P-133 Impaired blastocyst viability induced by vitrification during re-expansion after trophectoderm biopsy can be prevented by artificial shrinkage before vitrification
Authors: Miki T , Ezoe K , Aruga M , Narisawa M , Ueno S , Okimura T , Kato K 
Published: July 2024

Abstract

Study question
Does the duration between trophectoderm biopsy and vitrification or blastocyst re-expansion affect blastocyst adhesion and outgrowth competence?

Summary answer
Outgrowth was impacted when blastocysts were vitrified during re-expansion, especially at 2 h post-biopsy; however, this can be mitigated using artificial shrinkage pre-vitrification.

What is known already
Biopsied blastocysts are usually vitrified until the results of preimplantation genetic testing for aneuploidy are available. The viability of biopsied blastocysts after vitrification is affected by various factors; in fact, a rodent study reported that the duration from biopsy to vitrification and re-expansion at vitrification affected subsequent developmental competence. The vitrification of biopsied blastocysts immediately post-biopsy is recommended in the clinical setting. However, in some cases, embryologists cannot perform vitrification immediately after a biopsy because of other procedures. In this case, blastocysts start re-expanding, which requires embryologists to wait for the completion of the blastocyst re-expansion.

Study design, size, duration
Human blastocysts donated for research by consenting couples were randomly allocated into biopsied (n = 175) and non-biopsied (control; n = 50) groups. The re-expansion degree (%) at vitrification was calculated using the following formula: [1- (distance of perivitelline cavity at vitrification)/(distance of perivitelline cavity post-biopsy)] × 100. The blastocysts were categorized according to expansion degree: I, ≤0%; II, 1–49%; III, 50–99%; and IV, ≥100%.

Participants/materials, setting, methods
Biopsied and control blastocysts were vitrified 0–3 h post-biopsy. Some blastocysts were artificially shrunken in hypertonic solution before the vitrification. The blastocysts were warmed and plated on fibronectin-coated dishes and cultured for 96 h to assess the competence for blastocyst adhesion and outgrowth. The correlations of outgrowth outcomes with vitrification timing, re-expansion degree, and artificial shrinkage were retrospectively analysed.

Main results and the role of chance
No significant intergroup difference was observed in blastocyst adhesion rate. The multivariate linear regression analysis showed that the blastocyst outgrowth area was significantly smaller when the blastocysts were vitrified at 2 h post-biopsy (P = 0.0103). Re-expansion degree was positively correlated with duration after biopsy (Spearman’s rank correlation coefficient, 0.6921; P &lt; 0.0001). The proportion of expansion in group III was significantly higher at 2 h post-biopsy (P &lt; 0.0001). On multivariate linear regression analysis, the blastocyst outgrowth area was significantly smaller in group III (P = 0.0134). The outgrowth area of blastocysts vitrified at 2 h post-biopsy was significantly increased by artificial shrinkage before vitrification (P = 0.0025) and comparable with those of blastocysts vitrified at 0, 1, and 3 h post-biopsy. Multivariate linear regression analysis also demonstrated the effectiveness of artificial shrinkage for blastocysts vitrified at 2 h post-biopsy and for blastocysts for which re-expansion was initiated at vitrification.

Limitations, reasons for caution
Since this study used discarded vitrified blastocysts, double vitrification was performed. This was an experimental study; therefore, clinical studies are required to validate our findings.

Wider implications of the findings
The blastocysts are recommended to be vitrified before the initiation of re-expansion, which complicates the laboratory workflow. Our data demonstrate that blastocysts that expand post-biopsy can be vitrified after artificial shrinkage without decreasing their implantation competence. Our findings may contribute to improving the day-to-day practice in embryology laboratories.

Trial registration number
Not Applicable