Oxidative DNA damage in human sperm influences time to pregnancy

Steffen Loft 2 3 Tina Kold-Jensen 1 2 Niels Henrik Hjollund 0 2 Aleksander Giwercman 2 7 Jesper Gyllemborg 1 2 3 Erik Ernst 2 6 Jrn Olsen 2 5 Thomas Scheike 2 3 Henrik Enghusen Poulsen 2 4 Jens Peter Bonde 0 2 0 Department of Occupational Medicine, Aarhus University Hospital , Denmark 1 Department of Growth and Reproduction, The National University Hospital , Copenhagen 2 room 18-5-32 , Blegdamsvej 3, DK-2200 Copenhagen N , Denmark 3 Institute of Public Health, University of Copenhagen 4 Department of Clinical Pharmacology, The National University Hospital , Copenhagen , Denmark 5 The Danish Epidemiology Science Centre, University of Aarhus 6 Reproductive Toxicology Unit, Institute of Anatomy, University of Aarhus 7 Fertility Centre, Scanian Andrology Centre, Malmo University Hospital , Malmo , Sweden BACKGROUND: Oxidative stress and related DNA damage in human sperm may be important for fecundity and pregnancy outcome. METHODS: We studied the level of oxidative DNA damage in terms of 7-hydro-8-oxo-2-deoxyguanosine (8-oxodG) in sperm DNA among 225 first-pregnancy planners. Over the six menstrual cycle follow-up time, after cessation of contraception, 135 pregnancies were conceived. RESULTS: The likelihood of pregnancy occurring in a single menstrual cycle was inversely associated with the 8-oxodG level (P < 0.01). The odds ratio of pregnancy in each of the first three or all six follow-up menstrual cycles was 0.42 (0.23-0.78; 95% CI) and 0.61 (0.36-0.91) per unit increase in the log 8-oxodG/100 000 dG ratio after adjustment for potential confounders, (including sperm concentration) respectively. The intra-individual coefficient of variation of 8-oxodG in 2-6 monthly repeated sperm samples from 116 men was 19% for the 8-oxodG/dG ratio, whereas the inter-individual coefficient of variation was 49%. The 8-oxodG level was not significantly associated with smoking, consumption of alcohol or caffeine, exposure to welding fumes or the plasma levels of sex hormones. CONCLUSIONS: The data suggest that oxidative damage to sperm DNA influences fecundity and the level of damage is relatively constant within an individual and not influenced by smoking. Introduction The postulated secular, geographical and inter-individual variation in semen quality has been partly attributed to environmental factors, including estrogens, pesticides, pthalates, polycholorinated biphenyls (PCBs), air pollution and tobacco smoke acting in pre- and perinatal life or directly on sperm after puberty (Sharpe et al., 1993; Vine et al., 1994; Fisch and Goluboff, 1996; Vine, 1996). Besides potential hormone disrupting effects, most of the agents have the capacity to induce oxidative stress, which could damage sperm DNA. The need for study of oxidative stress in sperm and male infertility has recently been emphasised (Sharma et al., 1996). In human sperm DNA, substantial oxidative modification in terms of the oxidized deoxynucleoside, 8-oxo-7,8-dihydro2deoxyguanosine (8-oxodG), at the level of 24 per 100 000 deoxyguanosines (dG) has been demonstrated (Fraga et al., 1991; 1996; Shen et al., 2000). The level of 8-oxodG in sperm DNA has been reported to be increased in smokers and the level correlated with the intake and seminal plasma concentration of vitamin C, the most important antioxidant in sperm (Fraga et al., 1991; 1996; Shen et al., 1997). If not repaired, 8-oxodG modifications in DNA are mutagenic and may cause embryonic loss, malformations or childhood cancers (Fraga et al., 1991). Moreover, this modification could be a marker of oxidative stress in sperm which could also have negative effects on sperm function (Ni et al., 1997; Chen et al., 1997a;b; Shen et al., 2000). Accordingly, the level of oxidative modifications in seminal DNA may be a valuable biomarker of environmental factors affecting sperm. In a population of 266 healthy men from a cohort of 430 first pregnancy-planning couples, we studied oxidative modification in terms of 8-oxodG levels in DNA from up to six repeated semen samples and the relationship with life-style factors and semen quality in terms of volume, concentration and motility as well as with apparent male fecundity in terms of probability Table I. Characteristics of female partners of 225 men with successful analysis of sperm samples for 8-oxodG Age (years) Body weight (kg) Height (cm) Cycle length (days) Consumption of: Cigarettes (no/day) Alcohol (drinks/week) Caffeine (mg/week) Median 25 64 169 Interquartile range of pregnancy during six menstrual cycles follow-up after cessation of contraception. Materials and methods The present material was part of a two-centre study of fecundity and measures of semen quality in 430 first pregnancy-planning couples described in detail elsewhere (Bonde et al., 1998a;b). In brief, the study population was recruited by contacting, by means of a postal information brochure, 52 255 members of the trade unions for metal workers, office workers, nurses and daycare workers between 2035 years of age, who had no children and who were co-habiting with a person of the opposite sex and the same age range. Eligible couples were those planning to discontinue contraception to achieve a pregnancy and had no previous reproductive history in either partner. The protocol was approved by the local ethics committee and the subjects granted informed consent in accordance with the Helsinki declaration. The subjects of the present study consisted of 141 and 150 men recruited from the Copenhagen and Jutland areas respectively. Information regarding body weight, height, smoking, consumption of coffee, tea and alcohol and occupational exposures was obtained from a questionnaire. During follow-up over a period of six menstrual cycles, after discontinuation of contraception, occurrence of pregnancy was verified by a physician or commercial pregnancy tests. Semen samples were collected by masturbation. The subjects were asked to keep 23 days of abstinence prior to sample collection and report the actual abstinence period. The first sample was collected at entry and stored at 80 C. Subsequent samples were collected every month from cessation of contraception for at least 3 months and up to 6 months if conception had not occurred. Thus, each subject collected 36 samples. Except for the entry sample the samples were stored at 20 C for <3 months. The samples from the Copenhagen area and Jutland were stored for <12 and <30 months respectively at 80 C until analysis. The semen quality was determined in the entry sample in terms of volume, concentration, total number (volume 3 concentration), motility and morphology. Number of sperm cells were counted in a Burker-Tu rk or Makler chamber. The residual semen, after securing material for other analyses, was used for analysis of 8-oxodG in DNA. The repeated samples were available for 8-oxodG analysis from the Copenhagen area subjects only. A total of 475 repeated semen samples from 141 individuals from the Copen (...truncated)