A search for molecular mechanisms underlying male idiopathic infertility.

Infertility affects approximately 15% of the couples wanting to conceive. In 30 - 40% of the cases the aetiology of male infertility remains unknown and is called idiopathic male infertility. When assisted reproductive technologies are used to obtain pregnancy, an adequate (epi)genetic diagnosis of male infertility is of major importance to evaluate if a genetic abnormality will be transmitted to the offspring. In addition, there is need for better diagnostic seminal biomarkers to assess the success rates of these assisted reproductive technologies. This review investigated the possible causes and molecular mechanisms underlying male idiopathic infertility by extensive literature searches of: (i) causal gene mutations; (ii) proteome studies of spermatozoa from idiopathic infertile men;(iii) the role of epigenetics; (iv) post-translational modifications; and (v) sperm DNA fragmentation in infertile men. In conclusion, male infertility is a complex, multi-factorial disorder and the underlying causes often remain unknown. Further research on the (epi)genetic and molecular defects in spermatogenesis and sperm function is necessary to improve the diagnosis and to develop more personalized treatments of men with idiopathic infertility.

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.

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.

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.

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.

Is There a Relationship between Sperm DNA Fragmentation and Intra-Uterine Insemination Outcome in Couples with Unexplained or Mild Male Infertility? Results from the ID-Trial.

BACKGROUND: Sperm DNA fragmentation has been proposed as a candidate test for the assessment of sperm function on the premise that damage to the sperm chromatin is associated with a detrimental reproductive outcome. The objective of our study was to investigate whether sperm DNA fragmentation testing has a prognostic value, and thus can play a pivotal role in selecting future patients for intra-uterine insemination (IUI) therapy. METHODS: This was a prospective cohort study conducted in a University Hospital setting. SDF was measured through TUNEL assay on the fresh semen sample presented at diagnosis and at insemination in couples with idiopathic/mild male infertility undergoing natural cycle IUI treatment. The generalized estimating equation (GEE)-model and multivariable model were used to analyze the probability of live birth and clinical pregnancy, respectively. ROC analysis was carried out to determine an SDF cut-off. RESULTS: There was an inverse relationship between SDF in the ejaculate of the diagnostic semen sample and CP (p = 0.02; OR 0.94 95% CI (0.90, 0.989)) as well as LB (p = 0.04; OR 0.95 95% CI (0.90, 0.9985)). No significant association was found between SDF after gradient and IUI outcome in the diagnostic sample nor between SDF (ejaculate/after gradient) in the IUI samples. The ROC analysis proposed a cutoff of 17.5% as the best compromise between sensitivity and specificity in the diagnostic SDF for live birth; however, the test diagnostics are low, with an AUC of 0.576. CONCLUSIONS: Overall, this study strengthens the hypothesis of an inverse relationship between SDF and CP/LB. Furthermore, SDF taken together with other clinical characteristics might provide more insight into male reproductive potential and predicting IUI outcome. Couples with SDF ≥ 17.5% in the diagnostic semen sample did not reach live birth. Further research is necessary to establish the diagnostic and prognostic potential of SDF as an add-on test.

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.

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.

Is there a temporal trend in semen quality in Belgian candidate sperm donors and in sperm donors' fertility potential from 1995 onwards?

BACKGROUND: A recent meta-regression analysis reported a temporal trend in sperm count showing a significant decline in sperm count between 1973 and 2011. This decline is thought to affect fecundity. Moreover, semen quality is considered of key interest to public health given its association with all-cause male morbidity/mortality. The issue requires ongoing investigation due to geographical variation in semen quality and methodological errors in semen analysis. OBJECTIVE: To study whether there is a temporal trend in semen quality in Belgian candidate sperm donors and in sperm donors' fertility potential. MATERIALS AND METHODS: Retrospective analysis of samples provided by 439 candidate donors and pregnancy outcome in acceptors over a period of 23 years. RESULTS: A total of 807 specimens from 439 candidate donors were examined from January 1995 to December 2017 (Table S1). Sub-analyses performed with regard to TSC from 2010 onwards (weighing) revealed a significant negative trend (R2 =-0.033; ß=-0.18; CI: -0.16 to 0.07; p < 0.05). We found a statistically significant association between year of donation and morphology (R2 = 0.036; ß= -0.19; CI: -0.26 to -0.08; p < 0.0001). The mean (±SD) clinical pregnancy rate per effective donor recruited (n = 104), defined as the number of women with a clinical pregnancy, per number of women who initiated treatment with a donor's spermatozoa, was 68.5 (± 24.9) %. This measure did not show a significant change in function of year of donation. DISCUSSION: Candidate sperm donors represent a select group of men; as such, these results are not to be interpreted as representative for the general population. CONCLUSION: The study did not show a significant change in sperm concentration or fertility potential in sperm donors over a period of 23 years. However, a negative trend was found for TSC from 2010 onwards. Also, the results show a significant decrease in ideal morphology over time.

The role of sperm DNA fragmentation testing in predicting intra-uterine insemination outcome: A systematic review and meta-analysis.

There is a growing body of literature that recognizes the importance of sperm DNA fragmentation as a candidate test for the assessment of sperm function and thus male reproductive potential. Research on the subject has mostly been focused on couples undergoing IVF/ICSI treatment whilst much uncertainty still exists about the relationship between sperm DNA fragmentation and IUI. This study systematically reviews the literature, aiming to define the value of sperm DNA fragmentation measurement in predicting clinical pregnancy outcome in couples undergoing intra-uterine insemination From inception until March 2018, the relevant databases were searched for studies investigating the relationship between sperm DNA fragmentation as measured by SCSA, TUNEL, SCD or Comet assay and pregnancy outcome after IUI. The Quality in Prognosis Studies (QUIPS) tool was utilized for quality assessment. This review is reported according to the 2009 PRISMA statement. The literature search resulted in 433 studies of which we finally retained nine studies for the qualitative analysis and four studies for the meta-analysis, accounting for 940 IUI cycles. In summary, the observed effect of low sperm DNA fragmentation on clinical pregnancy after IUI as analyzed with the random effects model reveals a relative risk of 3.15 (95% CI: 1.46-6.79; I2 = 13.1%) and pooled sensitivity and specificity of respectively 94% (95% CI: 0.88; 0.97) and 19% (95% CI: 0.14; 0.26). Taken together, the included studies show a limited capacity of sperm DNA fragmentation in discriminating between couples who will benefit from the test, namely in either predicting IUI outcome or in advising for or against IUI as first choice of treatment instead of advancing to more invasive medically assisted reproduction. This review has thrown up many questions in need of further investigation. As such, future studies might explore issues such as determining relevant cut-off values for prediction of spontaneous pregnancy and pregnancy after IUI as well as the assessment of the stability of the test over time and before and after density gradient centrifugation.

Sperm nuclear maturity and chromatin stability in subfertile patients: Density gradient centrifugation is fair but non-discriminative in selecting the right population.

Multiple technologies exploring chromatin structure anomalies have been applied during the last decade to evaluate fertility disorders and to increase the predictive value of sperm analysis for procreation in vivo and in vitro. Our aim was to implement sperm nuclear maturity and nuclear chromatin stability as a functional test for male infertility diagnosis and to compare it with a fertile group. As semen processing is an integral part of assisted reproductive technologies the impact of density gradient centrifugation in selecting sperm based on nuclear maturity and stability was also analyzed. Flow cytometry combined with fluorescent dyes exhibiting affinity for DNA was implemented. Both nuclear parameters correlated significantly with semen parameters. The control fertile group had significantly higher mean condensed population and a significantly lower hypocondensed and hypercondensed fractions as compared to the subfertile study group. Density gradient centrifugation succeeded in selecting the condensed population in both the control and study groups, while reducing the hypocondensed percentage. The hypercondensed population which was ten-fold higher in the study group remained unchanged after selection, in both the control and the study groups. Sperm nuclear maturity and chromatin stability appears to be homogenous in the fertile sperm donors and heterogeneous in subfertile patients. Sperm preparation for assisted reproduction should aim to minimize the risk of abnormal spermatozoa being used for fertilization.

Metal concentrations, sperm motility, and RNA/DNA ratio in two echinoderm species from a highly contaminated fjord (the Sørfjord, Norway).

The present study evaluated the effects of field metal contamination on sperm motility and the RNA/DNA ratio in echinoderms. Populations of Asterias rubens and Echinus acutus that occur naturally along a contamination gradient of sediments by cadmium, copper, lead, and zinc in a Norwegian fjord (the Sørfjord) were studied. Sperm motility, a measure of sperm quality, was quantified using a computer-assisted sperm analysis system. The RNA/DNA ratio, a measure of protein synthesis, was assessed by a one-dye (ethidium bromide)/one-enzyme (RNase), 96-well microplate fluorometric assay. Although both species accumulate metals at high concentrations, neither sperm motility parameters in A. rubens nor the RNA/DNA ratio in both species were affected. The Sørfjord is still one of the most metal-contaminated marine sites in Europe, but even so, populations of A. rubens and E. acutus are able to endure under these conditions.

Human embryo research in Belgium: an overview.

OBJECTIVE: To present an overview of the numbers and types of human embryos used in research projects in Belgium from 2007 to 2015. DESIGN: Analysis of all research proposals approved by the Federal Commission for Medical and Scientific Research on Embryos In Vitro. SETTING: Not applicable. PATIENT(S): Not applicable. MAIN OUTCOME MEASURE(S): Number of embryos used for research, number of embryos created for research, and areas of embryo research. RESULT(S): Since 2007, 15,811 embryos were used for 36 research projects. In total, 10,492 (66%) fresh supernumerary embryos (unfit for transfer or freezing) were used, 4,083 (26%) frozen supernumerary embryos (donated by parents whose child wish was completed or abandoned), and 1,236 (8%) embryos created for research. Most projects focused on research into embryo development. Fresh supernumerary embryos were mainly used for human embryonic stem cell (hESC) research. Frozen supernumerary embryos were almost exclusively used for research into embryo development and for hESC research. Embryos created for research were used for research into embryo development, oocyte research, research into cryopreservation of oocytes, and hESC research. CONCLUSION(S): Having concrete data on embryo research is crucial for an informed debate. Moreover, these data are necessary to find out trends in research such as the numbers of embryos needed and the areas of research. Data collection requires a sufficiently clear definition of "research" and "embryo." These conceptual questions frequently reveal lack of clarity in legislation.