O-252 Genetic characterization of human sperm centrosome defects in infertile couples with complex embryo aneuploidy
Authors: Tsai S , Xie P , Cheung S , Kocur O , Aluko A , Rosenwaks Z , Palermo G 
Published: June 2023

Abstract

Study question
Can we characterize the genetic aspects related to sperm centrosome dysfunction in couples with high embryo aneuploidy that cannot exclusively be attributed to the oocyte?

Summary answer
We characterized structural and genomic abnormalities responsible for sperm centrosome dysfunction in infertile couples with complex embryo aneuploidy not exclusively related to the female partner.

What is known already
Conventional semen analysis provides only limited information on the proper function and embryo developmental competence of the male gamete. Therefore, an ancillary test, such as sperm chromatin fragmentation (SCF) and particularly double-stranded DNA breaks, has been claimed responsible for structural chromosomal abnormalities. It remains puzzling the source of complex embryo aneuploidy and chaotic chromosomal mosaicism that cannot solely be attributed to the female partner. The availability of genomic analysis may shed light on the integrity and function of the sperm centrosome responsible for ordaining chromosomal distribution at the first mitotic division.

Study design, size, duration
In the past 4 years, we identified 13 couples with unexpected high poor embryo cleavage resulting in a higher number of embryos with complex aneuploidy not solely attributable to the female gamete, resulting in extremely poor embryo implantation. Therefore, ancillary testing on the spermatozoa of the male partners was carried out to determine if the couple’s reproductive failure was eventually due to a stealth male gamete deficiency.

Participants/materials, setting, methods
Thirteen couples with poor clinical outcomes, characterized by poor embryo cleavage, high and complex embryo aneuploidy not exclusively due to the oocyte, with poor embryo implantation in multiple IVF/ICSI cycles, were offered adjuvant male partner testing including centrosome assessment through immunofluorescence staining on centrin (normal ≥ 60%), sperm chromatin fragmentation testing through terminal deoxynucleotide dUTP transferase nick-end labeling (TUNEL, normal ≤ 15%), transmission electron microscopy (TEM), and whole exome sequencing (WES) on spermatozoal DNA.

Main results and the role of chance
In 13 couples with an average paternal age of 38.1±2 years, semen analysis revealed a volume of 22.2±1.4mL, concentration of 58.2±37x10^6/mL, motility of 35.2±18% and normal morphology of 2.6±1%. They underwent with female partners (maternal age of 37.2±2 years) a total of 16 ICSI cycles with aneuploidy testing. Only 11 cycles yielded embryos for transfer due to poor embryo development with high aneuploidy rate, and none of them achieved a pregnancy.
Centrosome staining revealed that only 45.8±22% of the spermatozoa displayed this key organelle. An overall SCF was observed at 16.7±10%. TEM confirmed that approximately 70% of the spermatozoa had irregular proximal centrioles. Moreover, genomic analysis by WES revealed mutations on PLK4 gene responsible for centriole duplication, BRSK1 gene involved in centrosome duplication, TUBGCP6 gene responsible for centrosomal microtubule nucleation, MARK4 for centrosome localization, MAP1S for microtubule anchoring and DNA binding, RIC8A for chromosome distribution, and CALM3 for cytokinesis.
Of this cohort, 3 couples that subsequently underwent 3 ICSI cycles had their spermatozoa processed by microfluidics, aiming at selecting male gametes with higher genomic integrity. A fertilization rate of 78.3% (18/23) was achieved generating euploid embryos that resulted in 3 successful pregnancies.

Limitations, reasons for caution
We identified sperm centrosome dysfunction responsible for the peculiar and recurrent embryo aneuploidy. Although we identified shared gene mutations in those couples, these data are still preliminary. With exception of improving spermatozoa selection, there is a lack of a single effective treatment option to address sperm centrosome abnormality.

Wider implications of the findings
Ancillary tests with semen analysis were able to assess the function and competence of the male gamete. Corroborative genomic study can help provide insights into the reason for poor embryo implantation particularly those with high complex aneuploidy.

Trial registration number
Not applicable