Overall abstinence time is associated with sperm concentration in particular, but motility and morphology also show different associations in normal and pathological semen samples. The analysis of abstinence days as a continuous variable and the comparison of results in 0-2, 3-7 and > 7 days led to similar results in most but not in all analyses.
Concentration, volume, and TMSC: Our results for sperm concentration, volume, and TMSC are comparable with a cross sectional analysis of semen samples from normo- and oligozoospermic men providing ejaculates after abstinence times of 0-14 days 15. For the normozoospermic samples in our study, sperm concentration and TMSC peaked after 7 days of abstinence, thus on the upper end of the abstinence of 2-7 days recommended by WHO 12. The evaluation of 6 consecutive sperm samples after 1-11 days of abstinence in normozoospermic men also showed the highest sperm concentrations after >7 days of abstinence 18; smaller longitudinal studies confirmed these results 21, 22.In oligozoospermic samples, Levitas 15 found a higher sperm concentration after 3-5 days and peak TMSC after 4 days of abstinence; these findings are consistent with our results from the evaluation of abstinence time categories, but not with our results from the analysis of days of abstinence as a continuous variable. In addition to findings from available research, our study shows also that in astheno-, teratozoospermic, and OAT samples, semen concentration increases after longer periods of abstinence. The improvement of sperm quality is obligatory for fertility in cases of semen pathology; an increase in concentration may help to compensate deficits in motility and morphology by increasing the total number of sperm. Therefore, the selection of a period of abstinence at the higher end of the time frame recommended by WHO might contribute to the optimization of semen samples for natural and medically assisted conception. The lower sperm concentrations after shorter abstinence times for normozoospermic samples could be explained by depletion of the stored spermatozoa in the cauda epididymis when ejaculation occurs more frequently 18. A longer transport time through the epididymis (3 times longer in men with oligo- than with normozoospermia) likely influences sperm concentration in oligozoospermic men in relation to abstinence times 23. As previous results indicate, shorter abstinence times are associated with lower semen volume in normal and subnormal semen samples 15, 18, 21, 22.
Motility: When evaluating the days of abstinence as a continuous variable, longer abstinence time was associated with lower motility in normal and teratozoospermic samples. We found the highest values for total motility after 0-2 and 3-7 days of abstinence in the oligo- and the teratozoospermic samples. The normal, asthenozoospermic, and OAT samples did not show any significant difference in relation to abstinence time categories. In agreement with our findings, shorter abstinence time was associated with high motility for normal and some types of abnormal samples in other studies 15. However, longitudinal studies in smaller collectives did not find any association between abstinence period and changes in motility 21, 24, 25; this supports our findings that short abstinence times are not associated with higher motility in asthenozoospermic samples, either alone or in combination with other semen pathology, i.e. where an improvement of motility would be obligatory to improve fertility.
A possible explanation for a beneficial effect of short abstinence times is the transit time of spermatozoa through the epididymis and its consequences: more frequent ejaculation may lead to shorter storage times of spermatozoa in the epididymis. During their stay in the epididymis, spermatozoa are exposed to reactive oxygen species (ROS), produced by the spermatozoa and to a lesser extent by leukocytes. ROS can induce lipid peroxidation of unsaturated fatty acids in the plasma membrane of spermatozoa 26, 27. Various authors report unusually high levels of ROS in infertile men 26, 28. It is suggested that overly high levels of ROS cannot be counterbalanced by antioxidants in seminal plasma (such as catalase or superoxide dismutase) and consequently lead to oxidative stress, which has a negative impact on sperm motility 27. Interestingly, seminal total antioxidant capacity was significantly higher after 1 day than after 4 days of abstinence 25; this result supports the benefit of shorter abstinence time with regard to sperm motility.
Yet, there are more factors that have a modulatory effect on sperm motility: a positive correlation between levels of neutral alpha-glucosidase and motility has been demonstrated 29, 30. Elzanaty 29 reported higher levels of neutral alpha-glucosidase in semen samples produced after 4-7 days of abstinence in comparison with shorter abstinence times. Although we did not observe a clear benefit of short abstinence times in all types of sperm pathology, when an association could be demonstrated, shorter abstinence times were always related to higher motility and can therefore be recommended when higher motility is needed.
Morphology: In our study, a shorter abstinence period was associated with a higher percentage of morphologically normal spermatozoids in normal samples and in OAT samples when days of abstinence were evaluated as a continuous variable. Teratozoospermic samples showed a higher median percentage of morphologically normal spermatozoa after 0-2 days in comparison with 3-7 days of abstinence. According to our results and those of another large cohort study 15, shorter abstinence periods were associated with a higher percentage of morphologically normal spermatozoids. Again, results varied strongly between different pathologies, with teratozooic samples presenting best results in the 0 - 2 days abstinence window and OAT samples showing significant association with shorter abstinence time in the analysis of abstinence days as a continuous variable. A benefit of a short abstinence time was also observed in other studies investigating subnormal semen samples 6, 8, but there are also studies showing no association between abstinence time and changes in morphology in both normo- and oligozoospermic men 17, 25, 31, 32. In contrast, a study investigating semen samples from men who abstained sequentially for 1-10 days reported a significant increase of the normal spermatozoa after 2-5 days of abstinence and a significant decrease after 10 days 24. As ROS levels correlate negatively with the percentage of spermatozoa with normal and borderline morphology 33, increased exposure to ROS in the epididymis might be involved in the decreasing numbers of morphologically normal spermatozoa after prolonged abstinence periods.
Strengths and limitations: Our study reports data from one of the largest groups of men in infertile relationships. In comparison with existing studies, we not only investigated the difference between normal and oligozoospermic semen samples but also included asthenozoospermic, teratozoospermic, and OAT samples. Limiting factors of our study are the retrospective design and the small cohorts in some of the pathological categories. As we excluded men with 0% of motile sperms or sperms with normal morphology, our findings are not representative for men with severe cryptozoospermia or asthenozoospermia. As most of the men followed the advice to remain abstinent 2-7 days prior to sample production, there were few participants with an abstinence outside of this time window. Because of the small number of samples in certain categories, the results for OAT samples in particular should be interpreted with caution. False answers to fulfil the expectation of adherence to the recommended abstinence time likely result in underestimation of our findings. Another limitation is the cross-sectional design with only one sample per patient, as possible within-subject variability is not accounted for. Studies on this topic found that values for concentration, motility, percentage of normal forms, and sometimes volume might vary considerably between different samples of the same patient: within-subject coefficients of variation were calculated between 25-28% for ejaculate volume, 26-29% for sperm concentration, 18-34% for total motility, and 19-29% for morphology 34-36. However, in all 3 studies the between-subject coefficients of variation exceeded the within-subject coefficients of variation; intraclass correlation coefficients were ≥ 0.60 for most of the sperm parameters, indicating substantial reliability.