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.

Hexachlorobenzene exposure and the proportion of male births in Turkey 1935-1990.

Hexachlorobenzene (C(6)Cl(6), HCB) is a chemical that has been associated with significant immediate and long term adverse health effects in humans. It has been associated with both porphyria cutanea tarda and spontaneous abortions among survivors of widespread exposure in the 1950s in southeastern Turkey. HCB binds to the Ah receptor, albeit with lower affinity than dioxin. Dioxin exposure has been reported to lower human secondary sex ratio, putatively through a male mediated effect. We therefore wished to evaluate the impact of the HCB environmental event on the sex ratio of the progeny of the survivors. We undertook an assessment of 1) the effects of HCB exposure on the proportion of male births of individual subjects who had survived, 2) variables that significantly predicted the proportion of males among these individuals, and 3) the trend of the population sex ratio born in Turkey from 1935 to 1990. Women known to have been exposed to HCB in the 1950s did not have offspring with a significantly different sex ratio when compared to control populations. However, subjects reporting exposure at the peak of the episode (1955-57) had a significantly lower lifetime proportion of males than those exposed at a later date. The lifetime reported spontaneous abortion rate of these women also significantly predicted the percent males per subject. The available national data demonstrated a significant reduction in the calculated proportion of males from 1935 to 1970 that stabilized from 1970 to 1990. These data indicate that HCB exposure that was sufficient to induce clinical porphyria cutanea tarda may also have reduced the proportion of males in subjects over their reproductive life-span. The HCB episode does not explain the pattern of the national trend from a population perspective.

The effect of photomirex on the in vitro perfused ovary of the rat.

Photomirex, a photodegradation product of the insecticide mirex, is an environmental contaminant that has been identified in Great Lakes fish, soil, and human adipose tissue. Because of the potential for human exposure, the present study was designed to investigate the short-term effects of photomirex on the in vitro perfused ovary of the rat. Adult Sprague-Dawley rat ovaries were isolated and perfused for a total of 6 h with Medium 199. Following a 2-h baseline period, 10(-4) M of photomirex was administered to the medium. Control ovaries received medium or DMSO (vehicle control). Significant effects of perfusion and chemical intervention were identified using lactate dehydrogenase enzyme, glucose utilization, lactate, pyruvate, and flow:pressure ratio as markers of toxicity (P < 0.05). Lactate:pyruvate ratio, glutathione, and oxygen consumption did not demonstrate significant effects. Post hoc tests showed that there were significant differences between the DMSO + photomirex group and the control group (M199) using lactate dehydrogenase as a marker of toxicity. Pyruvate concentration was also reduced significantly after perfusion with DMSO + photomirex compared to M199 only and DMSO only (P < 0.05). Histopathologic changes were not discernible by light microscopy. These results suggest that metabolic and respiratory processes of the ovary are acutely sensitive to perturbation with photomirex in the in vitro perfused rat ovary model.

Declining sex ratios in Canada.

OBJECTIVE: To examine the trends in the proportion of annual live births that were male in Canada and to compare the trends with those in the United States. DESIGN: Analysis of census data. SETTING: Canada as a whole and 4 main regions (West, Ontario, Quebec and Atlantic). SUBJECTS: All live births from 1930 to 1990. OUTCOME MEASURES: Sex ratio (expressed as the proportion of total live births that were male [male proportion]) overall and by region. RESULTS: The male proportion in Canada decreased significantly after 1970 (p < 0.001); this represented a cumulative loss of 2.2 male births per 1000 live births from 1970 to 1990. Although a decrease was observed in all four regions studied, only that in the Atlantic region was significant (p < 0.001), representing a cumulative loss of 5.6 male births per 1000 live births from 1970 to 1990. A significant decrease in the male proportion was also observed in the United States from 1970 to 1990 (p < 0.001), although to a lesser degree than that observed in Canada, and represented a cumulative loss of 1.0 male births per 1000 live births. CONCLUSIONS: The decreased sex ratio in Canada adds to the growing debate over changes in biological markers and their potential causes. In addition, the study illustrates the potential use of the sex ratio as a widely available, unambiguous measure of the reproductive health of large populations.

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.

Developmental changes in the gonadotropin releasing hormone neuron of the female rabbit: effects of tamoxifen citrate and pregnant mare serum gonadotropin.

Developmental changes in immunostained gonadotropin releasing hormone neurons were demonstrated in female rabbits assigned to the following treatment groups: (i) tamoxifen citrate, 10 mg.kg-1 x day-1, in sesame seed oil (vehicle) (n = 24) or (ii) vehicle alone (control, n = 24) for 108 days; and (iii) 50 IU of pregnant mare serum gonadotropin on postnatal days 22 and 25 (n = 24) or vehicle on nontreatment days. Treatments had no effect on the total number of immunostained cells, but there was a significant (p = 0.0160) developmental shift from cells with smooth processes to rough. Group comparisons revealed that there was a significant (p < 0.001) age-related increase in the number of rough cells in pregnant mare serum treated rabbits between days 25 and 75, indicating an advancement in the shift from smooth to rough cells. Plasma gonadotropin levels, ovarian follicular development, and the developmental shift from smooth to rough cells were markedly suppressed by tamoxifen treatment compared with rabbits of the control group, while no difference in estradiol levels were found. Our results suggest that a developmental shift in gonadotropin releasing hormone cell morphology from smooth to rough precedes sexual maturity in the female rabbit.

In vitro fertilization and embryo transfer: a randomized controlled trial.

OBJECTIVE: The clinical indications for in vitro fertilization (IVF) have expanded to include many forms of infertility in addition to tubal disease. Pregnancies in IVF cycles are noteworthy but there is frequently a spontaneous cure for infertility among similar couples. The relative merit of IVF treatment over spontaneous cure or other forms of fertility treatment has not been rigorously evaluated. DESIGN: The study was a randomized controlled clinical trial comparing the clinical pregnancy rate among couples undergoing IVF with the rate among couples awaiting an IVF treatment. PATIENTS: Patients entering a provincially funded program of IVF were randomly allocated to a period of delay prior to IVF treatment (Control n = 194) or 1 or more cycles of IVF treatment (Experimental n = 205). MAIN OUTCOME MEASURES: Clinical pregnancy rate and adjusted time to pregnancy. RESULTS: In the Control group there were 13 pregnancies. In the Experimental group there were 13 pregnancies before treatment could be arranged. There were 20 additional pregnancies in treatment cycles. The intention-to-treat analysis showed an increase in the proportion of pregnancies from 8% to 17.4% and parturition from 4.9% to 11.6%. Substantially more patient-time (due to IVF) was required to achieve this increase. There was no difference between groups when time-to-event was considered by survival analysis, although a long-term trend in favor of the Experimental group was suggested. Low-event frequency and broad confidence intervals in Control patients prior to censor and transfer to IVF treatment prevented a conclusive assessment of the long-term benefits of IVF treatment. Generalizing these findings, improved effectiveness may be evident with delayed access to treatment (longer waiting lists), suitable candidates with appropriate primary clinical diagnoses and durations of infertility and higher rates of treatment over time (larger clinics). CONCLUSIONS: IVF treatment is effective in increasing, proportionally, the numbers of pregnancies, live births, and parturitions, but this occurred with significantly longer patient commitment.

Contamination of human ovarian follicular fluid and serum by chlorinated organic compounds in three Canadian cities.

OBJECTIVES: To determine the extent of contamination of ovarian follicular fluid and serum samples in women undergoing in-vitro fertilization and to study the effect of the contaminants on reproductive outcome. DESIGN: Inception cohort study. PATIENTS: Seventy-four women undergoing in-vitro fertilization at three regional clinics in Halifax, Hamilton, Ont., and Vancouver. MAIN OUTCOME MEASURES: Follicular fluid and serum levels of contaminants, cleavage rates and time to cleavage of first egg. RESULTS: Five chlorinated organic chemicals were frequently found in the two types of samples: alpha-chlordane (ALCH), dichlorochlorophenylethylene (DDE), heptachloroepoxide-oxychlordane (OXCH), hexachlorobenzene (HCB) and polychlorinated biphenyl (PCB). The levels were generally low. Regional differences between the three clinics were present. Samples from the Halifax clinic had the lowest frequency and level of contamination. The source of drinking water (well, bottled or municipal) was an important confounder. The concentrations of the five contaminants did not affect the cleavage rate or the time to cleavage of the first egg. CONCLUSION: Trace amounts of toxic and persistent chlorinated organic chemicals found in the follicular fluid of Canadian women undergoing in-vitro fertilization did not seem to have any adverse biologic effect on the rate of fertilization and the time to cleavage. Reasons for regional differences in the concentrations of contaminants require further study.

Hexachlorobenzene toxicity in the monkey primordial germ cell without induced porphyria.

Hexachlorobenzene is a persistent chlorinated organic chemical that has been detected in many tissues from a variety of species including human ovary and human ovarian follicular fluid. When administered in high dosage to nonhuman primates, hexachlorobenzene causes destruction of ovarian primordial germ cells in association with systemic toxicity. The purpose of these experiments was to assess relative ovarian germ cell sensitivity at much lower dosages of hexachlorobenzene that do not produce systemic effects and additionally to evaluate oocyte function by means of the response to superovulation, fertilization, and embryo cleavage during a cycle of in vitro fertilization in the cynomolgus monkey. Hexachlorobenzene in dosages of 0.1, 1.0, and 10.0 mg/kg/day was administered orally by gelatin capsule for 90 days. There was a dose-dependent accumulation of HCB in serum and other tissues without any change in the serum estradiol response to human menopausal gonadotropin, oocyte recovery, oocyte maturation, oocyte fertilization in vitro, and early embryo cleavage rate. There was a dose-related toxic effect observed in primordial germ cells at the lowest dose despite no evidence of systemic or hepatic effects. As there were no changes in the urinary porphyrin excretion, the mechanism of hexachlorobenzene ovotoxicity may be distinct from hexachlorobenzene-induced cytochrome P-450-dependent inhibition of uroporphobilinogen decarboxylase in the liver, although such intraovarian metabolism cannot be excluded.

Toxicity testing using the isolated in vitro perfused ovary.

This article discusses the use of in vitro perfusion techniques as a tool for toxicity testing in the ovary and how the rat ovary has been adapted for this purpose. A brief review of the development of in vitro ovarian perfusion is provided, focusing on steroidogenesis and physiology of ovulation. Adaptation of this model for use as a toxicologic model is discussed in the context of other isolated organ models, (that is, liver, heart, lung). Surgical procedures, perfusate and criteria for viability are outlined. Advantages of this technique are highlighted including ability to administer high doses of drugs directly to intact organ devoid of other influences. Applications of this model are discussed and data from studies of glutathione depleted ovaries perfused with hexachlorobenzene (HCB) are presented. Increased oxygen consumption after addition of HCB is suggestive of a disordered respiratory metabolism and is an example of future markers of ovarian injury using this innovative technique.

Hexachlorobenzene (HCB) suppresses circulating progesterone concentrations during the luteal phase in the cynomolgus monkey.

Hexachlorobenzene (HCB) is a known reproductive toxin. However, the full spectrum of its reproductive toxicity is unknown. Consequently, the effect of HCB on serum oestradiol (E2) and progesterone (P4) concentrations during the follicular (days 1-9), periovulatory (days 10-14) and luteal (days 15 to beginning of next menses) phases was investigated in the spontaneously cycling cynomolgus monkey. Adult female cynomolgus monkeys (n = 16) were randomly assigned to one of four treatment groups and orally doses with gelatin capsules containing HCB (0.0, 0.1, 1.0 and 10.0 mg kg-1 body wt. day-1) mixed with glucose. A 10-week acclimitization phase was followed by 13 weeks of dosing. HCB induced a dose-dependent suppression of serum P4 concentrations during the luteal phase. However, circulating levels of P4 were unaffected during the follicular and periovulatory phases of the menstrual cycle. Serum E2 concentrations, body weight, menstrual cycle length and duration of menses were not affected by HCB treatment. The range of menstrual cycle length and duration range of menses, however, were broader in the highest dose group. We conclude that HCB interfers with mechanisms regulating ovarian steroidogenesis and suppresses P4 levels during the luteal phase in the cynomolgus monkey.

Regulation of total ovarian glutathione content in the rat.

Glutathione (GSH) is an important intracellular thiol capable of altering metabolism following exposure to certain important biologic toxicants including radiation and cyclophosphamide. In order to evaluate the inhibition of glutathione synthesis in the ovary, 30-day-old Sprague Dawley rats were treated with either saline or 0.6 mumol/kg (0.133 mg/kg), 6.0 mumol/kg (1.33 mg/kg), or 4.5 mmol/kg (1000 mg/kg) buthionine sulphoximine (BSO) IP and sacrificed at 0, 1, 2, 4, 6, 8, and 24 h. There was an inhibition of glutathione synthesis with 4.5 mmol/kg (1000 mg/kg) BSO with a nadir at 8 h (P less than 0.001) and complete recovery at 24 h. In the subsequent experiments rats were divided into four groups. All animals received either saline or BSO 4.5 mmol/kg/day (1000 mg/kg/day) from day 27 to 30 of life and either saline or PMSG 5 IU IP on day 29 of life. BSO reduced ovarian content of GSH (saline-saline compared with BSO-saline, P less than 0.0001), which was countered by the prior administration of PMSG (BSO-saline compared with BSO-PMSG, P less than 0.005). Glutathione levels were as follows: saline-saline 4.3 +/- 0.04; saline-PMSG 5.0 +/- 0.4; BSO-saline 2.13 +/- 0.2; BSO-PMSG 3.24 +/- 0.2 nmol/mg ovary. These findings suggest the ovary is susceptible to GSH depletion by in vivo administration of BSO. Gonadotropin (PMSG) is capable of effecting a partial return of total ovarian GSH content.

The effect of estrous cycle and buthionine sulfoximine on glutathione release from the in vitro perfused rat ovary.

There is little known regarding the intracellular mechanisms of modification of damage in the ovary. Ovarian perfusion of en block dissections of the rat right ovary with aorta and vena cava were done to determine (a) if glutathione (GSH) is released by the ovary, (b) if the release is cycle dependent, and (c) if GSH released is the product of de novo ovarian synthesis. All perfused ovaries released GSH and the release was maximal at estrus and least at metestrus. Perfusion with buthionine sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase, resulted in a dose-dependent reduction in GSH released, indicating inhibition of de novo synthesis during perfusion.

Surface epithelium of the ovary following oral administration of hexachlorobenzene to the monkey.

Hexachlorobenzene (HCB) is a toxic and carcinogenic chemical that has been implicated in female reproductive dysfunctions, including destruction of ovarian follicles in primates. The purpose of this study was to determine the effects of HCB on ovary surface epithelium (SE). Gelatin capsules containing HCB mixed with glucose were given to 16 cynomolgus monkeys housed under controlled conditions in dosages of 0.0, 0.1, 1.0, or 10.0 mg/kg body weight daily, for 90 days; the first group served as the control. At necropsy one ovary from each animal was removed, fixed in glutaraldehyde, and processed by conventional methods for examination by transmission electron microscopy. SE from the animals in control group consisted of a single layer of squamous-to-cuboidal cells which possessed microvilli and contained cytoplasm rich in organelles; the nuclei were placed in middle of the cells. Although the types of alteration were similar in the treated groups, the degree of severity increased with increasing dose levels. In the lowest dose group (0.1 mg/kg) tested, stratification of cells was observed in some areas. Many cells were tall columnar, highly irregular in outline, and showed signs of degeneration. The nuclei had migrated toward the apical surface. Cytoplasm contained a large number of lysosomes, and numerous vesicles, which may have been swollen endoplasmic reticulum. In the 10 mg/kg group the affected cells were in advanced stages of degeneration. These observations support the evidence that HCB is a potent reproductive toxicant. Further studies are required to establish the effects of this damage on reproductive performance.

The short-term reproductive toxicity of cyclophosphamide in the female rat.

Cyclophosphamide (CTX) is a potent ovarian toxicant. Previous studies of the acute effects of CTX in the rat have demonstrated widespread ovarian follicle atresia, reduced serum estradiol, and progesterone with normal serum LH and FSH. The present investigations demonstrate that a single injection of CTX induces ovarian toxicity that reflects the loss of growing ovarian follicles. CTX induces a sensitization of serum FSH in response to GnRH within 24 h; this sensitization is lost by 7 days, and after 14 days the animals are capable of normal mating behavior. The observed protection of primordial follicles from the acute administration of CTX under these experimental circumstances may be related to the stage of the granulosa cell cycle of these follicles.

The agonist (d-leu-6,des-gly-10)-LHRH-ethylamide does not protect the fecundity of rats exposed to high dose unilateral ovarian irradiation.

Attempts to protect the ovary from the toxic effects of radiation and chemotherapy are relevant to the management of the young patient with cancer. Previous studies in a variety of animal species with several types of agonists of GnRH have shown promise in affording gonadal protection using indirect indices of reproductive function. The current investigations are based on these observations. Female rats were treated with (d-leu-6,des-gly-10) LHRH-ethylamide (GnRHa) from day 22 to day 37 of life. Sham-irradiation or unilateral irradiation of the left ovary was performed on day 30. The animals were mated following resumption of cycles and sacrificed on day 21 of pregnancy. There was no significant effect of ovarian artery ligation. Radiation reduced ovarian function ipsilateral to the radiation. GnRHa alone did not affect reproductive performance significantly. GnRHa and radiation combined resulted in no reproductive protection but augmented the damage done to the ipsilateral ovarian weight and to numbers of corpora lutea and fetuses. Under these experimental circumstances the agonist provided no protection.

The value of semen analysis and sperm function assays in predicting pregnancy among infertile couples.

Over a 2-year period, 227 couples were evaluated by an extended assessment of the male partner's ejaculate. This extended assessment comprised sperm penetration of denuded hamster oocytes, ability of sperm to penetrate synthetic mucus, and the adenosine triphosphate (ATP) content of whole semen. In proportional hazards analysis adjusting for the contribution of clinical and other seminal variables, the sperm penetration assay (SPA) test was a predictor of pregnancy in the subgroup with normal conventional seminal variables (greater than 40 million motile sperm per ejaculate). When the SPA result was 20% or more the probability of pregnancy was 3.7 times higher; the performance of the SPA as a diagnostic test was better in the couples with treated tubal disease than in other diagnostic categories.