Molecular analysis of sperm DNA fragmentation and apoptotic effects for unexplained infertility leading to recurrent pregnancy loss: a case-control study

Background: Conventional semen analysis often fails to uncover subtle paternal factors contributing to unexplained infertility (UI) and recurrent pregnancy loss (RPL), conditions traditionally attributed to maternal causes. Emerging evidence highlights the significance of sperm DNA integrity, particularly sperm DNA fragmentation (SDF) and apoptosis, as potential underlying factors. This study aimed to evaluate sperm molecular quality in men with UI or RPL despite normal semen parameters. Methods: A hospital-based prospective case-control study was conducted at an in vitro fertilization (IVF) centre in Delhi, India, involving 106 male participants: fertile controls (n = 52), RPL cases (n = 20), and UI cases (n = 34). All participants met the World Health Organization (WHO) 2021 semen reference standards and had no evidence of male factor infertility. Sperm DNA fragmentation was measured using the Sperm Chromatin Dispersion (SCD) test and Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assay, while apoptosis was analyzed by Annexin V-Fluorescein Isothiocyanate/Propidium Iodide (Annexin V-FITC/PI) staining with flow cytometry. Statistical analyses were performed using Analysis of Variance (ANOVA), Kruskal-Wallis H-test, and Chi-square tests, with statistical significance set at p < 0.05. Results: Despite normal semen profiles, DNA Fragmentation Index (DFI) was significantly higher in RPL (31.79 ± 24.23%) and UI (41.38 ± 16.32%) groups compared to controls (15.62 ± 5.18%) (p < 0.01). DFI correlated positively with paternal age (r = 0.277, p = 0.004) and negatively with progressive motility (r = −0.203, p = 0.043). Apoptosis levels were elevated in both patient groups, with RPL cases showing the most severe damage. Lifestyle factors, including higher alcohol use in UI and greater smoking prevalence in RPL, also showed associations. Conclusions: Sperm DNA fragmentation and apoptosis are overlooked contributors to male infertility in UI and RPL, underscoring the need for molecular sperm function tests beyond routine semen analysis to enhance diagnosis and management.

Sperm DNA fragmentation; Unexplained infertility; Recurrent pregnancy loss; Apoptosis; Sperm chromatin dispersion; TUNEL assay; Male factor infertility

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