Sperm DNA Fragmentation after Cryopreservation and Sperm Selection Has No Implications for Clinical Pregnancies and Live Births after Intrauterine Insemination with Donor Sperm.

Intrauterine insemination with donor sperm (IUI-D) requires multiple in vitro manipulations such as sperm selection and cryopreservation during which spermatozoa may be exposed to oxidative stress (OS) and other insults that may produce potential damage including sperm DNA fragmentation (SDF). High levels of SDF, referring to damage or breaks in the genetic material of sperm cells, are linked to an increased risk of reproductive failure. This retrospective, observational study set out to evaluate whether SDF assessment could predict clinical outcome in an IUI-D program, where sperm donors are selected on strict conventional semen parameters. A total of 18 donors and 106 recipients were matched for IUI-D. Out of 429 cycles, 100 (23.3%) resulted in clinical pregnancy. We counted 78 live births (18.2% of cycles), while 20 pregnancies ended in miscarriage (4.7% of cycles), 1 in extra-uterine pregnancy and 1 in stillbirth. Female age significantly influenced clinical pregnancy and miscarriage rates. SDF increased after cryopreservation (26.3 ± 14.5%; p < 0.001) and more so after post-thaw density gradient (34.9 ± 22.1%; p = 0.04) without affecting clinical pregnancy (OR [95% CI] 1.01 [0.99; 1.02]; p = 0.27), live birth (1.00 [0.99; 1.02]; p = 0.72) and miscarriage rates (1.02 [1.00; 1.05]; p = 0.08). The implications of our findings extend to a better selection of sperm donors and a better sperm preparation technique tailored to the donor semen's properties in order to maximize the chances of a favorable treatment outcome.

Antioxidants in Male Infertility-If We Want to Get This Right We Need to Take the Bull by the Horns: A Pilot Study.

Antioxidant therapy should be reserved for infertile patients who actually exhibit signs of oxidative stress (OS). Nevertheless, there is no consensus regarding the measure of the primary endpoint and the assay that should be used. The formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), an early marker of sperm DNA oxidation (SDO), was analyzed using flow cytometry, in men at a University hospital setup for infertility treatment. Similar to conventional semen parameters, 8-OHdG assay was validated on fresh semen samples to reduce the variability of results. SDO was associated with semen volume, sperm concentration, leucocytes and round cells, but not with age, body mass index, sperm DNA fragmentation (SDF) or OS. Whether the semen samples were normal or subnormal according to the WHO criteria, the expression of 8-OHdG was not different. Receiver operating characteristic curve analysis could discriminate two independent populations. Both SDF and SDO were independently expressed. A high SDF did not reveal a high SDO and vice versa. The thresholds for SDO have been established, but vary with the techniques used. The methodology for SDO needs to be further validated and optimized on a larger clinically defined patient population before the outcome measure is fit to monitor antioxidant therapy in male infertility.

Semen Thresholds of Normality Established by the WHO Do Not Reveal Genome Instability-A Potential Occult Male Factor.

Semen parameters are unable to inform on the function or fertilizing capacity of the male gamete. Standardized methods are provided by the WHO but, the lower reference limits have reduced sensitivity to predict chances of conception. Subfertile men may be falsely classified as "normal" and a male factor contributing to genome instability may be overlooked. Semen parameters, sperm DNA fragmentation (SDF), sperm chromatin maturity and stability, and sperm aneuploidy were assessed in fertile (F), subfertile normozoospermic (SN) and subfertile non-normozoospermic males (SN-N). Standardized assays employing flow cytometry were used to detect genome instability. Sperm DNA fragmentation did not differ significantly whether the semen samples were from a fertile (F), subfertile normozoospermic (SN) or subfertile non-normozoospermic male (SN-N). Chromatin decondensation was significantly reduced and hyperstability significantly increased in the SN group as compared to the F group. The frequency of diploidy was significantly different in the three study groups with significance between F and SN and between F and SN-N groups. Subfertile men with normal semen parameters are often excluded from extensive genetic testing. Genome instability might be an independent attribute of semen quality detecting problems not seen with semen analysis alone.

Variability in Sperm DNA Fragmentation in Men with Mild/Unexplained Subfertility in a Prospective Longitudinal Intrauterine Insemination Trial.

The biological variability of semen and sperm DNA fragmentation (SDF) parameters in a longitudinal intrauterine insemination (IUI) trial over multiple IUI cycles was investigated. A TUNEL assay was used for SDF testing, both before and after density gradient centrifugation. A significant age effect was observed: while semen parameters deteriorated with advancing age, on average, higher SDF values were observed for older males. There was quite some variability observed for both semen and SDF variables. Using fertile threshold values, three patient categories were distinguished: those with a high SDF in all samples, those with low SDF in all samples and those who fluctuated between high and low during the whole IUI trial. Density gradient centrifugation increases SDF. However, the three patient categories react differently after semen processing. A large percentage of those with high SDF retain their high SDF even after gradient centrifugation. The SDF fluctuaters react with a high SDF after gradient centrifugation. The low SDF category, on the contrary, distributes itself evenly between the three categories after gradient centrifugation. SDF testing after semen processing might be indispensable for therapeutic purposes, probably influencing medical decision-making. In order to isolate fluctuaters, a second SDF testing might be advocated in certain cases. SDF after semen processing is indispensable for therapeutic management.

Standards in semen examination: publishing reproducible and reliable data based on high-quality methodology.

Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.

Sperm as a Carrier of Genome Instability in Relation to Paternal Lifestyle and Nutritional Conditions.

Endogenous and exogenous factors can severely affect the integrity of genetic information by inducing DNA damage and impairing genome stability. The extent to which men with and without subfertility are exposed to several adverse lifestyle factors and the impact on sperm DNA fragmentation (SDF), sperm chromatin maturity (condensation and decondensation), stability (hypo- and hypercondensation) and sperm aneuploidy are assessed in this study. Standardized assays employing flow cytometry were used to detect genome instability in 556 samples. Semen parameters deteriorated with age, BMI, increased physical activity and smoking. Age and BMI were associated with increased SDF. Increased BMI was associated with increased hypocondensed chromatin and decreased decondensed chromatin. Increase in age also caused an increase in sex chromosome aneuploidy in sperms. Surprisingly, alcohol abuse reduced chromatin hypercondensation and drug abuse reduced SDF. Although genome instability was more pronounced in the subfertile population as compared to the fertile group, the proportion of men with at least one lifestyle risk factor was the same in both the fertile and subfertile groups. While one in three benefited from nutritional supplementation, one in five showed an increase in SDF after supplementation. Whilst the message of 'no smoking, no alcohol, no drugs, but a healthy diet' should be offered as good health advice, we are a long way from concluding that nutritional supplementation would be beneficial for male fertility.

The effect of human follicular fluid on bovine oocyte developmental competence and embryo quality.

In this study, the hypothesis that embryo development during routine IVF procedures is determined by the pre-ovulatory follicular fluid composition was tested. Follicular fluid from women with obesity ('obese') and a 'positive' or 'negative' IVF outcome was added during the in-vitro maturation of bovine oocytes. 'Negative' and 'obese' follicular fluid reduced bovine embryo development, compared with laboratory control embryo development (P < 0.05 or P < 0.1). The addition of follicular fluid also altered bovine blastocyst gene expression. Furthermore, LDHA and PPARGC1B gene expression differed between follicular fluid groups. Data suggest that pre-ovulatory follicular fluid can potentially affect oocyte developmental competence and embryo quality. Furthermore, the bovine model may be used as a screening tool.