O-020 Sperm phospholipase C zeta levels measured by flow cytometry are associated with sperm parameters
Authors: Duval C , Joao F , Calvé A , Montjean D , Bélanger M C , Benkhlaifa M , Miron P 
Published: June 2022

Abstract

Study question
Are the levels of phospholipase C zeta (PLCζ) in sperm associated with sperm parameters?

Summary answer
Yes, PLCζ levels are correlated with motility, vitality, concentration and morphology and decreased in samples with anormal sperm parameters.

What is known already
Sperm phospholipase C Zeta (PLCζ) is a protein mainly located in the sperm head and its major role is the induction of the Ca2+ oscillations necessary for oocyte activation following fertilization. During fertilization, PLCζ is released into the ovum cytoplasm and induces Ca2+ oscillations via the inositol 1,4,5-triphosphate signaling pathway. PLCζ deficiency have been shown to be associated with low or total failed fertilization after intracytoplasmic sperm injection. In such cases, artificial oocyte activation (AOA) using calcium ionophore has been shown to increase fertilization rates. Thus, finding easy method to quantify PLCζ in a clinical setting is crucial.

Study design, size, duration
Between May 2021 and September 2021, 92 patients already doing a spermogram gave their inform consent to donate their unused sperm sample to research. They were split into two groups based on the normality of the spermogram (following World Health Organization 2010 criteria), normal sperm parameters (NPS, n = 24) and abnormal sperm parameters (ASP, n = 68). Levels of PLCζ were also determined in a subgroup of patient before and after the passage in a microfluidic chamber (ZyMōt™)(n = 27).

Participants/materials, setting, methods
Age, volume, pH, concentration, total sperm count, motility, progressive motility, vitality, morphology, DNA fragmentation, chromatin compaction, level of PLCζ and fluorescence intensity were compared between the two groups and correlations were made to better understand the association between PLCζ levels and sperm parameters. PLCζ levels were assessed by flow cytometry after fixation and permeabilization of the cells, primary antibody (rabbit anti-human PLCζ) and secondary antibody (goat anti-rabbit coupled with the fluorochrome FITC).

Main results and the role of chance
Significantly lower PLCζ levels were observed in ASP patients compared to NSP patients (respectively 45.4% vs 56.9%, p = 0.0038). Positive correlations were also observed between PLCζ levels and sperm concentration, total sperm count, progressive motility, total motility, and sperm vitality (p&lt;0.001). ROC analysis revealed that a PLCζ levels cut-off of 64.4% could differentiate NSP from ASP patients with 83.8% sensitivity, 54.17% specificity, 54.2% PPV and 83.8% NPV (Area under the curve: 0.6829; p&lt;0.0079). Moreover, higher PLCζ levels were found after sperm migration using a ZyMōt™ device (before 45.96 vs after 60.15, p&lt;0.001). This method development also demonstrated the ability to detect low PLCζ levels in semen samples and those patients will be directed to artificial oocyte activation using a calcium ionophore to increase their chance of success.

Limitations, reasons for caution
Since there is a correlation between PLCζ levels and motility/vitality, it is expected that a sample treatment with a microfluidic chamber will increased the percentage of PLCζ. In addition, PLCζ is implicated in oocyte activation, so the real cut-off point should be determined with in-vitro fertilization (IVF) outcomes.

Wider implications of the findings
Demonstrating the association between PLCζ levels and sperm parameters established the relevance of using a microfluidic chamber for a better sperm quality for IVF or intracytoplasmic sperm injection (ICSI).

Trial registration number
Not applicable