P-190 Spontaneous blastocyst collapse during pre-vitrification equilibration is related to a lower live birth rate: a prospective cohort study
Authors: Murakami M , Tanaka K , Otsubo H , Mizumoto S , Nagao Y , Kuramoto T 
Published: June 2023

Abstract

Study question
This report provides updated data from a study investigating the association between the spontaneous collapse patterns of blastocysts during pre-vitrification equilibration and pregnancy success.

Summary answer
Live birth rates were lower for completely collapsed blastocysts, suggesting the possibility for including blastocyst collapse pattern as a criterion for selecting embryos for transfer.

What is known already
Previous time-lapse studies have found that blastocysts exhibiting strong contraction during culture have a low probability of hatching in animals and implanting in humans, suggesting blastocyst contraction has a negative impact on reproductive outcomes. Conversely, artificial shrinkage of human blastocysts before vitrification is considered to improve embryo survival and/or pregnancy rates by promoting cryoprotectant permeation inside the blastocoel. However, shrinkage is not induced in some vitrification protocols, including ours, as the viability is high; this demonstrates the potential of existing protocols to achieve sufficient cryoprotectant permeation. Moreover, the Alpha consensus meeting on cryopreservation did not issue recommendations regarding artificial shrinkage.

Study design, size, duration
This study included 798 patients who were undergoing their first autologous IVF/ICSI cycle followed by a freeze-all strategy, including blastocyst vitrification, at our clinic between June 2018 and November 2021. For patients with multiple vitrified blastocysts, embryos for transfer were selected hierarchically based only on morphological scoring during culture. To reduce bias, only data from a single blastocyst (day 5, Gardner score 4, excluding CC) transfer from each patient’s first warmed cycle was analyzed.

Participants/materials, setting, methods
Blastocysts were vitrified-warmed in in-house-prepared solutions using Rapid-i carriers. Prior to vitrification, intact blastocysts were equilibrated in 10% ethylene glycol (15 min, 37 °C). Their spontaneous collapse patterns were assessed under an inverted microscope before they were vitrified in 15% ethylene glycol + 15% dimethylsulfoxide + 0.5 M sucrose. Blastocyst collapse was defined as the separation of the trophectoderm cells from the zona pellucida. Collapsed blastocysts with/without a blastocoel cavity were defined as partially/completely collapsed blastocysts.

Main results and the role of chance
Non-collapsed (Nc), partially collapsed (Pc), and completely collapsed (Cc) blastocyst transfers (embryo survival rate, 99.6%) constituted 27.1%, 54.6%, and 18.3% of the 798 cycles, respectively. Live birth rates differed significantly between groups (Nc, 54.2%; Pc, 46.6%; Cc, 34.2%; P &lt; 0.001). Regarding perinatal outcomes of 366 singletons, the female rate tended to differ between groups (Nc, 35.3%; Pc, 44.3%; Cc, 53.1%; P  =  0.084). Significant differences in maternal age (years: Nc, 34.6 ± 4.0; Pc, 35.4 ± 3.7; Cc, 36.4 ± 4.0; P &lt; 0.001), MII oocyte number (Nc, 13.0 ± 6.2; Pc, 12.0 ± 6.6; Cc, 10.8 ± 6.9; P  =  0.009), blastocyst diameter (μM: Nc, 184 ± 18; Pc, 181 ± 20; Cc, 173 ± 23; P &lt; 0.001), and good-quality (4AA/4AB/4BA) blastocyst percentage (Nc, 70.4%; Pc, 61.2%; Cc, 47.9%; P &lt; 0.001) were found between groups. Upon logistic regression analysis including these potential confounders, only maternal age (odds ratio (OR), 0.89; 95% confidence interval (CI), 0.86–0.93; P &lt; 0.001) and collapse pattern (Cc/Nc: OR, 0.59; 95% CI, 0.37–0.93; P  =  0.023) significantly impacted the live birth rate, whereas blastocyst morphology (good/not: OR, 1.37; 95% CI, 0.97–1.94; P  =  0.069) tended to impact it.

Limitations, reasons for caution
The cryoprotectant concentration of our pre-vitrification equilibration solution is relatively low. Thus, caution is required when assessing the collapse pattern, as the solution osmolarity can influence blastocyst shrinkage. Follow-up studies with more participants are warranted to confirm these results and the health of the children born after vitrified-warmed embryo transfers.

Wider implications of the findings
A negative relationship was found between spontaneous blastocyst collapse during pre-vitrification equilibration and live birth rate. Studying blastocyst collapse pattern may assist selection of the most viable blastocysts after vitrification in ethylene glycol and dimethylsulfoxide—a widely used permeable cryoprotectant combination—thus, increasing IVF success rates.

Trial registration number
Not applicable