Marcello Spanò

Sperm chromatin damage impairs human fertility

Abstract Objective: To examine the relationship between sperm chromatin defects, evaluated by the flow cytometric (FCM) sperm chromatin structure assay (SCSA), and the probability of a pregnancy in a menstrual cycle (fecundability). Design: Follow-up study. Setting: The Section of Toxicology and Biomedical Sciences, ENEA Casaccia, Rome, Italy, and the Department of Occupational Medicine, Aarhus University Hospital, Aarhus, Denmark. Patient(s): Two hundred fifteen Danish first pregnancy planners with no previous knowledge of their fertility capability. Intervention(s): None. Main Outcome Measure(s): Semen samples were collected at enrollment to measure semen volume, sperm concentration, motility, and morphology (by microscopy), as well as chromatin susceptibility to in situ, acid-induced partial denaturation by the FCM SCSA. Time to pregnancy was evaluated during a 2-year follow-up period. Demographic, medical, reproductive, occupational, and lifestyle data were collected by questionnaire. Fecundability was correlated with SCSA-derived parameters. Result(s): Fecundability declines as a function of the percentage of sperm with abnormal chromatin and becomes small when aberrant cells are >40%. Conclusion(s): Optimal sperm chromatin packaging seems necessary for full expression of the male fertility potential. The SCSA emerged as a predictor of the probability to conceive in this population-based study.

Sperm count and chromatin structure in men exposed to inorganic lead: lowest adverse effect levels

Objectives: To obtain knowledge on male reproductive toxicity of inorganic lead at current European exposure levels and to establish lowest adverse effect levels, if any. Methods: A cross sectional survey of the semen of 503 men employed by 10 companies was conducted in the United Kingdom, Italy, and Belgium. The mean blood lead concentration was 31.0 μg/dl (range 4.6–64.5) in 362 workers exposed to lead and 4.4 μg/dl (range below the detection limit of 19.8) in 141 reference workers. Semen volume and sperm concentration were determined in a fresh semen sample according to an agreed protocol subject to quality assurance. The sperm chromatin structure assay (SCSA) was performed at a centralised laboratory. Extraneous determinants including centre, period of sexual abstinence, and age were taken into account in the statistical analysis. If appropriate, possible thresholds were examined by iterative threshold slope linear regression. Results: The median sperm concentration was reduced by 49% in men with blood lead concentration above 50 μg/dl. There was no indication of a linear trend of lower sperm concentration with increasing blood lead values, but threshold slope least square regression identified a blood lead concentration of 44 μg/dl (β=−0.037, F=4.35, p=0.038) as a likely threshold. Abnormal sperm chromatin structure was not related to blood lead concentration, but some indications of deterioration of sperm chromatin was found in men with the highest concentrations of lead within spermatozoa. Biological monitoring data did not indicate long term effects of lead on semen quantity or sperm chromatin. Conclusion: Adverse effects of lead on sperm concentration and susceptibility to acid induced denaturation of sperm chromatin are unlikely at blood lead concentrations below 45 μg/dl. Effects of low level exposure to lead on other measures of testicular function cannot be ruled out.

Does smoking during pregnancy affect sons' sperm counts?

Background. There has been an apparent decline in sperm density during the last 5 decades in Denmark, a country in which women have among the highest rates of smoking in Europe. We examined semen quality and sex hormones in men in relation to their mothers’ tobacco smoking during pregnancy. Methods. Male participants were selected from the population-based Danish Twin Registry and the Danish Civil Registration System as part of a study on hereditary and environmental determinants of semen quality. From November 1999 to May 2000 we collected one fresh semen and blood sample from each of 316 men. Data on prenatal tobacco exposure were obtained for 265 of these men from a questionnaire filled in by their mothers. Results. Adjusting for age, current smoking status and other factors, sperm density was 48% lower (95% confidence interval = −69% to −11) among sons of mothers who smoked more than 10 cigarettes per day during pregnancy. Total sperm counts and levels of inhibin-B were also reduced among this group, whereas follicular stimulating hormone levels were somewhat higher (16% increase; 95% confidence interval = −13% to 54%). These effects were not seen in the lower smoking category (1–10 cigarettes per day). Conclusions. High levels of smoking (>10 cigarettes per day) during pregnancy may be a partial explanation for the apparent secular decline and the geographic differences in sperm counts.

Integrating new tests of sperm genetic integrity into semen analysis: breakout group discussion.

The First International Conference on Male-Mediated Developmental Toxicity, held in September 1992, reported that the spermatozoon can bring genetic damage into the oocyte at fertilization and thereby contribute to subsequent abnormal pregnancy outcomes (Olshan and Mattison, 1994). At that time, laboratory tests for genetic defects in sperm were at an early stage of development and were relatively untested in the clinic and the field. A breakout group at that meeting discussed the need for improved sperm biomarkers of adverse reproductive effects and concluded that sensitive, reliable, and practical methods

Semen Quality and Exposure to Persistent Organochlorine Pollutants

Background: Inconsistent results have been found in previous human studies on male reproductive toxicity of persistent organochlorine pollutants. The majority of studies have been conducted among selected populations of infertility clients or among occupational cohorts including a limited number of participants. Methods: We conducted a cross-sectional study of semen quality and serum concentration of 2,2′,4,4′,5,5′-hexachlorobiphenyl (CB-153) and 1,1-dichloro-2,2-bis (p-chlorophenyl)-ethylene (p,p′-DDE) among 763 men. We included men from all regions in Greenland (n = 194), fishermen from Sweden (n = 185), inhabitants of the city of Kharkiv, Ukraine (n = 195), and inhabitants of the city of Warsaw, Poland (n = 189). Blood samples were analyzed for CB-153 and p,p′-DDE using gas chromatography–mass spectrometry and adjusted for serum lipids. Results: Sperm concentration was not impaired with increasing serum CB-153 or p,p′-DDE levels in any of the separate groups or overall. Similarly, the proportion of morphologically normal sperm was not associated with either CB-153 or p,p′-DDE blood concentration. However, sperm motility was inversely related to CB-153 concentration in Greenland and the Swedish fishermen population. Across all 4 regions, the sperm motility decreased on average by 3.6% (95% confidence interval = 1.7% to 5.6%) per one-unit increase in the log of blood CB-153 (ng/g lipid). The concentration of p,p′-DDE was negatively associated with sperm motility in the Greenlandic population and in the compiled dataset. Conclusion: Adult exposure to persistent organochlorine pollutants within the ranges observed in the present study is not likely to cause reduction in sperm concentration or morphology. However, higher exposure may be associated with impaired sperm motility.

Flow cytometric analysis for reproductive biology

Summary— Flow cytometric studies of spermatogenesis have been advanced by the need for: i) rapid, sensitive, objective and multiparameter measurements of reproductive effects due to environmental, occupational, and therapeutic exposure to toxicants; and ii) assessment of fertility potential of human and animal sperm. As a consequence, various flow cytometric techniques are already available to identify germ cell subpopulations undergoing both proliferative and maturative processes in normal and perturbed conditions. Significant improvements have been introduced in order to investigate the spermatogenic complex differentiation pathway and the apparent uniformity of mature sperm. Flow cytometry (FCM) has been applied to the measurement of both testis and sperm cells in a variety of species, including man. End points considered in toxicology studies are: altered testicular germ cell ratios, DNA and RNA content, increase of the coefficient of variation, induction of diploid elongated spermatids and diploid sperm, altered nuclear morphology, sperm cell viability, mitochondrial function and sperm chromatin structure. Precise DNA content measurements allow accurate analysis to determine the proportion of X‐ and Y‐chromosome bearing sperm and sorting of these subpopulations for gender preselection. FCM technology has reached a maturation level that allows its inclusion in the list of available and routine methods for reproductive studies in human and animal populations.

Exposure to PCBs and p,p′-DDE and human sperm chromatin integrity

Persistent organochlorine pollutants (POPs) such as polychlorinated biphenyls (PCBs) and dichlorodiphenyldichloroethylene (p,p′-DDE), the major metabolite of dichlorodiphenyltrichloroethane (DDT), are stable lipophilic compounds widely found in the environment and in the general population. They can enter the food chain, and their negative impact on male reproduction is currently under active scrutiny. To explore the hypothesis that environmental exposure to these compounds is associated with altered sperm chromatin structure integrity in human sperm, we conducted a study of 176 Swedish fishermen (with low and high consumption of fatty fish, a very important exposure source of POPs). We determined serum levels of 2,2′,4,4′,5,5′-hexachlorobiphenyl (CB-153) and p,p′-DDE, and we used the sperm chromatin structure assay (SCSA) to assess sperm DNA/chromatin integrity. When CB-153 serum levels (individual dose range, 39–1,460 ng/g lipid) were categorized into equally sized quintiles, we found an association with the DNA fragmentation index (%DFI). A significantly lower %DFI was found in the lowest CB-153 quintile (< 113 ng/g lipid) compared with the other quintiles; there was a similar tendency, although not statistically significant, between %DFI and p,p′-DDE. These results suggest that POP exposure may have a slight negative impact on human sperm chromatin integrity.

A longitudinal study of semen quality in pesticide spraying danish farmers

It was hypothesized that occupational exposure to pesticides during a spraying season causes changes in semen quality that might be detected in a longitudinal study. We analyzed the within-person changes in semen quality and reproductive hormones across a spraying season in groups of farmers using and not using pesticides. A total of 248 men collected two semen samples (participation rate: 32%). The median sperm concentration declined significantly from the first to the second sample in both groups, but there was no statistical difference in the decline between the two groups, unadjusted or adjusted. Only minor changes were found in sperm morphology, vitality, motility, sperm chromatin denaturation (SCSA), and reproductive hormones, and the differences in changes between the two groups were nonsignificant, or, in the opposite direction to the expected. There was no relation between the changes in sperm parameters in relation to pesticide exposure variables. In conclusion, use of pesticides by Danish farmers is not a likely cause of short-term effects on semen quality and reproductive hormones.

The significance of sperm nuclear DNA strand breaks on reproductive outcome.

Purpose of review A growing body of evidence indicates that ejaculated spermatozoa from men being treated with intracytoplasmic sperm injection contain nuclear abnormalities. Many of these nuclear anomalies manifest themselves as breaks in the sperm nuclear DNA. This review examines the mechanisms involved in generating DNA strand breaks during spermatogenesis in the human, the main techniques used to assess the sperm nucleus and the evidence, in relation to assisted reproduction, showing that sperm nuclear DNA strand breaks may impact on reproductive outcome. Recent findings Techniques such as the TUNEL assay and the sperm chromatin structure assay both show increased levels of DNA abnormalities in spermatozoa from men who have poor semen parameters. The reproductive parameters affected by an increased presence of DNA abnormalities in ejaculated spermatozoa include fertilization, blastocyst development, and pregnancy rates. Summary There is accumulating evidence linking sperm nuclear DNA anomalies to poor reproductive outcome in relation to assisted reproduction technologies. The tests currently available only provide an inkling of the impact of sperm nuclear DNA abnormalities on reproductive outcomes. Although the impact an abnormal paternal genome may have on reproductive outcome is unquestionably less than that of its female counterpart, it cannot be ignored.

Exposure to perfluorinated compounds and human semen quality in arctic and European populations

BACKGROUND Perfluorinated compounds (PFCs) have been suspected to adversely affect human reproductive health. The aim of this study was to investigate the associations between PFC exposure and male semen quality. METHODS PFCs were measured in serum from 588 partners of pregnant women from Greenland, Poland and Ukraine who provided a semen sample, using liquid chromatography tandem mass spectrometry. Perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorohexane sulfonic acid (PFHxS) and perfluorononanoic acid (PFNA) could be detected in >97% of the samples. The associations between levels of these compounds and semen volume, sperm concentration, total sperm count, motility and morphology were assessed. RESULTS Across countries, sperm concentration, total sperm count and semen volume were not consistently associated with PFOS, PFOA, PFHxS or PFNA levels. The proportion of morphologically normal cells was 35% lower [95% confidence interval (CI): 4-66%) for the third tertile of PFOS exposure as compared with the first. A similar reduction was found in relation to increasing PFHxS levels. At the third PFOA exposure tertile, the percentage of motile spermatozoa was 19% (95% CI: 1 to 39%) higher than in the first. CONCLUSIONS The most robust finding in the present study was the negative associations between PFOS exposure and sperm morphology suggesting adverse effects of PFOS on semen quality, possibly due to interference with the endocrine activity or sperm membrane function. It cannot be excluded that this association and the positive association between PFOA and semen motility, which was not consistent across countries, might represent a chance finding due to the multiple statistical tests being performed.