Single-center thorough evaluation and targeted treatment of globozoospermic men

Abstract

Purpose: To characterize, by specific biomarkers and nucleic acid sequencing, the structural and genomic sperm characteristics of partial (PG) and complete globozoospermic (CG) men in order to identify the best reproductive treatment.

Methods: We assessed spermatozoa from 14 consenting men ultrastructurally, as well as for histone content, sperm chromatin integrity, and sperm aneuploidy. Additional genomic, transcriptomic, and proteomic evaluations were carried out to further characterize the CG cohort. The presence of oocyte-activating sperm cytosolic factor (OASCF) was measured by a phospholipase C zeta (PLCζ) immunofluorescence assay. Couples were treated in subsequent cycles either by conventional ICSI or by ICSI with assisted gamete treatment (AGT) using calcium ionophore (Ionomycin, 19657, Sigma-Aldrich, Saint Louis, MO, USA).

Results: Ultrastructural assessment confirmed complete acrosome deficiency in all spermatozoa from CG men. Histone content, sperm chromatin integrity, and sperm aneuploidy did not differ significantly between the PG (n = 4) and CG (n = 10) cohorts. PLCζ assessment indicated a positive presence of OASCF in 4 PG couples, who underwent subsequent ICSI cycles that yielded a 36.1% (43/119) fertilization with a 50% (2/4) clinical pregnancy and delivery rate. PLCζ assessment failed to detect OASCF for 8 CG patients who underwent 9 subsequent ICSI cycles with AGT, yielding a remarkable improvement of fertilization (39/97; 40.2%) (P = 0.00001). Embryo implantation (6/21; 28.6%) and clinical pregnancies (5/7; 71.4%) were also enhanced, resulting in 4 deliveries. Gene mutations (DPY19L2, SPATA16, PICK1) were identified in spermatozoa from CG patients. Additionally, CG patients unable to sustain a term pregnancy had gene mutations involved in zygote development (NLRP5) and postnatal development (BSX). CG patients who successfully sustained a pregnancy had a mutation (PIWIL1) related to sperm phenotype. PLCZ1 was both mutated and underexpressed in these CG patients, regardless of reproductive outcome.

Conclusions: Sperm bioassays and genomic studies can be used to characterize this gamete's capacity to support embryonic development and to tailor treatments maximizing reproductive outcome.

Keywords: Assisted oocyte activation; Gene mutations; Globozoospermia; ICSI; Phospholipase C zeta; Proteomics; RNA expression.

Fig. 1

A total of 14 couples were enrolled in this study. Following standard semen analysis, spermatozoa from each patient were assessed ultrastructurally, as well as for histone content, sperm chromatin integrity, and aneuploidy. Patients were then allocated to two groups based on the proportion of round-headed spermatozoa. Those who exhibited 100% of the round-headed morphology were diagnosed as complete globozoospermic (CG), while those with a large fraction of spermatozoa exhibiting round-headed morphology were considered partial globozoospermic (PG). Additional genetics and transcriptomic analyses were performed on the spermatozoa from consenting men (n = 3) to further characterize the CG type. Of the 14 couples, 2 eventually elected to be treated elsewhere or not pursue infertility treatment. We then screened patients for OASCF by PLCζ assessment. Couples with a normal OASCF who were continuing at our center (n = 4) were treated with conventional ICSI, while consenting couples with an OASCF deficiency (n = 7) were treated by ICSI with AGT

Fig. 2

Global transcriptional change across the groups compared. All the genes are plotted, and each data point represents a gene. The log2 fold change of each gene is represented on the x-axis, and the log10 of its P-value is on the y-axis. Genes with a P-value < 0.1 and a log2 fold change greater than 1 are indicated by red dots, representing upregulated genes. Genes with a P-value < 0.1 and a log2 fold change less than 0 are indicated by blue dots, representing downregulated genes