P-066 A multi-bioassay approach to identify male gamete fertilization dysfunction
Authors: Kocur O , PIzzo E , Cheung S , Xie P , Ng L , Rosenwaks Z , Palermo G 
Published: July 2024

Abstract

Study question
Can a multi-bioassay approach identify a specific male gamete dysfunction often overlooked by a standard semen analysis?

Summary answer
In cases of persistent fertilization failure, additional testing may assist in pinpointing underlying factors contributing to compromised sperm embryo developmental competence and tailor treatment.

What is known already
Conventional semen analysis, while informative, ineffectively captures the complete spectrum of male gamete dysfunction. Despite adequate sperm concentration and motility, some couples may still experience fertilization failure with Intracytoplasmic Sperm Injection (ICSI). This highlights the complexity of male infertility, that extends beyond standard parameters. In addressing sperm-related fertilization challenges, various assisted oocyte activation (AOA) methods have been proposed to trigger intracellular Ca2+ release, leading to calcium spikes and meiosis initiation.

Study design, size, duration
In the past 10 months, we identified couples (n = 50) with a history of poor ICSI fertilization, exhibiting suboptimal head morphology. Screening for acrosomal dysfunction involved assessing the presence of PLCxand related gene mutations. We measured the degree of chromatin compaction by histone-to-protamine ratio and evaluated sperm chromatin fragmentation (SCF). To correct for eventual acrosomal dysfunction, couples were counseled to undergo subsequent ICSI cycles with AOA. Clinical outcome was compared between historical and treatment cycles.

Participants/materials, setting, methods
Fifty men with a history of poor ICSI fertilization were included. A standard semen analysis was conducted according to WHO guidelines. The presence of PLCx was measured through immunofluorescent staining (normal threshold, ≥ 30%), corroborated by gene mutation detection though DNA-seq. Sperm histone-to-protamine ratio was evaluated by Aniline Blue (normal threshold, &lt;15% histone retention). SCF was assessed utilizing the TUNEL assay (normal threshold, ≤15%). AOA was performed using ionomycin or recombinant PLCx protein.

Main results and the role of chance
Fifty men experienced a poor clinical outcome following ICSI with a fertilization of 7.4% (48/647), embryo implantation of 6.6% (1/15), clinical pregnancy of 6.6% (1/15), and pregnancy loss rate of 100% (1/1). Their semen analysis, interestingly, revealed the following semen parameters: 3.0±1 mL volume, 50.0±20 x106/mL concentration, 41.4±21% motility, and 1.9±1% normal morphology. Nonetheless, PLCx content was diminished, with values ranging from 2-27%. Genomic assessment confirmed mutations in PLCx, DPY19L2, SPATA16 and PICK1 genes, all implicated in sperm nuclear remodeling and acrosome formation. An elevated histone content at 29.7% was positively correlated with an increased SCF at 18.0% (R2=0.6, P&lt;0.001).
In subsequent cycles employing AOA, the normal fertilization increased to 42.3% (305/721), the embryo implantation reached 13.0% (9/69), the clinical pregnancy rose to 21.1% (8/38), and the delivery rate was 21.1% (8/38), without any pregnancy loss. (P&lt;0.001).

Limitations, reasons for caution
While this study affirms the effectiveness of a multifaceted assessment in pinpointing specific sperm dysfunctions and customizing treatment, it is still a retrospective observation requiring independent validation in a larger population.

Wider implications of the findings
Semen analysis alone fails to address specific aspects of male gamete dysfunction. Incorporating additional bioassays to assess chromatin structure, integrity, and acrosomal function offers valuable insights for fertility management in tailoring personalized treatments. This comprehensive approach enhances the understanding of male reproductive health, maximizing the potential for successful clinical outcome.

Trial registration number
not applicable