0026nbsp;\u003c/strong\u003eAll datasets used and analyzed in this study are available from the corresponding author on request.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests statement:\u003c/strong\u003e None of the authors have any relationships with industry or financial associations which may pose a conflict of interest. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eStorr, A., Venetis, C. A., Cooke, S., Kilani, S. \u0026amp; Ledger, W. Inter-observer and intra-observer agreement between embryologists during selection of a single Day 5 embryo for transfer: a multicenter study. \u003cem\u003eHum. 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Dev.\u003c/em\u003e \u003cstrong\u003e56\u003c/strong\u003e, 200\u0026ndash;207 (2010). \u003c/li\u003e\n\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Irregular cleavage, direct cleavage, clinical pregnancy rate, live birth rate, time-lapse imaging, morphokinetics","lastPublishedDoi":"10.21203/rs.3.rs-2746951/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2746951/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigates the incidence of irregular cleavage (IRC) among human embryos and their influence on IVF treatment outcomes. This study was designed as a prospective observational study in a single-centre IVF clinic including 1,001 women who underwent 1,976 assisted reproduction treatments during 2016\u0026ndash;2021. Morphokinetics of embryos was analysed and evaluated for the association between IRC and women\u0026rsquo;s characteristics, treatment characteristics, and pregnancy outcome. We found IRC incidence to be 17.5% (1,689/9,632 embryos). Of these, 85% embryos had one IRC, and 15% had multiple IRC. 35% of IRC events occurred during the embryo's first cell cycle. IRC embryos were found to correlate with male factor (p\u0026thinsp;=\u0026thinsp;0.01) and higher ICSI rate (p\u0026thinsp;=\u0026thinsp;0.01). Age, BMI, parity, basal FSH level, stimulation protocol and number of retrieved oocytes did not differ between groups. Embryos with early IRC or more than one IRC event had lower blastulation rate (p\u0026thinsp;=\u0026thinsp;0.01 and p\u0026thinsp;=\u0026thinsp;0.01, respectively). Fresh cycles with IRC embryos had a lower clinical pregnancy rate (p\u0026thinsp;=\u0026thinsp;0.01), and early IRC embryos had a lower live birth rate (p\u0026thinsp;=\u0026thinsp;0.04) compared to embryos without IRC. Frozen embryo transfer (FET) cycles of blastocyst embryos, with and without IRC, had comparable results. In conclusion, number of abnormal cleavage events and their timing are of great importance for the prognosis of the developing human embryo.\u003c/p\u003e","manuscriptTitle":"Behind the scenes: Cleavage patterns of 9600 embryos","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-04-17 16:56:44","doi":"10.21203/rs.3.rs-2746951/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4aaaea87-0efe-4e68-b61d-4a93bfc516e7","owner":[],"postedDate":"April 17th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-05-13T07:14:18+00:00","versionOfRecord":[],"versionCreatedAt":"2023-04-17 16:56:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2746951","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2746951","identity":"rs-2746951","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}