P–257 An unknown cause lead to polyspermy in IVF cycles and 0PN zygotes in ICSI cycles in male patient
Authors: Li X , Hou J , Shan X , Tian E , Wang Y , Xu W 
Published: August 2021

Abstract

Study question
The patient sperm has normal morphology and motility, which paternal factors cause the abnormal fertilization in IVF/ICSI and what is the underlying mechanism?

Summary answer
A genetic mutation of BEX1 and decreased PLC-zeta has been found in patient, which may provide novel insights of polyspermy and pronucleus formation during fertilization.

What is known already
In mammals, pronucleus formation, a landmark event for fertilization, is critical for embryonic development. Abnormal fertilization refers to the abnormal number of pronucleus and polar bodies in zygotes during in vitro fertilization, with an incidence of 5–15%, among which the incidence of polyspermy and 0PN is about 2–10% and 30%. However, the mechanisms underlying pronucleus formation still unclear. More research has focused on oocyte activation, while paternal relevant abnormal fertilizations have been rarely established. The mechanism of how sperm and/or substances carried by sperm influence the physiological process of fertilization is also unclear.

Study design, size, duration
In our study, we first work on the preliminary observation and analysis of sperm morphology, structure and sperm chromosome number, and then made further analysis at the genetic level to find out the cause of this particular phenotype in this patient. We use of zone-free golden hamster ova test the fertilizing capacity and rescue the pronucleus formation with SrCl2.

Participants/materials, setting, methods
The patient, golden hamster, Papanicolaou stain, scanning electron microscope (SEM), Transmission Electron Microscope (TEM), Fluorescence in situ hybridization (FISH), Whole Exome Sequencing (WES), IVF, ICSI, Assisted Oocyte Activation (AOA).

Main results and the role of chance
During 2016–2018, they did 4 cycle assistant reproduction technology. Cycle1, conventional IVF(C-IVF), 9 MII oocytes, 9 3PN zygotes; Cycle2, ICSI, 10 MII oocytes, 10 0PN zygotes; Cycle3, donor-oocytes C-IVF, 6 MII oocytes, 6 3PN zygotes, and the donor did C-IVF get normal zygotes and embryos; Cycle4, donor-sperm C-IVF, 7 MII oocytes, 4 2PN zygotes, 3 useable embryos. Remarkably, clinical examination about male shows normal sperm semen parameters. Papanicolaou stain and SEM shows that the sperm of the patient has normal morphology. The TEM data shows that the spermatozoa with normal head morphology and intact 9 + 2 sperm flagella structure. In the sperm FISH analysis, Chromosome ploidy is haploid. We performed WES on the male, after exclusion of frequent variants and application of technical and biological filters, two homozygous missense mutations were identified in BEX1 (c.191G&gt;A [p. W64X]), which has been few reports of male infertility. The western blot result show that the PLC-zeta was decreased in patient. After 10mM SrCl2 assisted oocyte activation, the zygote has the pronucleus formation in ICSI.

Limitations, reasons for caution
At present, we only observe sperm related factors (morphology, structure, chromosome number, genetic mutation). Next step is to detect the substances sperm carried (e.g. RNA-seq, proteomics). In this case, what is of great concern to us is the inconsistencies of the abnormal fertilization during the conventional IVF and ICSI cycles.
Wider implications of the findings: Many studies of fertilization mechanism, the main focus is on the maternal cytoplasmic factors, such as the Ca 2+ release initiate the fast block of oocytes. There are few reports about abnormal fertilization due to sperm factors. Our case may offer new insights for the study of fertilization.

Trial registration number
Not applicable