Human spermatozoa are produced in the seminiferous tubules, release into the rete testis, and subsequently enter the epididymis (1). During transit and storage in the epididymis, immature spermatozoa acquire motility and fertilizing potential, ultimately becoming functionally matured spermatozoa. Several factors correlate with semen quality, one of which is ejaculatory abstinence (2). During periods of ejaculatory abstinence, mature sperm mainly accumulate and greatly expose to reactive oxygen species (ROS) in the epididymis (3, 4). Excessive ROS impair semen quality by reducing sperm motility and causing DNA damage (5). Several studies have indicated that semen volume and sperm count increase, while progressive motility decreases, with longer periods of abstinence (6, 7).

The World Health Organization (WHO) recommends an abstinence period of 2-7 days prior to semen collection for standard analysis. This range is considered optimal for obtaining consistent and reliable sperm parameters, such as sperm count, motility, and morphology, without significantly affecting semen quality. In contrast, the European Society of Human Reproduction and Embryology (ESHRE) suggests a shorter abstinence period of 3-4 days. This recommendation is based on the idea that this specific window may better balance sperm concentration and motility while avoiding potential negative effects, such as reduced sperm motility and increased DNA fragmentation index (DFI), which can occur with longer abstinence periods (8). Current evidence regarding the correlation between short or long abstinence periods and semen quality remains unclear. In this large observational study of 3052 men, we conducted logistic analyses to evaluate the relationship between semen parameters and abstinence time categorized as short (0-1 day), recommend (2-7 days) and long (> 7 days).

Characteristics of the participants categorized by abstinence time (AT) are shown in Table 1 . The mean age of the subjects was 30.9 ± 5.0 years, with a mean BMI of 24.7 ± 3.6 kg/m². Among all participants, 1292 (42.3%) smoked tobacco and 1533 (50.2%) consumed alcohol. Educationally, 575 (18.9%) had completed middle school or lower levels, 538 (17.6%) had completed high school, and 1939 (63.5%) had completed college/university. The incidence rates of chronic diseases, urogenital infections and varicocele were 309 (10.1%), 420 (13.8%) and 318 (10.4%), respectively. No significant differences were observed between abstinence time and age, BMI, smoking, drinking, education, chronic diseases, urogenital infections and varicocele.

Compared to men with recommended AT, those with short AT had significantly lower semen volume (P < 0.001), sperm concentration (P < 0.001) and TMSC (P < 0.001), while men with long AT had significantly higher semen volume (P < 0.001), sperm concentration (P < 0.001), TMSC (P < 0.001) and DFI (P < 0.001) ( Table 2 ). Lower sperm progressive motility and total motility were shown in long AT than those in recommended EA. No significant differences were observed between AT and sperm normal morphology, semen leukocytes, AsAs and HDS. In terms of sperm kinematics, VCL, VSL, VAP, BCF and MAD significantly declined with AT, while ALH, LIN, WOB, and STR significantly increased with AT ( Table 3 ).

As shown in Table 4 , logistic regression analysis revealed a significantly positive association between short AT and low semen volume (OR = 3.1, 95% CI = 2.0-4.7, P < 0.001), low sperm concentration (OR = 1.7, 95% CI = 1.1-2.6, P = 0.02) and low TMSC (OR = 2.0, 95% CI = 1.3-3.1, P < 0.001). After adjusting for potential confounders including age, BMI, educational levels, smoking, alcohol consumption, chronic diseases, urogenital infections and varicocele), a high risk of poor semen quality was still observed in men with short AT. Additionally, long AT was significantly negatively associated with low sperm concentration (OR = 0.5, 95% CI = 0.3-0.8, P = 0.007) and positively associated with low sperm progressive motility (OR = 1.5, 95% CI = 1.2-1.9, P = 0.002), low sperm total motility (OR = 1.4, 95% CI = 1.1-1.8, P = 0.007) and high sperm DFI (OR = 2.8, 95% CI = 1.7-4.6, P < 0.001), and those relationship were still observed after adjusting for several confounders. No significant associations were observed for sperm normal morphology (all P > 0.05).

The dose-response relationships between continuous AT and abnormal semen parameters after adjusting for confounders were further explored using RCS ( Figure 1 ). When AT was lower than the median AT in men with recommended AT (4 days), risk of low semen volume (P non-linear < 0.001), sperm concentration (P non-linear = 0.039) and TMSC (P non-linear < 0.001) appeared to decrease with increasing AT, while there was no significant difference for AT longer than 4 days. No significant non-linear relationships were observed between AT and risk of abnormal sperm progressive motility, total motility and sperm DFI.

Semen analysis, including sperm concentration, motility and morphology, has been as the commonly test for evaluating male fertility (9, 10). Several pre-analytical factors, such as time of abstinence and method of semen collection, have been shown to affect semen parameters (11). In this observational study with a relatively large sample size, we assessed the relationship between AT and semen quality in men who undergoing fertility examination. Our results indicate that shorter AT was positively associated with abnormal semen volume, sperm concentration and TMSC, and long AT was positively associated with abnormal sperm progressive motility, total motility and DFI.

The recommended duration of abstinence by the WHO laboratory Manual for the Examination and Processing of Human Semen ranges from 2 to 7 days. The association between AT and semen quality remains controversial (12, 13). Several studies have shown that abstinence duration is significantly linked with semen volume, sperm concentration, motility, normal morphology, and DFI (14–16). However, other studies have not reported significant associations, particularly concerning sperm morphology. Recently, a systematic review suggested that shorter abstinence durations improve semen quality, including lower sperm DFI, higher motility and normal morphology (17). In addition, several guidelines address the beneficial effects of short abstinence duration in reducing sperm DFI (18, 19). However, another review concluded that the effects of short-term abstinence on semen parameters are contradictory and inconclusive (20).

On the other hand, prolonged AT was associated with increased seminal volume, semen leukocytes, sperm concentration, DFI and decreased sperm motility (15). Given that semen volume increases with abstinence duration, the change in sperm count exceeded that in sperm concentration. Recent studies have suggested improvements in sperm count with abstinence periods longer than 5 or 7 days (17, 20), while our study observed TMSC exhibited the most significant changes with increasing AT. As long-term AT appear to reduce sperm motility, TMSC is recommended as a preferred evaluation of semen parameters.

The pathophysiological mechanisms linking abnormal abstinence duration to semen quality remains unclear. Shorter abstinence periods have been reported to affect the duration of sperm retention in the epididymis, which may improve sperm antioxidant capacity and seminal metabolites (21). Prolonged retention of sperm in the epididymis increases susceptibility to damage by ROS (7, 22, 23). ROS at normal physiological levels is essential for the processes of maturation, hyperactivation, capacitation, acrosome reaction, zona pellucida binding and oocyte fusion (24–26). However, an imbalance between ROS and antioxidants leads to oxidative stress, impairing acrosome, mitochondrial, and DNA integrity.

One major strength of our study is its relatively large sample size, which provides high statistical power and precision. The current study also included dose-response relationship between abstinence duration and semen parameters. In addition, we also considered several potential confounding factors, such as age, BMI, drinking, and smoking, that have been previously reported to be associated with semen quality. However, several limitations need to be discussed. First, all semen parameters were obtained from men who undergoing fertility evaluation at a single clinic center and were unable to generalize men with reproductive age. Second, the level of ROS was not assessed in this study, which might affect the relationship between abstinence duration and semen quality. Third, as our study is a retrospective design, potential selection bias is unavoidable. Finally, although we adjusted for several potential confounders, there is still the possibility of residual confounding by unknown factors.

In conclusion, our study showed that both short and long AT were significantly associated with lower sperm quality, which indicated the importance of maintaining a recommended AT for semen analysis. Additionally, short abstinence periods short abstinence periods may be recommended to maintain optimal sperm DNA integrity.