Jean Clair Sadeu

Alcohol, drugs, caffeine, tobacco, and environmental contaminant exposure: Reproductive health consequences and clinical implications

Reproductive function and fertility are thought to be compromised by behaviors such as cigarette smoking, substance abuse, and alcohol consumption; however, the strength of these associations are uncertain. Furthermore, the reproductive system is thought to be under attack from exposure to environmental contaminants, particularly those chemicals shown to affect endocrine homeostasis. The relationship between exposure to environmental contaminants and adverse effects on human reproductive health are frequently debated in the scientific literature and these controversies have spread into the lay press drawing increased public and regulatory attention. Therefore, the objective of the present review was to critically evaluate the literature concerning the relationship between lifestyle exposures and adverse effects on fertility as well as examining the evidence for a role of environmental contaminants in the purported decline of semen quality and the pathophysiology of subfertility, polycystic ovarian syndrome, and endometriosis. The authors conclude that whereas cigarette smoking is strongly associated with adverse reproductive outcomes, high-level exposures to other lifestyle factors are only weakly linked with negative fertility impacts. Finally, there is no compelling evidence that environmental contaminants, at concentrations representative of the levels measured in contemporary biomonitoring studies, have any effect, positive or negative, on reproductive health in the general population. Further research using prospective study designs with robust sample sizes are needed to evaluate testable hypotheses that address the relationship between exposure and adverse reproductive health effects.

Effect of in vitro exposure to benzo[a]pyrene, a component of cigarette smoke, on folliculogenesis, steroidogenesis and oocyte nuclear maturation.

We previoulsy quantified the concentration of benzo[a]pyrene (B[a]P) in the follicular fluid of women exposed to mainstream and/or sidestream cigarette smoke. The objective of this study was to quantify the effects of B[a]P-exposure, at concentrations representative of follicluar fluid concentrations, on folliculogenesis, on gonadal steroid and anti-müllerian hormone (AMH) output, oocyte growth, and nuclear maturation. Follicles (100-130 μm) isolated from ovaries of F1 hybrid (C57BL/6j×CBA/Ca) mice were cultured for 13 days in increasing concentrations of B[a]P (0 ng/ml (control) to 45 ng/ml). B[a]P treatment inhibited (p < 0 .05) antral follicle development, decreased estradiol output and follicle survival at the 45.0 ng/ml dose. B[a]P exposure decreased AMH output overall during preantral (p = 0.014) and antral (p = 0.026) follicle development but had no effect on progesterone output or oocyte growth and nuclear maturation in surviving follicles. These data suggest that B[a]P is an important toxic component of cigarette smoke that adversely affects follicular development and survival.

In Vitro Oocyte Maturation: Current Status

Due to its numerous clinical applications, in vitro maturation (IVM) has emerged as a significant topic in the field of assisted reproduction. IVM of germinal vesicle breakdown/metaphase I and germinal vesicle stage oocytes collected from in vitro fertilization (IVF) superovulation cycles are commonly applied with unsatisfactory results. The biological aspect of this so-called rescue in vitro oocyte maturation greatly differs from the actual IVM practice. In the latter, immature oocytes are obtained from small antral follicles of unprimed or minimally stimulated cycles aiming to avoid ovarian hyperstimulation syndrome in high-risk patients or simply as an alternative to conventional IVF in normo-ovulatory patients. Over the past decade, cases reports regarding IVM have been sporadically reported, with ~25 peer-reviewed articles currently available. These studies present variable outcomes and deal with clinical approaches about selecting the most appropriate patient population that could benefit from IVM technology. Although some of the studies are encouraging, the vast majority includes small sample sizes, thus making the data rather inconclusive. As such there is a certain reserve in the IVF community to embark on treatment cycles for IVM in routine use. Laboratory parameters play an important role in the success of IVM, and research for optimal culture conditions is warranted. Existing data from newborns assure us that IVM may be a safe procedure provided in assisted reproductive technology. When optimized, it will serve, not only for infertile patients, but also as a more patient-friendly alternative than standard controlled ovarian stimulation to obtain oocytes for donation or preservation of fecundity.

Cigarette smoke condensate exposure delays follicular development and function in a stage-dependent manner

OBJECTIVE To determine the effects of cigarette smoke condensate (CSC) on follicular development and function from the early preantral stage through ovulation. DESIGN Prospective laboratory study. SETTING Academic research environment. ANIMAL(S) Female F1 hybrid (C57BL/6j×CBA/Ca) mice. INTERVENTION(S) Mouse early preantral follicles (100-130 μm) were exposed to increasing concentrations of CSC (0 μg/mL [control] to 130 μg/mL) during in vitro growth and ovulation. MAIN OUTCOME MEASURE(S) Follicular development, follicle survival, gonadal steroid output, expansion of the cumulus-oocyte complex, oocyte growth, and maturation. RESULT(S) Cigarette smoke condensate exposure significantly inhibited follicular development in the preantral and antral stage and decreased follicle survival at 90 μg CSC/mL and higher. Estradiol output was significantly lower in CSC-exposed (90 and 130 μg/mL) follicles. Before ovulation, CSC significantly increased P output, which decreased thereafter. Cigarette smoke condensate exposure reduced cumulus-oocyte complex expansion and subsequently reduced the number of polar body oocytes. CONCLUSION(S) Cigarette smoke condensate exposure inhibits follicle development and leads to premature luteinization of the preovulatory follicle, with decreased oocyte maturation in a mouse isolated follicle culture system that mimics murine folliculogenesis in vivo.

The cigarette smoke constituent benzo[a]pyrene disrupts metabolic enzyme, and apoptosis pathway member gene expression in ovarian follicles.

Benzo[a]pyrene (B[a]P) is a prototypical polycyclic aromatic hydrocarbon (PAH) present in cigarette smoke. We previously showed that B[a]P adversely affects follicular development and survival. The objective of this study was to identify the key molecular pathways underlying B[a]P-induced abnormal follicular development. Isolated follicles (100-130 μm) from ovaries of F1 hybrid (C57BL/6j×CBA/Ca) mice were cultured for 8 (preantral/antral follicles) and 12 (preovulatory follicles) days in increasing concentrations of B[a]P (0 ng/mL [control] to 45 ng/mL). Expression of aryl hydrocarbon receptor (AhR), aryl hydroxylase steroidogenic enzyme, cell-cycle, and apoptotic genes were quantified. B[a]P exposure significantly (P<0.05) increased mRNA expression of Cyp1a1 in preantral/antral follicles and Cyp1b1, Bax and Hsp90ab1 in preovulatory follicles. No significant effect on mRNA expression of StAR, Cyp11a1, aromatase, Cdk4, Cdk2, Ccnd2, cIAP2, and survivin was observed. In conclusion, this study suggests that B[a]P exposure significantly affects the phase I enzymes and cell death genes during preantral/antral and preovulatory growth, and thus highlight the AhR signaling and apoptotis pathways in delayed follicle growth and decreased viability.

Effect of in utero and lactational nicotine exposure on the male reproductive tract in peripubertal and adult rats

The objective of this study was to determine the effect of in utero and lactational exposure to nicotine on the male reproductive tract. Dams were randomly assigned to receive saline or nicotine bitartrate (1mg/kg-d s.c.) daily for two weeks prior to mating until weaning (postnatal day 21). Male offspring were sacrificed at 7 (peri-pubertal) and 26 (adult) weeks of age. Nicotine-exposure resulted in retention of spermatids after stage VIII, tubular vacuolation, degeneration of pachytene and round spermatids at stage VII in the testes; and lymphocyte infiltration, germ cell exfoliation, and hypospermia in epididymides, at 7 weeks of age. Nicotine-exposure had no effect on testis or epididymal morphology, daily sperm production, epididymal sperm reserve, sperm viability at 26 weeks of age, and circulating testosterone levels at either age examined. We conclude that maternal nicotine-exposure during pregnancy and lactation can induce transient structural changes in the testis and epididymis of male offspring.

Neurotrophins (BDNF and NGF) in follicular fluid of women with different infertility diagnoses.

Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are intra-ovarian signalling peptides that are important in follicle development and oocyte maturation. In the ovary, neurotrophin expression is regulated by gonadotrophins. Therefore, this study postulates that aetiology of infertility will affect follicular-fluid BDNF and NGF concentrations. Follicular fluid from the first follicle aspirated from 190 infertile women attending a university-affiliated fertility programme (McMaster University and ONE Fertility, Burlington, Ontario) was collected between February 2004 and November 2010. The relationship between follicular-fluid BDNF and NGF concentration and age, day-3 FSH and peak serum oestradiol concentrations and antral follicle count was determined. Participants were aged between 24 and 44 years (mean±SEM, 35.2±0.3years) of age. The median concentrations of BDNF and NGF in the follicular fluid was 19.4pg/ml and 344.6ng/ml, respectively. The concentrations of BDNF and NGF were significantly related (P=0.028) but only the BDNF concentration was significantly higher (P<0.05) in women with unexplained infertility compared with other causes of infertility. It is concluded that, apart from unexplained infertility, the underlying cause of infertility did not affect ovarian output of BDNF and NGF in response to ovulation induction.

In vitro exposure to cigarette smoke induces oxidative stress in follicular cells of F1 hybrid mice

This study assessed the influence of cigarette smoke condensate (CSC) and benzo(a)pyrene [B(a)P] on the levels of two oxidative stress biomarkers [8‐isoprostane (8‐IsoP) and 8‐hydroxy‐2‐deoxy Guanosine (8‐OH‐dG)], in in‐vitro spent media of follicle cells. Follicles (100–130 µm) isolated from ovaries of F1 hybrid (C57Bl/6j × CBA/Ca) mice were cultured for 13 days in media exposed to B(a)P [0 ng ml–1 (control) to 45 ng ml–1] or CSC [0 µg ml–1 (control) to 130 µg ml–1]. The concentrations of oxidative stress biomarkers in spent media were quantified by enzyme‐linked immune sorbent assays (ELISA). CSC and B(a)P treatment induced a significant, dose‐dependent increase in the concentrations of 8‐IsoP and 8‐OH‐dG in the spent media. We conclude that CSC and B(a)P exposure can induce oxidative stress in ovarian follicles, an effect that may contribute to the previously documented decline in follicle development and premature ovarian failure in women who smoke.

Screening for DNA adducts in ovarian follicles exposed to benzo[a]pyrene and cigarette smoke condensate using liquid chromatography-tandem mass spectrometry.

A rapid mass spectrometric method was applied to non-targeted screening of DNA adducts in follicular cells (granulosa cells and theca cells) from isolated ovarian follicles that were exposed in-vitro to benzo[a]pyrene (B[a]P) and cigarette smoke condensate (CSC) for 13days of culture. The method employed a constant neutral loss (CNL) scan to identify chromatographic peaks associated to a neutral loss of deoxyribose moiety of DNA nucleosides. These peaks were subsequently analyzed by a product ion scan in tandem mass spectrometry to elucidate structures of DNA adducts. The identification was further confirmed through synthesis of proposed DNA adducts where possible. Three DNA adducts, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide-dG (BPDE-dG), phenanthrene 1,2-quinone-dG (PheQ-dG) and B[a]P-7,8-quinone-dG (BPQ-dG) were identified in the follicular cells from isolated ovarian follicles exposed to B[a]P. Along with these three, an additional DNA adduct, 4-aminobiphenyl-dG, was identified in the follicular cells from isolated ovarian follicles exposed to CSC. The amounts of the identified DNA adducts in follicular cells increased in a dose-dependent manner for both B[a]P (0, 1.5, 5, 15 and 45ng/mL) and CSC (0, 30, 60, 90 and 130μg/mL). The results revealed that B[a]P-related DNA adducts were the major adducts in the ovarian follicular cells exposed to CSC. The results also revealed that two oxidative biomarkers, 8-hydroxy-2-deoxy guanosine (8-OH-dG) and 8-isoprostane (8-IsoP), in both B[a]P-exposed and CSC-exposed ovarian follicles had strong correlations with the three DNA adducts, BPDE-dG, BPQ-dG and PheQ-dG. A pathway to describe formation of DNA adducts was proposed based on the DNA adducts observed.

Ovarian Toxicity of Environmental Contaminants: 50 Shades of Grey

Exposure to environmental contaminants is thought to be important in the development of adverse effects on reproductive health. While the adverse effects of environmental contaminants on semen quality and testicular function have been well studied, effects on ovarian function are less well defined. Epidemiological studies have linked exposure to environmental contaminants with adverse effects on menstrual cycle characteristics, infertility, and earlier age of menopause onset; yet direct evidence of effects on ovarian function is lacking. Environmental contaminant concentrations have been quantified in human ovarian follicular fluid establishing target tissue exposure; however, such data is sporadic and limited to women undergoing assisted reproductive therapies making generalization of results to the broader population of women difficult. We note that the relationship between serum and follicular fluid concentrations can be orders of magnitude different and thus target tissue distribution requires further study. Animal studies revealed effects of environmental contaminants on ovarian follicle dynamics, oocyte maturation, steroidogenesis, and epigenetic changes. Issues of dosing such as concentration of test chemicals used, route of administration, and use of multiple dose groups remain important limitations of the current literature. While animal studies establish a basis for biological plausibility of effects and support conclusions of reproductive hazard, we conclude that exposures in the general human population are too low to present a demonstrable risk to human ovarian function.