Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men.

OBJECTIVE: To evaluate two different assays of human sperm DNA integrity, DNA denaturation (DD) and DNA fragmentation (DF), and to correlate these with standard semen parameters. DESIGN: Prospective, observational study. SETTING: University infertility clinic. PATIENT(S): Forty consecutive semen samples from 33 nonazoospermic men presenting for infertility evaluation and 7 fertile men presenting for vasectomy. INTERVENTION(S): Assessment of sperm concentration, motility, morphology, DD and DF. MAIN OUTCOME MEASURE(S): Sperm DD and DF in fertile and infertile men. RESULT(S): The mean (+/-SE) rates of DD and DF were significantly higher in infertile subjects compared to fertile controls, respectively: 25.4 +/- 3.0 vs. 10.2 +/- 2.3 (P=.028) and 27.6 +/- 2.5 vs. 13.3 +/- 2.5% (P=.016). DF and DD correlated strongly (r = 0.71, P<.0001). Also, DD and DF correlated negatively with standard semen parameters (concentration, motility, and morphology), the strongest correlation being with sperm motility. CONCLUSION(S): The strong correlation between sperm DD and DF, and the higher levels of sperm DNA damage in infertile compared with fertile men, indicate that male infertility is associated with poor sperm DNA integrity. Although infertile men may father children with assisted conception, fertilization with DNA-damaged spermatozoa may increase the risk of genetic disease in the offspring.

Effects of chilling to 0 degrees C on the morphology of meiotic spindles in human metaphase II oocytes.

OBJECTIVE: To determine the effects of chilling to 0 degrees C on the meiotic spindle of human metaphase II oocytes, as observed by optical sectioning microscopy. DESIGN: Laboratory study. SETTING: Academic research laboratory in a medical school. PATIENT(S): Seventy-two women undergoing infertility treatment donated a total of 108 oocytes. INTERVENTION(S): Metaphase II oocytes were stripped of their cumulus cells, cooled directly to 0 degrees C, and held for periods of 1 to 10 minutes. They were then fixed at 37 degrees C, stained for immunofluorescence, and examined microscopically. MAIN OUTCOME MEASURE(S): Morphology of the meiotic spindle in chilled and control oocytes. RESULT(S): Microscopic evaluations of 46 chilled oocytes revealed various time-dependent changes in microtubules compared to 9 control oocytes. After 1 minute at 0 degrees C, spindle damage was negligible, but in oocytes cooled for 2 or 3 minutes, there was obvious shortening of the spindle and loss of polarity. Cooling to 0 degrees C for 4 to 9 minutes resulted in increasingly more drastic changes; by 10 minutes the spindles had totally disappeared. Despite depolymerization of microtubular tubulin at 0 degrees C, the chromosomes did not become dispersed, but remained anchored even in the absence of spindles. CONCLUSION(S): Even brief exposure of human oocytes to temperatures near 0 degrees C causes profound alterations of the meiotic spindle.

Smoking and reproduction: gene damage to human gametes and embryos.

Assisted conception is a useful methodology for detecting disturbances in clinical outcome, meiotic maturation, and genetic integrity of human gametes. Germinal cells are vulnerable to genetic damage from smoking, but can repair damage during meiosis. In ejaculated spermatozoa, repair capacity declines drastically. Smoking alters the meiotic spindle of oocytes and spermatozoa, leading to chromosome errors which affect reproductive outcomes. Smoking is associated with reduced numbers of retrieved oocytes, leading to early age of menopause. Oocyte elimination occurs preferentially during meiosis I, a period sensitive to genetic damage. Smoking inhibits embryo fragmentation; inhibition may confer survival advantage to embryos genetically altered. Smoking is associated with low sperm quality, but clinical effects are not recognized. Cadmium (a heavy metal), nicotine (a toxic alkaloid), and its metabolite cotinine, are detectable in gonadal tissues and fluids in association with smoking. Cotinine incorporates into ovarian granulosa-lutein cells, compromising the developmental potential of follicles. Benzo[a]pyrene is a carcinogenic polycyclic aromatic hydrocarbon resulting from cigarette combustion. Its reactive metabolite binds covalently to DNA, forming adducts. Smoking-related adducts were detectable in ovarian granulosa-lutein cells, oocytes, spermatozoa and preimplantation embryos. Transmission of altered DNA from smoking by spermatozoa was demonstrated in preimplantation embryos and in association with increased risk of childhood cancer.

Detection of benzo(a)pyrene diol epoxide-DNA adducts in sperm of men exposed to cigarette smoke.

OBJECTIVE: To determine whether the adducts formed when benzo(a)pyrene, a diol epoxide derivative, binds covalently to DNA (BPDE-DNA adducts) are detectable in the sperm of men who smoke cigarettes. DESIGN: Prospective study. SETTING: The Toronto Hospital IVF-ET program. PATIENT(S): Twenty-three patients with normal seminal parameters: 11 smokers (20.6 +/- 0.7 cigarettes per day) and 12 nonsmokers. INTERVENTION(S): Semen samples obtained by masturbation. MAIN OUTCOME MEASURE(S): Seminal plasma samples were assessed for cotinine by RIA. Sperm were treated with dithiothreitol to release disulfide bonds and allow for DNA binding, then exposed to an anti-BPDE monoclonal antibody, a biotinylated antibody, and streptavidin-conjugated peroxidase. Staining intensity scores, determined in 100 cells per individual, were correlated with seminal plasma cotinine levels, a marker of smoking. RESULT(S): Cotinine levels correlated highly with the number of cigarettes smoked per day. Mean cotinine levels and mean staining intensity scores were higher in smokers than in nonsmokers. Staining intensity correlated highly with cotinine levels. CONCLUSION(S): We demonstrated, for the first time, that BPDE-DNA adducts in sperm cells are increased by smoking; we also detected comparatively high levels in nonsmokers, which indicates that environmental exposure also is substantial. The formation of adducts in spermatozoa is a potential source of transmissible prezygotic DNA damage.

Detection of benzo[a]pyrene diol epoxide-DNA adducts in embryos from smoking couples: evidence for transmission by spermatozoa.

Tobacco smoking is deleterious to reproduction. Benzo[a]pyrene (B[a]P) is a potent carcinogen in cigarette smoke. Its reactive metabolite induces DNA-adducts, which can cause mutations. We investigated whether B[a]P diol epoxide (BPDE) DNA adducts are detectable in preimplantation embryos in relation to parental smoking. A total of 17 couples were classified by their smoking habits: (i) both partners smoke; (ii) wife non-smoker, husband smokes; and (iii) both partners were non-smokers. Their 27 embryos were exposed to an anti-BPDE monoclonal antibody that recognizes BPDE-DNA adducts. Immunostaining was assessed in each embryo and an intensity score was calculated for embryos in each smoking group. The proportion of blastomeres which stained was higher for embryos of smokers than for non-smokers (0.723 versus 0.310). The mean intensity score was also higher for embryos of smokers (1.40+/-0.28) than for non-smokers (0.38+/-0.14; P = 0.015), but was similar for both types of smoking couples. The mean intensity score was positively correlated with the number of cigarettes smoked by fathers (P = 0.02). Increased mean immunostaining in embryos from smokers, relative to non-smokers, indicates a relationship with parental smoking. The similar levels of immunostaining in embryos from both types of smoking couples suggest that transmission of modified DNA is mainly through spermatozoa. We confirmed paternal transmission of modified DNA by detection of DNA adducts in spermatozoa of a smoker father and his embryo.

Immunodetection of benzo[a]pyrene adducts in ovarian cells of women exposed to cigarette smoke.

Benzo[a]-pyrene (B[a]P) is a potent mutagen and carcinogen present in cigarettes. We report here on immunodetection and quantification of B[a]P-DNA adducts in granulosa-lutein cells of patients undergoing in-vitro fertilization (IVF) and embryo transfer, who were exposed to cigarette smoke. Follicular fluids (FF) and granulosa-lutein cells were obtained from the same follicular aspirate from 32 women self-reported as active smokers, passive smokers, or non-smokers. Cells were immunostained with 5D11, an anti-B[a]P diolepoxide monoclonal antibody that recognizes DNA adducts. Cotinine, a reliable marker for recent smoke exposure and dose, was assessed by radioimmunoassay in 32 FF samples. Individual scores of cell immunoreactivity were highly correlated with FF cotinine concentrations. Evaluations of immunostaining intensity in 9770 granulosa-lutein cells from the 32 women revealed higher average scores in active and passive smokers, relative to non-smokers. In passive smokers the average level of cell immunostaining was 63% of that of active smokers. These relationships provide quantitative evidence that B[a]P-DNA adduct levels are related to smoke exposure and dose, both recent and long term. Immunostaining was confined to the nucleus, suggesting adduct formation by covalent binding to DNA. Presence of adducts in granulosa-lutein cells from women exposed to cigarette smoke may increase the risk for DNA damage.

Interovarian differences in levels of cotinine, a major metabolite of nicotine, in women undergoing IVF who are exposed to cigarette smoke.

PURPOSE: Our purpose was to determine whether there is variation in levels of follicular fluid (FF) cotinine between the two ovaries of women undergoing IVF-ET who are exposed to cigarette smoke. METHODS: In 61 women, there were two to four determinations of FF continine levels for each of two follicles, one from each ovary. For each woman a t test for significant difference between the means of both ovaries was done to test for interovarian variation. RESULTS: Thirty-seven nonsmokers, 8 passive smokers, and 16 active smokers differed greatly (P < 0.0001) in mean FF cotinine levels: 13.0, 91.1, and 420.3 ng/ml, respectively. Fourteen women had significant differences, at the P = 0.025 level or below, between their two ovaries. Five of them had differences significant at the 0.001 level. Even so, the correlation between the cotinine levels of the two ovaries was high. CONCLUSIONS: Cotinine uptake between the two ovaries of a woman may differ approximately one-fourth of the time. In spite of these differences, the overall correlation between ovaries is high. The clear distinction in levels of FF cotinine among active, passive, and nonsmokers demonstrates the reliability of FF cotinine testing. Detection of cotinine in a large proportion of nonsmokers shows how pervasive nicotine is in the environment.

Effects of cigarette smoking and age on the maturation of human oocytes.

We investigated whether cigarette smoking, measured by follicular fluid concentrations of cotinine (a major metabolite of nicotine), affects the maturity of oocytes from women undergoing in-vitro fertilization (IVF) and embryo transfer. In 234 women, follicular fluid samples were assessed for cotinine and their 2020 oocytes were assessed for maturity stage. Data on individual proportions of oocytes which were mature (OM) and were fertilized (OF) were analysed by regression in relation to age and follicular fluid cotinine. OF gave an independent assessment of oocyte maturity. Both age and follicular fluid cotinine entered the OM and OF regressions and were significant. The age-adjusted regression coefficients for log cotinine were positive; greater cotinine concentrations usually accompanied greater OM and OF. The cotinine effect on OM was positive in younger women, but it became negative (decreased OM with increasing cotinine concentrations) in older women (> or = 40 years). We further found in older women an average reduction of approximately 50% in the number of mature oocytes; this reduced number was lower than the number of embryos usually transferred. Smoking can reduce the number of mature oocytes even further, therefore risking a negative IVF-embryo transfer outcome. This may be the reason why the negative effects of smoking become clinically detectable in older women.

Immunodetection of cotinine protein in granulosa-lutein cells of women exposed to cigarette smoke.

OBJECTIVE: To detect immunoreactivity to cotinine protein, a major metabolite of nicotine, in granulosa-lutein cells from patients exposed to cigarette smoke, as measured by levels of cotinine in follicular fluid (FF) samples. DESIGN: Controlled immunocytochemical study. SETTING: Hospital IVF-ET program treating infertile patients. PATIENT(S): Twenty-eight women classified by self-reported smoking habits: active smokers (n = 17), passive smokers (n = 4), and nonsmokers (n = 7). INTERVENTION(S): Ovarian hyperstimulation. MAIN OUTCOME MEASURE(S): Grades of immunostaining intensity were assessed in granulosa-lutein cells. Patient scores of cell immunostaining were calculated and regressed on levels of FF cotinine. RESULT(S): Cotinine levels in FF were higher in active smokers than in passive smokers or nonsmokers. Cotinine immunostaining was visualized in the nucleus and cytoplasm of granulosa-lutein cells. Mean grades and mean scores of immunostaining intensity were higher in active smokers than in passive smokers or nonsmokers. There was a strong positive correlation between scores of cell immunostaining and FF cotinine levels. CONCLUSION(S): The association between cotinine expression in granulosa-lutein cells and FF cotinine provides reliable evidence for a dose-related effect. This constituent of cigarette smoke appears to interact directly with and incorporate into these ovarian cells. Our approach seems useful for monitoring ovarian exposure to environmental toxins.

Cotinine, a major metabolite of nicotine, is detectable in follicular fluids of passive smokers in in vitro fertilization therapy.

OBJECTIVE: To assess cotinine, a metabolite of nicotine, in follicular fluids (FF) of women who smoke either actively or passively or not all. DESIGN: Controlled clinical study. SETTING: Infertile patients in a hospital IVF-ET program. PATIENTS: One hundred eleven women classified by smoking habits: active smokers (n = 44), passive smokers (n = 17), or nonsmokers (n = 50). INTERVENTIONS: Ovarian hyperstimulation. MAIN OUTCOME MEASURE: Cotinine levels in FF. RESULTS: A strong correlation between number of cigarettes smoked and levels of FF cotinine was found. The levels of FF cotinine were: active smokers 710.4 +/- 128.2, passive smokers 76.3 +/- 56.5, and nonsmokers 4.2 +/- 2.0 ng/mL (mean +/- SEM). The level in active smokers was significantly greater than in other groups. The levels of FF cotinine in passive smokers differed significantly from nonsmokers. Eighty-four percent of nonsmokers actually were exposed to nicotine, with a mean value of 5.0 ng/mL. CONCLUSIONS: Cotinine was detectable in a dose-dependent manner in active and passive smokers. It was detected in all active smokers and in a majority of passive smokers and self-reported nonsmokers. A strong interindividual variation suggests differences in metabolism and smoking habits. Follicular fluid cotinine assessments are useful for infertility studies.

Cigarette smoking may affect meiotic maturation of human oocytes.

There is a strong association between cigarette smoking and reduced fecundity, reduced fertility, and early mean age of menopause, suggesting that smoking may impair oocyte function and viability. We analysed the effect of smoking on meiotic maturation of oocytes. A total of 156 women undergoing in-vitro fertilization therapy, classified as non-smokers (n = 102), passive smokers (n = 21), light smokers (< 15 cigarettes/day; n = 19), and heavy smokers (> or = 15 cigarettes/day, n = 14), participated in this study. Tubal factor infertility was more frequent in smokers, in agreement with the literature, and showed a significant dose effect (P = 0.008). Smokers had decreased numbers of retrieved oocytes compared with non-smokers, suggesting reduced fertility. Cytogenetic data from 286 oocytes apparently unfertilized showed similar proportions of haploid (normal) and aneuploid chromosome complements among groups. In contrast, oocytes with diploid complements were more frequent among smokers. Regression of individual proportions of diploid oocytes on number of cigarettes smoked per day was very significant (P = 0.0003). The increased frequency of oocyte diploidy in smokers, probably resulting from prevention of first polar body extrusion, indicates meiotic immaturity. Also, triploid zygotes occurred more frequently in smokers (P = 0.03), suggesting preferential digynic fertilization. Our study shows that external factors, like cigarette smoking, may be hazardous to the viability and function of developing oocytes and their resulting embryos.

Cadmium accumulation in follicular fluid of women in in vitro fertilization-embryo transfer is higher in smokers.

OBJECTIVE: To assess cadmium, a heavy metal in cigarette tobacco, in follicular fluid (FF) of women in IVF-ET, who smoke. DESIGN: Controlled clinical study. SETTING: Infertile patients in a hospital IVF-ET program. PATIENTS: Fifty-one women selected in groups according to smoking habits: nonsmokers (n = 10), passive smokers (n = 17), light smokers (< 15 cigarettes per day, n = 19), and heavy smokers (> or = 15 cigarettes per day, n = 5). INTERVENTIONS: Ovarian hyperstimulation with GnRH agonist. RESULTS: The mean +/- SEM level of FF cadmium was higher in smokers (7.93 +/- 0.16 ng/mL) than in nonsmokers (6.73 +/- 0.31 ng/mL), and with a dose-effect of smoking. The individual levels in passive, light, and heavy smoking women also were higher than in nonsmoking women. CONCLUSIONS: Despite lack of vascularization of the follicle, cadmium accumulation was detectable in FF. Cadmium also could accumulate in oocytes of smokers; it does so, in a dose-dependent manner, in oocytes of cadmium-treated rats. Access to cadmium and other contaminants of cigarette smoke in FF may compromise the quality of oocytes, becoming a risk factor.

Chromosome status of untransferred (spare) embryos and probability of pregnancy after in-vitro fertilisation.

Many spontaneous abortions are associated with chromosomal abnormality of the fetus. In in-vitro fertilisation (IVF) the chromosome status of untransferred ("spare") embryos and subsequent fate (pregnancy or not) of the transferred sibling embryos might be related. Since the spare and transferred embryos of a patient's cycle genetically are full siblings, the inherited chromosomal abnormalities in spare embryos have a 50% probability of also appearing in transferred embryos. We have tested whether chromosome analysis of spare embryos has predictive power for transferred embryos. 48 couples with a total of 437 embryos were selected because their spare embryos (1-4 per couple; 76 total) were successfully analysed for chromosome status. 16 patients became pregnant. These women produced a higher proportion of chromosomally normal spare embryos (9/24; 37.5%) than those who did not achieve pregnancy (1/52; 1.9%). The proportion of patients who had only normal embryos was significantly higher (p = 0.012) in the pregnant group than in the non-pregnant group, and the proportion of patients who had only abnormal embryos was significantly higher (p = 0.001) in the non-pregnant group. Patients with preclinical and clinical pregnancy losses had only chromosomally abnormal spare embryos; by contrast, 50% of spare embryos from patients with ectopic pregnancies were normal. The proportion of spare embryos that were normal (13%, 10/76), was similar to the livebirth rate of 11% per transferred embryo (19 infants from 171 transferred embryos). These results suggest that chromosome analysis of spare embryos may have predictive value for their transferred sibling embryos. We conclude that improving detection of chromosomally normal embryos for transfer should improve the success rate in IVF.

Cytogenetics of human oocytes, zygotes, and embryos after in vitro fertilization.

Chromosome errors, inherited or arising de novo during gametogenesis and transmitted at fertilization to the conceptus, may be a major cause of embryonic mortality. The in vitro fertilization and embryo transfer (IVF/ET) procedure provides extra material--oocytes, zygotes, and embryos--to investigate the contribution of chromosomal abnormality to implantation failure. This paper reviews the results of cytogenetic studies on such material. Estimates from a total of 1120 oocytes from 11 studies give an overall proportion of chromosomal abnormality of 35%. Single and multiple nullisomies and disomies are found, involving nonrandom chromosome gain or loss. Hypohaploid complements are more frequent than hyperhaploid complements. The higher rate of chromosome loss of hypohaploid karyotypes was found to be largely artifactual. The estimated overall frequency of aneuploidy is 13%. In embryos the level of chromosomal abnormality is 23%-40%. Errors of fertilization are responsible for a substantial number of triploid embryos, many of which develop into mosaics. Factors extrinsic to the conceptus, such as infertility, advanced maternal age, and ovarian hyperstimulation, may increase the level of chromosomal abnormality. More refined methods for accurately recognizing and selecting chromosomally normal embryos for transfer are needed to improve the success rate of this reproductive technology.

Evidence for maternal predisposition to chromosome aneuploidy in multiple oocytes of some in vitro fertilization patients.

OBJECTIVES: To assess the rate of chromosome aneuploidy (e.g., extra or missing chromosomes) in oocytes remaining unfertilized in our in vitro fertilization (IVF) program. To determine whether two parameters of the IVF technique, advanced maternal age and hormonal follicle stimulation, affect this rate. DESIGN: Data on oocyte retrieval, fertilization, and aneuploidy rates are analyzed to test for possible relations with maternal age and two hormonal stimulation regimens. SETTING: Patients of our IVF program from 119 stimulated cycles over 8 months. PATIENTS, PARTICIPANTS: In vitro fertilization patients selected for having oocytes (1 to 18) remaining unfertilized after insemination in vitro. RESULTS: Advanced maternal age decreases both the number of retrieved oocytes and the fertilization rate, but hormonal treatments have no effect. Aneuploidy (rate 27%), involving group G most frequently, appears associated with advanced age. Patients who were previously parous produced significantly reduced numbers of aneuploid oocytes compared with the nonparous group. A significant excess (P = 0.01) of patients had multiple oocytes all alike (all haploid or all aneuploid), showing correlation among multiple oocytes of a patient in chromosome status. CONCLUSIONS: Maternal age affects reproductive performance and is related to specific chromosomal aneuploidy. Women who were previously parous are more likely to produce normal oocytes than nonparous women; oocyte normality therefore may improve the chance for a future successive pregnancy. Nonrandomness in chromosome abnormality of some patients' multiple oocytes is evidence for maternal predisposition to meiotic nondisjunction. Consequently, these patients are at risk for failed IVF cycles.

Non-poisson distribution of sperm from grandfathers in zona-free hamster ova.

Earlier reports indicated that sperm from 25% of patients from infertile couples, but not from normal or fertile donors, show deviations from the theoretical Poisson distribution of the number of sperm penetrating zona-free hamster ova. Using semen samples from 15 grandfathers (aged 60 to 84 years) and 24 young fathers (aged 25 to 36 years), this study analyzed whether age also has an effect on the distribution. It was found that the overall fit to the Poisson distribution of the samples from grandfathers was very poor; in contrast, the samples from young fathers fit well. The observed deviations from the Poisson distribution among grandfathers may be a consequence of their long periods of sexual abstinence. Decrease in sexual activity produces age-different populations of sperm that probably differ in penetrating ability. Samples from older fathers also show a worse fit to the Poisson distribution than do those from younger fathers. These results suggest that the duration of sperm storage in the genital tract after maturation has an effect on sperm function.

Abnormalities of sperm chromosome condensation in the cytoplasm of immature human oocytes.

This study analysed data from 27 couples in an IVF-ET programme. The maternal age range was 28-43 years. Statistical analyses on 182 oocytes showed no maternal age effect on the number of oocytes, their stage of maturation or their fertilization rate. There was also no effect of age of either partner or of seminal parameters on the fertilization rate. In contrast, occurrence of diploid oocytes was confined to three of the older women. The proportion of failures of fertilization was significantly higher in immature oocytes. These failures, which included 18 uncleaved, multipronuclear or fragmented zygotes, were related to disturbances of oocyte maturation. Four (out of five) oocytes re-inseminated with fresh semen produced polyspermy. One zygote showed marked asynchrony in the development of the two pronuclei. In eight zygotes the paternal complements had an allocyclic pattern of chromosome condensation between and within chromosomes or chromosome regions. In two other zygotes the paternal complement showed one chromosome prematurely condensed. This single-chromatid chromosome would be lost in the following cleavage division, suggesting that aneuploidy due to 'anaphase lag' is not a rare event during embryo cleavage.

Confirmation of an abnormal (non-Poisson) distribution of sperm from some infertile men in the hamster-ovum test.

The present study was carried out to investigate an earlier report that stated that some men of infertile couples (patients), but not normal donors, have an abnormal (non-Poison) distribution of penetrating sperm among ova in the hamster-ovum test. Semen samples from 60 men, 24 proven fathers and 36 patients, were analyzed for agreement with the theoretical Poisson distribution (PD). Most of the fathers (23 of 24) fit PD well, but 10 of the patients did not. The overall (group) fit of fathers is good, but that of the patients is poor. Patients, but not fathers, are heterogeneous in their agreement with PD; about 25% fit poorly whereas more than 50% fit well. The 25% fitting poorly may often be those patients who are truly infertile (even when their wives are actually fertile).

Sperm from some infertile men may consist of several populations.

In the hamster ovum penetration (HOP) test, when ova have equal penetrability and sperm have equal penetrating ability, the distribution of zona-free hamster ova classified by number of penetrating human sperm is expected to follow the Poisson distribution (PD). This study reports tests for PD in HOP tests on 9 infertile patients and 11 normal controls. The data, presented in detail, show the expected PD in the control group. In contrast, 3 patients had HOP tests with definite non-PDs, whereas 5 patients have clearcut PDs. The cause of non-PD is unknown, but could result from differences among motile sperm in penetrating ability.

Testicular function in eight patients with seminoma after unilateral orchidectomy and radiotherapy.

Testicular function was monitored in eight patients with low stage seminoma who were treated with radiotherapy following unilateral orchidectomy. The absorbed gonadal radiation dose ranged from 15 to 157.5 rad. At 10-24 months after radiotherapy, serum hormone levels, sperm analysis, sperm penetration into zona-free hamster ova (HOP-test) and lymphocyte chromosome abnormalities were evaluated. Two patients were azoospermic with elevated serum levels of LH and FSH. The remaining six patients had slightly decreased (n = 3) or normal (n = 3) seminal parameters. Their HOP rates were within the normal range. A low incidence of polyspermy (i.e. only one penetrating sperm per ovum) was found in the patients, suggesting low penetrability of motile sperm. A highly significant correlation was found between sperm count or sperm penetrability and time post-irradiation. The results indicate that restitution of testicular function is time-dependent.