Ovotoxicity of cigarette smoke: A systematic review of the literature

Accepted Manuscript Title: Ovotoxicity of cigarette smoke: a systematic review of the literature Authors: Maria Cristina Budani, Gian Mario Tiboni PII: DOI: Reference:

S0890-6238(17)30136-3 http://dx.doi.org/doi:10.1016/j.reprotox.2017.06.184 RTX 7529

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Reproductive Toxicology

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14-3-2017 25-6-2017 28-6-2017

Please cite this article as: Budani Maria Cristina, Tiboni Gian Mario.Ovotoxicity of cigarette smoke: a systematic review of the literature.Reproductive Toxicology http://dx.doi.org/10.1016/j.reprotox.2017.06.184 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Ovotoxicity of cigarette smoke: a systematic review of the literature

Maria Cristina BUDANI and Gian Mario TIBONI

Department of Medicine and Aging Sciences, University “G. d’Annunzio” of Chieti-Pescara, Italy

Correspondence to: Gian Mario Tiboni, MD Department of Medicine and Aging Sciences University “G. d’Annunzio” of Chieti-Pescara Via dei Vestini 66013 Chieti, Italy. Tel: +39 0859172390 e-mail: [email protected]

Highlights

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Cigarette smoke negatively affects ovarian folliculogenesis.

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The hypothesized causes of ovarian damage include increased oxidative stress, increased cellular apoptosis or autophagy, DNA damage and abnormal crosstalk between oocyte and granulosa cells.

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Cigarette smoke may hamper the outcome of in vitro fertilization programs

Abstract This study reviews the scientific literature on the noxious effects of cigarette smoke on the ovarian follicle, and the cumulative data on the impact of smoking on in vitro fertilization (IVF) cycle outcome. There is a close association between tobacco smoke and accelerated follicle loss, 1

abnormal follicle growth and impairment of oocyte morphology and maturation. There is an increasing amount of evidence indicating that smoke can directly derange folliculogenesis. Increased cellular apoptosis or autophagy, DNA damage and abnormal crosstalk between oocyte and granulosa cells have been implicated in the demise of ovarian follicles. It becomes increasingly clear that maternal smoking can exert multigenerational effects on the ovarian function of the progeny. Growing evidence suggests that cigarette smoke is associated with decreased results after IFV. Further research is needed to better define the molecular mechanisms behind smoking-induced ovarian disruption.

Key words: cigarette smoke, ovarian toxicity, follicle depletion, premature ovarian failure, in vitro fertilization.

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Introduction Smoking is a worldwide issue. About 5.8 trillion cigarettes were smoked worldwide in 2014 [1]. It has been estimated that approximately 30 % of women and 35% of men of reproductive age smoke cigarettes in the U.S.A [2]. About 177 million of women worldwide are smokers [3]. Cigarette smoke contains about 4000 chemical substances, with several of them showing toxic effects, including mutagenic and carcinogenic properties. Toxicants present in cigarette smoke include polycyclic aromatic hydrocarbons (PAHs), such as benzoapyrene (BaP), nitrosamises, heavy metals (cadmium), alkaloids (nicotine and its major metabolite, cotinine) and aromatic amines. Owing to the different properties and targets of the chemical compounds contained in cigarettes, smoke appears to exert hazardous effects on the entire reproductive system in women and on each stage of the reproductive function [4]. It has been documented that cigarette smoke exposure can affect steroidogenesis [4, 5], folliculogenesis [6-11], embryo development [4, 12, 13] and implantation [14, 15]. Toxic effects of cigarette smoke on tubal and endometrium function [4] and on endometrial angiogenesis [16] have been also documented. Active smoking was associated with delayed conception [17-19] and early menopause [20-23]. Among the 4000 toxic chemicals contained in cigarettes, nicotine with its metabolite (cotinine), PAHs and the heavy metal cadmium are the most intensively studied in relation to the issue of smoke-induced ovarian toxicity [9,10, 24, 25, 26, 27]. The deranging effects of smoking on fertility are of particular concern not only for couples naturally attempting to conceive [28], but also for women undergoing IVF. Several studies investigated whether cigarette smoke exposure influences IVF outcome, reporting controversial results. While several reports have expressed support to the concept that smoke exposure can impair IVF success at different levels [14, 29-34], others [35-38] failed to find an impact. This study aims to systematically review the effects of tobacco smoke on folliculogenesis and oocyte quality, and on 3

molecular mechanisms behind cigarette smoke-mediated ovotoxicity. The literature concerning the influence of smoking on IVF outcome was also reviewed. Methods A systematic search of electronic literature through MEDLINE, EMBASE and Google Scholar databases was performed in order to collect studies about the toxic effects of cigarette smoke on the ovary. In particular, human clinical studies and experimental studies with animals or cell cultures were selected. The following headings and text strings were used alone or in combination: ‘folliculogenesis’, ‘oocytes’, ‘granulosa cells’, ‘ovary’, ‘fertility’, ‘infertility’, ‘premature ovarian failure’, ‘ovary’, ‘cigarette smoke’, ‘smoke components’ ’polyciclic aromatic hydrocarbons’, ‘nicotine’, ‘cotinine’, ‘cadmium’, ‘benzo[a]pirene’, ‘dimethylbenz[a]anthracene’, 3methylcholanthrene’, ‘in vitro fertilization’, ‘assisted reproductive technology’. The authors focused on publications examining the effects of cigarette smoke on folliculogenesis and IVF procedures outcome. Only publications written in English were considered. Studies assessing alternative types of smoking (nargile) were excluded. Results Studies selection and characteristics The studies selection process is summarized in Figure 1. The search strategy yielded 1124 publications. 1011 papers were not considered relevant after review of titles and abstracts. Of the 113 remaining publications, 5 were excluded for the following reasons: one study was excluded since it investigated the effects of nargile smoke on female reproduction [39]. Four studies were written in languages different from English [40-43]. 108 studies were included in the systematic review. The studies assessing the effects of cigarette smoking on folliculogenesis are shown in Table 1 and Table 2. Results of recent studies focusing on the effects of cigarette smoke on IVF

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procedures are illustrated in Table 3. Figure 2 illustrates the potential mechanisms involved in cigarette smoke-induced follicle loss. Cigarette smoking and folliculogenesis Cigarette smoking components in follicular fluid Follicular fluid is a liquid produced during ovarian folliculogenesis and filling the follicular antrum. It provides an important microenvironment rich in metabolites, hormones, enzymes, electrolytes, and antioxidant, serving crucial roles for the optimal maturation of granulosa cells and oocytes. Follicular fluid originates by the secretion of granulosa cells and by diffusion from the network of capillaries in the theca interna [44]. Analysis of follicular fluid can offer information about the growth and differentiation of the follicle, the oocyte quality and may contribute to predict embryo quality and early embryonic development [45]. The “so called” blood-follicle barrier, which serves as ‘molecular sieve’, mediates the transport from theca to the follicular antrum [46]. Several studies quantified the presence of tobacco components in follicular fluid. The major metabolite of nicotine, cotinine, was identified in follicular fluid samples of smokers [47-50]. There was an evident correlation between the levels of follicular fluid cotinine and the number of cigarette smoked [51]. Cadmium is a heavy metal present in cigarettes and detectable not only in follicular fluid [52,53], but also in the oocytes of smokers [52]. The tobacco-specific N-nitrosamine 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone (NNK) is carcinogenic in animal models, and may be an important determinant in the initiation smoke-induced lung cancer [54]. Measurable amounts of NNK were detected in cervical mucus of smokers, strengthening the postulation that cigarette smoke may increase the risk of cervical cancer [55]. On the contrary, only small amounts of NNK metabolites appears to reach the follicular compartment, as suggested by Matthews et al. [54] who detected NNK metabolites only in one of the twenty-two follicular fluid samples tested.

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BaP, a polycyclic aromatic hydrocarbon present in cigarette smoke [56], was quantified in serum and follicular fluid of women undergoing IVF treatments exposed to mainstream smoke and nonsmokers [24]. Higher levels of BaP were present in follicular fluids of women exposed to cigarette smoke. BaP levels increased with the increasing daily number of cigarettes [24]. Moreover, women exposed to mainstream smoke had higher levels of BaP in their follicular fluids in comparison to sidestream exposed or non-smoking women. BaP levels were higher in follicular fluids of women who did not conceive versus those who achieved pregnancy [6]. Cigarette smoking effects on follicle growth and steroidogenesis There is convincing evidence that cigarette smoke and tobacco constituents can affect ovarian steroidogenesis and follicle growth. As compared to non-smokers, smokers had urinary estrogen excretion during the luteal phase that was three-time lower [57]. Assessments carried out on human granulosa cells showed that aromatase, the enzyme converting androgens in estrogens, can be inhibited by components of cigarette smoke [58]. Using bovine cells (theca interna and granulosa cells isolated from follicles), Sander et al. [59] demonstrated significant disruptive effects of nicotine on androgen production in theca interna. This effect was noted under exposure levels that were within the µM range detectable in the blood of smokers [59]. The evidence that BaP is detectable in follicular fluid of smokers, prompted research looking at its potential influence on follicle growth and maturation [9, 24]. Neal et al. [24] tested the effects of BaP on isolated rat follicles using a level of exposure representative of the BaP concentration measured in human follicular fluid (1.5 ng ml -1). A significant decrement in follicle growth in vitro was evidenced. None of the follicles treated with BaP reached the stage of pre-antral formation. A gradual increase in concentration of BaP used (1.5, 5.0, 15.0, 45.0 and 135.0 ng ml -1 BaP) for 0-3-5 days of exposure resulted in a dose and time-dependent decrease in the percentages of proliferating follicles. E2 concentrations in the culture media were lower in treatment groups compared with 6

controls. In addition, BaP exposure led to DNA adducts formation in rat ovaries after oral administration of 5.0 mg BaP/kg body weight [8]. The capacity of BaP to form DNA adducts in ovaries was associated with lower implantation and pregnancy rates in smokers. Excess generation of DNA adducts in embryos may also contribute to implantation failure after normal fertilization [60, 61]. Sadeau and Foster [9] tested the effects of BaP and cigarette smoke condensate (CSC) [10] on follicle development and steroidogenesis. Follicles isolated from mice ovaries, and exposed at increasing concentrations of BaP (1.5 ng/ml to 45.0 ng/ml) for 12 days, showed an inhibition of antral follicle development and a decrease of E2 and AMH output. No effects on oocyte growth and nuclear maturation in the surviving follicles were noted [9]. CSC derives from the particulate phase of cigarette smoke, and is composed of toxicants such as nicotine, phenol, nitrosamines and trace metals such as cadmium. Using a mouse isolated follicle culture system mimicking follicle development, early pre-antral follicles were exposed to graded CSC concentrations through ovulation (30, 60, 90 or 130 µg/ml) for 12 days [10]. Findings showed a dose- and time-dependent correlation between CSC exposure and delay of follicle development progression. In addition, steroidogenesis was disrupted in a dose-dependent manner. Treatment-related effects also included a reduction in cumulus-oocyte complex (COC) expansion in both low and high CSC exposure groups, and a concentration-dependent decrease in the percentage of metaphase II oocytes [10]. While BaP alone failed to induce harmful effects on oocytes [9], CSC impaired oocyte maturation [10]. 7,12-dimethylbenzanthracene (DMBA) is another polycyclic aromatic hydrocarbon that exerts disruptive effects on all ovarian follicle types [62,63]. Exposure to this toxicant occurs not only trough cigarette smoke, but also via car exhaust fumes, and burning of organic matter [7]. Treatment of mice with a single high dose of DMBA (80 mg/kg), 3-methylcholanthrene (3-MC) (80 mg/kg), or BaP (80 mg/kg) depleted primordial and primary follicles, with 50% of primordial follicles being 7

loss within one day of DMBA exposure, and within three days of BaP and 3-MC exposure [64]. Since smokers are chronically exposed to tobacco-derived toxic chemicals, Borman et al. 2000 [65] tested the ovotoxicity of low and repeated exposure to DMBA, 3-MC or BaP in B6C3F mice and Fisher 344 rats, revealing that this chronic exposure is more toxic for the ovary than the single high dose exposure. In agreement with previous reports [64], mice were more susceptible than rats [65]. While in mice PAH mainly targeted primordial follicles, secondary follicles were primarily disrupted in rats. It is known that PAHs must undergo metabolic conversion into toxic intermediates to affect ovarian health [7,8, 66-68]. This occurs via activation of the aryl hydrocarbon receptor (AhR) which activates the pro-apoptotic gene Bax and concomitantly increases the expression of the P450 isoforms (mainly cyp1A1 and cyp1B1) catalyzing PHAs bioactivation. This metabolic process take places in the liver, but also in the ovary [7,8,66-68]. Among the PAHs assessed, DMBA was the most potent ovotoxicant. Studies carried out by Rajapaksa et al.[68] and Igawa et al.[7] using an in vitro ovarian culture system, highlighted the key role played by ovarian microsomial epoxide hydrolase (mEH) to form the ovotoxic metabolite DMBA-3,4-diol-1,2epoxide [7,68]. The competitive inhibition of mEH by cyclohexene oxide, prevented DMBAmediated follicle depletion in mice [68] and rats [7]. In the Igawa study, using cultured F344 rat ovaries, DMBA decreased the number of healthy primordial follicles at concentrations ≥ 75.0 nM. Concentrations ≥ 375.0 nM decreased the number of healthy primary follicles, and concentrations ≥ 750.0 nM caused a depletion of all the types of healthy follicle population [7]. Madden et al. [69] focused on the impact of low-level acute DMBA exposures (12.5 nM or 75.0 nM) on postnatal day four F344 rat ovaries. While primordial or small primary follicles were not disrupted after both four and eight additional days of culture, a loss of large primary follicles (both concentrations) and secondary follicles (only low concentration) was seen [69]. The larger follicles may thus be more sensitive to DMBA than primordial and small primary follicles. Increased

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primordial follicle recruitment incited by the loss of larger follicles may play thus a role in DMBAmediated ovotoxicity [69]. Cumulus expansion of the oocyte complex occurs in the periovulatory follicle in response to the gonadotropin surge. The expansion process plays a crucial role in the fertilization process and embryo development [70]. A study conducted on isolated oocyte-cumulus complexes (COC) from porcine ovaries evaluated the effects of cadmium, nicotine, cotinine and anabasine on FSH-induced COC expansion [71]. Cumulus expansion was suppressed by exposure to different concentrations of cadmium, anabasine and nicotine, but not cotinine. There was a concomitant decrease of progesterone synthesis by cumulus cells [71]. There is evidence that cadmium can interfere with the ovarian steroidogenic pathway [72-73]. Exposure of rat ovarian follicles to concentration of CdCl2 ≥ 1.2 µg/ml was followed by impaired follicle growth and steroidogenesis [74]. Besides the studies assessing the effects of specific cigarette smoke constituents, an important contribution in the characterization of the impact of tobacco on folliculogenesis was given by in vivo animal models mimicking human exposure to cigarette smoke [75, 76]. When C57BL/6 mice were exposed to mainstream cigarette smoke a significant reduction in the number of primordial follicles, but not of the growing or antral follicles in comparison to controls resulted [75]. According to the data provided by Gannon et al. [77], a significant decrease in C57BL/6 mice primordial follicles is observable after 4 weeks of cigarette smoke exposure. Effects of cigarette smoke on oocytes Paixao et al. [76] noted a smaller oocyte diameter in incipient antral follicles under smoke exposure and, an increase of oocyte size after smoking cessation. Granulosa cells count decreased after, but not during the smoke exposure period. These data suggested that cigarette smoking elicits negative effects, which lasts after exposure withdrawal [76]. Mouse in vivo (0, 5.0, 7.5 or 10 mg/kg) and in vitro (0, 1.0, 5.0 or 10 mmol/l) assessments showed a role of nicotine in inducing alterations in oocyte maturation and in reducing 9

ovulation rate [78]. In vivo nicotine exposure was associated to increased premature centromere separation, premature anaphase and reduced the number of oocytes ovulated [78]. Unfortunately, in vitro experiments failed to confirm these effects [78]. The toxic potential of nicotine (1.0, 2.5, 5.0, or 10 mmol/L) on cultured mouse oocytes was probed by Zenzes et al. [79], who observed increased spindle and chromosome damages, abnormal chromosome alignment, and errors in meiotic maturation in exposed eggs [79]. Oocytes collected from mice exposed to nose-only cigarette smoke showed a high proportion (19%) of oocytes with one more sister chromatids non-aligned on the metaphase plate compared with the percentage of abnormal oocytes in non-smoking mice (2%) [80]. These disruptive effects on DNA may contribute to explain the defective oocyte nuclear maturation associated to cigarette smoke exposure. The main focus of the study carried out by Liu et al. [81] was to determine the effects of nicotine (0.5, 1.0, 2.5, 5.0, or 10.0 mmol) on bovine oocytes maturation. Nicotine interfered with the maturation process of the oocytes in a dose- and stage-dependent manner. The treatment related effects noted included anomalous oocyte cumulus cell expansion, decreased maturation rate, and reduced haploid complements of matured oocytes. Later findings revealed a negative impact of nicotine on meiotic spindles of bovine oocytes that manifested asymmetrical meiotic spindles and disordered microfilaments [81]. The second meiotic spindles seemed to be more sensitive to nicotine exposure [82]. A direct effect of nicotine on meiotic maturation of gametes was also observed in cultured hamster oocytes [83]. The highest concentration used (5mM), distressed both the first and second meiotic divisions, resulting in aberrant chromatin configurations, while exposure at or below 0.5 mM had no effect [83]. The effect of cadmium on oogenesis was also assessed using an in vivo Xenopus model, revealing altered percentage of oocytes at all stages of oogenesis and increased number of atretic gametes 10

[84]. The harmful influence of cadmium on Xenopus laevis fertilization rates and embryo development was recently characterized [85]. The fertilization rate decreased in a concentrationrelated manner (from 0.25 mg/ml to 25 mg/ml), and the embryogenesis was perturbed [85]. Following in vitro exposure to CSC (0.02 mg/ml continuously or 0.1 mg/ml for 20 hours) and cadmium (5-100 microM), mouse embryos displayed reduced cleavage, increased ROS generation, telomeres shortening and loss, chromosome instability, and apoptosis [86]. In agreement with Shilo et al. [12] findings, Jennings et al. [80] observed a significant increase in zona pellucida thickness in smoking mice compared to non-smoking. Cigarette smoke did not influence the location of the spindle, but induced changes in spindle size and shape. Oocytes from smoking mice had a shorter pole-to-pole spindle length but wider spindle equators compared with non-smoking mice, and manifested errors in chromosome alignment. Notably, human oocytes and embryos from smoking women also showed increased thickness of the zona pellucida [12]. Cigarette smoke extract (CSE) was linked to altered zona pellucida thickness, increased perivitelline space size and increased frequency of polar bodies with small size, strip-like shape and rough surface in a murine model [87]. Molecular and cellular mechanisms implicated in folliculogenesis impairment Several studies looked at the mechanisms mediating cigarette smoke-induced follicular impairment. Oxidative stress, DNA damage, abnormal crosstalk between oocyte and granulosa cells, and increased cellular apoptosis or autophagy have been implicated. Oxidative stress The term redox balance indicates the physiological stability between free radicals and antioxidant defense. An alteration of this balance toward an increase of pro-oxidants than anti-oxidants establishes a condition called oxidative stress [88]. Heavy metals contained in cigarette smoke, such as cadmium, and different components of cigarettes may play a role in promoting oxidative stress 11

and thereby in altering the integrity of membranes. The interfering effects of cadmium and lead exposure on redox balance in rat granulosa cells have been documented [26]. Lead and cadmium were used alone (0.05 mg/kg body weight given intra-peritoneally for 15 days), or in combination using the same final dose (0.05 mg/kg body weight for 15 days), and taking half of the concentration of each metal. Alone or in combination, lead and cadmium administration decreased SOD activity and GSH content and increased catalase activity. Cadmium appeared to be the most important effector of these alterations [26]. The impact of cigarette smoking on the ovarian transcriptome was investigated later [89, 90], with findings indicating that BaP exposure can upregulate the genes involved in different pathways, especially genes controlling nitric oxide and ROS production, and increased levels of mitochondrial ROS in oolemma membrane [89]. Mitochondrial ROS level alterations were associated to a considerable increase in plasma membrane lipid peroxidation and to perturbed fertilization [89]. Exposure of female C57BL/6 mice to nose-only cigarette caused an altered ovarian expression of genes involved in different crucial pathways, including proliferation and cell growth, apoptosis, primordial follicle activation, folliculogenesis, detoxification and oxidative stress [90]. Other toxicological responses noted in smoke-exposed oocytes included activation of the antioxidant defense pathway, and an increase in mitochondrial ROS and in lipid peroxidation in the oolemma membrane. Overall, smoke-exposed females had an increased time to conception and a significant reduction of litter sizes [90]. A recent study looked at the influence of cigarette smoke on mRNA levels of antioxidant enzymes, in mural granulosa cells of smoker women undergoing an IVF program, and found a significant modification towards an overexpression of SOD2 and catalase in smoker women in comparison to non-smokers [34]. It may be relevant that increased ROS formation was detected not only in smokeexposed granulosa cells and in oocyte membranes [26, 89,90], but also in follicles cultured in vitro, and in mice ovaries exposed to cigarette smoke components. Siddique et al. [91] measured the levels of two oxidative stress biomarkers, namely 8-IsoP and 8-OH-dG, in media of mice follicles 12

exposed in-vitro to CSC and BaP for 13 days at concentrations of 0-130.0 µg/ml of CSC and 0-45.0 ng/ml of BaP. A significant dose-dependent increase in the two stress biomarkers suggested a causal relationship between CSC and BaP exposure and increased oxidative stress in ovarian follicles [91]. C57BL/6 mice exposed to cigarette smoke extract (CSE) manifested and altered ovarian gene expression associated to a decrement of mRNA expression levels of the cytosolic antioxidants GSTM2 and GSTA3 [87]. These genes are important in protecting oocytes from toxic substances, and their altered expression has been linked to ovarian oxidative damage [92]. The capacity of DMBA to initiate apoptosis in granulosa and theca cells of cultured rat follicles has been documented [93]. Increased ROS generation appeared a key determinant of DMBA-induced follicle loss, as pointed out by the observations that: a) ROS increment preceded apoptosis; b) GSH depletion potentiated the proapoptotic effects of DMBA; c) GSH supplementation prevented DMBA-induced apoptosis [93]. Overall, current data support the notion that increased ROS formation is a main causal factor in smoke-mediated ovotoxicity [26, 87, 89-91]. Redox balance alterations induced by cigarette smoke can initiate lipid peroxidation [94], thereby interfering with several crucial processes, including granulosa cells maturation, binding of gonadotropins to their receptors [26], and egg fertilizing capacity [89, 90]. DNA damage and abnormal crosstalk between oocyte and granulosa cells There is fair evidence that cigarette smoke can induce DNA damage, and an altered communication between the oocyte and granulosa cells. Sinko et al. [95] reported a significant increase in DNA damage in human cumulus cells derived from smoking women, and a correlation between smoking-induced DNA damage and a lower IVF success rate [95]. DNA adducts are strong chemical interactions between toxicants and DNA. The lower implantation and pregnancy rates observed in smokers undergoing IVF techniques were attributed to the ability of BaP to form DNA adducts in ovaries [24,60,61]. Ramesh et al.[8], 13

showed that BaP can form DNA adducts in rat ovaries and liver 1, 7, 14, 21 and 28 days after administration of BaP (5.0 mg/kg body weight orally given in a single gavage). The ovarian tissue showed greater concentrations of DNA adducts when compared to the liver tissue 1-day after BaP exposure. With the increment of time interval after exposure cessation, adducts levels in ovaries and liver tissues declined rapidly. Levels of the parent compound and BaP metabolites in plasma, ovaries and liver were also measured. Concentrations of unmetabolized BaP peaked 24h after BaP exposure in plasma, liver and ovaries. A similar trend was observed with respect to BaP metabolites. Unmetabolized BaP and metabolites concentrations were lower on day 14, 21, and 28 versus day 1, after BaP exposure. The capacity of Bap to induce DNA damage in mice oocytes and cumulus cells was also recently assessed by Einaudi et al [96]. In vivo acute BaP administration (13 mg/kg) revealed an increase in DNA damages in both oocytes (4-6 days after the exposure) and cumulus cells ( 2, 4, and 6 days after exposure) and the formation of benzoapyrene-7,8-9,10 diolepoxideDNA adducts in cumulus cells after 2, 4, and 6 days after the exposure [96]. DNA perturbations were also noted in neonatal rat ovaries after exposure to DMBA at low and high concentrations (1.0 µM for 2 days, and 12.5 nM or 75 nM for 4-8 days) [25]. The literature concerning the repercussions of cigarette smoke on the crosstalk between oocyte and granulosa cells has especially focused on disruption of ovarian gap junctions [97]. Gap junctions play important roles in regulating growth, metabolism and differentiation during folliculogenesis [98,99]. PAHs can promote formation of DNA adducts by generating highly reactive intermediates which bind to DNA [100]. Moreover, PAHs are able of inhibiting gap-junctional intracellular communication in cultured cells. Sharovskara et al. [101] assessed the consequences of carcinogenic and non-carcinogenic PAHs on gap junction intercellular communication (GJIC). Carcinogenic PAHs influenced this communication in hepatoma cells, in particular, GJIC was strongly inhibited after one hour of exposure to BaP (20 µM). Using a neonatal rat whole ovary 14

culture method, Ganesan et al. [102] revealed that DMBA (12.5 nM, 75.0 nM for 4-8 days) can alter the expression of the major gap junction genes Cx37 and Cx43 at transcriptional and posttranscriptional levels. Follicle loss: apoptosis or autophagy? Cigarette smoke has been associated to premature menopause and depletion of ovarian follicles, but the mechanisms underpinning these effects remain not fully defined. Recent evidence suggests that both apoptosis [90,93] and autophagy of primordial follicles [77, 103] may play a role. Using a model of transplanted hamster ovarian follicles, Bordel et al. [104] showed that nicotine dose-dependently inhibits follicular growth [104]. Follicles were isolated from donors and then transplanted in recipient animals dosed daily with a subcutaneous injection of 0.2 or 1.0 mg/kg body weight of nicotine. Treatment with the higher dose impaired follicle growth and induced granulosa cells apoptosis. DMBA (1.0 µM for 2 days and 12.5 or 75.0 nM for 4-8 days) induced apoptosis in neonatal rat ovaries, as shown by increased caspase-3 levels [25]. The markers of early follicular atresia, p53 and caspase 3, were searched in granulosa cells of antral follicles collected from mice exposed to 1.5 or 3.0 mg/kg of BaP given i.p. for 7 days [89], or exposed to nose-only cigarette smoke [90]. Markers of apoptosis were present in smoke-exposed secondary follicles, but not in primordial and primary follicles. Starting from previous findings [9, 10, 24, 77, 105] indicating that a diminished number of primordial follicles are observable in cigarette smokeexposed ovaries versus controls, a complex series of mechanistic events leading to follicular depletion was elaborated [89, 90]. Based on the detection of apoptotic markers in the latter stage of follicular development, it was proposed that ovotoxic agents present in cigarette smoke, target maturing follicles and that their destruction stimulates the primordial follicle population to activate and replace lost follicles. This seems to be a main causal factor in cigarette smoke-mediated loss of primordial follicles.

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A study focusing on apoptosis markers in ovaries of mice exposed to mainstream smoke showed a significant reduction in the number of follicles at different stages of development, but a lack of influence on apoptosis [75]. Autophagy is a recently discovered mechanism of cell death and a possible determinant of premature ovarian failure. The consequences of cigarette smoke exposure on granulosa cells of ovarian follicles, and in particular the role of autophagy in primordial follicle depletion has been the subject of several investigations [77, 103, 106]. Autophagy is activated in response to different insults, including nutrient starvation, genotoxic agents, and oxidative stress [107]. Briefly, autophagy is a process that removes long-lived proteins and damaged organelles (mitochondria and ER) through lysosomial degradation. The activation of Beclin 1 (part of the class III phosphoinositide 3-Kinase complex) is involved in membrane nucleation. During the autophagic process, LC3 is processed in LC3-I and LC3-II, involved in the sequestration of organelles in the autophagosome. Bcl-2 is a key gene important in both apoptosis and autophagy and Beclin 1 is inhibited by Bcl-2 [108]. A mouse study on the relationship between whole-body exposure to cigarette smoke and follicle loss, revealed a depletion of the primordial follicle pool and a concomitant profound increase in the number of autophagosomes and in the expression of Beclin-1 and LC-3 in granulosa cells [77]. Reduced Bcl-2 expression with no change in the levels of the pro-apoptotic Bax was another treatment-related effect. These findings promoted the concept that autophagy is a cellular determinant of smoking-induced ovarian follicle loss. Gannon et al. [103] tested the hypothesis that cigarette smoke causes mitochondrial damage leading to the activation of autophagy pathway and follicle demise. Deregulation of mitochondrial dynamics was examined in the homogenates of ovaries from smoke-exposed and control mice, and was confirmed by an increased expression of PARK2 and a decreased expression of MFN1. These findings, combined with data showing an overexpression of the autophagic proteins Beclin 1 and LC3, and an underexpression of the autophagy inhibitor Bcl-2, suggested that cigarette smoke induces mitochondrial dysfunction 16

culminating in mitochondrial specific autophagy (mitophagy). Increased generation of ROS, and consequent dysfunction of mitochondria was postulated to play a central role in this process [109]. Additional evidence for this assertion, originated by toxicity assessments carried out in whole ovary homogenates after whole-body exposure to cigarette smoke, twice daily, 5 days a week for 8 weeks, and showing increased autophagy via activation of AMPK pathway together with an inhibition of anti-autophagic markers AKT and mTOR [118]. AMPK pathway serves an important role in regulating cellular energy metabolism in response to low energy levels [110]. There is evidence suggesting that tobacco smoke can elicit mitochondrial dysfunction and increased ROS generation, leading to the inability of granulosa cells to meet its energy need, and culminating in the activation of the reparative autophagy cascade via AMPk pathway [106]. Chloroquine and its derivative hydroxychloroquine, used as anti-malarial and anti-inflammatory therapies, are the most clinically relevant autophagic inhibitors [111]. Interestingly, Furlong et al. [111] demonstrated that hydroxychloroquine attenuates the autophagic signaling incited by wholebody exposure of mouse ovary to cigarettes (twice daily, for 5 days a week, for 8 weeks). Maternal cigarette smoking and effects on the next generation There is experimental evidence suggesting a strong impact of maternal cigarette smoke on the fertility of female offspring. Exposure of female mice to BaP and DMBA (1.0 mg/kg s.c. each per week for 3 weeks) prior to pregnancy or during lactation caused a remarkable depletion of primordial and primary follicle pool in the female offspring [105]. When postnatal day 4 ovaries were exposed to PAHs, the expression of Hrk significantly increased in primordial and primary oocytes and in granulosa cells in comparison to controls within 24 hours of exposure. Hrk (Bcl-2 interacting protein) is an Ahr-regulated cell death gene whose activation is crucial in PAHs-mediate follicle depletion. Reduced ovarian reserve in rat female offspring followed maternal smoking exposure starting from the proestrus period and during the entire pregnancy [112]. In vitro exposure

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of mouse embryonic ovaries to PAHs promoted a high level of germ cell loss [113]. Moreover, increment of fetal ovarian germ cell loss also followed in utero exposure to PAHs [113]. Exposure of mice to cigarette smoke throughout pregnancy and lactation altered the ovarian size and the number of follicles in the offspring [114]. Other effects noted included a significant elevation of the markers of apoptosis in neonatal ovaries, a reduced number of MII eggs ovulated, and a poor oocyte quality due to oxidative stress [114]. The deletion of the modifier subunit of glutamate cysteine ligase (Gclm) and, consequently, a deficiency in GSH synthesis led to destruction of oogonia, premature ovarian failure, and ovarian tumorigenesis after BaP exposure [115]. Gclm−/− prenatally BaP-treated females had a significant decrements in follicle numbers, offspring production and increased frequency of ovarian tumors in response to prenatal BaP exposure [115]. Prenatal BaP exposure seemed to influence puberty age of female offspring. In particular, F1 daughters of mothers administered with BaP during gestation reached puberty 5 days earlier in comparison to controls [116]. A recent evaluation of the multigenerational (F2) and transgenerational (F3) effects of cigarette smoke exposure showed multigenerational effects on female fertility, including changes on oocyte quality, increased time to conception, and lag time between pregnancies in F2 generation [117]. Deleterious effects of maternal cigarette smoke on the future generations were also described in the human. Jensen et al. [118,119] reported reduced fecundability in women exposed to maternal cigarette smoke during prenatal life. Of particular relevance is the observation that prenatal exposure to maternal cigarette may lead to an earlier age of menopause, as suggested by a report where smoking information was obtained from the mothers of 4025 participants to a U.S. project analyzing the health effects of prenatal diethylstilbestrol exposure [120]. In utero exposure to cigarette smoke caused a depletion of somatic cells, but not of oogonia, in first-trimester ovaries obtained from legal abortions [121], and an earlier age of menarche of daughters [122]. Angenard et al. [123] demonstrated a negative influence of cadmium on the number of germ cells in human 18

fetal gonads in both males and females. The impact of an active metabolite of DMBA (9,10dimethyl-1,2-benzanthracene-3,4-dihydrodiol) was tested on cultured of human fetal ovaries, with data suggesting that PAHs exposure, via Ahr activation, is associated with a reduced germ cell proliferation [124]. A study carried out on second trimester human fetal ovaries collected from smokers after medical termination of pregnancy, demonstrated a complex impairment of normal ovarian fetal physiology [125]. While ovarian morphology remained unchanged, the fetal endocrine signaling and follicle progression were heavily compromised together with key fetal ovarian transcripts and expression of mediators of Ahr pathway. Cigarette smoke and IVF outcome Understanding the possible impact of cigarette smoke on IVF outcome remains an important issue. Early studies carried out after the birth of IVF techniques reported conflicting results in term of oocytes retrieved [126-128], fertilization rates [126,127,129] and pregnancy rates [128-130]. More recent investigations addressing the issue, provided data that collectively support the notion that cigarette smoke has a negative influence on IVF success rate. In a retrospective study involving 111 patients undergoing IVF cycles (71 non-smokers and 40 smokers) the number of oocytes retrieved was significantly lower in smokers in comparison to nonsmokers [14]. Active smoking was causally linked to impaired ovarian response, as indicated by the lower number of oocytes retrieved [14], and a modified follicle size repartition in antral follicle count [14]. A correlation between cigarette smoke exposure and decreased AMH concentration was also documented [14]. Cigarette smoke was also associated to a lower clinical pregnancy rate (29. 6% in non-smokers and 10% in smokers) [14]. A cohort prospective study including 166 patients (33 recent smokers and 133 non-smokers) undergoing a first cycle of IVF, showed a direct association between the levels of cotinine concentration and the number of cigarettes smoked [36]. Cotinine concentrations in serum also correlated positively with cotinine measured in follicular fluids. Patients were divided into two groups according to the cotinine concentrations in follicular 19

fluid: recent smoker women with a cotinine concentration ≥ 10 ng/mL, and non-smoking women with undetectable levels of cotinine. The mean number of oocytes retrieved was 7.78 ± 0.81 in smokers and 9.55 ± 0.46 in non-smokers but this difference was not statistically significant. There were no statistical differences in the quality of embryos transferred among women of the two studied groups. A non-significant trend toward a lower implantation rate in smokers (7.4%) than in non-smokers (14.81%) was also reported. A study conducted by Cinar et al. [37] provided results that are in overt contrast with previous studies [14, 32, 36]. This comparative prospective analysis included 171 non-smokers and 43 smokers. Cotinine levels were analyzed by ELISA technique in follicular fluid of women undergoing ART procedures. Based on cotinine levels, patients were grouped in smokers (cotinine concentration > 20 ng/mL) and non-smokers (cotinine level ≤ 20 ng/mL). The ovarian stimulation protocols was also considered and patients were divided into three groups: group A (gonadotropin releasing hormone agonist), group B (gonadotropin releasing hormone antagonist) and group C (others stimulation protocols including micro-dose flare up, natural cycle, etc). Intriguingly, the total number of collected oocytes and mature oocytes was significantly higher in smokers in comparison to non-smokers. Within the gonadotropin releasing hormone antagonist group, the mean age of smokers was lower and the total number of retrieved oocytes was significantly higher in comparison to non-smokers. Another important aspect to be borne in mind when assessing the impact of smoke on IVF outcome is the fertilization rate. This parameter resulted affected in smokers [13, 29, 131]. Tiboni et al. [29] examined the impact of cigarette smoking on follicular and plasma concentrations of vitamin A, vitamin E, lycopene and beta-carotene and on IVF outcome. In this study consisting of 70 female patients undergoing IVF treatments (17 smokers and 43 non-smokers), HPLC assay revealed that smokers had lower β- carotene follicular fluid concentrations compared to non-smokers. Findings also revealed that smokers had a significantly lower fertilization rate compared to non-smokers (71.5% in non-smokers and 55. 9% in smokers). In a retrospective study, including 130 patients 20

undergoing intracytoplasmic sperm injection (ICSI) procedure due to male factor infertility (72 smokers and 58 non-smokers) the number of non-fertilized oocytes was significantly lower in smokers than in non-smokers [13]. Literature data indicate that albeit cigarette smoke is not affecting of embryo developmental rate [35, 37], it is associated with lower implantation rates [30, 36, 34, 132]. Neal et al. [30] found a correlation between cigarette smoke and decreased implantation and pregnancy rates. In this study, 225 female patients undergoing IVF were included and divided into three groups based on cigarette smoke exposure types: mainstream and sidestream smoke, and no-smokers. While there were no differences between mainstream and sidestream exposure, a striking difference was noted in terms of implantation and pregnancy rates of mainstream and sidestream smokers compared to nonsmokers. With respect to the pregnancy rate, cigarette smoke appeared to lower the probability to achieve pregnancy in several studies [14, 30-33, 34, 131,133]. Smoking patients had significantly lower odds of clinical pregnancy per cycle (OR 0.56, 95% CI 0.43–0.73) in a meta-analysis with a computerized search [31]. Ben-Haroush et al. [33] piloted a cohort study, including 42 smokers and 195 non-smokers, in order to elucidate the effects of passive and active smoking on pregnancy rates after IVF treatments and after transferring high quality embryos. Again, the clinical pregnancy rate was lower in smokers compared to non-smokers, but there was no difference between passive and active smokers. A recent study, evaluating the IVF outcome of 20 smokers and 20 non-smokers undergoing ICSI procedures, showed a significantly lower fertilization, implantation, and pregnancy rates in smokers compared to non-smokers [34]. In particular, with regard to the pregnancy rate, only 2 of 20 smoker women achieved a pregnancy compared to 11 of 20 nonsmokers [34]. For additional complexity, other authors failed to associate cigarette smoke with reduced pregnancy rates [35, 36, 37, 38]. In a retrospective analysis involving 389 patients undergoing first IVF cycle and where smoke habit was detected by a questionnaire, Wright et al. 21

[35] failed to associate cigarette-smoking with a poorer IVF outcome. After testing urinary cotinine of 127 couples undergoing IVF procedures, Kim et al. [38] found no association between the presence of urinary cotinine and poorer outcomes of infertility treatments, including IVF and intrauterine insemination (IUI). A lower live birth rate is another parameter that some authors associated with an exposure to cigarette smoke [31, 33]. Conclusion Cigarette smoke is causally linked to a number of diseases and health problems, including reproductive health impairment. There is compelling evidence that cigarette smoke is associated to prolonged time to pregnancy, disrupted ovarian steroidogenesis, and accelerated depletion of ovarian reserve leading to early menopause [134, 135, 136]. Compared to nonsmokers, women that smoke showed an overall odds ratio for risk of infertility of 1.60 (95% CI 1.34-1.91) [137]. Several toxicants derived from cigarette smoke, including nicotine, cotinine, BaP and cadmium have been quantified in follicular fluids of women undergoing IVF, indicating that cigarette smoke constituents can directly target ovarian follicles. In general, the amount of toxicants present in the follicular fluid was related to the daily number of cigarette smoked. It is important to note that the ovary is equipped with the enzymatic systems needed to bioactivate the parent compound to the ovarotoxic intermediate [7,67,68]. This seems an important point considering that toxic reactive intermediates generally are short-lived molecules, and thus the reactive molecules that are crucial for the initiation of toxic effects are likely those that are generated in the target tissue. In this respect, it may be noteworthy to note that the ovary displays the capacity of converting PHAs in to more ovotoxic intermediates [7, 67, 68]. Ovarian enzymes are also able to convert nicotine into its major metabolite cotinine [66]. The fact that smoke is a mixture of chemical classes including hydrocarbons, alcohols, phenols, aldehydes, ketones, alkaloids, acids and heavy metals has represented a difficulty for the understanding of the molecular pathways behind tobacco-mediated ovarian injury. Among the 22

several components present in the cigarette smoke, PAH (including BaP, DMBA, 3-MC), nicotine and cadmium were identified as the main effectors of ovarian toxicity. Insight gained by in vitro and in vivo experimental models have shown that cigarette smoke can affect both oocyte quantity and quality and derange ovarian steroidogenesis. Loss of primordial and primary follicles followed the intraperitoneal injection of a single high dose of PAHs in rats and mice [64]. A more toxic ovarian response resulted by the chronic administration of PAHs at low doses in mice and rats, with DMBA resulting the more toxic among the PAHs tested [65]. Intraperitoneal injection of BaP to neonatal mice resulted in primordial follicle depletion that was attributed to a mechanism involving developing follicle atresia and accelerated resting pool activation [89]. Oral administration of CSE to mice was associated with a shrink size and poor quality eggs [87]. In-vitro studies using oocytes, follicles and granulosa cells, yielded interesting insights in on the toxicity potential of specific components of cigarette smoke. For instance, BaP induced demise and altered growth of rat and mouse follicles [7, 9]. Cultured granulosa bovine cells manifested decreased E2 production following exposure to nicotine but not to cotinine [59]. Levels of oxidative stress biomarkers increased in in vitro spent media of follicle cells exposed to CSC and BaP [91]. Increased ROS generation may result in DNA damages [18, 19], and disruption of the communication via gap junctions between granulosa cells and the oocyte [97, 102]. Cultured rat ovaries cultured in presence of BaP at concentrations representative of the levels detected in human follicular fluid impaired follicular growth [24]. A study [9] quantified the concentration of BaP in follicular fluids of women exposed to mainstream and sidestream smoke, and then evaluated the effects of increasing BaP levels on cultured mice ovaries. In recent years, investigators have concentrated on animal models recapitulating human exposure to cigarette smoke. Mouse inhalation models simulate human exposure to cigarettes. In particular, the nasal exposure most accurately mimics direct human contact with cigarettes. Undoubtedly, 23

inhalation models provided important information on several mechanisms potentially involved in cigarette smoke-mediated ovotoxicity, including oxidative stress [26, 87, 89, 90], DNA damages [80] and follicle loss through autophagy/apoptosis [75, 77, 103, 106]. When considering human studies, it is important to highlight their important contribution in providing evidence for multigenerational effects of maternal smoking on the ovarian function of the progeny [122, 125], due to an impact on zona pellucida thickness [12] and on DNA integrity [95]. There is an increasing amount of evidence indicating that cigarette smoke exposure can have negative repercussions on IVF success. A negative correlation between cigarette smoke exposure and number of retrieved oocytes at ovum pick-up has been documented by several studies [14, 32, 36, 37]. Moreover, some studies recorded lower fertilization [13, 29, 34], and implantation rates [30, 34, 36, 132] in smokers in comparison to non-smokers. With respect to the pregnancy rate, conflicting data have been reported. While some studies suggested that smoke is associated with lower clinical pregnancy rates in smokers [30-33, 34], others provided data questioning the concept that cigarette-smoking habit affects IVF outcomes [35-38]. Several possible explanations exist for the inconsistencies presented by the IVF studies, including the heterogeneous indications to IVF among studies, lack of information about patient’s life style like the assumption of alcohol, caffeine, illicit drugs, and harmful environmental exposures [13, 37, 124]. Another potential methodological issue is that in the majority of the studies, smoking habit was assessed by questionnaires [13, 29, 30, 33, 35], and not by quantification of cotinine serum levels. It is also notable that no consensus has been reached on the cotinine cut-off level be adopted in smoker detection. Indeed, while some studies considered positive a value of cotinine ≥ 10 ng/ml in serum or follicular fluid [36, 132], others considered women as smokers if they had a value of cotinine ≥ 20 ng/ml in serum or follicular fluid [37]. There are thus several reasons accounting for the lack of consistency across studies. 24

In summary, a wealth of scientific evidence indicates that cigarette smoke derange ovarian function, and decrease the ovarian reserve by intervening at different stages of folliculogenesis. Data presented in this review offer a valuable support to discourage women to smoke in order to preserve their reproductive potential. Particularly worrying and calling for further research are the emerging data supportive of the likelihood that cigarette smoke can cause transgenerational effects. Understanding the molecular mechanisms underpinning cigarette-induced ovarian damage remains a crucial question.

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Declaration: The authors report no financial or commercial conflicts of interests.

37

Legend to figures. Figure 1. Flow chart of studies selection and characteristics.

IDENTIFICATION

SCREENING

Articles identified through database searching: n= 1124

Studies excluded after screening titles and/or abstracts: n= 1011

Studies retrieved for detailed evaluation: n= 113 ELIGIBILITY

Studies excluded (n=5) for these reasons: - Other types of smoke: n=1 - Other languages: n=4

INCLUDED

Studies included in systematic review: n= 108

38

Figure 2. Possible mechanisms involved in cigarette smoke-induced follicle loss.

CIGARETTE SMOKE SMOKE EXPOSURE CIGARETTE EXPOSURE PAH AHR

AHR PATHWAY ACTIVATION

ACTIVATION OF DIFFERENT PATHWAYS ARNT

nucleus

XRE

ALTERED CROSSTALK OOCYTE-GRANULOSA

DNA DAMAGE

ALTERATION OF REDOX BALANCE

OXIDATIVE STRESS

CELL DEATH

MEMBRANE LIPID PEROXIDATION

ALTERED FERTILIZING CAPACITY OOCYTES ALTERED GONADOTROPINS BINDING

DNA ADDUCTS, DSBS, CHROMOSOMAL ABERRATION

AUTOPHAGY

ALTERED OOCYTE DEVELOPMENT

OR APOPTOSIS?

FOLLICLE LOSS

Table 1. Effects of cigarette smoke components on folliculogenesis.

Authors Ramesh et al. [8]

Model and age Rat (tenweek-old)

Exposure

Agents

Oral administra tion

BaP

Dose 5 mg/kg body weight (single oral gavage)

Effects BaP concentration in plasma, ovaries and liver higher on day 1 post BaP administration. Gradual decrease thereafter (7, 14, 21 and 28 days post administration) DNA adducts higher in ovarian than in liver tissue (day 1 post administration). Gradual decrease thereafter (7, 14, 21, and 28 days post administration)

Sadeau and Foster. [9]

Mouse (13day-old)

Cultured mouse ovaries

BaP

1.5 to 45 ng/ml for 12 days

Inhibition of antral follicle development (45.0 ng/ml from day 8) Significant reduction in E2 output (45.0 ng/ml, day 8) and increase between day 8 and 12

39

Lower anti Mülleran hormone output on day 4, 8 but not on day 12 Reduction in follicle survival at all doses (day 12), in particular with 45 ng/ml (day 12) Neal et al. [24]

Rat (22-to 26day-old)

Cultured rat ovaries

BaP

1.5 to 135 ng/ml (0-3-5 days)

Decrease of follicle growth in vitro in a dose-dependent manner starting at 1.5 ng/ml (37% of control) in 5-days culture period Lower E2 level in spent media of treated ovaries beginning at 5.0 ng/ml

Sobinoff et al. [89]

Mouse (4day-old)

Intraperitoneal administra tion

BaP

Cultured mouse ovaries

In vivo:1.5mg/kg/dai ly, 3mg/kg/daily (7 days)

Up-regulation of genes involved in follicular growth and development, Dna repair, follicular atresia and oxidative stress, AhR signaling

In vitro:1 µM (4 days)

Significant decrease in primordial follicles and increase in the number of activating follicles Increased levels of mitochondrial ROS /lipid peroxidation in oolemma membrane Apoptotic markers in the latter stage of follicular development

Einaudi et al. [96]

Mouse (4week-old)

Oral administra tion

BaP

13 mg/kg (single dose)

DNA damages in both oocytes (4-6 days after the exposure) and cumulus cells (2, 4, 6 days after exposure) Benzoapyrene-7, 8-9, 10 diolepoxideDNA adducts in cumulus cells after 2, 4, 6 days from exposure

Siddique et al. [91]

Igawa et al. [7]

Mouse (age not mentioned)

Rat (PND4)

Cultured mouse follicles

Cultured rat ovaries

CSC, BaP

CSC: 30 to 130 µg /ml for 13 days BaP: 1.5 to 45 ng/ml for 13 days

DMBA, DMBA3,4-diol + mEH inhibitor (CH0)

DMBA: 12.5nM1µM (15 days) + CHO (2 µM) DMBA-3,4-diol: 12.5nM-1µM for 15 days

8-Iso-P concentration higher in spent media of the BaP (15 and 45 ng/ml) and CSC (130 µg /ml) dose groups 8-OH-dG higher in the spent media of all BaP doses groups except for the lowest group (1.5 ng/ml) Ovotoxicity of DMBA-3,4- diol at lower concentrations (12.5 nM-primordial follicle; 75 nM-primary follicle) than DMBA (75 nM-primordial follicle; 375 nM-primary follicle) after 15 days of incubation Primordial and primary follicle loss (1µM DMBA-4 days) CHO (2 µM) prevented the loss of follicles induced by DMBA (1µM DMBA-4 days)

40

DMBA (1µM) increased the level of mEH mRNA (2 and 4 days) Ganesan et al. [25]

Rat (PND4)

Cultured rat ovaries

DMBA

12.5 nM- 75 nM for 4-8 days 1µM for 2 days

Increased caspase 3 protein level (both DMBA treatments-8 days) and γH2AX protein levels (DMBA 1µM-2 days) Increased levels of mRNA encoding Atm, Xrcc6, Brca1 and Rad51 after DMBA (12.5 nM DMBA- 4 days) Increased Atm, PARP1 mRNA levels after 8 days of both DMBA exposures. PARP protein level increased after 4 days of 12.5 nM DMBA and by both DMBA exposures after 8 days Increased levels of ATM protein in a concentration-dependent temporal pattern (75 nM DMBA after 4 days; 12.5 nM DMBA after 8 days) Increased levels of pATM (in large primary and secondary follicles) after 8 days of 75 nM DMBA exposure

Mattison and Thorgeis son. [64]

Mouse (4 to 6 weeks old)

Intraperitoneal administra tion

DMBA, 3-MC, BaP

DMBA: 80 mg/kg, 3-MC: 80 mg/kg, BaP: 80 mg/kg (single dose)

Primordial oocytes destroyed by carcinogenic polycyclic hydrocarbons: DMBA (1 day after administration), 3MC (2-3 days after administration) and BaP (2-3 days after administration) No effect induced by non-carcinogenic polycyclic hydrocarbons

Borman et al. [65]

Mouse (28 days) Rats (28 days)

Intraperitoneal administra tion

DMBA, 3-MC, BaP

Mouse: DMBA: 0.007 mg/kg to 3.5 mg/kg, 3-MC: 0.0015 mg/kg to 0.75 mg/kg, BaP: 0.0075 mg/kg to 15 mg/kg for 15 days

Wide range of ovotoxic effects in mice and rats mediated by DMBA, 3-MC and BaP

Rat: DMBA: 0.35 mg/kg to 7.0 mg/kg, 3-MC: 0.0015 mg/kg to 60.0 mg/kg, BaP: 0.0075 mg/kg to 60.0 mg/kg for15 days

Rajapaks ka et al. [68]

Mouse (PND4)

Cultured mouse ovaries

DMBA + mEH inhibitor (CH0)

12.5 nM, 25 nM, 50 nM, 75 nM, 125 nM, 250 nM, 500 nM, 1 µM for 15 days + CHO (2 µM)

Healthy primordial follicles loss at concentrations ≥ 12.5. Healthy primary follicles were reduced at concentrations ≥ 25nM DMBA. Healthy secondary follicles loss at all concentrations. All healthy follicle populations depleted by

41

DMBA at concentrations ≥ 250nM (after 15 days of incubation) Healthy primordial and primary follicle loss after 6 hours of DMBA 1µM DMBA effect (1 µM after 2 days of exposure) on the level of mEH mRNA Prevention of loss of primordial and primary follicles after addition of 2mM CHO to DMBA incubation (6 hours) Madden et al. [69]

Rat (PND4)

Cultured rat ovaries

DMBA

12.5 nM-75nM for 1,2,4,8 days

No primordial or small primary follicle loss at both the DMBA concentrations. Large primary follicle loss (both concentrations) and induction of secondary follicle depletion (lowconcentration) after 8 days Increased levels of Cyp2E1 (both DMBA treatments- after 4 days), Gstpi (12.5 nM75nM- after 1 day and 4 days respectively), Sod1 mRNA (75nM- after 2days), Sod2 (75nM-after 2 days). Becn1 mRNA increased (low DMBA after 2 days and 4 days), reduced Atg7 mRNA (12.5 nM – after 2days).

TsaiTurton [93]

Rat (25-daysold)

Cultured rat ovaries

DMBA

DMBA: 0,0.1,1, 10, 100 µM (12h24h-48h) DMBA: 0,0.5,1, 5 µM (0-48h) + 5mM GEE,0.5mM DTT, 0.2mM BHT

DMBA-induced apoptosis in preovulatory follicles (10 µM, 48 h) Increased ROS after 12 h in the 1 and 10µM (DCF and DHR fluorescences) groups and after 24 h in the 0.5 µM group (DCF fluorescence) GEE protection against DMBA-induced apoptosis GSH depletion (BSO treatment) + DMBA 0.5 µM+ FSH 75 ng/ml caused greater granulosa cell apoptosis

Ganesan and Keating [102]

Rat (PND4)

Cultured rat ovaries

DMBA

12.5 nM- 75 nM for 4-8 days

Cx37 increased by 12.5 nM DMBA (4 days) and decreased at 75nM (4 days) and 12.5 nM (8 days) Cx43 increased at 4 days by both DMBA exposures and decreased at 8 days (both concentrations) P53 and Bax increased after 8 days by both DMBA exposures

42

Sanders et al. [59]

Bovine (age not mentioned)

Granulosa and thecal cells culture

Nicotine , cotinine

6, 60, 600 µM (24h)

Inhibitory effects of nicotine on androgen production by theca interna

Liu et al. [70]

Bovine (age not mentioned)

COC culture

Nicotine

0, 0.5, 1.0, 2.5, 5.0, and 10.0 mmol

Decreased cumulus oocyte complex development in a dosedependent manner and decreased perivitelline space formation (5mmol) Altered oocytes maturation rate (2.5 and 5.0 mmol) and microfilament organization (2.5 mmol) Reduced oocytes haploid state (2.5 -5.0 mmol)

Vrsanska et al. [71]

Bovine (age not mentioned)

COC culture

Nicotine , Anabasi ne, Cotinine , Cadmiu m

Nicotine: 2x10 -4 to 10-6 M, Anabasine: 10-4 to 10-6 M, Cotinine: 10-4 to 10-6 M, Cadmium: 0.5x 10-4 to 10-6 M (24 hours)

Decreased cumulus oocyte complex development (cadmium, nicotine and anabasine-induced) and progesterone (cadmium, nicotine, cotinine, anabasinemediated) and hyaluronic acid synthesis (nicotine, cadmiummediated)

Mailhes et al. [78]

Mouse (8weeks-old)

In vivo

Nicotine

In vivo: 0, 5.0, 7.5, 10 mg/kg at 3,0, +3 relative to an injection of hCG

Increased premature centromere separation (10 mg/kg)

In vitro: 1.0, 5.0, 10.0 mmol/l (16 hours)

Reduction (50%) of ovulated oocytes (10 mg/kg)

1-2.5-5-10 mmol/L (8-16 hours)

Effect on meiotic spindle function in a dose-related manner. Dose and timerelated blockage in MI phase

Zenzes et al. [79]

Mouse (8-20 weeks)

Cultured mouse oocytes

Cultured mouse oocytes

Nicotine

Increased premature anaphase (5.0, 10.0 mg/kg)

No effects in in vitro experiments

Reduction of the proportion of oocytes reaching metaphase II stage (10 mmol/L), dose-related proportion of abnormal metaphase II oocytes Abnormal diploid oocytes (2.5 mmol/L) but not at higher concentrations Liu et al. [81]

Bovine (age not mentioned)

Cultured bovine oocytes

Nicotine

0.01-1.0mM, 2.06.0 mM

Altered oocytes maturation rate and lower haploid oocytes in the 2.0 to 6.0 mM groups Altered microfilaments organization, meiosis progression and subsequent embryonic development

43

Liu et al. [82]

Bovine (age not mentioned)

COC culture

Racowsk y et al [83]

Hamster

Cultured hamster oocytes

Nicotine

0.5mM-5.0 mM

Perturbations at both the first and second meiotic divisions (5mM, 24 hours) with oocytes block at metaphase I and altered chromosomes segregation

Bordel et al. [104]

Hamster (6to-8 weeksold)

Subcutane ous administra tion

Nicotine

0.2 mg/kg body weight-1.0 mg/kg body weight

Inhibition of follicle growth nicotinemediated (1.0 mg/kg body weight)

Rat (age not mentioned)

Intraperitoneal administra tion

Lead acetate (LA), Cadmiu m acetate (CA)

In vivo: LA-CA: 0.05 mg/kg body weight on a daily basis (15 days), LA+CA: a total dose of 0.05 mg/kg body weight taking half of the concentration of each metal (15 days)

Reduced glutathione content, SOD activity and elevated catalase activity and lipid peroxidation in cells of cadmiumtreated animals

Nampoot hiri et al. [26]

Nicotine

0.01-0.5 mM 2.0-4.0 mM

Altered polymerization of microfilaments and impaired blastocyst chromosomal composition

COC culture

In vitro: Concentration of metal that reach the ovary when exposed to 0.05 mg/kg body weight for 15 days ( 1 hour) Wan et al. [74]

Rats (PND 12 to 14)

Decrease of the cleavage rates (2.0 and 4.0 mM)

Cultured rat follicles

cadmiu m

0.1- 1.6 µg/ml (12 days)

Increased levels of caspase3 in granulosa cells in transplanted follicles of high-dose nicotine-treated animals

LA+CA manifested intermediate changes, LA alone induced minimum changes in antioxidant defense Decrease glutathione content in in vitro CA and LA+CA groups. Elevated lipid peroxidation in in vitro CA group

Follicle growth, differentiation and steroidogenesis altered with 1.2 µg/ml Reduced survival rate and rate of antral follicles (1.6 µg/ml, day 2) Dose-dependent alteration of germinal vesicle breakdown (≥ 1.6 µg/ml)

Lienesh et al. [84]

Xenopus laevis

Dorsal limph sac injection

Cadmiu m

0.5, 0.75, 1.0, 3.0, 5.0 mg/kg (21 days)

Altered percentage of oocytes at all stage of oogenesis and increasing number of atretic gametes

Huang et al. [86]

Mouse (age non mentioned)

Cultured mouse embryos

CSC, Cadmiu m

CSC: 0.02mg/ml continuously or 0.1 mg/ml for 20 h

Increased oxidative stress and telomeres shortening with chromosomal instability

44

Cadmium: 5-100 microM

Table 2. Effects of cigarette smoke on folliculogenesis.

Authors

Model and age

Exposure

Agents

Dose

Effects

Sadeau and Foster. [10]

Mouse (13day-old)

Cultured mouse ovaries

Cigarett e smoke condens ate (CSC)

Serial concentrations: 30, 60, 90,130 µg/ml for 12 days

Inhibition of follicle development at all stages Lower E2 levels (90-130 µg/ml, day 8) CSC increase P output before ovulation induction Reduced COC complex in the low (30, 60µg/ml) and high (90,130 µg/ml) groups Altered oocyte maturation in a dosedependent manner

Mai et al. [87]

Mouse (fourweek-old)

Oral administrat ion

Cigarett e smoke extract (CSE)

2mg/ml CSE solution orally daily for four weeks

Reduction in the zona pellucida thickness Alteration in the first polar body morphology Reduction in the levels of antioxidants GSTM2-GSTM3

Tuttle et al. [75]

Mouse (6-8 weeks old)

Exposure chambers

Cigarett e smoke

Mouse (PND4)

Cultured mouse ovaries

BaP

Nose-only exposure to two cigarettes daily, 5 days per week for a total of 8 weeks. Initial 2-week leadup in which mice were exposed to one cigarette in the first week and two cigarettes in the second week

Reduction in the number of primordial follicles but not growing or antral follicles No effect on markers of apoptosis after smoke exposure Increase in Bcl-2 expression and no overall change in Bax levels (100 ng/ml BaP in vitro for 6 h)

In vitro: BaP 1–10 000 ng/ml (15 days) Paixao et al. [76]

Mouse (35 days)

Exposure chambers

Cigarett e smoke

Whole body exposure to cigarette smoke 8h/day, 15 days, 7 days/week

Alteration of oocytes development in incipient antral follicles Inadequate granulosa cells proliferation after smoke exposure cessation

45

Gannon et al. [77]

Mouse (8weeks-old)

Exposure chambers

Cigarett e smoke

Whole-body exposure to cigarette smoke, twice daily, 5 days/week for 4,8,9, 17 weeks

Reduction in the number of primordial follicle pool as early as 4 weeks of exposure Increased level of Hsp25 and decreased level of SOD2 after 8 weeks of exposure Increased levels of Beclin1 and LC3 in granulosa cells Increased number of autophagosomes in granulosa cells

Jenning et al. [80]

Sobinoff et al. [90]

Gannon et al. [103]

Mouse (6weeks-old)

Mouse (5weeks-old)

Mouse (8weeks-old)

Exposure chambers

Exposure chambers

Exposure chambers

Cigarett e smoke

Cigarett e smoke

Cigarett e smoke

Nose-only exposure to mainstream cigarette smoke for 1 h, twice daily, for a total of 12 weeks.

Smoking mice oocytes with a thicker zona pellucida and with a shorter pole-to-pole spindle length and wider meiotic spindles compared to the controls

Nose-only exposure cigarette smoke for 1 h, twice daily, 4 times/week for a total of 12-18 weeks. Each exposure lasted 60 minutes

Significant reduction in primordial and primary follicle number

Whole-body exposure to cigarette smoke, twice daily, 5 days/week for 8 weeks

Mitochondrial damages in ovaries

Errors in chromosomes alignment

Apoptosis indicators localized to growing and antral follicles Oxidative stress in ovulated oocytes Increased levels of mitochondrial ROS and lipid peroxidation

Increased number of autophagosomes in granulosa cells Overexpression of BECN1 and LC3 in smoke-exposed mice BCL2 significantly lower in the ovaries of mice exposed to cigarette smoke Increased parkin levels, decreased expression of the genes Mfn1 and Mfn2

Furlong et al. [106]

Mouse (8weeks-old)

Exposure chambers

Cigarett e smoke

Whole-body exposure to cigarette smoke, twice daily, 5 days/week for 8 weeks

Increase in the expression of proautophagic genes in ovarian homogenates

Furlong et al. [111]

Mouse (8weeks-old)

Exposure chambers

Cigarett e smoke + implanta tion of cloroqui ne (CQ) pellets

Whole-body exposure to cigarette smoke, twice daily, 5 days/week for 8 weeks

Effect of cloroquine (CQ) in reduction of autophagy in ovaries

46

Hassa et al. [131]

Mouse (14-16 weeks-old)

In-vitro fertilizatio n mouse model

Cigarett e smoke

In vivo cigarette smoke exposure + 50 mg/kg of vitamin E

Effect of cigarette smoke- exposed female on fertilization and cleavage rates No impact of vitamin E on fertilization, cleavage and embryo development among cigarette smoke-exposed male and female mice

47

Table 3. Effects of cigarette smoke on IVF outcome. Authors

Gruber et al. [13]

Type of study

Retrospective

Study group

n=130 Non-smokers: n= 58 Smokers: n=72

Freour et al. [14]

Retrospective

n=111

Number of cigarettes smoked

Cotinine dosage

6 women smoked 6 cigarettes per day, 23 (10 cigarettes per day), 26 women (15-20 cigarettes per day) and 17 smoked 20 or more cigarettes per day.

Not performed

Lower fertilization rate in smokers compared to non-smokers

9 cigarettes daily

Not performed

Decreased anti Mülleran hormone concentration in smokers

Non-smokers: n= 71

Lower number of oocytes retrieved and reduced clinical pregnancy rate in smokers

Smokers: n=40

Neal et al. [24]

Retrospective

n=29 Non-smokers: n= 10

Effects

11.5± 1.5 cigarettes per day (range 3-25)

Not performed

Decrease in implantation and pregnancy rates in mainstream smokers compared with non-smokers

5 to 20 cigarettes per day

Not performed

Lower levels of β-carotene in follicular fluid of smokers than non-smokers

Mainstream Smokers: n=19 Tiboni et al. [29]

Prospective

n=60 Non-smokers: n= 43

Negative impact of smoking habits on fertilization rates

Smokers: n=17 Neal et al. [30]

Retrospective

n=225 Non-smokers: n= 146

Mainstream smokers: 10.7 ± 2 cigarettes per day

Not performed

Significant difference in pregnancy rate per embryo transfer between

48

Mainstream smokers: n=39

mainstream, sidestream and nonsmokers.

Sidestream smokers= 40 Waylen et al. [31]

Meta-analysis with a computerized search

-

-

-

Association between cigarette smoking and lower clinical pregnancy rates

Freour et al. [32]

Prospective

n=277

Not specified

Not performed

Association between cigarette smoke and lower clinical pregnancy rates

10.4 ± 7.4 cigarettes per day

Not performed

Lower clinical pregnancy rates in smokers than non-smokers

Non-smokers: n= 197 Smokers: n=80 Ben Haroush et al. [33]

Cohort

n=237 Non-smokers: n= 195

Lower live birth rate in smokers than non-smokers

Smokers: n= 42 Budani et al. [34]

Prospective

n=40

13 ± 6 cigarettes/day

Non-smokers=20

Not performed

Lower fertilization, implantation and pregnancy rates in smokers

Smokers=20 Wright et al. [35]

Retrospective

n= 389 Non-smokers: n= 159 (<35 yrs), n=147 (> 35 yrs) Smokers: n= 36 current smokers (<35 years)

Increased levels of SOD2 and catalase mRNA in GCs of smokers

Current smokers: Twothirds of women (21/30) smoked 10 cigarettes or fewer per day (information not available for six women).

Not performed

No association between smoking status and fertility or pregnancy outcomes

n= 47 had smoked in the past (≥ 35 years)

49

Fuentes et al. [36]

Cohort prospective

n=166 Non-smokers: n= 133

17.8 ± 8.94 cigarettes per week

Recent smokers: cotinine > 10 ng/ml in follicular fluid

Recent smokers: n=33

Association between cigarette smoking and decline in the number of oocytes retrieved during IVF cycles Lower implantation rate among recent smoker women Association between recent male smoking and decreased in live birth rates

Cinar et al. [37]

Comparative prospective

n=214

Not specified

Non-smokers: n= 171

Smoker: cotinine > 20 ng/ml in FF

Smokers: n=43

Higher number of collected oocytes in smokers treated with antagonist stimulation No association between male smoking and reduced IVF success

Kim et al. [38]

Retrospective

n= 127

Not specified

Urinary cotinine concentration ≥ 200 ng/ml

No association between positive urinary cotinine tests and IVF/IUI outcomes

Zenzes et al. [60]

Prospective

n= 234

30 cigarettes per day (9.9±0.7)

Follicular fluid cotinine dosage

Positive cotinine effect on oocyte maturation in younger women, negative effect (decreased oocyte maturation with increasing cotinine concentrations) in older women

Non-smokers: n= 130 Passive smokers: n=30 Active smokers: n=74

El-Nemr 98 [126]

Retrospective

n=173 Non-smokers=108

Smoking negative effect on the proportion of fertilized oocytes in women >35 years of age 3 women were smoked 5 or less cigarettes per day, 24 (10 cigarettes per day), 20 (15 cigarettes daily) and

Not performed

Higher mean basal FSH levels in smokers

50

Smokers=65

Van Voorhish 92 [128]

Retrospective, cohort syudy

n=54 Non smokers=36

18 (20 or more cigarettes daily). Other women smoked less than 10 cigarettes. Smokers: ≥ 10 cigarettes per day

Lower numbers of retrieved oocytes and higher fertilization failure in smokers Lower clinical pregnancy rate in smokers not statistically significant Not performed

Smokers=18

Sterzick et al. [129]

Retrospective

n=197

No differences in the amounts of gonadotropin administration between smokers and non-smokers Lower E2 levels, follicles, oocytes retrieved and embryos per cycle in smokers

Not specified

Non smokers=68 Passive smokers=26 Active smokers=103

Non-smoker, cotinine concentration ≤ 2 0 ng/mL

No differences in fertilization and pregnancy rates between the three groups

Passive smoker: cotinine concentration >20 ng/mL and≤ 50 ng/mL

E2 levels alteration between active and passive smokers

Active smoker, cotinine concentration >50 ng/mL. Pattinson et al. [130]

Retrospective

n=360 couples Non-smoker female partners=236 Smoker female partners=124

17 women smoked < 10 cigarettes/day, 46 women (10 to 20 cigarettes/day) and 62 women smoked ≥ 20 cigarettes/day.

Not performed

No differences in E2 levels, number of eggs, fertilization and implantation rates between smokers and non-smokers. Lower pregnancy rates in smokers. Higher spontaneous abortions in smokers.

51

Benedict et al. [132]

Retrospective

n=1909

Not specified

Never smoker: n= 1319

Smoker: cotinine concentration ≥10 ng/ml in follicular fluid

Ex-Smokers: n= 590 Klonoff-Kohen et al. [133]

Prospective

Risk of implantation failure among women exposed to secondhand smoke exposure. Reduced odds of a live birth in smokers.

n=221 couples

Not specified.

Female (221): NonSmokers: 47.1%, Smokers: 50.2%, Smoking status unknown: 2.7%

Mean period of smoking by female: 3.77 ± 6.7 Mean period of smoking by men: 4.2 ± 7.31

Not performed

Major risk of not achieving a pregnancy for couples who smoke for > 5 years Decrease number of oocytes in couples who smoke during the week of the visit for IVF or GIFT

Men (166): Nonsmokers 42.1%, smokers 42.5%, smoking status unknown 15.4%

52