Beyond Conventional Semen Analysis: Targeted Sperm Gene-Expression Markers Associated with Semen Quality and IVF/ICSI Outcomes

Background: Conventional semen analysis remains the cornerstone of male infertility diagnostics; however, its predictive value for fertilization potential and assisted reproductive technology (ART) outcomes remains limited. Molecular alterations in spermatozoa may contribute to impaired reproductive outcomes even in men with normozoospermia. Objective: To investigate the associations between targeted sperm expression profiles of AURKA, HDAC4, and CARHSP1 and conventional semen quality, fertilization, embryo development, and pregnancy outcomes in IVF/ICSI programs. Materials and Methods: This combined prospective-retrospective observational study included 173 men undergoing IVF treatment between March 2024 and January 2025 at the International Clinical Center for Reproductive Medicine PERSONA, Almaty, Kazakhstan. Sperm expression levels of AURKA, HDAC4, and CARHSP1 were analyzed by RT-qPCR using the EurobioPlex Sperm Messenger 4 Fertility assay. Based on the manufacturer-defined Sperm Function Index (SFI), samples were categorized as having normal, intermediate, or reduced gene-expression profiles. Among 99 normozoospermic men, two with intermediate expression profiles were excluded from the comparative IVF/ICSI outcome analysis, resulting in a final subgroup of 97 men (70 normal expression and 27 reduced expression). Embryological outcomes were analyzed using generalized estimating equations (GEE) with patient-level clustering to account for multiple oocytes and embryos originating from the same patient. Results: Among the 173 analyzed samples, 56% had normal, 42% reduced, and 2% intermediate gene-expression profiles. Among the individual target transcripts, CARHSP1 was significantly associated with conventional semen quality, with higher expression in normozoospermic men than in men with abnormal semen parameters (p < 0.001). Notably, 27.2% of normozoospermic men exhibited reduced gene-expression profiles despite normal conventional semen parameters. Within the normozoospermic subgroup, reduced expression was associated with lower fertilization (68.2% vs. 81.8%; OR = 0.48, 95% CI 0.36–0.63; p < 0.000001), blastocyst formation (38.7% vs. 53.5%; OR = 0.60, 95% CI 0.42–0.84; p = 0.0034), and good-quality day-5 blastocyst development (9.9% vs. 19.4%; OR = 0.50, 95% CI 0.30–0.85; p = 0.0106). Among patients with an evaluable embryo transfer, positive hCG was observed in 14.3% of the reduced-expression group compared with 51.7% of the normal-expression group (p = 0.0065); given the limited number of events, this finding should be considered exploratory. Conclusions: Targeted sperm gene-expression analysis identified a molecularly distinct subgroup of normozoospermic men with reduced expression profiles associated with poorer fertilization, blastocyst development, good-quality day-5 blastocyst formation, and biochemical pregnancy outcomes. These findings suggest that targeted sperm gene-expression assessment may provide additional information beyond conventional semen analysis. However, these associations should not be interpreted as evidence of causality, and larger prospective multicenter studies incorporating relevant male, female, and cycle-related factors and live-birth outcomes are required before clinical implementation.