P-527 What is the influence of blastocyst hatching position on clinical pregnancy rates?
Authors: Gonçalves Pinto G , Reis I , Erberelli R , Chehin M B , Alegretti J R , Lorenzon A 
Published: June 2023

Abstract

Study question
Is there a relationship between the hatching zone in the blastocyst and its gestational potential?

Summary answer
Blastocysts that presented a hatching area opposite to the inner cell mass showed lower clinical pregnancy rates.

What is known already
Embryo implantation in the uterine endometrium is dependent on blastocyst hatching out of the zona pellucida, allowing the cells of the trophectoderm to establish contact with the uterine epithelium. Hatching begins with the continuous expansion of the blastocoel, thinning the zona pellucida. The blastocyst then exits through a rupture, called the hatching zone. In humans, trophoblast cells close to the inner cell mass (ICM) – polar trophoectoderm – rapidly proliferate and differentiate to follow endometrial invasion, being thus distinct from the remaining trophoectoderm. The association between the hatching zone (according to ICM localization) and implantation rates is scarce.

Study design, size, duration
This is a prospective observational cohort study with 588 patients that underwent in vitro fertilization (IVF) cycles with autologous oocytes and had a fresh or frozen hatching blastocyst embryo transfer between January/2018 and August/2022 in a private ART center. All embryos were cultured in a time-lapse system (Embryoscope, Vitrolife) and were fertilized by ICSI or IVF. Only embryos with known reproductive outcomes (the presence/absence of gestational sac and heartbeat) were included in this analysis.

Participants/materials, setting, methods
Embryos with evidence of natural hatching (either by penetration of trophectoderm cells, or by regular disruption of the trophectoderm followed by embryo extrusion) were included. Using the ICM as reference, blastocysts were classified according to its hatching position in four categories: mass, opposite to mass, intermediate (between mass and opposite) and multiple hatching. ANOVA, Fisher and chi-squared tests were applied properly for statistical analysis. p &lt; 0,05 was considered significant.

Main results and the role of chance
The majority of embryos were classified as intermediate (n = 540, 77.4%), followed by opposite to mass (n = 88, 12.6%), mass (n = 50, 7.2%) and multiple hatching sites (n = 20, 2.8%), in a total of 698 embryos analyzed. Maternal age was similar between groups (36,22±3,65; 36,03±3,3; 36,07±3,23; 37,35±3,57, p = 0.35, for mass, intermediate, opposite to mass and multiple hatching respectively). The majority of embryos presented hatching by penetration of trophoectoderm cells (n = 685, 98.1%), regardless of hatching position and mode of insemination (IVF or ICSI). Biopsied embryos were included since they had reached natural hatching before trophoectoderm biopsy. There was no difference in the proportion of euploid (64%) and non-biopsied (36%) embryos transfers according to hatching position subgroups (68% vs 32%; 64.3% vs 35.7%; 63.3% vs 36.4%; 50% vs 50% for mass, intermediate, opposite and multiple hatching respectively, p = 0.56). Positive pregnancy rate were lower in the opposite to ICM group (47.7%, p = 0.04) when compared to intermediate group (59.3%). Positive pregnancy rates in mass and multiple hatching sites were 54% and 55% respectively. Clinical pregnancy rate were also lower in the opposite to ICM group (45.5%, p = 0.04) when compared to intermediate group (57%). Clinical pregnancy rates in mass and multiple hatching sites were 52% and 50% respectively.

Limitations, reasons for caution
Two experienced senior embryologists classified the blastocyst hatching position; however, operator subjectivity cannot be excluded. The influence of the ICSI hole in the hatching type (penetration or disruption) and position is debatable; nevertheless, both types and all hatching position classification were present in ICSI and IVF insemination.

Wider implications of the findings
The hatching zone of the embryo is potentially a variable of attention for embryo selection. The proliferation and differentiation of trophoblast cells opposite to the inner cell mass may affect the ability of the embryo to progress in the apposition, adhesion and, mostly importantly, invasion of the uterine endometrium.

Trial registration number
Not applicable.