Approximately 15% of reproductive-age couples suffer from infertility globally, with male factors accounting for half of these cases (1, 2). Conventional semen analysis is a standard method for evaluating semen quality, including parameters such as volume, sperm concentration, motility and morphology. However, this method is limited to evaluating basic parameters and additional sperm functions, such as DNA integrity, acrosome reaction and capacitation, require further assessment. Sperm DNA integrity is a critical parameter for male reproduction. Due to the complex organization of haploid genome, the DNA within the sperm nucleus is often altered by protamine-mediated compaction (3). Previous studies have demonstrated that sperm DNA fragmentation can adversely affect fertilization and embryo development (4–6). DNA Fragmentation Index (DFI) is a parameter used to assess the integrity of sperm DNA. Specifically, DFI measures the percentage of spermatozoa exhibiting DNA fragmentation, which can be an indicator of sperm quality and fertility potential. High DFI values are associated with increased sperm DNA damage, which may result from factors like oxidative stress, apoptosis, or autophagy (7).

Recurrent pregnancy loss (RPL) is defined as two or more clinically recognized pregnancy losses before 20-24 weeks of gestation, including both embryo and fetal loss (8). According to previous epidemiological studies, the prevalence of RPL is 1-4% among all patients who achieve pregnancy (9). Several factors have been suggested to contribute to pathogenesis of RPL, including maternal age, genetic defects, uterine pathology, endocrine disorders, and infectious agents. However, the exact etiology of RPL in approximately 50-75% of patients remains unclear (10). Although most known causes of RPL are related to female factors, recent studies have also indicated that semen quality may influence the risk of PRL. A systematic review and meta-analysis including 16 studies and 2969 couples suggested that increased DFI was associated with an increased risk of RPL (11). Additionally, several studies reported male partners of couples with RPL were more likely to have high DFI compared with those with low DFI (12, 13). Importantly, new guidelines from the European Society of Human Reproduction and Embryology (ESHRE), the American Urological Association (AUA), and the American Society for Reproductive Medicine (ASRM) recommend sperm DFI as a parameter for evaluating RPL (14–16).

Assays for analyzing sperm DNA integrity have been developed in recent years, including the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, the comet assay, the sperm chromatin dispersion (SCD) assay, and the sperm chromatin structure assay (SCSA). In a recent review, the authors provided a comprehensive summary of the techniques, advantages, and disadvantages of the TUNEL, SCSA, SCD, and Comet assays (17). The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay is noted for its sensitivity, reliability, and minimal interrater variability. However, its disadvantages include high cost, the need for specialized equipment, and extensive training requirements. The sperm chromatin dispersion (SCD) assay is advantageous due to its simplicity and minimal equipment requirements, though it is hindered by a high degree of interobserver variability. The comet assay offers sensitivity and reproducibility, but suffers from interobserver variability, the necessity for an experienced operator, and the use of variable protocols. Finally, the sperm chromatin structure assay (SCSA) is characterized by a standardized protocol, reproducibility, and the ability to examine a large number of cells, though it also faces limitations in terms of high cost, the need for specialized equipment, and specific training requirements. Among these tests, SCSA is utilized for assessing the DFI in several large cohorts, although there is an ongoing effort to establish a consensus approach that is both highly reliable and cost-effective. Date from SCSA indicate that DFI has a strong positive correlation with pregnancy outcomes and miscarriage (18). In this study, which includes a relatively large sample size, we measured sperm DFI using SCSA to investigate the association between DFI and RPL risk.

Clinical characteristics of 634 men from infertile couples affected by RPL and 851 men from couples who underwent fertile evaluation are reported in Table 1 . Age and BMI did not show any statistically significant differences between the case and control groups. Lifestyle factors, including smoking and alcohol consumption, were similar between the two groups. No significant differences were observed in educational level, sex frequency, history of chronic diseases and urogenital infections. The duration of infertility (P < 0.001) and the prevalence of varicocele (P = 0.004) were significantly higher in the case group compared to the control group.

Semen parameters were further compared between two groups. No statistically significant differences in semen volume, sperm concentration, progressive motility, total motility, TMSC, morphology, AsAs, DFI, HDS and leukocytes were found between men in the case group and those in the control group ( Table 2 ). In addition, the proportion of men with abnormal semen parameters did not differ significantly between the two groups. A higher proportion of men with DFI > 30% was observed in the case group compared to the control group (6.3% vs. 5.2%), but this difference did not reach statistical significance. Similarly, the study groups were comparable in terms of sperm kinematics, including VCL, VSL, VAP, BCF, ALH, MAD, LIN, WOB, and STR ( Table 3 ).

The association between abnormal semen parameters and the risk of RPL was further investigated using logistic regression analysis ( Table 4 ). No significant associations were observed in either the crude or adjusted models. We further performed multivariate regression models for evaluating the association between sperm DFI and RPL. However, no correlation between DFI and RPL were found (adjusted OR 1.01, 95% CI (1.00-1.03), P = 0.09, Table 5 ). RCS was performed to assess the dose-response relationships between RPL and continues DFI ( Figure 1 ). The DFI was included as a natural cubic spline function, adjusting for age, BMI, education, smoking, drinking, sex frequency, chronic disease, duration of infertility, urogenital infections, varicocele, semen volume, sperm concentration, total motility, progressive motility, TMSC and normal morphology. No significant non-linear relationships were observed between continuous DFI and the risk of RPL.

Recent studies have suggested a positive correlation between sperm DFI and abnormal pregnancy history. In this study, a total of 1485 men from couples with RPL and those without RPL was recruited, and semen quality, including sperm DFI was evaluated. However, our results indicated that no significant relationship was observed between sperm DFI and the risk of RPL.

Several clinical characteristics, including lifestyle and medical history, have been associated with RPL. A recent study by Esquerre-Lamare et al. recruited 60 participants (33 men from couples with RPL and 27 men from couples with a history of live birth less than the one year) and assessed environmental and family factors as well as semen parameters (21). The results showed that the RPL group were more likely to have a higher male BMI and a family history of infertility. They also found no significant differences in andrological histories and clinical examinations (e.g., cryptorchidism, varicoceles, vas deferens, epididymal position) between cases and controls. However, our study showed that the mean BMI of men did not differ between the study groups, which is consistent with several previously published results (13, 22). In addition, our study found that men from couples with RPL were more likely to have varicocele compared to men from couples without RPL. This finding is consistent with Busnelli et al., who reported a higher prevalence of varicocele in men with RPL compared to the men without RPL(14.9% vs. 8.0%), although this difference did not reach statistical significance (23).

Traditional semen analysis provides a quantitative assessment of sperm concentration, motility and morphology (24). Several studies have shown a trend of low sperm motility and high abnormal sperm morphology in men from couples with RPL (12, 25, 26). However, similar to our findings, many studies that examined the correlation between routine semen parameters and RPL found no significant correlation (27, 28). Since normal sperm genetic material is essential for successful fertilization, as well as embryo and fetal development, sperm DNA integrity is considered a key parameter for evaluating clinical pregnancy outcomes. Several meta-analyses have reported that sperm DFI is associated with RPL (29, 30). However, these meta-analyses may be underpowered due to the limited number of studies and sample sizes included (20). Results from our study are consistent with Zhang et al. that reported no significant difference in DFI between RPL group and control group (27). Additionally, the researchers followed up with the participants for one year and observed a trend toward higher DFI in men from couples with subsequent abnormal pregnancy outcomes compared to those from couples with ongoing pregnancies. This suggests that DFI may be a potential semen parameter for predicting future reproductive outcomes.

A cut-off DFI > 30% was reported as a severe DNA fragmentation in several studies (13, 31, 32). Consistent with other studies, our study also showed a trend of a higher proportion of sperm DFI ≥ 30% in men from the RPL group compared to those from the control group (22, 33). Interestingly, the RCS model indicated that when DFI exceeded 30%, the risk of RPL appeared to increase. Damaged DNA in the sperm may cause embryos to fail in their early stages, contributing to pregnancy loss (34). In addition, sperm DNA contributes to the development of placenta. Fragmented DNA can impair trophoblast development, leading to inadequate placental function, which is a known cause of pregnancy loss (35). Furthermore, DNA fragmentation may disrupt epigenetic marks in sperm, leading to errors in gene expression regulation during embryogenesis. This can result in developmental abnormalities and increase the risk of miscarriage (36). These findings suggest that a threshold DFI, rather than continuous DFI, may be more useful for assessing the correlation between DFI and RPL.

This study has several strengths. First, the relatively large sample size provides substantial statistical power. Additionally, our study included potential confounding factors (e.g. lifestyles and medical history) that have been previously reported to be associated with RPL. However, this study has several limitations. First, the study is limited by its retrospective design and potential selection bias. Second, all participants were recruited from couples undergoing fertility evaluation at a single clinic. Third, although several potential confounders were adjusted for, residual confounding and unknown factors could not be ruled out. It is note that the relationship between sperm DNA fragmentation and recurrent pregnancy loss cannot be fully understood without considering female factors. Maternal age, uterine environment, oocyte quality, and immune response all play significant roles in moderating the impact of DFI on pregnancy outcomes. Forth, the SCSA assay cannot measure DNA strand breaks directly. Finally, different cut-off values for DFI may produce biased results.

In conclusion, our study found no significant relationship between sperm DFI and the risk of RPL. Further prospective studies are needed to investigate the impact of DFI on fertility outcomes in couples experiencing RPL.