P-146 Larger time intervals between pronuclear fading and the first mitosis are strongly associated with trichotomous mitoses and cleavage arrest in human embryos
Authors: Lagalla C , Marchio L , Bartolacci A , Piccioni C , Zacà C , Tarozzi N , Spadoni V , Patria G , Borini A , Coticchio G 
Published: June 2023

Abstract

Study question
Does the duration of the interval between pronuclear fading and first cell division affect preimplantation embryo developmental competence?

Summary answer
Larger pronuclear-fading to first-division intervals (t2-tPNf) are associated with greater risk of direct unequal cleavage (DUC) and lower blastulation rate, but not of blastocyst aneuploidy.

What is known already
During the last decade, time lapse technology (TLT) has made possible detailed and dynamic observation of in vitro embryo development, offering a powerful non-invasive tool for improving embryo selection. Recent evidence suggests that the phase between pronuclear fading and the first cleavage is a perilous bridge between the zygote and the cleaving embryo. Following pronuclear breakdown, delayed progression through mitosis caused by incomplete DNA replication may result in chromosome lagging, whole and segmental segregation errors, and breakage. In this analysis, we specifically tested the hypothesis that delays in a short final phase of fertilization have an impact on embryo development.

Study design, size, duration
Retrospective study of 1316 zygotes cultured until day 5 using TLT and derived from first ICSI cycles (111 no PGT-A and 94 PGT-A) performed between 2018 and 2022. Testicular sperm or cryopreserved gametes cycles were excluded. DUC was defined as the cleavage of one zygote or blastomere into three daughter blastomeres or cell cycle interval shorter than five hours at the first (DUC1), second (DUC2) or third (DUC3) cell division cycle.

Participants/materials, setting, methods
t2-tPNf intervals were clustered into quartiles (Q1, &lt;2hpi; Q2, 2–2.5hpi; Q3, 2.5–3hpi and Q4,  &gt; 3hpi), which were compared based on embryo development and live birth by linear and logistic regression. The Q1 cluster was set as control, while all other quartiles groups were considered as dummy variables. Multivariate analysis was conducted using the generalized estimating equations, to control for variable numbers of zygotes from each patient. The primary outcome was DUC rate.

Main results and the role of chance
Overall, rates of DUC1, DUC2 and DUC 3 rates were 10.3%, 8% and 2.9%, respectively. After adjusting for major confounders (maternal age, paternal age, maternal body mass index, cause of infertility, anti-Müllerian hormone, follicle stimulating hormone) we observed a higher DUC 1 and DUC 2 rate with increasing times of t2-tPNf. We observed abnormal distribution of DUC1 and DUC2 rate between quartiles groups. Specifically, 89% of DUC1 was shown among Q3 (21%) and Q4 (78%) and 61% of DUC2 among Q3 (22%) and Q4 (39%). The percentage of DUC1 was higher for Q3 (multivariate-OR = 3.29, 95%CI 0.99-10.85, adjusted-p = 0.01) and Q4 (multivariate-OR = 22.79, 95%CI 7.80-66.62, adjusted-p&lt;0.01]. The percentage of DUC2 was higher only in Q4 (multivariate-OR = 2.58, 95%CI 1.13-5-57, adjusted-p = 0.01). Moreover, we observed a reduced blastulation rate in Q3 (multivariate-OR = 0.68, 95%CI 0.49-0.95, adjusted-p = 0.01) and Q4 (multivariate-OR = 0.27, 95%CI 0.19-0.38, adjusted-p&lt;0.01). No significant differences were observed in DUC3 rate, top quality blastocyst, euploidy rate (PGT-A cycles analysis) and live birth rate.

Limitations, reasons for caution
The retrospective single-centre design and the limited sample size are limitations of the study

Wider implications of the findings
The study is consistent with the emerging view that the final phases of fertilization - although short - are crucial for development. Delays in progression towards mitosis probably express chromosome integrity loss and perturbances of chromosome segregation. In most embryos, such perturbances appear to cause trichotomous mitoses and cleavage arrest.

Trial registration number
Not applicable