P-302 Correlation of blastocyst shrinkage-pattern during vitrification and post-warming embryo performance
Authors: Pavlidou T , Panagiotidis Y , Lamprianidis C , Goudakou M , Margariti C , Eleftheriadou A , Krontiris G , Prapa M , Prapas N , Prapas Y 
Published: June 2023

Abstract

Study question
Can blastocyst shrinkage-pattern during vitrification be predictive of post-warming survival and reproductive potential?

Summary answer
Blastocysts assuming a specific shrinkage-pattern during vitrification are liable to lower post-warming morphology and, potentially, degeneration.

What is known already
Blastocyst vitrification is a highly standardized technique. However, there are still possible factors than can compromise the outcome of the technique which should be minimized. For example, the degree of blastocoel expansion before vitrification may negatively influence embryo survival post-warming. There are studies which demonstrate that expanded blastocysts exhibit lower survival rate because of their higher blastocoel volume and amount of water, which is more prone to the detrimental ice crystal formation during vitrification. Moreover, shrinkage of expanded blastocysts during vitrification differs as some of them shrink in a uniform, spherical way while others shrink in a non-canonical way

Study design, size, duration
A retrospective study was conducted at IAKENTRO fertility clinic, Thessaloniki. A total of 2167 blastocysts vitrified between November 2018-December 2022, out of which 532 blastocysts warmed during the same period, were included in the present study. No biopsied or PGT-a tested embryos were included. Embryo vitrification was performed following the same protocol during the whole period of the study. For the clinical outcomes, 178 single-embryo transfers and 123 double-embryo transfers were analyzed

Participants/materials, setting, methods
During blastocyst exposure to the vitrification solution two shrinkage patterns were detected, allocating embryos into Group-1 for blastocysts presenting shrinkage without any detachment of the trophectoderm cells from the zona pellucida, leading to a ZP reforming which eventually resembled a “deflated” ball and into Group-2 for blastocysts that collapsed in a uniform way, having their TE cells fully detached from the ZP, which remained spherical throughout the exposure to the vitrification solution

Main results and the role of chance
In the total cohort of 2167 assessed blastocysts, 437 were allocated in Group-1 and 1730 in Group-2, which can be translated as 20,2% event occurrence. The Group-1 blastocysts were mostly of expansion degree 4 (76,7%) and degree 3 (23,3%), according to the Gardner system, while no smaller blastocysts were assigned in this group. As primary outcome was examined post-warming embryo survival with blastocysts in Group-1 presenting significantly higher degeneration rate (22/187, 11,7%) respect to blastocysts in Group-2 (7/345, 2%) (χ2 test, p &lt; 0.0001, OR = 5,798, 95%CI = 2,511 to 14,55). Moreover, in order to assess any potential decline in embryo morphology post-warming, we corresponded Gardner’s system evaluation to a numerical score and we calculated the integrity in morphology before and after vitrification. Before vitrification, embryos’ morphology score was similar in both groups (74,8% and 73,1% for Group-1 and Group-2, respectively) while, after warming, Group-1 blastocysts exhibited significantly lower integrity of their morphology (86,52% integrity in score) respect to Group-2 blastocysts (93,82% integrity in score) (t-test, p &lt; 0,0001). However, when analyzing PR and CPR no significant differences were observed between the two groups

Limitations, reasons for caution
The retrospective aspect of the study is per se a general limitation. Moreover, given that the analysis of the data was not limited to freeze-all cases, the embryos chosen for the fresh embryo-transfers, which means those with the highest implantation potential, were excluded from the study’s cohort

Wider implications of the findings
Our results show that blastocysts resembling a “deflated” ball during vitrification have compromised post-warming survival. Given that this phenomenon is exclusively observed in expanded blastocysts, whose ZP is thin and TE cells are stretched, identifying the expansion grade over which is happening could be beneficial in clinical practice

Trial registration number
Not applicable