Several epidemiological studies have shown a strong association between cigarette smoking and impaired ovarian function. In fact, smoking women may experience increased rates of pregnancy loss and earlier age at menopause. Although the link between cigarette smoking and complications of pregnancy has been largely proved, very little is known about the effect of smoking on fertility. Cigarette smoking is associated with a reduced monthly probability of conception in natural cycles and with a reduced chance for pregnancy in assisted reproduction cycles.

Nicotine and its major metabolites have been found in follicular fluids of women undergoing assisted conception in a dose-related association with cigarette consumption. Interestingly, cotinine, a nicotine metabolite, was found in ovarian granulosa-luteal cells in a dose-dependent relationship with its follicular fluid levels. Binding of this alkaloid to nuclear and cytoplasmatic proteins could affect the developmental potential of maturing follicles and lead to perturbation in meiotic maturation of oocytes. Viagra Super Active

Whereas the developing follicle as well as the oocyte have been extensively indicated as possible targets of nicotine, the effects of nicotine on the luteal phase remain mostly unexplored. Because CL is the main endocrine gland supporting both the luteal phase and early pregnancy, we wanted to investigate the possible effect of nicotine on human luteal steroidogenesis. Interestingly, we found that both nicotine and M-nicotine were able to affect steroidogenesis significantly by inhibiting the release of progesterone by human luteal cells. This effect was demonstrated statistically even at the lowest tested concentrations. In these experiments, nicotine and M-nicotine concentrations ranged between the cotinine levels observed in the blood and follicular fluid of smoking women. However, neither nicotine nor M-nicotine was able to affect the hCG-induced production of progesterone.

The mechanism through which hCG stimulates progesterone production is well known and widely described. Hormone/receptor interaction results in cAMP formation and arachidonic acid release; these molecules act in a synergistic manner to induce steroid production by increasing steroidogenic acute regulatory protein (StAR) gene expression.

On the other hand, a variety of nonneuronal tissues have been demonstrated recently to express nicotinic acetylcholine receptors (nAChRs). In human umbilical vein endothelial cells, nAChR-induced action has been described as being completely dependent on the phosphati-dylinositol 3-kinase and mitogen-activated protein kinase pathways. Despite that report, the mechanisms underlying nAChR signaling and its downstream pathways remain mostly unknown. Therefore, new studies regarding signaling cascades are necessary to understand if the inhibitory effect of nicotine on progesterone production is not able to overcome the hCG-activated pathway or if nicotine and hCG act through two different, noninterfering metabolic pathways.

The next step was to investigate the mechanisms through which nicotine reduced progesterone production. The PGs are widely described as playing a key role in the complex regulation of CL physiology. Human CL produces both PGE2 and PGF2a in different ways during the distinct luteal phases. In a large number of species, including humans, PGF2a seems to be important for luteolysis, but accumulating evidence suggests a luteotropic effect of PGE2. In the present study, we demonstrated that both nicotine and M-nicotine were able to significantly increase PGF2a release and to reduce PGE2 production by luteal cells, even at the lowest tested doses.

It has been demonstrated previously that PGF2a from 1011 to 10~6 M is able to reduce significantly progesterone production by granulosa-luteal cells in vitro. Release of PGF2a induced by nicotine and M-nicotine in human isolated luteal cells ranged between 10~9 and 10~8 M according to our results. Therefore, it is tempting to speculate that nicotine and its metabolite could influence luteal steroidogenesis by modulating the PG system.

Other in vitro studies have investigated the effect of smoking on human granulosa-luteal cells and produced conflicting final data. Barbieri et al. demonstrated that in vitro exposure to low-molecular-weight constituents of tobacco-smoke extracts inhibited granulosa cell aromatase. In contrast, Weiss and Eckert found that neither nicotine nor cotinine influenced progesterone or estradiol secretion by granulosa cells, whereas Bodis et al. found a dose-dependent increase in estradiol secretion and a decrease in progesterone production by granulosa cells in response to nicotine treatment.

Our results, obtained using isolated human luteal cells, are in agreement with those of Bodis et al. The discrepancies with the other reports could depend on the different cell type used in each study. Although granulosa-luteal cells share some similarities with human luteal cells, these two cell types differ for both anatomical and biochemical properties.

Finally, we investigated VEGF mRNA expression in luteal cells in response to nicotine and M-nicotine treatments. In human ovaries, angiogenesis is associated with follicular development and CL formation. Among the several factors involved in angiogenesis, VEGF has been described to be a potent proangiogenic factor In addition to its stimulating effect on endothelial cell growth in vitro, this cytokine is also a blood vessel permeability inducer in vivo. The presence of VEGF and its specific receptor, the fms-like tyrosine kinase (Flt-1), has been demonstrated recently in human granulosa and theca-luteal cells. Concentrations of VEGF in luteal extracts were higher in the early and midluteal phases and tended to decrease toward the late luteal phase, suggesting a VEGF steroidogenic support through vascularization promotion. Interestingly, VEGF correlation with cell hypoxic conditions has been widely reported, and Ferrara et al. demonstrated VEGF mRNA up-regulation in rat hypoxic luteal cells. On the other hand, the hypoxic in vivo nicotine effect is well known, even though the mechanism by which it occurs is still not completely understood.

In the present study, we found that nicotine significantly increased VEGF mRNA expression in human luteal cells but that M-nicotine did not. How nicotine can actually induce VEGF expression on human isolated luteal cells has not yet been clarified; therefore, more details about molecular pathways are necessary to address the discrepancy between nicotine and M-nicotine regarding the induction of VEGF expression.

In conclusion, we demonstrated the ability of nicotine and M-nicotine to affect production of progesterone and PGs as well as expression of VEGF in human isolated luteal cells. Based on these results, we can speculate that nicotine and M-nicotine could influence CL physiology by modulating both its steroidogenic activity (probably through the PG system) and its vascularization. Furthermore, we can speculate that a CL deficiency may be one of several mechanisms through which nicotine can cause infertility and early pregnancy loss.