Reproductive toxicology of environmental toxicants: emerging issues and concerns.

Environmental toxicants comprise a number of man-made organic chemicals which may resist metabolism or their metabolites may persist in the environment and accumulate in the food chain. Some of these persistent chemicals are carried over long distances via the atmospheric transport and can have biological effects in fish, wildlife and humans. In this review the relationship between structure of these chemicals, their mode of action and their possible roles in adverse developmental and reproductive processes in humans will be discussed. The focus will be on model polychlorinated biphenyls and dioxins, organochlorines, phthalates, a constituent of cigarette smoke (benzo-a-pyrene), synthetic polymers (polybrominated diphenyl ethers and polyfluorinated compounds), and a fungicide (vinclozolin).

Influence of dichlorodiphenylchloroethylene on vascular endothelial growth factor and insulin-like growth factor in human and rat ovarian cells.

1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE, DDE), a metabolite of DDT is a persistent hormonally active environmental toxicant present in human serum and follicular fluid. The objective of this study was to investigate the effects of DDE on the expression of the ovarian vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF-1) in primary cultures of human granulosa cells and in the rat ovary. Granulosa cells were obtained at the time of oocyte retrieval for in vitro fertilization and cultured with environmentally relevant concentrations of DDE. Immature female rats were treated with 100 microg DDE/kg body weight or vehicle at 28 and 31 days of age and then euthanized at 50 days of age for collection of ovarian tissue. Expression of VEGF, the VEGF receptor fetal liver kinase (Flk-1) and IGF-1 were determined by Western blotting analysis of protein lysates from granulosa cell cultures and by immunohistochemistry in the rat ovary. DDE at concentrations of 100-1000 ng/mL increased the expression of VEGF, Flk-1 and IGF-1 in vitro in primary cultures of human granulosa cells, with the highest expression occurring at 1000 ng/mL. Similarly, acute administration of DDE resulted in a significant increase in immunoreactive VEGF, Flk-1 and IGF-1 in the rat ovary. We conclude that DDE, at levels, which have been detected in humans, alters the expression of the ovarian growth factors VEGF and IGF-1 both in vivo and in vitro. This alteration in expression of growth factors may lead to altered ovarian function as seen in polycystic ovaries and impaired fertility.

Non-genomic action of estradiol and progesterone on cytosolic calcium concentrations in primary cultures of human granulosa-lutein cells.

BACKGROUND: The present study examined whether the sex steroids, estradiol and progesterone, could alter cytoplasmic calcium concentrations ([Ca(2+)](cyt)) in human granulosa-lutein cells. METHODS: Human granulosa cells were obtained at the time of oocyte retrieval for IVF and cultured for 3-7 days. Cells were loaded with Fura-2 AM and changes in [Ca(2+)](cyt) of single cells were studied using a dynamic digital Ca(2+) imaging system. RESULTS: Both estradiol and progesterone stimulated elevations of [Ca(2+)](cyt) in Ca(2+)-containing medium within seconds of exposure of the granulosa-lutein cells to the steroid, but only estradiol caused an increase in [Ca(2+)](cyt) in Ca(2+)-free medium. Both ICI-182780 and RU 486 stimulated [Ca(2+)](cyt) increases and inhibited the effects of estradiol and progesterone, respectively. Tamoxifen also induced transient increases in [Ca(2+)](cyt) concentrations but inhibited the effects of both estradiol and progesterone. The inhibitory effects of tamoxifen, ICI-182780 and RU 4486 on [Ca(2+)](cyt) responses to estradiol and progesterone could be reversed with higher concentrations of estradiol and progesterone, respectively. The [Ca(2+)](cyt) effects induced with tamoxifen could not be eliminated by prior treatment with RU 486 or ICI-182780. CONCLUSION: These results provide strong evidence that both estradiol and progesterone as well as the steroid antagonists, tamoxifen, RU 486 and ICI-182780, can act on human granulosa-lutein cells through a non-genomic mechanism.

Dichlorodiphenylchloroethylene elevates cytosolic calcium concentrations and oscillations in primary cultures of human granulosa-lutein cells.

1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), a metabolite of DDT (1,1-dichlorodiphenyltrichloroethane), is a persistent hormonally active environmental toxicant that has been found in human serum and follicular fluid. The objective of this study was to determine whether DDE can alter free calcium ion concentrations in the cytosol ([Ca(2+)](cyt)) of human granulosa cells. Changes in [Ca(2+)](cyt) in single cells loaded with Fura-2 were studied using a dynamic digital Ca(2+) imaging system. At a concentration of 100 ng/ml, DDE stimulated small elevations of [Ca(2+)](cyt) accompanied by Ca(2+) oscillations. At 1 microg DDE/ml, there was a biphasic Ca(2+) response with marked elevations of [Ca(2+)](cyt) over time. In Ca(2+)-free medium, cells showed an initial small elevation of [Ca(2+)](cyt), which was magnified after addition of Ca(2+) to the medium. Washing the cells after DDE treatment failed to remove the elevated [Ca(2+)](cyt) and oscillations, both of which were eliminated by addition of EGTA. ATP also induced [Ca(2+)](cyt) elevations and oscillations, and these effects were potentiated when DDE was added. FSH induced transient [Ca(2+)](cyt) elevations, whereas hCG caused a prolonged elevation and marked oscillations in [Ca(2+)](cyt). These results suggest that DDE at concentrations normally found in human tissues induces elevations in [Ca(2+)](cyt) in granulosa-lutein cells. Our data therefore highlight a novel mechanism through which DDE can alter endocrine homeostasis and possibly act as an endocrine toxicant.

Levels of environmental contaminants in human follicular fluid, serum, and seminal plasma of couples undergoing in vitro fertilization.

Environmental chemicals are thought to adversely affect human reproductive function, however there are no studies that have explored the association between failed fertilization and exposure of both partners to environmental contaminants. Therefore, we collected blood and follicular fluid from the female partner and seminal plasma from the male partner of 21 couples attending an in vitro fertilization (IVF) program, in order to determine the extent of the existence of environmental chemicals in these fluids. Any relationship to the outcome of IVF was also considered. Sera and fluids were analysed for a variety of contaminants, including polychlorinated biphenyls, pesticides, cotinine, and the steroids progesterone and estradiol. Of the couples examined, 18 had fertilizations, three of whom became pregnant. There were no fertilizations in three other couples. The contaminants most frequently found in follicular fluid, more than 50% of the samples tested, were p,p'-DDE, mirex, hexachloroethane, 1,2,4-trichlorobenzene, PCB 49, PCB 153, and PCB 180. Cadmium was detected in eight of 21 (38.1%) samples of follicular fluid whereas cotinine was detected in 18 (85.7%). Residue levels of p,p'-DDE, endosulfan I, PCB 99, PCB 138, PCB 153, PCB 180 were quantified in more than 50% of the sera samples examined. Seminal plasma was relatively free of pollutants with mirex being the most frequently detected contaminant found in seven of 21 (33.3%) samples. Mirex could not be detected in the seminal plasma of the husbands whose partner's oocytes failed to fertilize whereas significant levels of mirex were found in the seminal plasma of all couples who had a pregnancy. Cadmium was also found in the follicular fluid of these pregnant subjects. No relationship was found between follicular fluid cotinine in pregnant and non-pregnant subjects. Where identical contaminants were found in both sera and follicular fluids, the levels were about twofold higher in serum and were positively correlated in both fluids. Fertilization was negatively correlated with serum and follicular fluid p,p'-DDE whereas pregnancy was positively correlated with follicular fluid PCB 49. These data reveal that more than 50% of the population of women attending a fertility program have had exposure to environmental chemicals sufficient to produce detectable concentrations in their serum and ovarian follicular fluid. Of the chemical contaminants detected in the serum and follicular fluid of these women, p,p'-DDE was the most frequently detected, had the highest residue levels, and was associated with failed fertilization.

Sperm swim-up techniques and DNA fragmentation.

BACKGROUND: Swim-up techniques for sperm separation may have detrimental effects on sperm DNA. We wished to determine whether the normal swim-up method with centrifugation used in our laboratory, which involves a centrifugation step, was harmful to sperm compared with swim-up without centrifugation. METHODS: Semen samples were obtained from patients undergoing IVF or andrology assessment. An aliquot was removed for fixation and subsequent DNA fragmentation determination. The remaining sample was divided into two equal parts, which were subjected to swim-up either with (normal swim-up) or without (direct-swim-up) centrifugation. Semen analysis was performed both before and after swim-up. DNA fragmentation, in spermatozoa previously fixed in 4% paraformaldehyde, was assessed by the terminal transferase-mediated DNA end-labelling procedure (TUNEL). The percentage of spermatozoa with DNA damage after each swim-up technique was compared with that in the original semen sample. RESULTS: DNA damage was <5% in most samples. No significant change in DNA fragmentation was observed between the two swim-up procedures, although the 'normal' swim-up sample had significantly less DNA fragmentation than the pre-swim-up sample. CONCLUSIONS: We conclude that our normal swim-up technique caused no more DNA damage to spermatozoa from normal semen samples than a direct swim-up technique that involved no centrifugation step.

Relationship between steroid concentrations in ovarian follicular fluid and oocyte morphology in patients undergoing intracytoplasmic sperm injection (ICSI) treatment.

The objective of this study was to investigate the relationship between oocyte morphology and follicular fluid steroid concentrations in patients being treated with intracytoplasmic sperm injection. A total of 82 IVF cycles were evaluated in patients aged 24-40 years. Oocytes at metaphase II were graded into four groups according to the status of the first polar body and the size of the perivitelline space. The proportion of oocytes at the germinal vesicle and germinal vesicle breakdown stages, and the proportion of degenerated oocytes and oocytes with a large polar body were compared with different concentrations of oestradiol, progesterone and testosterone in the follicular fluid. The association between these oocyte characteristics and the ratio of oestradiol:testosterone and oestradiol:progesterone was also analysed. The results showed that oocyte morphology, as assessed by the status of the first polar body and the size of the perivitelline space, is associated with the ratio of oestradiol:testosterone and oestradiol:progesterone but not with the absolute concentrations of oestradiol, progesterone and testosterone in the follicular fluid. A ratio of oestradiol:testosterone > 200 is the best indicator for a small proportion of grade 1 and 2 oocytes (poor quality), a large proportion of grade 3 and 4 oocytes (good quality), and a small proportion of oocytes with cytoplasmic inclusions. These results will be of clinical use in evaluating oocyte quality.

Melatonin receptor mRNA expression in human granulosa cells.

We have shown that the melatonin receptor agonist, 2-[125I] iodomelatonin, binds to high-affinity guanine nucleotide-sensitive sites on human granulosa (HG) cell membranes. In order to confirm the presence of melatonin receptors in HG cells, we have now used a reverse transcriptase-polymerase chain reaction (RT-PCR) procedure to examine receptor subtype expression. RT-PCR studies revealed the presence of the mt1 (Mel1alpha) melatonin receptor subtype in ten single or pooled HG cell samples which were obtained from 14 patients. In contrast, expression of MT2 ( Mel1b) mRNA was observed in only two of these HG samples. DNA sequencing of the mt1 PCR product confirmed its identity with the reported human mt1 melatonin receptor. The expression of mt1 and MT2 receptor mRNA in HG cells and the reported presence of melatonin in human follicular fluid indicate a potentially important role for this hormone in regulating human ovarian and reproductive function.

Hypotension following mild bouts of resistance exercise and submaximal dynamic exercise.

Our purposes were (1) to examine resting arterial blood pressure following an acute bout of resistance exercise and submaximal dynamic exercise, (2) to examine the effects of these exercises on the plasma concentrations of atrial natriuretic peptide ([ANP]), and (3) to evaluate the potential relationship between [ANP] and post-exercise blood pressure. Thirteen males [24.3+/-(2.4) years] performed 15 min of unilateral leg press exercise (65% of their one-repetition maximum) and, I week later, approximately 15 min of cycle ergometry (at 65% of their maximum oxygen consumption). Intra-arterial pressure was monitored during exercise and for 1 h post-exercise. Arterial blood was drawn at rest, during exercise and at intervals up to 60 min post-exercise for analysis of haematocrit and [alphaANP]. No differences occurred in blood pressure between trials, but significant decrements occurred following exercise in both trials. Systolic pressure was approximately 20 mmHg lower than before exercise after 10 min, and mean pressure was approximately 7 mmHg lower from 30 min onwards. Only slight (non-significant) elevations in [alphaANP] were detected immediately following exercise, with the concentrations declining to pre-exercise values by 5 min post-exercise. We conclude that post-exercise hypotension occurs following acute bouts of either resistance or submaximal dynamic exercise and, in this investigation, that this decreased blood pressure was not directly related to the release of alphaANP.

Steroidogenesis in luteinized granulosa cell cultures varies with follicular priming regimen.

During follicular development, a co-ordinated gonadotrophin and endocrine environment is believed to be essential for normal function of the resulting corpus luteum. Whether differences in the gonadotrophins used to promote follicular development can have lasting effects on granulosa cells after they have undergone luteinization and culture, remains to be studied. We measured steroid production under basal and human chorionic gonadotrophin (HCG) stimulation in short and long term cultures of luteinizing granulosa cells obtained from normal ovulatory women undergoing assisted folliculogenesis with either human menopausal gonadotrophin (HMG) or follicle stimulating hormone (FSH). Basal progesterone and oestradiol production by luteinized granulosa cells obtained from follicles stimulated to develop with FSH was significantly greater than that from HMG derived follicles (P < 0.001). In short term cultures, treatment with 10 IU HCG caused a 10-fold increase in progesterone release by cells from FSH stimulated follicles, whereas cells of HMG origin produced only 5-fold more progesterone (P < 0.0001). In cultures that were maintained for 2 weeks, progesterone secretion was reduced, but a similar trend in HCG responsiveness was observed. These experiments demonstrate that the composition of the gonadotrophins used to promote follicular development in vivo leads to differences in granulosa cell steroidogenesis which are evident after luteinization and culture. They additionally support the notion that the environment of follicular development will be reflected in the resulting corpus luteum.

Canadian semen quality: an analysis of sperm density among eleven academic fertility centers.

OBJECTIVE: To determine whether sperm quality has declined among Canadian men during the past 13 years and whether there are regional differences in sperm quality. DESIGN: Retrospective temporal series of cross-sectional studies. SETTING: University fertility centers across Canada. PATIENT(S): Men being investigated as part of the normal infertility work-up. MAIN OUTCOME MEASURE(S): Sperm concentrations among all the samples were compared on an annual basis to assess any changes over 13 years from 1984 through 1996. RESULT(S): There were regional differences and trends in both up and down directions. Linear regression analysis of the means of each center for each year showed no significant trend. However, when all the samples were analyzed by regression analysis there was a significant downward trend. CONCLUSION(S): Linear regression analysis showed a significant downward trend in sperm concentration among 48,968 samples from Canadian men obtained from 1984 through 1996. A significant difference was seen in the mean concentrations between centers, ranging from 48.6 to 104.5 X 10(6)/mL. Secular trends in sperm density are dependent on the statistical method used for analysis.

Acrosin activity in pelleted frozen sperm does not correlate with in vitro fertilization of oocytes.

Spermatozoa pelleted after swim-up were frozen and then analysed in batches for acrosin activity, using a spectrophotometric method, and expressed as microIU micrograms DNA-1. A total of 259 sperm samples were analysed and the acrosin activity compared with fertilization in vitro. Of these, 224 samples fertilized at least one oocyte and 35 samples failed to fertilize any oocyte. Analysis by Student's t-test indicated that there was a statistically significant difference (P = 0.02) in acrosin activity between the two groups. However, when samples that failed to fertilize were matched for concentration, motility and normal morphology with samples that fertilized, this significance was lost (P = 0.77). It is concluded that total acrosin in pelleted sperm frozen after regular swim-up, does not correlate with fertilizing ability of spermatozoa used for insemination.

Semen parameters as predictors of in-vitro fertilization: the importance of strict criteria sperm morphology.

This study evaluated 120 couples undergoing in-vitro fertilization treatment to determine which semen parameter(s) predicted fertilization and whether there was any consistent relationship between strict criteria and standard assessment of sperm morphology. Strict criteria morphology was the only significant predictor of fertilization (P = 0.0006, r2 = 0.09), with a sensitivity of 94% and a specificity of 40%. A 12% cut-off point presented a negative predictive value of 98% and a positive predictive value of 22%. The probability of satisfactory fertilization is 40% with morphology <4%, which increases to 97% with normal morphology (>=12%). The receiver operating characteristic curve deviated significantly from the diagonal with a 76% area under the curve, making this a superior predictive test. This was augmented by likelihood ratios (LR) of 8. 25 (LR+) for results with <4% normal morphology and 0.15 (LR-) for results with >/= 12% normal morphology by strict criteria. While there was some correlation between strict criteria and standard assessment of morphology (r = 0.35), the former explained only 12% (r2 = 0.12) of the variability in the latter. This study concludes that strict criteria morphology predicts fertilization, while other semen parameters do not. A 12% cut-off point makes strict criteria morphology an excellent predictor of satisfactory fertilization, while a value <4% is a good predictor of poor fertilization.

Polyploidy and failed fertilization in in-vitro fertilization are related to patient's age and gamete quality.

A review of 392 cycles of in-vitro fertilization (IVF) was carried out in order to identify whether any factors such as sperm concentration at insemination, sperm motility or morphology, oocyte grading, number of oocytes retrieved, patient age, follicular stimulation protocols or duration of follicular growth, could be associated with the incidence of polyploidy or completely failed fertilization. The majority of polypronuclear fertilizations occurred in mature oocytes and in patients < 37 years of age and the incidence of polyploidy was strongly associated with fertilization and pregnancy rates. Fertilization rates were highest with mature oocytes. A significant linear trend was observed with failed fertilization and sperm concentration, morphology and motility. High rates of failed fertilization were found in patients > 37 years of age and with one to five oocytes retrieved. No significant difference was seen in stimulation protocols and oocyte grading between oocytes that fertilized and those that did not. Cycles with good sperm morphology and motility, the presence of mature oocytes, the retrieval of a large number of oocytes and younger maternal age seem to provide the best chance for IVF success.

Alterations in circulating ovarian steroids in hexachlorobenzene-exposed monkeys.

Hexachlorobenzene (HCB) is a global pollutant that has been identified in human serum and ovarian follicular fluid, and its effect on ovarian function has not been adequately defined. Thus, the effects of HCB on ovarian steroidogenesis and menstrual cycle characteristics were investigated in cynomolgus monkeys (n = 16) orally dosed by gelatin capsule (0.0, 0.1, 1.0, and 10.0 mg HCB/kg b.wt./d) for 90 d (approximately three menstrual cycles). Analysis of change in menstrual cycle length for each animal revealed a dose-dependent increase (P = 0.02) in cycle length. Ovulatory levels of estradiol (E2) were significantly reduced (P = 0.02) in the highest treatment group. During ovulation induction, the area under the E2 concentration curve (AUC) was significantly (P = 0.03) suppressed in the highest treatment group. Our data demonstrate that HCB treatment, under the conditions of the present study, alters both ovarian function and menstrual cycle characteristics with a no observable adverse effect level of 1.0 mg/kg.

Immune response to changes in training intensity and volume in runners.

We examined the acute and chronic effects of changes in training volume and intensity on the blood lymphocyte percentages and immunoglobulin levels in runners. Twelve runners participated in four 10-d phases of low volume/low intensity (LV/LI), high volume/low intensity (HV/LI), or high volume/high intensity (HV/HI) running. Subjects were assigned to one of two different training group orders: 1) LV/LI, HV/LI, LV/LI, HV/HI; or 2) LV/LI, HV/HI, LV/LI, HV/LI. Venous blood was drawn at rest on days 1, 4, and 7; and 5 min post-exercise on days 1 and 7 of each 10-d phase. Lymphocyte subsets were determined by flow cytometry for CD3+, CD4+, CD8+, and HLA-DR+. IgG, and IgM levels were obtained by ELISA analysis. Immunoglobulin, CD8+ and HLA-DR+ levels, and pre-exercise plasma cortisol concentrations were not significantly affected by alterations in volume or intensity. A transient decrease (P < 0.05) was observed in CD4+ and the CD4/CD8 ratio 5 min post-exercise during the HV/LI and HV/HI phases. Results indicate that the exercise-induced lymphocyte subset reduction is transient and suggest that it is more dependent upon training intensity than volume, and the training order of exposure to the high-intensity stimulus may determine the magnitude of subsequent responses.

Natural cycles for in-vitro fertilization: cost-effectiveness analysis and factors influencing outcome.

Improvements in oocyte culture technique, sperm preparation, oocyte retrieval method and ovarian stimulation regimens have produced higher pregnancy rates with in-vitro fertilization (IVF) treatment. However, because ovarian stimulation is expensive and not without risk, there is increasing interest in the option of using natural cycles for IVF. This study was performed to document the experience and outcome in 240 natural cycles. Cancellation occurred in 28 cycles (12%), and LH surge was observed in 56 (23%), leaving 156 (65%) cycles which progressed to oocyte retrieval. No oocytes were retrieved in 26 cycles. Among the successful oocyte retrievals, the majority yielded one oocyte. There was no evidence of fertilization in 26 cases, and triploid fertilization was observed in 12 cases. Embryos suitable for transfer were available in 92 cycles in which 11 (12%) clinical pregnancies were confirmed. Despite the high failure rate at each step in the process, natural cycles are more cost-effective than stimulated cycles which incur an incremental cost per live birth of $48,000. Natural cycles offer a low-cost alternative that may be more accessible to patients.

Melatonin receptors on human granulosa cell membranes.

Putative melatonin binding sites were detected in the membrane fraction of gonadotropin-stimulated human granulosa cells using the melatonin analogue 2-[125I]-iodomelatonin (125I-IML). Saturation studies and Scatchard analysis revealed the presence of a major binding site with a Kd of 99 pM. Guanosine triphosphate shifted the receptor affinity to 380 pM. In competition studies, the rank order of potency of indoles for inhibition of 125I-IML binding at these sites was typical of melatonin receptors: 2-iodomelatonin > melatonin > N-acetylserotonin > 5-methoxytryptamine > serotonin. Culture of cells for 7 days in vitro increased receptor density but not the affinity. These findings strongly suggest that melatonin found in follicular fluid may have a physiological role.