P-206 Unveiling the elasticity of zona pellucida: shifting focus to a neglected embryonic feature
Authors: De Girolamo S , Alteri A , Barbagallo B  M , Rabellotti E , Papaleo E , Pagliardini L 
Published: July 2024

Abstract

Study question
Is it feasible to quantitatively assess the elasticity of the zona pellucida, and how does this measurement correlate with blastocyst features?

Summary answer
The elasticity of the zona pellucida can be quantitatively evaluated, demonstrating a correlation with distinct developmental stages and morphological features of blastocysts.

What is known already
Embryologists have primarily focused on assessing morphokinetic and morphological features of embryos to enhance success rates in assisted reproduction technology cycles. However, the zona pellucida (ZP) has often been overlooked as a potential marker of embryonic quality. Previous studies have established a correlation between the thickness of the ZP and implantation rates in cleavage-stage embryos. Yet, the comprehensive attributes of the ZP in blastocysts remain under-explored, with critical aspects such as its elasticity receiving scant attention.

Study design, size, duration
A retrospective analysis was conducted on 254 blastocysts from 103 patients. For each blastocyst, two images were captured: one prior to and another following lased-induced artificial shrinkage. This study focused on assessing ZP elasticity by comparing its thickness during blastocele pressure-induced distension and subsequent to blastocyst collapse. Variations in thickness were interpreted as reflections of pressure release. To ensure the reliability and accuracy of data, measurements were performed by two independent operators.

Participants/materials, setting, methods
Measurements were performed using ImageJ software. Operators executed twelve radial assessments of ZP thickness at 30-degree intervals and three additional measurements of the area within the ZP. These measurements were averaged for analysis. The ZP re-expansion percentage post-laser collapse was determined by comparing the change in thickness relative to the initial thickness. Linear regression and Spearman’s correlation were employed to examine the relationships among ZP re-expansion, thickness, enclosed area, patient age, and blastocyst features.

Main results and the role of chance
The average ZP thickness prior to freezing (Pre-ZPT) was 5.1 ± 2.0 micrometers, increasing to 8.6 ± 2.0 micrometers following collapse. The mean blastocyst diameter at the time of freezing was 192 ± 17 micrometers, with ZP re-expansion averaging 82.1%. A strong correlation was observed between Pre-ZPT and post-collapse thickness, with a coefficient of 0.75 (P = 4.8 × 10−47).
Pre-ZPT showed a significant inverse and non-linear relationship with pre-collapse blastocyst diameter (Pre-BD), as assessed by Spearman’s correlation (P value: 2.9 × 10−³¹). Additionally, a linear correlation was observed between Pre-BD and the percentage of ZP re-expansion post-collapse (P value: 3.8 × 10−²¹).
Given the strong interdependence of these parameters, reflecting expected behavior from a semi-elastic sheath undergoing stretching, linear correlation between Pre-BD and re-expansion percentage was used to define ZP elasticity. Deviations from average behavior were normalized into percentiles and analyzed against patient age and blastocyst characteristics.
Multivariate regression, adjusted for variables including Pre-ZPT, revealed a strong inverse association between blastocyst elasticity and freezing day, with a decrease of 16.8 percentiles on average (P value: 4.9 × 10−6). Conversely, a positive association was identified with poor-quality blastocysts, which showed an average increase of 12.4 percentiles in elasticity (P value: 1.5 × 10−³). No significant correlation emerged between ZP elasticity and patient age

Limitations, reasons for caution
This preliminary study was limited to the evaluation of the morphological and developmental characteristics of blastocysts, without delving into clinical outcomes. It is critical to note that blastocyst quality assessment is influenced by operator interpretation, which introduces a degree of variability into the morphological evaluation process.

Wider implications of the findings
Evaluating the elasticity of the ZP, a relatively unexplored aspect, might provide insights into its predictive value of blastocyst competence and subsequent successful clinical outcomes following embryo transfer. Such an analysis could significantly enrich our understanding of embryonic reproductive competence.

Trial registration number
not applicable