Örebro University
School of Medical Sciences Degree project, 15 ECTS January 2019
Impact of maternal smoking on
mRNA expression in placental tissue
- a systematic literature survey
Version 1
Author: Felix Roxenlund Supervisor: Maria Lodefalk, MD, PhD Örebro, Sweden
1
Abstract
Introduction: Maternal smoking during pregnancy is becoming less common. However, it is still a significant predictor of adverse outcomes. Smoking during pregnancy has been
associated with impaired fetal development and growth. The purpose of this systematic litera-ture study was to describe the placental molecular targets that have been identified as poten-tially affected by maternal smoking, with emphasis on alterations in placental gene expres-sion.
Objective: To decribe the impact of maternal smoking on mRNA expression in placental tissue.
Design: Systematic literature survey
Methods: We searched for studies in PubMed. To be included in the survey the studies had to be written in English and asses mRNA expression in isolated placental cells, animal, or hu-man placental tissue in conjunction with exposure to some or all elements of tobacco smoke. Out of 54 studies found, 14 met the eligibility criteria. One reviewer assessed the eligibility and quality of the studies.
Results: Eight studies investigated human placental tissue, two investigated rodent placental tissue, and five studies investigated isolated placental cells. Two studies profiled the global gene expression, while the other studies examined the expression of the CYP genes, or genes involved in angiogenic regulation, cellular proliferation, or amino acid metabolism and transport in relation to cigarette smoking.
Discussion: We conclude that even though the influence of cigarette smoking on gene expres-sion in placenta has only been investigated in a few studies, they all found transcriptional al-terations in relation to smoke exposure. Future investigations are necessary for the examina-tion of how these changes may impact fetal development and growth.
2
Keywords: Tobacco, smoking, placenta, pregnancy, mRNA, literature survey.
Abbreviations: 11β-HSD2, 11β-hydroxysteroid dehydrogenase 2; AHRR, aryl hydrocarbon receptor re-pressor; AHQR, Agency for Healthcare Research and Quality; Ang1, angiopoi-etin-1; Ang2, angiopoietin-2; CSE, cigarette smoke-extract; CYP, Cytochrome P450; FGR, fetal growth restriction; Flt1, fms-like tyrosine kinase-1; GM-CSF, granulocyte-macrophage colony-stimulating factor; GOT, aspartate aminotransferase; HIF-1α, hypoxia-inducible fac-tor-1α; LAT, linker for activation of T-cells family members; MDRP1a, multidrug resistance protein 1a; NOS, Newcastle-Ottawa scale; PlGF, placental growth factor; RT-qPCR, real time quantitative reverse transcription PCR; sENG, soluble endoglin; sFlt-1, soluble fms-like tyro-sine ki-nase-1; sFRP1, secreted frizzled-related protein; SIDS, sudden infant death syndrome; SYRCLE’s RoB tool, SYRCLE’s tool for assessing risk of bias; TC, trophoblast cell; Tie2, tyrosine kinase receptor-2; VEGF, vascular endothelial growth factor.
3
Index
1. Introduction 4
2. Material and Methods 6
2.1 Inclusion criteria 6
2.2 Study procedure 6
2.3 Selection of eligible articles 6
2.4 Quality assessment 7
2.5 Ethics 8
3. Results 9
3.1. Characteristics of the included studies 9
3.2. Genome-wide profiling 11
3.3. Cytochromes P450 11
3.4. Angiogenic regulation 12
3.5. Cellular proliferation 13
3.6. Amino acid metabolism and transport 13
3.7. Cadmium 13
4. Discussion 15
4.1. Cytochromes P450 15
4.2. Vascular integrity 15
4.3. Cellular proliferation 16
4.4. Amino acid exchange and metabolism 17
4.5. Cadmium 17
4.6. Offspring sex 18
4.7. Reliability of data regarding tobacco exposure 18
4.8. Methods for the evaluation of gene expression 19
4.9. Therapy development 19 4.10. Future perspectives 19 4.11 Study limitations 20 5. Conclusions 21 6. Acknowledgments 22 7. References 23
4
1. Introduction.
Smoking during pregnancy has been associated with impaired fetal development and growth [1]. Specifically, maternal smoking has been shown to decrease birth weight, reduce fetal head, abdominal circumference, and femur length [2]. Along with the most morbid outcomes, a 20-30% increased probability for stillbirth, a 40% elevation of the risk of infant mortality and a 2-fold increase in the incidence of sudden infant death syndrome (SIDS) have been associated with prenatal smoking [3]. Maternal smoking in pregnancy has also been
associated with the development of postnatal morbidities such as asthma, obesity, and cardio-vascular disorders. [4-6]. In addition to debilitative effects on the fetus, smoking during preg-nancy has also been found to adversely affect the mother during childbirth predisposing to conditions such as placenta previa, placenta accreta, and placental abruptions, as well as in-creasing the risk for infections and ectopic pregnancies [7-10]. Although rarely life-threaten-ing to the mother, these adverse conditions, are imminently more so for the fetus [7-10]. According to the Centers for Disease Control and Prevention, maternal smoking is becom-ing less common; however, it is still a significant predictor of adverse outcomes such as prematurity, low birth weight and by extension, fetal mortality [11,12].
Although the association between maternal smoking and the mentioned adverse outcomes has been made clear, a consensus regarding how maternal smoking elicits them has not yet been achieved.
The placenta, which is the primary link between the mother and fetus has many functions; transport and metabolism of nutrients, exchange of blood gasses, endocrine signaling, and protection against xenobiotics [13]. The exchange of blood gasses is mainly bottlenecked by the rate of blood flow through the umbilical arteries and placental perfusion capacity, as op-posed to the transport of nutrients, which relies heavily on the viability of cytotrophoblasts and syncytiotrophoblasts [14,15]. As a major endocrine organ which lacks innervation, it is left to interconnect with fetal and maternal tissue with the help of blood-borne paracrine and autocrine factors [14]. As portrayed, placental functions are heavily dependent on adequate vascularization, and perfusion, and as such has a well-developed barrier function hindering the passage of potentially harmful substances from the mother to the fetus [14].
Smoking during pregnancy has been found to adversely affect several placental functions, including the ones mentioned above. Even though numerous efforts have been made to ex-plain the pathologic process by which smoking-induced placental alterations occur, the mech-anisms behind it are still mostly unknown. It has also been speculated that theses
smoking-5
induced placental alterations may be implicated in the morbid fetal outcomes which were cited earlier [16].
We aimed at summarizing the current knowledge on alterations in gene expression in pla-cental tissue evoked by exposure to cigarette smoking.
6
2. Material and Methods.
2.1. Inclusion criteria.
Studies investigating effects of cigarette smoking on mRNA expression in isolated placental cells, animal or human placental tissue with ethical approval and written in English were included in this literature survey.
2.2. Study procedure.
The search was conducted in PubMed the 30th of June 2018. The following terms were used: (1) smoking, (2) placenta, (3) mRNA, and (4) messenger RNA in all fields. No manual search was performed. No restriction based on time of publication was applied.
2.3. Selection of eligible articles.
One reviewer (FR) assessed the eligibility and quality of the articles found. The search identi-fied 54 publications as presented in Table 1. Their titles and abstracts were vetted for identifi-cation of studies fulfilling the inclusion criteria. After this procedure, 26 of the 54 publica-tions were chosen and read in full text. Out of them, 14 were selected for the full review and included in this thesis. The remaining 12 were excluded as they did not meet the inclusion criteria (Figure 1).
Table 1. Search results.
Date of search: 30th of June 2018
ID Search terms Hits Time
#1 smoking 255304 05:45:16
#2 placenta 90201 05:45:40
#3 mRNA 614729 05:46:01
#4 messenger RNA 410021 05:46:33
#5 (mRNA) OR messenger RNA 614729 05:47:00 #6 (((mRNA) OR messenger RNA)) AND smoking 2113 05:47:20 #7 (((((mRNA) OR messenger RNA)) AND smoking)) AND placenta 52 05:48:11 #8 ((((((mRNA) OR messenger RNA)) AND smoking)) AND placenta)
Sort by: Best Match
7
Figure 1. Flowchart for study eligibility. 2.4. Quality assessment.
Quality assessment was performed using two risk of bias (RoB) tools: SYRCLE’s RoB tool and The Newcastle-Ottawa scale (NOS). Both were used according to the instructions pro-vided by respective manufacturers [17-20].
SYRCLE’s RoB tool is designed to help assign RoB in animal intervention studies and con-sist of 10 items relating to selection bias, performance bias, detection bias, attrition bias, re-porting bias and other biases. Scoring is reported as low, high or unclear RoB [17]. SYR-CLE’s RoB tool was used to assess the RoB in both animal and in vitro studies due the rela-tive similarities in how these two types of studies are conducted and reported [21].
NOS was used to assess RoB in human studies.NOS uses a “star system” whereby studies are judged on three domains; selection of the study groups, the comparability of the groups, and the finding of either the exposure or outcome of interest for case-control or cohort studies. A maximal score of 4 points for selection, 2 points for comparability, and 3 points for out-comes can be allotted [18-20]. The Agency for Healthcare Research and Quality (AHQR) acts to improve the safety and quality of America's health care system. In order to clarify the results generated by the usage of NOS, threshold conversion was applied by which
cumulative scores were converted to AHQR standards of good (≥3 stars in selection domain,
Studies identified through database searching
(n = 54)
Additional studies identified through other sources
(n = 0)
Studies included for vetting of ti-tles and abstracts
(n = 54)
Studies excluded af-ter vetting titles and
abstracts (n = 27)
Full-text articles assessed for el-igibility (n = 26)
Full-text articles excluded due to:
Lack of ethical approval (n=2).
Lack of statistical data (n=2).
Lack of gene expression investigation
(n = 8). Studies included for full review
in this thesis (n =14) Study not available in
Eng-lish and therefore excluded (n =1)
8
plus ≥1 star in comparability domain, plus ≥2 stars in outcome/exposure domain), fair (≥2 stars in selection domain, plus ≥1 star in comparability domain, plus ≥1 star in
outcome/exposure domain), and poor (<2 stars in selection domain, plus <1 star in comparability domain, plus <2 stars in outcome/exposure domain) [22].
2.5. Ethics.
In order to minimize the risk of including studies with ethical insufficiencies, only studies which reported to have been ethically approved by a board or committee were included in this systematic literature survey.
9
3. Results.
3.1. Characteristics of the included studies.
The main characteristics concerning the methodology of the included studies are summarized in table 2. The results of them are presented in the text below.
Table 2. Main characteristics of the included studies sorted by targets studied.
Article Targets studied Placental tissue model Methods Quality Bruchova,
2010 [23]
Gene expression pro-files determined with 18,216 gene probes.
Quantitative
verification of selected genes using RT-qPCR: CYP1B1, COX412, PLA2G5, EDNRA, and PROCR.
Human full-term placen-tas from smokers (n=12) and non-smokers (n=64). Smoking was assessed using a questionnaire, and measurements of plasma cotinine levels in maternal venous and cord blood.
mRNA: Microarray assay (HumanRef-8 v2 Expression BeadChips), complemented with RT-qPCR. Good1 Huuskonen, 2008 [24]
CYP enzyme family expression.
Human full-term placen-tas. Controls: n=5. Tobacco-exposed: n=5. Reports on how tobacco exposure was confirmed could not be found.
Microarray complemented with RT-qPCR.
Fair1
Yan, 2005,
[25]
CYP1A1 expression. Wistar rats. Controls: n=79. Tobacco exposed: n=76.
RT-qPCR. Low2
Fa, 2018
[26]
CYP1A1 and AHRR expression, and DNA methylation of their re-spective promoter genes.
Human first-trimester placentas (6-12 weeks). Smoking was assessed with the help of a ques-tionnaire.
Controls: n=22. Tobacco exposed: n=17
mRNA: RT-qPCR.
DNA methylation: Bisulfite conversion of DNA followed by PCR amplification.
Fair1
Whyatt, 1995 [27]
CYP1A1 expression. Human full-term placen-tas from ex-smokers (n=36), current smokers (n=15), and non-smokers (n=104).
Smoking was assessed using a questionnaire and measurement of plasma cotinine levels in mater-nal venous blood.
Slot-blot analysis Good1
Chen, 2017 [28]
VEGF, Tie2, Flt1, Ang1 and Ang2 ex-pression. SPF Sprague Dawley rats. Control: n=8. Tobacco-exposed: n=29. RT-qPCR Low2 Romani, 2011 [29]
Effects of nicotine and cotinine on gene and protein expression of sFlt-1, sENG, PlGF, and VEGF.
TCs collected from full-term human placentas (n=24).
mRNA: RT-qPCR.
Protein: ELISA.
10
Article Targets studied Placental tissue model Methods Quality Zhao, 2017
[30]
Effects of nicotine on VEGF, HIF-1α and sFlt1 gene and protein expression.
TCs obtained from a hu-man choriocarcinoma cell line (BeWo).
mRNA: RT-qPCR.
Protein: Western blotting complemented with ELISA.
Low2
Wang,
2015 [31]
SFRP1 gene and pro-tein expression.
Human full-term placentas. Smoking was assessed by self-report-ing. mRNA: Controls: n=14. Tobacco exposed: n=7. protein: Controls: n=8. Tobacco exposed: n=9. mRNA: RT-qPCR.
Protein: Immunoblot assay with densitometry. Fair1 Kraus, 2014[32] Effects of adrenomedullin on TC invasiveness. Adrenomedullin gene (ADM) and protein ex-pression
TCs: Immortalized first-trimester human cyto-trophoblast cell line (HTR-8/SVneo).
Human full-term placen-tas from smokers (n=11) and non-smokers (n=11). Smoking was assessed by self-reporting.
Cellular invasiveness: Im-munohistochemistry. ADM expression: RT-qPCR Adrenomedullin expression: Immunohistochemical-staining. Low2 Fair1 Fu, 2012 [33] Effects on GM-CSF expression in TCs treated with CSE.
TCs: Immortalized normal human tropho-blast cell line (B6Tert-1).
RT-qPCR Low2 Day, 2015 [34] Expression of amino acid exchange transporters (LAT-1/2/3, GOT-1/2).
Human full-term placen-tas from pre-pregnancy smokers (n=26) versus never-smokers (n=76). Human full-term placen-tas from current smokers (n=14) versus non-smok-ers (n=81).
Smoking was assessed using a questionnaire.
RT-qPCR Good1
Stasenko
2010[35]
Cadmium concentra-tions. Effects of cad-mium on leptin expres-sion.
Human full-term placen-tas from smokers (n=99) and non-smokers (n=109), as assessed by self-reporting of smoking habits.
Cytotrophoblasts were collected from full-term human placentas (n=4).
Cadmium levels: Atomic absorption spectroscopy. Leptin mRNA: RT-qPCR. Good1 Low2 Yang, 2006 [36] Effects of cadmium on 11β-HSD2 enzyme ac-tivity, promoter activ-ity, protein levels, and mRNA levels in iso-lated human tropho-blast cells.
Primary human tropho-blast cells isolated from full-term human placen-tas obtained from un-complicated pregnancies after elective cesarean section.
Enzymatic activity:
Radiometric conversion assay.
Promoter activity: Reporter gene assay.
Protein levels: Western blot. mRNA levels: RT-qPCR.
11
1NOS, Newcastle-Ottawa scale. 2SYRCLE’s RoB tool, SYRCLE’s tool for assessing risk of bias. Abbreviations: 11β-HSD2, 11β-hydroxysteroid dehydrogenase 2; AHRR, aryl hydrocarbon receptor re-pressor; Ang1, angiopoietin-1; Ang2, angiopoietin-2; CSE, cigarette smoke-extract; CYP, Cytochrome P450; Flt1, fms-like tyrosine kinase-1; GM-CSF, granulocyte-macrophage colony-stimulating factor; GOT, aspar-tate aminotransferase; HIF-1α, hypoxia-inducible factor-1α; HUVEC, human umbilical vein endothelial cell, LAT, linker for activation of T-cells family members; PlGF, placental growth factor; RT-qPCR, real time quantitative reverse transcription PCR; sENG, soluble endoglin; sFlt-1, soluble fms-like tyrosine kinase-1; sFRP1, secreted frizzled-related protein; TC, trophoblast cell; Tie2, tyrosine kinase receptor-2; VEGF, vascu-lar endothelial growth factor.
3.2. Genome-wide profiling.
A microarray analysis of 18,216 genes found that 10,590 genes were expressed at a detectable level in human placental tissues. Of them, 241 genes showed a significant differential expres-sion in current smokers compared with non-smokers after adjustment for multiple testing us-ing the Benjamini and Hochberg method [23]. Of the differentially expressed genes, 178 were significantly up-regulated, and 63 were significantly down-regulated in smoking women. The up-regulated genes included genes coding for xenobiotic-metabolizing enzymes (CYP1A1,
CYP1B1, CYB5A and COX412), collagen (COL6A3, COL1A1, COL1A2, COL6A1, COL3A1, COL4A1, COL5A1 and COL6A2), extracellular matrix (EFEMP1, MXRA5, SPON1, EFEMP2
and FRAS1), coagulation factors (F5, F13A1), endothelial components (EDG2, EDNRA,
LIPG, LYVE1 and PECAM1), vascular factors (CTHRC1, AGTR1) and inflammatory factors
(PLA2G5, AFAP1L2 and IL33). The down-regulated genes included genes related to adipo-cytes (LEP, LPL), anion transport (SLCO4A1, SLC39A6, and SLC7A1), polyamine biosynthe-sis (AMD1), pregnancy maintenance (PROCR) and genotoxic stress (NEK11). The microarray findings were confirmed by RT-qPCR analyses of five chosen genes [23].
In another genome-wide study, regulation of the CYP1 family in placentas from smoking women was examined. No significant up or down-regulation of members of the aryl hydrocar-bon receptor complex or nuclear receptors were observed in smoking women as compared to non-smoking women [24].
3.3. Cytochromes P450.
Female rats not exposed to cigarette smoking showed no CYP1A1 expression in their placen-tas. However, expression of that gene was detected in placentas of rats exposed to cigarette smoking. The expression increased by gestational age. Furthermore, the level of multidrug
re-sistance protein 1a (MDRP1a) expression was significantly increased at gestational day (GD)
21, but not at GD 14, in rats exposed to cigarette smoking [25].
In first trimester placentas of female fetuses of smoking women, a significant increase of the methylation of aryl hydrocarbon receptor repressor (AHRR) was observed compared to first
12
trimester placentas of female fetuses of non-smoking women. However, no association was found between AHRR DNA methylation and gene expression. The expression of CYP1A1 in placentas of exposed fetuses was significantly increased, even though no differences in DNA methylation of that gene were observed compared to placentas of un-exposed fetuses [26]. Placentas from both current smokers and mothers who had quit smoking ≥ 1 month before delivery showed a significant increase in CYP1A1 mRNA levels. In contrast, environmental tobacco smoke exposure resulted in only a marginal increase in CYP1A1 expression in pla-cental tissue [27].
3.4. Angiogenic regulation.
Rats exposed to smoking with normal-litter-size pregnancies (NP; 13±3 fetuses per gestation) but low birth weight (LBW) showed a tendency towards decreased placental mRNA levels of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang1) and tyrosine kinase recep-tor-2 (Tie2). However, a significant decrease of these mRNA levels was observed in rats ex-posed to smoking with small-litter-size pregnancies (SP; 2±1 fetuses per gestation) but normal birth weight (NBW). Concomitantly, a tendency for decreased placental mRNA levels of fms-like tyrosine kinase-1 (Flt1) and angiopoietin-2 (Ang2) was observed in rat with SP and LBW of their offspring compared to rats with NP and NBW their offspring [28].
Trophoblast cells (TCs) treated with nicotine and cotinine respectively showed a significant decrease in soluble fms-like tyrosine kinase-1 (sFlt1), placental growth factor (PlGF), and sol-uble endoglin (sENG) secretion, while VEGF121 and 165 isoform expressions remained un-changed [29].
TCs cultured under hypoxic conditions demonstrated a significant increase in sFlt1 secre-tion, which was inhibited when the TCs were treated with a low dose of nicotine. When TCs were treated by a low dose of nicotine under normoxic conditions, no significant changes of the secretion of sFlt1 were found. Furthermore, nicotine treatment induced a significant in-crease of VEGF expression in TCs under hypoxic conditions. Comparatively, TCs in
normoxic conditions did not respond to nicotine treatment. Decreasing sFlt1 by the usage of a neutralizing antibody was found to significantly improve VEGF concentrations in TCs under both hypoxic and normoxic conditions [30].
Under normoxic conditions, TCs were found to express a negligible amount of hypoxia-in-ducible factor-1α (HIF-1α), but the HIF-1α protein concentrations increased significantly un-der hypoxic conditions. Treatment of TCs with HIF-1α small interfering RNA (siRNA), which is a posttranscriptional gene down-regulator of HIF-1α, significantly inhibited HIF-1α
13
protein expression under hypoxic, but not, normoxic conditions. Treatment of TCs with a low dose of nicotine significantly up-regulated HIF-1α expression under hypoxic conditions, but not under normoxic conditions. Further, pre-treatment with HIF-1α siRNA significantly in-hibited nicotine-mediated VEGF expression [30].
3.5. Cellular proliferation.
Placentas of smoking women showed a significant (>10-fold) up-regulation of both mRNA and protein levels of secreted frizzled-related protein 1 (sFRP1) [31].
Gene expression of the angiogenic and anti-inflammatory peptide adrenomedullin (AM) was significantly increased (3.3-fold) in placentas of smokers compared to the placentas of nonsmokers. TCs treated with AM, or 1% cigarette-smoke extract (CSE) showed a significant increase in cellular invasiveness. When TCs were co-treated with an AM inhibitor and AM or CSE, cellular invasiveness was significantly attenuated. The intensity of AM-staining was significantly higher in placentas of smoking women [32].
TCs cultured with 10% CSE in the medium showed a significantly increased GM-CSF ex-pression (5.7-fold) and significantly increased GM-CSF protein level (4.3-fold) [33]. 3.6. Amino acid metabolism and transport.
Pre-pregnancy smoking was found to significantly increase the human placental mRNA ex-pression of linker for activation of T-cells family (LAT) member 2, y+LAT2, and significantly decrease the placental mRNA expression of aspartate aminotransferase (GOT) 1 compared to placentas from women who never have smoked. Pre-pregnancy smoking was also found to significantly increase the placental mRNA expression of y+LAT1 in placentas of female but not male fetuses [34].
Maternal smoking during pregnancy was found to significantly increase the placental mRNA expression of LAT3 and y+LAT2 in placentas of female but not male fetuses [34]. 3.7. Cadmium.
A doubling of the cadmium concentration was observed in placentas from smoking mothers when compared to non-smoking mothers (22.1 ng g-1 wet weight ± 6.79 and 10.3 ng g-1 ± 4.26 wet weight, respectively, P<0.05). A dose-dependent decline in transcription of leptin mRNA in TCs co-cultured with 10 μM or 20 μM cadmium chloride (CdC12) (P<0.025) was also found [35].
14
TCs treated with 1.0 μM CdCl2 for different intervals (3, 6, 12, or 24 hours) showed a time-dependent decrease in 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) activity. Significant reduction occurred already after three hours of treatment [36].
TCs treated with varying doses of CdCl2 (0.25 μM to 1.0 μM) showed a significant concen-tration-dependent decrease in 11β-HSD2 activity with peak effect at 0.75 μM CdCl2 [36].
15
4. Discussion.
4.1. Cytochromes P450.
Cigarette smoke contains over 7000 chemical compounds, of which numerous can potentially be transferred from the mother to the fetus through the placenta (e.g., polycyclic aromatic hy-drocarbons [PAHs]) [37,38].
Several PAHs have been classified as toxic and potentially carcinogenic. But they can be detoxified by the action of CYP enzymes [38,39]. However, with detoxification emanates a consequent formation of potentially genotoxic DNA adducts and free radicals, which also can be harmful to the fetus [39]. A few studies aimed to establish that placental upregulation of CYP transcripts, primarily CYP1A1, is a direct consequence of tobacco exposure. Upregula-tion of CYP1A1 expression was confirmed in placental tissue in both rat and human studies; however, the mechanism governing this up-regulation could not be determined [25,26]. Up-regulated CYP1A1 expression was associated with tobacco-induced FGR [26]; however, the mechanism by which the generation of harmful byproducts impedes fetal growth remains to be further evaluated.
4.2. Vascular integrity.
As stated earlier, adequate vascularization and perfusion is essential for the placenta to func-tion optimally [14,15]. Several of the reviewed studies presented data indicating aberrant sig-naling in reference to angiogenesis, thrombogenesis, and vasoconstriction in placentas ex-posed to cigarette smoking, which probably leads to suboptimal placental function.
VEGF and its receptor Flt1 play a significant role in endothelial cell proliferation, migra-tion, and tube formation in the placenta [40]. Ang1, Ang2 and their receptor Tie2 are essential for vascular remodeling and stabilization of newly formed blood vessels [41]. VEGF, Flt1, Ang1, Ang2, and Tie2 placental mRNA expression were decreased or tended to be decreased in rats exposed to cigarette smoking [28]. This supports the hypothesis that smoking may dis-rupt placental vascularization and predispose for FGR.
EDNRA encodes for the receptor for endothelin-1, a vasoconstrictive peptide, and PLA2G5 encodes for a calcium-dependent phospholipase [42]. Both transcripts were shown to be significantly up-regulated in placentas from smoking women [23]. PROCR, which encodes an endothelial protein C receptor (PROCR) was shown to be significantly down-regulated in placentas from smoking women when investigated by microarray assay; however, RT-qPCR analysis of PROCR failed to show statistically significant down-regulation [23].
16
Both the up-regulation of EDNRA and PLA2G5 and down-regulation of PROCR could po-tentially predispose for placental abruption, placental insufficiency and/or FGR by increasing the production of thrombotic factors as well as increasing the rate for thrombi formation [23]. Studies performed on TCs treated with nicotine showed ambiguous results. Nicotine was shown to potentiate the angiogenic effects of VEGF and PlGF in TCs by decreasing the secre-tion of the competitive inhibitor sFlt-1, while simultaneously decreasing the secresecre-tion of PlGF [29]. TCs studied in hypoxic conditions showed significantly increased expression of HIF-1α, and co-treatment with nicotine further amplified the HIF-1α expression. Interestingly pre-treatment with HIF-1α siRNA significantly inhibited nicotine-mediated VEGF expression, which points to a potential HIF-1α-mediated increase in VEGF expression by nicotine. This imbalance of vasoactive factors has been proposed to be one of the mechanisms by which maternal smoking leads to FGR [30].
4.3. Cellular proliferation.
In order to sufficiently supply the fetus with vital nutrients, adequate trophoblastic invasion and placental tissue remodeling must be achieved. Three studies specifically studied the ef-fects of smoking on trophoblastic proliferation, out of which two focused on placental cellular proliferation induced by smoking as a potential defense mechanism [31-33].
The WNT signaling pathway promotes embryonic and placental development by regulating cellular differentiation, proliferation and apoptosis [43]. In placentas of smoking women, pro-tein and mRNA levels of the WNT signaling pathway antagonist sFRP1 were found to be significantly up-regulated. This up-regulation is suggested to contribute to abnormal placenta-tion, and as a consequence, an increased risk of perinatal and neonatal morbidity [31].
The angiogenic and anti-inflammatory peptide AM was significantly up-regulated in TCs treated with CSE [32,44] and in placentas of smoking women [32]. It is suggested that AM could potentially improve placental function and vascularization, annulling the undesirable effects of tobacco exposure [32]. A mechanism by which CSE increases AM mediated cellu-lar invasion was however not addressed in these studies.
Paralleled to the effects of AM, GM-CSF has been suggested as a critical regulator of em-bryonic implantation and development, as well as placental development, through cell number ruling and improved blastocyst viability [45]. This is supported by the observed impaired fer-tility, FGR and fetal loss in mice lacking the GM-CSF gene [46]. TCs cultured with CSE showed a significantly increased GM-CSF mRNA and protein expression suggesting a com-pensatory mechanism by which TCs are tasked to restore damaged tissue [33].
17 4.4. Amino acid exchange and metabolism.
Placental amino acid transfer is vital for fetal growth, while gene expression of facilitated amino acid transporters LAT3 and TAT1 has been positively correlated with fetal growth [47]. One of the included studies investigated the effects of smoking on amino acid exchange and metabolism [34]. Interestingly, genes for amino acidexchange transporters y+LAT1, LAT2 and y+LAT2, and facilitated transporter LAT3 were variably up-regulated in placentas from past and current smokers when compared to non-smokers. Also, maternal smoking during pregnancy was found to significantly increase the placental mRNA expression of LAT3 in pla-centas of female but not male fetuses [34]. It remains however to be explained how these changes are expressed on a post-transcriptional level.
4.5. Cadmium.
Cadmium is a major component of tobacco smoke and has been associated with small for ges-tational age (SGA) infants even in the absence of maternal smoking during pregnancy [48]. However, the underlying mechanisms for how fetal growth restriction (FGR) is attained is un-known [2]. Cadmium can act as a metalloestrogen and an endocrine disruptor in reproductive tissues. This can have a detrimental effect regarding the synthesis of both steroids, such as progesterone, and polypeptide hormones, such as human chorionic gonadotropin (hCG) and leptin, in the placenta [49,50].
Several of the reported results accent that cadmium exposure due to tobacco smoking may contribute to FGR. Firstly, cadmium concentrations were double as high in placentas from smoking women as in placentas from non-smoking women [35]. Secondly, cadmium was ob-served to significantly decrease leptin mRNA transcription in TCs, which may mitigate lep-tin’s tropic, mitogenic, and angiogenic effects in placental tissue [35,51]. Thirdly, cadmium significantly decreased the activity of 11β-HSD2 in TCs. Glucocorticoids are known to inhibit growth and 11β-HSD2 protects the fetus from exposure to high levels of maternal glucocorti-coids by converting maternal cortisol to its inactive metabolite cortisone. Placental 11β-HSD2 activity has been positively correlated with birth weight, while 11β-11β-HSD2 deficiency resulting from mutations in the HSD11B2 gene has been associated with FGR [36,52].
18 4.6. Offspring sex
Only two of the studies included in this survey adjusted their findings for fetal sex [26,34]. As, stated earlier, Day et al., found fetal sex-specific changes in the expression of amino acid exchange and metabolism genes in placental tissue in relation to cigarette smoke exposure [34]. Placental gene expression is fetal sex-specific, which highlights the importance of ad-dressing fetal sex when conducting studies on placental tissue [53].
4.7. Reliability of data regarding tobacco exposure.
In order to reliably evaluate tobacco exposure, a viable indicator should be used. All included human studies needed data on smoking habits to be able to correctly dichotomies obtained tis-sues into two groups, those from smokers and those from non-smokers. Information on smok-ing status was collected in these studies in three different ways: questionnaire together with measurements of plasma cotinine levels in maternal venous and cord blood [23,27], question-naire only [26,34], or self-reporting [31,32,35]. One study did not report how tobacco expo-sure was confirmed [24].
Bruchova et al. mentioned that in the initial phase of their study ten women declared to have been exposed to tobacco smoke, but the questionnaire data seemed to be unreliable and there-fore smoking status was further confirmed using the measurement of plasma cotinine levels in maternal venous and cord blood. This led to two non-smoking women being reclassified as smokers [23]. Conversely, Fa et al. refer to a study similar to their own of a maternal smok-ing/non-smoking cohort, where the correspondence between smoking status among mothers and presence of cotinine in fetal organs supported high reliability of self-reported smoking habits [26,54]. From the studies at hand we suggest that a combination of different methods for collecting reliable data on maternal smoking should be employed as each method carries its own weaknesses. In the past, questionnaires have shown to be reliable in assessing smok-ing habits; however, a reluctance towards disclossmok-ing smoksmok-ing status can be expected in certain populations, including pregnant women whose smoking is often regarded as socially unac-ceptable [55]. Measuring plasma cotinine levels provides a marker for recent cigarette smoke exposure. However, the cotinine concentration in body fluids of pregnant women has been shown to differ from that of the average adult population due to altered metabolism and distri-bution of nicotine and cotinine in body fluids during pregnancy. Higher clearance rates of co-tinine in pregnant compared with nonpregnant women has been suggested [56]. A strong case has been made for the collection of cord blood as cotinine levels obtained discriminate not
19
only smokers and non-smokers but also non-smokers which have been exposed to secondhand smoke [57].
4.8. Methods for evaluation of gene expression.
Two studies investigated tobacco smoke-induced alterations in the global transcriptome from human placentas, and both of them used micro-array assays. An alternative to microarray as-says is deep sequencing, which has the advantage of not being restricted to a preset number of genes, but the disadvantage of being quite expensive. Most studies included in this survey used RT-qPCR, which is a useful method because of its ability to produce quantitative results, however, it can only be utilized when examining a restricted number of genes.
4.9. Therapy development.
The studies included in this literature survey primarily focused on genomic alterations since that was the objective of the survey, and as such they provided little information regarding how observed findings could be useful in the future for the potential development of therapy. Smoking cessation is still the mainstay of treatment. Studies investigating the use of nico-tine replacement therapy have been conducted, however, without consistent results concern-ing positive or negative impacts on fetal outcome [58]. Future studies may use findconcern-ings from studies included in this survey in the development of new strategies for the treatment of FGR or poor fetal development.
4.10. Future perspectives.
Studies attempting to explain the effects of tobacco-smoke exposure on the developing fetus are met with the chief obstacle of limited knowledge on which components that principally mediate the adverse effects. The number of byproducts generated from tobacco-exposure is still left to be investigated. Human studies are further complicated by confounding factors, such as poor maternal psychological, social and physical status, which are associated with maternal smoking during pregnancy and by themselves can negatively impact fetal develop-ment and health [59,60].
Seven of the human studies included in this survey examined gene expressions in full term placentas, while only one examined gene expressions in first trimester placentas. Comparing previous studies performed on placental and fetal tissue, variable results can be observed de-pending on fetal age [26,61]. This highlights the complexity of performing studies on placen-tal tissue and marks the risk of drawing false conclusions. Alterations in gene expression at
20
term could potentially be misleading considering how fetal development from a zygote to a fully developed newborn is an extremely interchangeable process.
The included studies examined various components found in tobacco and potentially toxic byproducts. All studies presented data supporting an association between tobacco-exposure and transcriptional alterations. However, further investigations are necessary to pin down how these alterations are elicited. The mechanisms by which tobacco exposure decreases 11β-HSD2 activity, increases CYP1A1 mRNA expression, dysregulates expression of angiogenic and vascular remodeling factors and increases AM and GM-CSF expression in placental tis-sue remain to be determined. Also, the implications of these alterations are largely hypothet-ical, and the potential impact they may have on fetal development is unknown.
4.11 Study limitations.
Several weaknesses should be addressed. Only one reviewer conducted the study, which con-sequently increased the risk of bias in the selection and assessment of studies. Limitations associated with a systematic literature search include the possibility of missing published work. PubMed was the only database searched, and without the addition of manual searches, the risk of missing published work increased further. As positive results are more easily published, studies presenting negative results were more probable to be missed. The search terms and inclusion criteria were customized to generate a moderately wide range of studies to attain a reasonable amount of data for one reviewer to explore under a limited time period. Furthermore, there are other tools availabe to determine RoB of studies. Due to the relative dissmiliarites in the design of included studies we found the need to use two tools; SYRCLE`s RoB tool for animal intervention and in vitro studies, and NOS for human cohort studies.
21
5. Conclusions.
The literature on gene expression alterations in placental tissue exposed to cigarette smoking is limited, even though the placenta is crucial for fetal development and growth and prenatal smoking is clearly associated with adverse pregnancy outcomes. However, available data shows that cigarette smoking during pregnancy elicits several important changes in gene ex-pression in the placenta. These include essential pathways, such as metabolism of xenobiotics, angiogenesis, cellular proliferation, and amino acid transport and metabolism. More studies on this topic are needed for increasing our understanding of how cigarette smoking negatively affects the fetus. This knowledge may increase our possibility to treat fetuses at risk for FGR more efficiently in the future.
22
6. Acknowledgments.
First and foremost, I would like to thank Dr. Maria Lodefalk of the School of Medical Sci-ences at Örebro University whose guidance and support have been invaluable in the making of this paper. Secondly, I would like to thank Dr. Anna Green and Sanja Farkas whose inputs on this thesis have been momentously appreciated. Last but not least, I would like to
acknowledge Sara Johansson as the second reader of this thesis, as her comments on this the-sis were of great value.
23
7. References.
[1] Horta BL, Victora CG, Menezes AM, Halpern R, Barros FC. Low birthweight, preterm births and intrauterine growth retardation in relation to maternal smoking. Paediatr Perinat Epidemiol. 1997 Apr;11(2):140-51
[2] Reeves S, Bernstein I. Effects of maternal tobacco-smoke exposure on fetal growth and neonatal size. Expert Rev Obstet Gynecol. 2008 Nov 1;3(6): 719-730. doi:
10.1586/17474108.3.6.719.
[3] Salihu HM, Wilson RE. Epidemiology of prenatal smoking and perinatal outcomes. Early Human Development. 2007 Nov;83(11):713-20.
[4] Jaakkola JJK, Gissler M. Maternal Smoking in Pregnancy, Fetal Development, and Child-hood Asthma. American Journal of Public Health. 2004 Jan;94(1):136-40.
[5] Ino T. A meta-analysis of association between maternal smoking during pregnancy and offspring obesity. Pediatrics International, 2010;52(1):94-9.
[6] Somm E, Schwitzgebel V, Vauthay D, Aubert M, and Hüppi P. Prenatal nicotine exposure and the programming of metabolic and cardiovascular disorders. Molecular and Cellular En-docrinology, 2009; 304(1-2):69-77.
[7] Ananth CV, Savitz DA, Luther ER. Maternal cigarette smoking as a risk factor for placen-tal abruption, placenta previa, and uterine bleeding in pregnancy. Am J Epidemiol
1996;144:881-9.
[8] Miller DA, Chollet JA, Goodwin TM. Clinical risk factors for placenta previa, placenta accreta. Am J Obstet Gynecol 1997;177:210-4.
[9] Goujard J, Rumeau C, Schwartz D. Smoking during pregnancy, stillbirth and abruptio pla-centae. Biomedicine 1975;23:20-2.
[10] Roelands J, Jamison MG, Lyerly AD, James AH. Consequences of Smoking during Pregnancy on Maternal Health. Journal of Women’s Health. 2009 Jun;18(6):867-72. [11] Curtin SC, Mathews T.J. Smoking Prevalence and Cessation Before and during Preg-nancy: Data From the Birth Certificate, 2014. National Vital Statistics Reports 2016 Feb 10; 65(1):1-14.
[12] CDC. The health consequences of smoking—50 years of progress: A report of the Surgeon General. Atlanta, GA: National Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health. 2014.
[13] Donnelly L, Campling G. Functions of the placenta. Anaesthesia & Intensive Care Medi-cine. 2014 Mar;15(3):136-9.
24
[14] Gude NM, Roberts CT, Kalionis B, King RG. Growth and function of the normal human placenta. Thrombosis Research. 2004 Jan;114(5-6):397-407
[15] Carter AM. Placental Gas Exchange and the Oxygen Supply to the Fetus. Compr Phys-iol. 2015 Jul 1;5(3):1381-403.
[16] Zdravkovic T, Genbacev O, McMaster MT, Fisher SJ. The adverse effects of maternal smoking on the human placenta: a review. Placenta. 2005 Apr;26 Suppl A:S81-6.
[17] Hooijmans CR, Rovers MM, de Vries RB, Leenaars M, Ritskes-Hoitinga M, Langendam MW. SYRCLE’s risk of bias tool for animal studies. BMC Medical Research Methodology. 2014 Dec;14(1).
[18] The Ottawa Hospital research institute. The Newcastle-Ottawa Scale (NOS) for assessing the quality of nonrandomised studies in meta-analyses [Internet]. Ottawa: The Ottawa Hospi-tal research institute [cited 2018 October 25] Available from
http://www.ohri.ca/pro-grams/clinical_epidemiology/oxford.asp
[19] The Ottawa Hospital research institute. The Newcastle-Ottawa Scale (NOS) [Internet] Ottawa: The Ottawa Hospital research institute [cited 2018 October 25] Available from http://www.ohri.ca/programs/clinical_epidemiology/nosgen.pdf
[20] The Ottawa Hospital research institute. Coding manual for the Newcastle-Ottawa Scale (NOS) [Internet] Ottawa: The Ottawa Hospital research institute [cited 2018 October 25] Available from http://www.ohri.ca/programs/clinical_epidemiology/nos_manual.pdf
[21] Verhagen H, Aruoma OI, van Delft JHM, Dragsted LO, Ferguson LR, Knasmüller S, et al. The 10 basic requirements for a scientific paper reporting antioxidant, antimutagenic or anticarcinogenic potential of test substances in vitro experiments and animal studies in vivo. Food and Chemical Toxicology. 2003 May;41(5):603-10.
[22] Viswanathan M, Patnode CD, Berkman ND, Bass EB, Chang S, Hartling L, et al. Assessing the Risk of Bias of Individual Studies in Systematic Reviews of Health Care Interventions [Internet]. Agency for Healthcare Research and Quality; 2017 [cited 2018 Octo-ber 25]. Available from:
https://effectivehealthcare.ahrq.gov/topics/methods-bias-up-date/methods/
[23] Bruchova H, Vasikova A, Merkerova M, Milcova A, Topinka J, Balascak I, et al. Effect of maternal tobacco smoke exposure on the placental transcriptome. Placenta. 2010
Mar;31(3):186-91. doi: 10.1016/j.placenta.2009.12.016. Epub 2010 Jan 25.
[24] Huuskonen P, Storvik M, Reinisalo M, Honkakoski P, Rysä J, Hakkola J, et al. Microarray analysis of the global alterations in the gene expression in the placentas from cigarette-smoking mothers. Clin Pharmacol Ther. 2008 Apr;83(4):542-50. Epub 2007 Oct 27.
25
[25] Yan Y, Wang H, Feng Y. Alterations of placental cytochrome P450 1A1 and P-glycopro-tein in tobacco-induced intrauterine growth retardation in rats. Acta Pharmacol Sin. 2005 Nov;26(11):1387-94.
[26] Fa S, Larsen TV, Bilde K, Daugaard TF, Ernst EH, Lykke-Hartmann K, et al. Changes in first trimester fetal CYP1A1 and AHRR DNA methylation and mRNA expression in response to exposure to maternal cigarette smoking. Environ Toxicol Pharmacol. 2018 Jan;57:19-27. doi: 10.1016/j.etap.2017.11.007. Epub 2017 Nov 16.
[27] Whyatt RM, Garte SJ, Cosma G, Bell DA, Jedrychowski W, Wahrendorf J, et al. CYP1A1 messenger RNA levels in placental tissue as a biomarker of environmental expo-sure. Cancer Epidemiol Biomarkers Prev. 1995 Mar;4(2):147-53.
[28] Chen Z-Y, Yao Y. A synergistic negative effect of gestational smoke-exposure and small litter size on rat placental efficiency, vascularisation and angiogenic factors mRNA ex-pression. PLoS One. 2017 Jul 18;12(7):e0181348. doi: 10.1371/journal.pone.0181348. eCollection 2017.
[29] Romani F, Lanzone A, Tropea A, Tiberi F, Catino S, Apa R. Nicotine and cotinine affect the release of vasoactive factors by trophoblast cells and human umbilical vein endothelial cells. Placenta. 2011 Feb;32(2):153-60. doi: 10.1016/j.placenta.2010.11.010. Epub 2010 Dec 9.
[30] Zhao H, Wu L, Wang Y, Zhou J, Li R, Zhou J, et al. Nicotine promotes vascular
endothelial growth factor secretion by human trophoblast cells under hypoxic conditions and improves the proliferation and tube formation capacity of human umbilical endothelial cells. Reprod Biomed Online. 2017 Apr;34(4):406-413. doi: 10.1016/j.rbmo.2016.12.014. Epub 2017 Jan 17.
[31] Wang A, Zsengellér ZK, Hecht JL, Buccafusca R, Burke SD, Rajakumar A, et al. Excess placental secreted frizzled-related protein 1 in maternal smokers impairs fetal growth. J Clin Invest. 2015 Nov 2;125(11):4021-5. doi: 10.1172/JCI80457. Epub 2015 Sep 28.
[32] Kraus DM, Feng L, Heine RP, Brown HL, Caron KM, Murtha AP, et al. Cigarette smoke-induced placental adrenomedullin expression and trophoblast cell invasion. Reprod Sci. 2014 Jan;21(1):63-71. doi: 10.1177/1933719113488456. Epub 2013 May 7.
[33] Fu Y-Y, Nergard JC, Barnette NK, Wang Y-L, Chai KX, Chen L-M. Proteasome inhibi-tion augments cigarette smoke-induced GM-CSF expression in trophoblast cells via the epi-dermal growth factor receptor. PLoS One. 2012;7(8):e43042. doi:
26
[34] Day PE, Ntani G, Crozier SR, Mahon PA, Inskip HM, Cooper C, et al. Maternal Factors Are Associated with the Expression of Placental Genes Involved in Amino Acid Metabolism and Transport. PLoS One. 2015 Dec 14;10(12):e0143653. doi:
10.1371/journal.pone.0143653. eCollection 2015.
[35] Stasenko S, Bradford EM, Piasek M, Henson MC, Varnai VM, Jurasović J, et al. Metals in human placenta: focus on the effects of cadmium on steroid hormones and leptin. J Appl Toxicol. 2010 Apr;30(3):242-53. doi: 10.1002/jat.1490.
[36] Yang K, Julan L, Rubio F, Sharma A, Guan H. Cadmium reduces 11 beta-hydroxysteroid dehydrogenase type 2 activity and expression in human placental trophoblast cells. Am J Physiol Endocrinol Metab. 2006 Jan;290(1): E135-E142. Epub 2005 Sep 6.
[37] Autrup H, Vestergaard AB, Okkels H.Transplacental transfer of environmental genotox-ins: polycyclic aromatic hydrocarbon-albumin in non-smoking women, and the effect of ma-ternal GSTM1 genotype. Carcinogenesis. 1995 Jun;16(6):1305-9.
[38] Rodgman A, Perfetti TA. The composition of cigarette smoke: a catalogue of the polycy-clic aromatic hydrocarbons. Beiträge zur Tabakforschung International. 2006;22(1):13-69. [39] Nielsen, C.H., Larsen, A., Nielsen, A.L., 2016. DNA methylation alterations in response to prenatal exposure of maternal cigarette smoking: a persistent epigenetic impact on health from maternal lifestyle? Arch. Toxicol. 90, 231-245.
[40] Maisonpierre PC, Suri C, Jones PF, Bartunkova S, Wiegand SJ, Radziejewski C, Comp-ton D, et al. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science. 1997 Jul 4;277(5322):55-60.
[41] Jeyabalan A1, Powers RW, Durica AR, Harger GF, Roberts JM, Ness RB. Cigarette smoke exposure and angiogenic factors in pregnancy and preeclampsia. Am J Hypertens. 2008 Aug;21(8):943-7.
[42] O'Leary NA, Wright MW, Brister JR, Ciufo S, Haddad D, McVeigh R, et al. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional an-notation. Nucleic Acids Res. 2016 Jan 4;44(D1):D733-45.
[43] Tepekoy F, Akkoyunlu G, Demir R. The role of Wnt signaling members in the uterus and embryo during pre-implantation and implantation. Journal of Assisted Reproduction and Genetics. 2015 Mar;32(3):337–46.
[44] Caron KM, Smithies O. Multiple roles of adrenomedullin revealed by animal models. Microsc Res Tech. 2002;57(1):55-59.
27
[45] Sjöblom C, Roberts CT, Wikland M, Robertson SA (2005) Granulocyte-macrophage col-ony-stimulating factor alleviates adverse consequences of embryo-culture on fetal growth tra-jectory and placental morphogenesis. Endocrinology 146: 2142-2153.
[46] Seymour JF, Lieschke GJ, Grail D, Quilici C, Hodgson G, et al. (1997) Mice lacking both granulocyte colony-stimulating factor (CSF) and granulocyte-macrophage CSF have im-paired reproductive capacity, perturbed neonatal granulopoiesis, lung disease, amyloidosis, and reduced long-term survival. Blood 90: 3037-3049
[47] Cleal JK, Glazier JD, Ntani G, Crozier SR, Day PE, Harvey NC, et al. Facilitated trans-porters mediate net efflux of amino acids to the fetus across the basal membrane of the pla-cental syncytiotrophoblast: Plapla-cental amino acid transport. The Journal of Physiology. 2011 Feb 15;589(4):987–97
[48] Wang H, Liu L, Hu Y-F, Hao J-H, Chen Y-H, Su P-Y, et al. Maternal serum cadmium level during pregnancy and its association with small for gestational age infants: a population-based birth cohort study. Scientific Reports [Internet]. 2016 Sep [cited 2018 Dec 3];6(1). Available from: http://www.nature.com/articles/srep22631.
[49] Chedrese P, Piasek M, Henson M. Cadmium as an Endocrine Disruptor in the Reproduc-tive System. Immunology‚ Endocrine & Metabolic Agents in Medicinal Chemistry. 2006 Feb 1;6(1):27-35.
[50] Kawai M, Swan KF, Green AE, Edwards DE, Anderson MB, Henson MC. Placental En-docrine Disruption Induced by Cadmium: Effects on P450 Cholesterol Side-Chain Cleavage and 3-beta-Hydroxysteroid Dehydrogenase Enzymes in Cultured Human Trophoblasts. Biology of reproduction 2002;67:178-83.
[51] Gabory A, Ferry L, Fajardy I, Jouneau L, Gothié J-D, Vigé A, et al. Maternal Diets Trig-ger Sex-Specific Divergent Trajectories of Gene Expression and Epigenetic Systems in Mouse Placenta. Aguila MB, editor. PLoS ONE. 2012 Nov 5;7(11):e47986.
[52] Cottrell EC, Seckl JR, Holmes MC, Wyrwoll CS.Foetal and placental 11β-HSD2: a hub for developmental programming. Acta Physiol (Oxf). 2014 Feb;210(2):288-95. doi:
10.1111/apha.12187. Epub 2013 Dec 12.
[53] Gabory A, Ferry L, Fajardy I, Jouneau L, Gothié J-D, Vigé A, et al. Maternal Diets Trig-ger Sex-Specific Divergent Trajectories of Gene Expression and Epigenetic Systems in Mouse Placenta. Aguila MB, editor. PLoS ONE. 2012 Nov 5;7(11):e47986.
[54] Fa S, Larsen TV, Bilde K, Daugaard TF, Ernst E.H, Olesen, RH Mamsen, et al. Assess-ment of global DNA methylation in the first trimester fetal tissues exposed to maternal ciga-rette smoking. Clin Epigenetics. 2016;8, 128.
28
[55] Florescu A, Ferrence R, Einarson T, Selby P, Soldin O, Koren G. Methods for Quantifi-cation of Exposure to Cigarette Smoking and Environmental Tobacco Smoke: Focus on De-velopmental Toxicology: Therapeutic Drug Monitoring. 2009 Feb;31(1):14-30.
[56] Rebagliato M, Bolumar F, du Florey CV, et al. Variations in cotinine levels in smokers during and after pregnancy. Am J Obstet Gynecol. 1998;178:568-571.
[57] Pichini S, Basagaña X, Pacifici R, Garcia O, Puig C, Vall O, et al. Cord Serum Cotinine as a Biomarker of Fetal Exposure to Cigarette Smoke at the End of Pregnancy. Environmental Health Perspectives. 2000;108(11):5.
[58] Coleman T, Chamberlain C, Davey MA, Cooper SE, Leonardi‐Bee J. Pharmacological interventions for promoting smoking cessation during pregnancy. Cochrane Database of Sys-tematic Reviews 2015, Issue 12. Art. No.: CD010078. DOI:
10.1002/14651858.CD010078.pub2.
[59] Mund M, Louwen F, Klingelhoefer D, Gerber A. Smoking and Pregnancy — A Review on the First Major Environmental Risk Factor of the Unborn. Int J Environ Res Public Health. 2013 Dec; 10(12): 6485-6499.
[60] Lyerly AD, Mitchell LM, Armstrong EM, Harris LH, Kukla R, Kuppermann M, et al. Risk and the Pregnant Body. Hastings Center Report. 2009;39(6):34–42.
[61] Fa S, Larsen TV, Bilde K, Daugaard TF, Ernst EH, Olesen RH, et al. Assessment of global DNA methylation in the first trimester fetal tissues exposed to maternal cigarette smoking. Clinical Epigenetics [Internet]. 2016 Dec [cited 2019 Jan 5];8(1). Available from: http://clinicalepigeneticsjournal.biomedcentral.com/articles/10.1186/s13148-016-0296-0
