Binding of human chorionic gonadotrophin to rat ovary during development.

Ovarian tissue of prenatal, newborn, and 5-day-old rats does not specifically bind 125I-labelled HCG. Specific binding of HCG was first observed in ovaries of 10-day-old animals and binding increased with age. These results indicate that, contrary to rat testis, the HCG receptor in the rat ovary is not present during foetal and early postnatal development. Thus, the insensitivity of the ovary to endogenous and exogenous LH or HCG during this developmental period is due to the lack of specific receptors.

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).

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.

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.

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.

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.

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.

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.

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.

Cytologic investigation of human in vitro fertilization failures.

A cytogenetic study of unsuccessfully fertilized oocytes was done for information on the frequency and type of chromosomal abnormalities leading to preimplantation loss. Of 72 oocytes, 44 were assessed as mature, 19 as intermediate, and 9 as immature. The mean fertilization rate was 68%. This rate was significantly lower in immature oocytes than in mature ones (22% versus 79%). Immature oocytes completed maturation in vitro up to metaphase II of meiosis. Intermediate oocytes became fertilizable, however, a significant proportion showed morphologic abnormalities after fertilization. Thus, eggs with three pronuclei were hypotriploid or had three prophasic chromosome sets, together with polyspermy and with first polar body chromosomes. Eggs with developmental arrest showed marked asynchrony in pronuclear morphogenesis. One had a chromosome complement of 24,XX. It is concluded that insemination prior to completion of maturation leads to abnormal development.

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.

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.

H-Y antigen negative germ cells in gonadal sex organization in vitro: a morphological study.

Dissociation-reorganization experiments were done with gonadal cells of newborn rats. Rotation cultures consisted of mixtures of somatic and germ cells of opposite sex. Somatic cells, ovarian or testicular, determined a female or male type respectively, of gonadal histomorphic organization. Germ cells did not affect the type of organization of somatic cells. Accordingly, suspensions containing somatic cells of one sex together with germ cells of both sexes, reorganized in rotation culture, into either a) follicles containing XX or XY germ cells, or b) tubules containing XX or XY or both types of germ cells. These results give morphological evidence for heterosexual germ-somatic cells interactions. Based on morphological and H-Y antigen studies, failure of germ cells to bind and express H-Y antigen is considered as a possible factor for this failure of germ cells to affect gonadal sex.

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.

Ageing of the female gamete. 2 Lactate dehydrogenase activity in fragmented oocytes and ova of the rat.

LDH enzyme activities were determined in atretic and fragmented oocytes. The enzymatic activities in fragmented oocytes and ova were compared to those already known in early rat cleavage stages. Atretic oocytes are totally devoid of LDH activity. However, fragmented oocytes and ova exhibit nearly identical LDH activities to early cleavage stages. This result supports our earlier assumption that fragmentation and cleavage are homologus processes, induced by the same maternally transmitted factors in the oocyte cytoplasm.

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.

Variability in serologically detected male antigen titer and some resulting problems: a critical review.

Seroologically detected male antigen" (also called H-Y antigen) was first described in normal male mammals but now appears to occur in normal female mammals as well. "Serologically detected male predominant" (SDMP) antigen is a more appropriate name since the titer in normal males usually exceeds that of normal females. As we show, in each sex there is a considerable inter-individual variability in SDMP antigen titer, and in moderate-to-large size samples the low end of the male range of titers usually coincides with the high end of the female range. Several major problems arise from failure to recognize and/or to deal adequately with this normal variation in SDMP antigen titer. The chief problem is that the "controls" used (often a single individual) may be inadequate and misleading, leading to unjustified designation of samples as "positive", "negative", or even "deviant" ("intermediate", "reduced") for SDMP antigen titer. Other problems include deficiencies in technique and lack of statistical control for test and sample variability. Adequate attention to these problems, especially to the normal variability in SDMP antigen titer, could reduce the contradictions and inconsistencies which have troubled this field.