Cigarette Smoking and Pregnancy I: Ovarian, Uterine and Placental Effects

Placenta (1999), 20, 265–272

CURRENT TOPIC Cigarette Smoking and Pregnancy I: Ovarian, Uterine and Placental Effects K. T. Shivericka,c and C. Salafiab a

Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, USA Department of Pathology, Columbia University College of Physicians and Surgeons, USA Paper accepted 9 November 1998

b

This review examines the major observations and principal controversies relating to the effects of smoking and the constituents of tobacco on ovarian, uterine and placental tissues. Maternal exposure is assessed relative to specific tobacco-related chemicals and the feto-placental impact of mutagenic products, in addition to nicotine replacement as a pharmacological intervention for smoking cessation. Important new information is being learned from clinical in vitro fertilization and assisted reproduction technologies regarding the effects of smoking on fertility. Present evidence supports an adverse effect of smoking on ovarian function which is prolonged and dose-dependent, whereas there appear to be more reversible effects on implantation and ongoing pregnancy. The anti-oestrogenic effect of smoking is reviewed in terms of direct effects of nicotine, cadmium and polyaromatic hydrocarbons on oestrogen synthesis and metabolism, oocytes and granulosa-luteal function. Innovative new models provide evidence that smoking may alter fertility through effects on uterine-fallopian tube functions which mediate gamete and conceptus transport. It is of interest that smoking is associated with a decreased incidence of uterine fibroids, endometriosis and uterine cancer, which may reflect inhibitory effects of smoke constituents on uterine cell proliferation and extracellular matrix interactions. The increased miscarriage rate among mothers who smoke may be related to direct adverse effects of nicotine, cadmium and polyaromatic hydrocarbons on trophoblast invasion and proliferation. In this respect, alterations in trophoblast differentiation along invasive or proliferative pathways may explain the changes in endocrine function and vascular morphology that are observed in smokers. In summary, significant advances are being made in the understanding of cellular and molecular mechanisms which underlie the differential effects of cigarette smoking on reproductive tissues.  1999 W. B. Saunders Company Ltd Placenta (1999), 20, 265–272

INTRODUCTION Maternal smoking during pregnancy is known to be associated with adverse pregnancy outcomes, including low birth weight, premature delivery, spontaneous abortion, placental abruption, perinatal mortality and ectopic pregnancy (Cnattingius et al., 1985; Mattison et al., 1989; U.S. Department of Health and Human Services, 1990; Ananth, Savitz and Lutther, 1996; Andres, 1996; Ahluwalia, Grummer-Strawn and Scanlon, 1997; Wang et al., 1997). Infants born to mothers who smoke are an average of 200 g lighter at birth than infants of non-smokers, even after adjustment for gestational age. Although this may seem only a modest decrease in birthweight, there are also increased postnatal morbidity and mortality relating to deficits in pulmonary function and neuroc

To whom correspondence should be addressed at: Dept. Pharmacology & Therapeutics, University of Florida, Box 100267, 1600 SW Archer Rd, Gainesville, FL 32610-0267, USA. E-mail: [email protected] 0143–4004/99/040265+08 $12.00/0

cognitive development (Picone et al., 1982; Naeye, 1992; Economides and Braithwaite, 1994; MacDorman et al., 1997), topics which are not the subject of this review. An intriguing aspect of the association between smoking and reduced birthweight is the dose relationship and reversibility, in that women who cease smoking before 30 weeks gestation have heavier infants (MacArthur and Knox, 1988). This review of pregnancy and smoking is in two parts. This first section focuses on the recent evidence for effects of smoking and the constituents of tobacco on ovarian, uterine and placental function. Important new information is being learned from clinical in vitro fertilization and assisted reproduction technologies regarding the effects of smoking on fertility (Pattinson, Taylor and Pattinson, 1991; Hughes and Brennan, 1996; van Voorhis et al., 1996; Zenzes, Reed and Casper, 1997). The second part focuses on the vascular effects of smoking related to the obstetric complications of spontaneous abortion, placental abruption and pre-eclampsia during pregnancy. It is of particular interest to the authors that smoking is associated with a  1999 W. B. Saunders Company Ltd

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decreased incidence of uterine fibroids, endometriosis and uterine cancer (Cramer et al., 1986; Baron, 1996), as well as with a decreased risk of pre-eclampsia, one of the most serious complications of pregnancy (Klonoff-Cohen, Edelstein and Savitz, 1993; Baron, 1996). This review will explore possible mechanisms which may underlie the seemingly ‘protective’ effects of cigarette smoking on uterine disease and pregnancy. The question of whether passive exposure to environment tobacco smoke (ETS) is a risk for adverse pregnancy outcomes is of importance, but has not been resolved sufficiently to be addressed in this review.

EXPOSURE Epidemiological research into the reproductive effects of cigarette smoking has been limited in that assessment of exposure in most studies is based on self-reported smoking patterns. In this regard, questionnaire-based assessments may introduce ascertainment bias into the data, but usually in the direction of under-reporting of tobacco use. As the serum half-life of nicotine is only 2 h compared with 16 h for cotinine, the major metabolite, levels of cotinine are used as a more sensitive and reliable indicator for daily smoke exposure. Cotinine levels in maternal and neonatal hair show significant differences between active smokers, passive smokers and non-smokers (Eliopoulos et al., 1996), and cotinine concentrations in maternal urine are inversely related to infant birth size (Wang et al., 1997). At least 60 toxic compounds are among the 4000 chemical constituents identified in tobacco smoke and include polycyclic aromatic hydrocarbons (PAHs), nitroso compounds, aromatic amines and protein pyrolysates (Koop, 1984). Two major classes of mutagenic agents are nitrosamines and PAHs. Mean concentrations of benzo(a)pyrene, a major PAH, and declared tar, nicotine and carbon monoxide levels per cigarette have been documented in 35 major brands (Kaiserman and Rickert, 1992). The presence of NNK, the tobaccospecific carcinogenic nitrosamine 4-(methyl-nitrosamino)-1-(3pyridyl)-1-butanone, in cervical mucous of smokers (Prokopczyk et al., 1997) has been linked with findings of DNA damage in cervical epithelium and cervical dysplasia, as well as increased risk for cervical cancer in smokers (Winkelstein, 1990; Simons, Phillips and Coleman, 1993). Cigarette use during pregnancy has been related to the presence of smoking and benzo(a)pyrene-related DNA adducts in human term placentas (Everson et al., 1986; Manchester et al., 1988), indicating environmental exposure that damages human DNA. Potent tobacco-related carcinogens also appear to cross the placenta based upon evidence that 4-aminobiphenyl binds covalently to fetal haemoglobin in significantly higher concentrations in smokers (Myers et al., 1996), and a derivative of the carcinogen NNK was found in the urine of infants born to smokers (Hecht et al., 1998). Moreover, tobacco smoke induces placental and fetal enzyme systems capable of bioactivation of pro-carcinogens to carcinogenic and mutagenic products (Pasanen et al., 1988; Sanyal et al., 1993).

Increased emphasis is being placed on smoking cessation during pregnancy using pharmacological intervention with nicotine replacement, yet little has been reported regarding safety or efficacy of nicotine replacement in pregnant women or their fetuses. When compared to hourly cigarette smoking, the short-term use of a transdermal nicotine patch (21 mg) resulted in similar maternal plasma nicotine levels and similar changes in fetal cerebral artery resistance indices, an indirect measure of fetal hypoxia (Oncken et al., 1997). The finding that salivary cotinine concentrations during transdermal replacement were lower in pregnant women than reported in smoking or non-smoking non-pregnant adults (Wright et al., 1997) suggests that nicotine may be less rapidly metabolized in pregnant women. While it is assumed that the benefits of nicotine replacement far outweigh the known risks of maternal cigarette smoking, animal studies show that nicotine alone has significant adverse effects on fetal development (Navarro et al., 1989; Slotkin et al., 1995; Slotkin, 1998). In this context, it has been proposed that episodic nicotine replacement (i.e. gum, inhalers, overnight removal of transdermal patches) may be advantageous in producing lower peak blood levels and more frequent recovery periods between fetal exposures (Oncken et al., 1996; Slotkin, 1998). Evidence supports several hypotheses to explain associations between maternal smoking and fetoplacental alterations (Cnattingius and Nordström, 1996; Soothill et al., 1996). First, changes in uteroplacental or fetoplacental blood flow may be related to the vasoconstrictive effects of nicotine (Andersen and Hermann, 1984). Doppler studies suggest that cigarette smoke increases resistance in fetoplacental circulation, but reduces resistance in uteroplacental vessels (Morrow, Ritchie and Bull, 1988). In this respect, placentae of smokers have morphological features consistent with underperfusion (van der Velde et al., 1983; Burton, Palmer and Dalton, 1989; Naeye, 1989; Jauniaux and Burton, 1992). Second, chemicals within tobacco smoke exert direct toxic effects on fetal and placental cells as evidenced by the presence of altered enzyme expression and the presence of protein and DNA adducts (Everson et al., 1986; Manchester et al., 1988; Pasanen et al., 1988; Sanyal et al., 1993; Myers et al., 1996). Third, smoking induces fetal hypoxia through diffusion of carbon monoxide, nicotine and thiocyanate across the placenta. The enhanced ability of fetal haemoglobin to form carboxy-haemoglobin decreases the fetal transport of oxygen (Soothill et al., 1996).

FERTILITY Hughes and Brennan (1996) recently conducted a metaanalysis to assess the effects of female and male smoking on natural and assisted fertilization. In 13 studies of natural conception, all but one demonstrated a negative association between smoking and time to conception and live birth rate. Smoking one pack of cigarettes per day and starting to smoke before 18 years of age were further associated with an increased risk of infertility, providing evidence of dose- and age-related

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effects on natural fertility (Laurent et al., 1992). In this regard, assisted reproduction cycles offer a unique opportunity to test ovarian function and fertility in smokers and non-smokers. Ovarian function can be assessed by measuring blood steroid hormone levels, oocyte numbers and oocyte quality following the administration of a known stimulatory dose of gonadotropins. In the meta-analysis by Hughes and Brennan (1996), seven studies of sub-fertile women undergoing assisted reproductive technologies showed small but consistent reductions in pregnancy rates among smokers. Recent assisted reproduction studies have begun to identify differential effects of smoking on ovarian function compared to implantation and the maintenance of pregnancy. Using questionnaires to document tobacco use, van Voorhis et al. (1996) found that current and past smokers had reduced serum oestradiol concentrations, numbers of retrieved oocytes, and numbers of embryos. Moreover, women who smoked during their treatment cycle (current smokers) had approximately a 50 per cent reduction in implantation rate and on-going pregnancy rate compared with women who had never smoked, despite the transfer of an equal number of embryos during their cycle. At the same time, women who quit smoking before their treatment cycle had the same pregnancy rate as nonsmokers, suggesting some reversible effects. In comparison, a retrospective analysis of early IVF trials by Pattinson et al. (1991) found no significant difference in peak oestradiol levels, the number of eggs retrieved, fertilization rate or implantation rate. The incidence of spontaneous abortion was higher in smokers than in non-smokers, however, making the delivery rate per cycle (successful pregnancies) significantly lower in smokers. In a third type of study, Sterzik et al. (1996) evaluated tobacco use based upon cotinine concentrations in follicular fluid and included only women with normal ovulatory cycles whose infertility was strictly due to a tubal factor. Under these selection criteria, serum oestradiol levels were decreased significantly in smokers, yet no significant differences were found in fertilization and pregnancy rates. Older women undergoing IVF had an average reduction of 50 per cent in the number of mature oocytes and smoking, as measured by cotinine concentrations in follicular fluid, further reduced the number of mature oocytes (Zenzes, Reed and Casper, 1997). As seen with these representative studies, evaluation of the influence of smoking status on fertility in women undergoing IVF is subject to a number of confounding variables, including methods for assessment of tobacco exposure, the cause of infertility, and the success rates of each individual laboratory, as well as age-related changes in the number and quality of retrieved oocytes. Despite substantial variability in patient cohort selection and clinical parameters for assisted reproductive cycles, some interesting themes have developed from studies on the influence of tobacco use on fertility. First, smoking appears to be related to a prolonged and dose-dependent adverse effect on ovarian function in past and current smokers. Second, smoking may have a more transient effect on implantation and ongoing pregnancy in that current smokers had a markedly decreased

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pregnancy rate after treatment cycle when compared to nonsmokers, as well as to women who quit smoking before initiating treatment (van Voorhis et al., 1996).

OVARIAN AND ANTI-OESTROGENIC EFFECTS Epidemiological studies indicate that cigarette smoking is associated with an anti-oestrogenic effect in women (see Baron, La Vecchia and Levi, 1990, for review). It is well accepted that menopause occurs at least 1 to 1.5 years earlier in current smokers compared with women who have never smoked. Several mechanisms have been proposed for tobacco effects on the ovary. Both extracts of cigarette smoke and nicotine produced a direct inhibition of granulosa cell aromatase activity in vitro (Barbieri, McShane and Ryan, 1986), an effect which has been suggested to be responsible for the decrease in serum oestradiol concentrations observed in women undergoing ovarian stimulation. However, others have not found an inhibitory effect of cotinine or nicotine on oestradiol secretion by human granulosa cells obtained from IVF patients (Weiss and Eckert, 1989). Pregnant women who smoke have decreased blood and urine levels of oestriol and oestradiol (Boyce et al., 1975; Mochizuki et al., 1984; Bernstein et al., 1989). In this regard, nicotine and cotinine directly inhibited aromatase in human trophoblasts in culture in a dose-dependent fashion (Barbieri, Gochberg and Ryan, 1986). In addition, both aromatase activity and cytochrome P-450 aromatase concentration in term placentae from smokers were significantly decreased (Kitawaki et al., 1993). Thus, there is evidence for acute and chronic inhibition of steroidogenic function and oestrogen synthesis by cigarette smoke and its constituents. It has been proposed that inhibition of granulosa-luteal cell function may lead to corpus luteal deficiency which could underlie the increase in early pregnancy loss observed in smokers. Smoking also appears to affect steroid hormone metabolism. The observation that the 2-hydroxylation of oestradiol is increased in smokers has lead to the proposal that increased catechol oestrogen formation is a mechanism for the antioestrogenic effect of smoking (Michnovicz et al., 1986). Placental microsomes of smokers have increased 2- and 4-hydroxylation of oestradiol (Juchau, Namkung and Chao, 1982). In this respect, PAHs in cigarette smoke may induce microsomal cytochrome P-450s which metabolize steroid hormones, possibly enhancing the formation of catechol metabolites of oestradiol (Chao et al., 1981; Juchau, Namkung and Chao, 1982). Cigarette smoke extracts cause ovarian atresia in rodents (Mattison et al., 1989; Mattison, 1980). A potential toxin to the ovary is the heavy metal cadmium which is present in high concentrations in tobacco and accumulates in ovarian tissue of smokers (Varga et al., 1993). Both cadmium and cotinine levels in ovarian follicular fluid of women in IVF therapy were correlated with the level of cigarette smoking (Zenzes et al., 1995). In rats, cadmium is a potent ovarian toxin associated with alterations in the meiotic maturation of oocytes (Watanabe

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and Endo, 1982). PAHs present in cigarette smoke represent another potential source of chemicals toxic to the ovary. In this regard, benzo(a)pyrene was detected as a DNA adduct in granulosa-lutein cells of women undergoing IVF and embryo transfer who were exposed to cigarette smoke (Zenzes, Puy and Bielecki, 1998). The prototype PAHs 7,12dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene cause oocyte destruction in mice and alter the formation of corpora lutea (Mattison, 1980; Mattison, White and Nightingale, 1980). Recent in vitro studies with human and mouse oocytes further demonstrated that DMBA produced a dose-dependent induction of programmed cell death (apoptosis) in germ cells, as well as in mouse granulosa cells (Tilly et al., 1997). The observation that mouse oocytes deficient in expression of bax, a key regulatory gene in the apoptotic pathway, were resistant to DMBA toxicity is further evidence that PAH-mediated oocyte destruction involves activation of genes involved in programmed cell death (Perez et al., 1997; Tilly and Perez, 1997). Thus, ovotoxicants may activate the same cell death regulatory pathways utilized by ovarian cells under normal physiological conditions, which may explain the early menopause in women who smoke cigarettes.

UTERINE AND IMPLANTATION EFFECTS There is increasing evidence that smoking may alter fertility by effects on uterine-fallopian tube functions which mediate gamate and conceptus transport. Smoking increased the resting tone of the uterus and the amplitude of uterine contraction in women (Neri and Eckerling, 1969). Nicotine administered to rhesus monkeys significantly altered oviductal tone and contraction amplitude (Neri and Marcus, 1972). Talbot and co-workers recently demonstrated an in vivo effect of both mainstream and sidestream smoke exposure on oviductal ciliated cells and corpora lutea in hamsters (Magers et al., 1995). Further in vitro studies showed an inhibitory effect of smoke solutions on ciliary beat frequency and oocyte pick-up rate which was correlated to the presence of potassium cyanide at relevant concentrations (Knoll and Talbot, 1998; Talbot et al., 1998). Thus, innovative new models are providing evidence that smoking may impair fertility through alterations in utero-tubal motility or the rate of oocyte, sperm or conceptus transport, factors which may underlie the reported increase in risk for ectopic pregnancy in smokers (Saraiya et al., 1998). Little is known regarding the effects of smoking on the response of the human uterine endometrium to steroid hormones, nor on the ability of the conceptus to implant. In this respect, smokers have as much as a 50 per cent lower risk of endometrial cancer compared with non-smokers, as well as a decreased incidence of endometriosis and uterine fibroids (Cramer et al., 1986; Baron, La Vecchia and Levi, 1990; Baron, 1996). Although factors such as weight, diet, parity, alcohol use and early menopause in smokers may exert an antioestrogenic effect, post-menopausal women who smoke still exhibit a substantially lowered risk of endometrial cancer

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(Franks et al., 1987). It is intriguing to ask how cigarette smoke is seemingly ‘protective’ against benign and malignant uterine disorders, while at the same time being associated with impaired implantation and pregnancy rate. In some studies, infertility in women who smoke has been linked to fallopian tube dysfunction and pelvic infections (Phipps et al., 1987). There is, however, some evidence for direct effects of constituents of cigarette smoke on uterine tissue. Nicotine administration to rats produced an adverse effect on the decidualization process (Card and Mitchel, 1978), as well as delayed implantation and altered spacing of implantation sites (Yoshinaga et al., 1979). These nicotine effects, however, may also reflect delayed utero-tubal transport or altered steroid hormone levels in pregnant rats. Our in vitro data show that exposure of human RL95-2 endometrial adenocarcinoma cells to benzo(a)pyrene markedly decreased proliferation, cell attachment and invasion of basement membrane (Charles, Grant and Shiverick, 1998). The benzo(a)pyrene-mediated alterations in endometrial cell attachment appear to be linked with a loss of cell surface expression of receptors for epidermal growth factor (EGF) and decreased levels of E-cadherin, a cell adhesion molecule essential for cell to cell communication (Shiverick, Charles and McGarry, 1999). The observed inhibitory effect of benzo(a)pyrene on endometrial cell proliferation and interaction with extracellular matrix and adjacent cells could explain the decreased incidence of uterine cancer and endometriosis found in cigarette smokers. The RL95-2 human uterine epithelial cell line is distinct from other endometrial cell lines in that it exhibits an adhesiveness of its apical pole for trophoblast and thereby serves as an in vitro model for human uterine epithelium which is receptive for implantation (Thie et al., 1997). In this regard, it is tempting to speculate that the benzo(a)pyrene-mediated loss of cell adhesion molecules serves as a biomarker for alterations in uterine epithelial cell polarization which may have an impact on trophoblast implantation.

PLACENTAL EFFECTS ON TROPHOBLAST INVASION AND PROLIFERATION The increased miscarriage rate among mothers who smoke may be as high as 33 per cent (Cnattingius et al., 1985; Economides and Braithwaite, 1994; Ananth et al., 1996; Andres, 1996; Ahluwalia, Grummer-Strawn and Scanlon, 1997; Wang et al., 1997). Jauniaux and Burton have described increased syncytial necrosis and increased thickness of the syncytio/cytotrophoblast membrane in early pregnancy in mothers who smoke (Poppe et al., 1995). During early pregnancy, stem cells either fuse to form the syncytium or aggregate to form cell columns that first adhere to and then invade the uterus. Experimental data indicate that smoking appears to induce a generalized dysfunction of both villous and invasive trophoblasts in early pregnancy. Biochemical markers of placental function show that maternal levels of oestriol, oestradiol, human chorionic gonadotropin (hcG), and human

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placental lactogen (hPL) were lower in smokers compared to non-smokers (Boyce et al., 1975; Mochizuki et al., 1984; Bernstein et al., 1989). As described above, placental aromatase activity has been found to be decreased in vivo in smokers, as well as in vitro by exposure to nicotine and cotinine (Barbieri, Gochberg and Ryan, 1986, Kitawaki et al., 1993). The heavy metal cadmium is a known placental toxin in both animals and humans (Eisenmann and Miller, 1996). Placental levels of cadmium are increased in smokers (Kuhnert et al., 1982). In the perfused human placenta model, cadmium produced ultrastructural changes and decreased the secretion of hCG (Wier et al., 1990). Cadmium was further found to inhibit cell proliferation of trophoblastic choriocarcinoma cells through interactions with calmodulin, an intracellular calcium binding protein (Powlin, Keng and Miller, 1997). A consistent finding has been the presence of smokingrelated alterations in placental receptors for EGF, a potent mitogen and differentiation factor for trophoblasts. Placentae from smokers show a marked decrease in EGF receptor tyrosine kinase activity, whereas insulin receptor phosphorylation was normal or increased (Lucier et al., 1987; Wang et al., 1988; Gabriel, Alsat and Evain-Brion, 1994). Exposure of human placental cells in primary culture to PAHs directly resulted in concentration-dependent loss of EGF-receptor binding and kinase activity, which was greatest in cells from first trimester placentae (Guyda et al., 1990). In first trimester placental villus explants grown on Matrigel, benzo(a)pyrene significantly decreased EGF receptor levels as well as hCG secretion (Andjelkovic et al., 1995). Further studies with human trophoblastic choriocarcinoma cell lines have shown that the benzo(a)pyrene-mediated loss of EGF receptors is linked not only with decreased trophoblast proliferation and hCG secretion (Zhang et al., 1995), but also with decreased expression of the proto-oncogene c-myc, whereas TGF-â1 expression is increased (Zhang and Shiverick, 1997a). Thus, evidence is mounting to indicate that constituents of tobacco smoke have the potential to alter trophoblast gene expression in the direction of downmodulation of positive regulators (EGF-R, c-myc) and upmodulation of negative regulators (TGF-â1) of trophoblast function. In the study of chorionic villous samples from early gestation, a key observation is that the formation of trophoblast cell columns was markedly reduced in smokers, and this defect was not reversed when villous explants were grown in culture (Genbacev et al., 1995). When chorionic villi from nonsmokers were exposed to nicotine in vitro, there was a doserelated inhibition of cell column formation and reduced invasion of basement membrane, apparently related to decreased cytotrophoblast synthesis and activation of the 92 kDa Type IV collagenase (Genbacev et al., 1995). Recent studies with cultured trophoblasts demonstrated that hypoxic conditions inhibit the ability of cytotrophoblasts to differentiate along the invasive pathway (Genbacev et al., 1997). In this model, cells from pre-eclamptic placentae were less invasive and failed to modulate integrin, collagenase and HLA-G expression in culture (Lim et al., 1997). Given the evidence that

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oxygen tension determines whether cells proliferate or invade, it is likely that smoking-induced hypoxia may be a factor in altered trophoblast differentiation. In addition, in vitro exposure of trophoblastic choriocarcinoma cells to benzo(a)pyrene significantly inhibited cell invasion of basement membrane (Zhang and Shiverick, 1997b). In this respect, alterations in trophoblast differentiation along invasive or proliferative pathways may explain the changes in endocrine function as well as vascular morphology that are observed in smokers.

IMMUNE EFFECTS While there is little evidence to date, smoking effects on maternal immune function may have a negative impact on pregnancy. Cigarette smoke is associated with increased peripheral leukocyte counts in adults (Schwartz and Weiss, 1991), and pregnant women who smoke have elevated levels of neutrophils, monocytes and lymphocytes (Mercelina-Roumans et al., 1994). The maternal haematological profile, however, is in direct contrast with that observed in neonates of smoking mothers in that total leukocyte counts in cord blood were not altered, while neutrophil levels were significantly decreased (Mercelina-Roumans et al., 1996). Uterine cervical mucous of smokers contains constituents of cigarette smoke, including the nitrosamine NNK (Prokopczyk et al., 1997), and smoking is associated with a decreased number of cervical Langerhans’ cells which mediate local immune responses in the genital tract (Poppe et al., 1996). Recent concern has been expressed as to whether the association between cigarette smoking and obstetric complications of uterine bleeding, placental abruption and premature rupture of membranes provides a basis for maternal-fetal transfer of infectious agents. Several large studies have found the risk of vertical transmission of HIV-1 to be significantly increased among women who smoke during pregnancy, which was independent of illicit drug use (Burns et al., 1994; Turner et al., 1997). However, the association between smoking and HIV transmission was not demonstrated in a third recent study in which smoking was strongly linked with the use of hard drugs (Kalish et al., 1998). Thus, further research is critical to elucidate the extent to which smokingrelated alterations in maternal immune function and placental lesions may be risk factors in perinatal transmission of infectious disease.

CONCLUSIONS This review has attempted to be representative of the major observations and principal controversies relating to the effects of maternal smoking on ovarian, uterine and placental tissues. In this respect, new information is becoming available regarding the effects of hypoxia on human placental cytokine expression (Conrad and Benyo, 1997) and cytotrophoblast differentiation/invasion pathways (Lim et al., 1997) which will serve to define mechanisms by which constituents of cigarette

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smoke may alter placental gene expression and signal transduction pathways. It may be more difficult to determine whether the anti-oestrogenic effects of smoking alter adaptive mechanisms of pregnancy, including placental perfusion. Finally, an unresolved issue is how smoking is associated with an increased risk of cervical cancer concurrent with a decreased

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incidence of benign and malignant uterine disease. A significant advance will be made with the identification of the cellular and molecular mechanisms which underlie these differential effects of cigarette smoking on reproductive tissues, which most certainly have an impact on fertility and pregnancy.

ACKNOWLEDGEMENTS The authors wish to acknowledge support from the National Institute of Health (K.T.S. and C.S.) and the NIEHS Superfund Basic Research Program (K.T.S.).

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