P-734 Blastocysts derived from tripronuclear zygote including small pronuclear zygote have high diploid and euploid rate with NGS and SNP analysis.
Authors: Mizuta S , Lee Y , Kato Y , Matsubayashi H , Ishikawa T , Shibahara H 
Published: June 2023

Abstract

Study question
What is diploid and euploid frequency of blastocysts derived from tripronuclear zygote (3PN) including a small pronucleus (2.1PN) with NGS and SNP analysis?

Summary answer
Diploid rate of the blastocysts derived from 3PN including 2.1PN was 91.3%, in which euploid, aneuploid and mosaic embryos were 18.7%, 61.3% and 11.3%, respectively.

What is known already
A 3PN zygote is thought to be produced when two sperm enter into the same oocyte during in vitro fertilization (IVF) or whenthe second polar body is not extruded on both IVF and intracytoplasmic sperm injection (ICSI). It has been reported that 3PN is observed around 3-10%, and the embryos are usually discarded by the reason for genetic risks for triploid. However, some studies reported that blastocyst derived from 3PN and/or 2.1PN (defined as small PN &lt; 20μm) contained normal diploid and euploid embryos by the preimplantation genetic testing, and that normal healthy babies were born from these embryos.

Study design, size, duration
We conducted a cross-sectional study between July 2019 and June 2022 in single reproduction center. We included 80 blastocysts in this study which generated from 3PN zygotes including 2.1PN zygotes. Of these, 71 blastocysts were from ICSI and 9 blastocysts were from IVF. These blastocysts were assessed for chromosomal ploidy by next generation sequencing (NGS) and single nucleotide polymorphism (SNP) analysis and embryonic development of these blastocysts were monitored by time-lapse imaging system.

Participants/materials, setting, methods
Average age of women was 39.2 years (22-47 years). The smallest pronuclear diameter who provided blastocysts were 12.7±2.8μm (8.5-19.7μm) in average. The blastocysts were divided between inner cell mass (ICM) and trophectoderm (TE) using laser system under inverted microscope, followed by NGS and SNP. The t-test was performed to analyze differences between triploid and diploid. Statistical significance was defined as P &lt; 0.05. All participants provided written informed consent, and Institutional Review Board approval was obtained.

Main results and the role of chance
Diploid rate of the blastocysts derived from 3PN including 2.1PN was 91.3%, in which euploid, aneuploid and mosaic embryos were 18.7%, 61.3% and 11.3%, respectively. Of 80 blastocysts, 64 blastocysts (80%) were able to be divided ICM from TE. In terms of triploid and diploid, the results of ICM and TE were completely concordant, although 15.6% discordancy was observed between ICM and TE. Average ages of the patients with triploid, euploid, aneuploid, and mosaic embryos were 41.1±5.1 years, 36.0±6.3 years, 40.6±3.5 years, and 35.2±3.6 years, respectively. The patients’ age with aneuploid embryos was significantly higher than those with euploid and mosaic embryos (P = 0.015 and P &lt; 0.001, respectively). Mean diameter of the smallest pronucleus of triploids and diploids were 15.2±4.1μm (8.5-19.7μm) and 12.7±2.8μm (8.5-18.9μm), respectively. Of diploid embryos, euploid, aneuploid, and mosaic were comparable in mean diameter of the smallest pronucleus. There were no differences in insemination methods (IVF and ICSI), frequency of abnormal blastomere division, embryonic developmental speed, and blastocyst grade, between triploid and diploid embryos.

Limitations, reasons for caution
Since this was a cross-sectional study by cytogenetics analysis of blastocysts, causal association between incidence of 3PN (including 2.1PN) and postnatal findings and significance of extra micro pronucleus are unknown. We need to assess longitudinal-study with large sample size of embryo transfer with PGT-A.

Wider implications of the findings
We revealed that blastocyst derived from 3PN including 2.1PN had high diploid and euploid rate by NGS and SNP analysis. Therefore, 3PN including 2.1PN embryos could be used for embryo transfer after chromosome ploidy analyses.

Trial registration number
Not applicable