Evaluation of HPL, Leptin, and IGF-1 in Placental Tissues of Maternal Smoking During Pregnancy

Evaluation of HPL, Leptin, and IGF-1 in Placental Tissues of Maternal Smoking During Pregnancy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of HPL, Leptin, and IGF-1 in Placental Tissues of Maternal Smoking During Pregnancy Hayrunnisa Yeşil Sarsmaz, Seren Gülşen Gürgen, Kemal Sarsmaz, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6139249/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Maternal smoking during pregnancy although it is known to be associated with fetal growth restriction, it’s effect on postpartum breastfeeding is not yet clear. The aim of the present study was to examine the impacts of smoking in pregnancy on breastfeeding as well as its impacts on placental immunoreactivity. The study was conducted on 70 women who gave birth via spontaneous vaginal delivery. Groups were classified as Smokers (n=35) and Non-smokers (n=35). Cross-sections were taken from the placenta tissues after which their IGF-1, Leptin, HPL immunoreactivities were examined. A significant difference was identified between the smoking status of the pregnant women and findings on their IGF-1, Leptin and HPL immunoreactivities (p<0,05). Lower IGF-1, Leptin and HPL median scores were observed for smoker pregnant women compared with non-smoker pregnant women. Lower placental IGF-1 Leptin and HPL expressions in smoker groups were found to be correlated with the lower values related with the baby and breastfeeding. Lower HPL expression values of the placentas of smoker mothers led us to think that it is related with insufficient mother’s milk during the postpartum nursing period. Smoking cigarette placenta IGF-1 Leptin HPL breastfeeding Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction It is known that 20-50 % of women in developed countries smoke starting from the onset of pregnancy. [1] Intrauterine growth restriction (IUGR) caused on the fetus by the cigarettes smoked by the mother during pregnancy is the most frequently encountered complication. [1-3] Chronic and acute hypoxia along with the presence of carbon monoxide in maternal circulation leads to changes in the transmission of oxygen to the fetus and hence lower birth weights.[2] Even though this impact on fetal growth takes place subject to dose during the third trimester of pregnancy, the specific impact of maternal smoking on the placenta and thus on the fetus along with the correlations between the various factors expressed by the placenta have not yet been properly understood. [2, 3] Insulin Growth Factor (IGF-1) has a polypeptide structure and it is a hormone comprised of 70 amino acids that structurally resembles proinsulin. The primary synthesis location of IGFs that are synthesized in almost all tissues is known as the liver, it is important for cell growth and differentiation and is included in the functioning of many organs[4]. Maternal tissue development and placental blood flow are induced by the combination of steroids, eritropoetin, renin, aldosteron and IGF-1. Nutritional support for fetal and neonatal development is provided through uteroplacental blood flow and postpartum milk release.[5] Insulin resembling growth factors make an impact on maternal tissue growth and metabolism thus playing a critical role in fetal growth and development. Maternal IGF that regulates placenta formation, substrate transportation and hormone release affects the usability of maternal substrate or the provision of nutrition to the fetus thus playing a role in fetal development. [6] Leptin is a hormone that is primarily synthesized in adipose tissue and less in placental tissue.[7] It is thought to play a role in eating habits and the controlling of the energy balance through its impacts on the central nervous system. It reflects the fat reserve in the body and is synthesized from white and brown fat tissue. Leptin overcomes the blood-brain barrier through its special receptors after mixing in with the blood thus affecting the central nervous system to reduce nutrition intake while increasing energy consumption. All adipocytes have leptin release ability, however large adipocytes release more hormones compared with the smaller ones. [8] Placenta is in effect during the fetal period as well as a source of leptin.[9] Following its breaking off from the placenta after birth, milk releasing breast epithelium cells become the leptin source and thus mother’s milk that is the source of leptin during postpartum period for the newborn enables a significant amount of leptin intake.[10] It is known based on previously conducted studies that smoker women give birth to babies with less birthweight compared with nonsmoker women.[11, 12] Mantzoros et al. put forth as a result of the comparison between women who smoked during their pregnancy and those who did not that the varying leptin concentrations may be related with the decrease in fetal growth. [13] Human Prolactin Lactogen (HPL) is a single chain polypeptide molecule generated in the placenta by syncytiotrophoblasts, with a short half-life but even so with a high level in circulation. [14] It appears in the mother’s serum during the 6 th -7 th week of pregnancy. The HPL level in maternal circulation is related with fetal and placental weight and slowly increases until the last 4 weeks of pregnancy. It mixes in with the fetal circulation in a very low amount. Placental lactogen and HGH hormones display molecular similarities. The major biological impact of HPL is due to the growth hormone and prolactin similarity. HPL release acts as the growth hormone thus supporting fetal growth. [15] Some studies in the literature have shown that IGF-1 deficiency significantly increases the risk of cardiovascular diseases, and leptin reduces vasodilation through the expression of inducible NO synthase (iNOS) in the vascular wall. [16, 17] No study was observed in literature that has previously examined the correlation between IGF-1, Leptin and HPL expressions in the placentas of smoker and nonsmoker pregnant women. Thus, the current study aimed to examine the effects of IGF-1, Leptin and HPL expressions in the placental tissues of smoker and nonsmoker pregnant women. Materials and Method This study was approved by Manisa Celal Bayar University Health Science Ethics Committee on 02/ 08/ 2017 with the number 20.478.486. Participants Our study was conducted with 70 women[18] (case: 35 smokers, control: 35 nonsmokers) [18]who applied to the T.R. Ministry of Health Manisa Merkezefendi State Hospital Delivery Room Unit during the dates of March-May and who gave birth through spontaneous vaginal birth. In this investigation was received a statement that the subject gave “informed consent”. Inclusion criteria for research Pregnant women who have started smoking at least 100 cigarettes in her lifetime and who now smokes every day[19], pregnant women who have never smoked throughout their life, including pregnancy[19],pregnant women who gave birth at term, multiple pregnancy, preeclampsia, gestational diabetes, thyroid dysfunction, those who do not have a systemic disease and due to systemic disease during pregnancy, those who do not use, pregnant women who had spontaneous vaginal delivery, in order not to encounter communication barriers and because cultural differences may vary, the pregnant woman is not a foreign national, pregnant women who do not have any barriers to communication by phone, those who do not have any health problems that prevent breastfeeding. Exclusion Criteria for research Multiple pregnancy, insulin dependent diabetes, pregnant women who have had a cesarean section, preeclampsia, thyroid disease. Groups were classified as Group 1(case): Smokers “before and during pregnancy” (n=35), Group 2(control): Non-smokers “lifetime and during pregnancy” ( n=35). Placenta Tissue Selection The present study was carried out at the Manisa Merkez Efendi State Hospital during October 2017-April 2018 for a period of about 6 months by taking the necessary ethical and official permissions. It was aimed to relationship between histopathological changes of the placenta tissues acquired from “pregnant women who have started smoking at least 100 cigarettes in her lifetime and who now smokes every day” and from “pregnant women who have not smoked and non exposed second-hand smoke” at all in addition to examining umbilical cord bloods[19]. It took about 6 months between October 2017-April 2018 to collect the placentas. Immunohistochemical Staining on placenta The placenta tissues separated from the mothers were weighed rapidly. Following these processes, a sample tissue with dimensions of 1 cm3 was taken from the larger placenta tissue which was then placed in 10% formaldehyde and fixed for 24 hours. They were then subject to dehydration processes and embedded in paraffin blocks. The embedded tissues were transferred as 5 μ sections onto a microscope slide via a microtome (Leica RM 2135; Bensheim, Germany). For immunohistochemical staining, the sections were first incubated at 60 ºC overnight and were then incubated in xylene for 30 min. After washing with serial concentrations of ethanol (95, 80, 70, and 60%), the sections were washed with distilled water and phosphate-buffered saline (PBS) for 10 min. They were then held in 2% trypsin in Tris buffer at 37 C for 15 min and were washed with PBS three times for 5 min. The sections were incubated in a solution of 3% H2O2 for 15 min to inhibit endogenous peroxidase activity. After that, the sections were washed with PBS and were incubated with blocking solution, Large Volume Ultra V Block (Thermo Fisher, USA) for 1 h. The sections were then incubated at 4 C with primary antibodies overnight: HPL (Mouse monoclonal, Thermo Fisher, USA), Leptin (Bioss, Woburn, Massachusetts, USA), IGF-1 (Bioss, Woburn, Massachusetts- USA) Then, they were washed three times for 5 min, each with PBS, followed by incubation with biotinylated IgG and then with streptavidin–peroxidase conjugate (Thermo Fisher, CA, USA). After washing three times for 5 min with PBS, the sections were incubated with DAB substrate containing diaminobenzidine (Thermo Fisher, CA, USA) for 5 min to detect immunoreactivity and then with Mayer’s hematoxylin. The sections were covered with mounting medium and were analyzed on a light microscope with a CX 31 microscope (Olympus, Tokyo, Japan). Control samples were processed in an identical manner, but in the absence of primary antibody step. All of the immunostained sections were reviewed by two histologists, who had no knowledge of the groups. Slides were examined under low power (X10 objective) to identify the regions containing HPL, Leptin and, IGF-1 staining ten areas selected at random were scored and in sections, where all of the staining appeared intense, one random field was selected. The proportion of placenta in each selected field was determined by counting at high magnification. At least 100 cells were scored per X 40 field for each group. All the sections were scored in a semiquantitative manner, which considers both the intensity and the percentage of cells, staining at each intensity. The intensities were classified as 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining), whereas 10% groupings were used for the percentage of cells that stained positive. For each slide, a value designated H-SCORE was obtained by the application of the algorithm: H-SCORE: Σ Pi.(i+1), where I and PC represent the intensity and the percentage of cells that stain at each intensity, respectively, and the corresponding H-SCOREs were calculated separately. Numbers were calculated on 10 randomly selected areas using the image analysis system (Leica Q Win V3 Plus Image), and the mean values were employed for analysis.[20] Umbilical cord blood biochemistry analysis Whole blood taken from the umblical cord was collected into 5.5 ml tubes with yellow caps and serum separator gel for biomarker analyses. After waiting at least 30 minutes for the coagulation reaction, the tubes were centrifuged at 1000g x 15 min at +4°C. The serum samples obtained will be portioned separately into polypropylene 0.5 ml eppendorf tubes for each biomarker. Samples were stabilized at -80 and stored in a deep freezer until the working day. When the planned number of patients is reached, serum samples will be raised to +4°C the night before the working day and all samples will be thawed homogeneously.[21] Analyzes of Leptin and HPL biomarkers were performed by ELISA method. When the number of all blood samples reaches 35 patients in the case group and 35 patients in the control group, all of them were studied at the same time to be statistically significant. Therefore, the half-lives of the biomarkers weren’t affected. Statistics SPSS 25.0(SPSS Inc.; Chicago, IL, USA) software was used for data evaluation. Statistically significant results were defined as those with a P value less than 0.05. The data analysis focused on the "mean ±SD" values. Independent t test and Mann Whitney U test were used for this study. Results A total of 70 parturients who presented to the labor ward of the one study centers participated in the study and all the samples taken (100%) were adequate and analyzed. The study found that 57.1% of pregnant women who smoke are aged 25 or younger, 94.3% are married, 42.9% are literate, 80% are housewives, 91.4% have a similar income status, 68.6% do not have social security, 74.3% have had multiple pregnancies, 42.9% have given birth to two or more children, 77.1% have not had a miscarriage, and 37.1% have two or more living children. The study found that 60% of pregnant women who do not smoke are over the age of 26, 100% are married, 62.9% have an education level of primary school or higher, 97.1% are housewives, 94.3% have a similar income status, 65.7% have social security, 74.3% have had multiple pregnancies, 45.7% have had one birth, 85.7% have not had a miscarriage, and 42.9% have one living child (Table 1). Table 1: Sociodemographic characteristics/variable of participants (n=70) Variable Smokers (n=35) Nonsmokers (n=35) Total (n=70) x 2 /p Frequency (n) % Frequency (n) % Frequency (n) % Age (years) <26 ≥26 20 15 57.1 42.9 14 21 40 60 34 36 48.6 51.4 2.05/0.15 Marital status Married Single 33 2 94.3 5.7 35 0 100 0 68 2 97.1 2.9 -/- Level of education No formal education Formal education Primary school and above 6 15 14 17,1 42.9 40 3 10 22 8.6 28.6 62.9 9 25 36 12,9 35.7 51.4 3.77/0.15 Social Security Yes No 11 24 31.4 68.6 23 12 65.7 34.3 34 36 48.6 51.4 8.23/0.004* Parity Primipar Multipar 9 26 25.7 74.3 9 26 25.7 74.3 18 52 25.7 74.3 0.00/1.00 Number of births None 1 2 and above 10 10 15 28.6 28.6 42.9 10 16 9 28.6 45.7 25.7 20 26 24 28.6 37.1 34.3 2.88/0.23 Number of abortions None 1 2 or more 27 6 2 77.1 17.1 5.7 30 3 2 85.7 8.6 5.7 57 9 4 81.4 12.9 5.7 -/- Number of living children None 1 2 and above 11 11 13 31.4 31.4 37.1 11 15 9 31.4 42.9 25.7 22 26 22 31.4 37.1 31.4 1.34/0.51 *p<0,05 x2: Chi-square test Immunohistochemical Findings Whereas IGF-1 immunoreaction was stained at intensities ranging from moderate to strong in the villous stromal cells of the nonsmoker group, it was at intensities ranging from weak to moderate in syncytiotrophoblast cells (Figure 1 A1,A2). Whereas weak IGF-1 reaction was observed in both villous stromal cells and syncytiotrophoblast cells in the smoker group (Figure 1 B1, B2). While leptin immunoreaction was stained strongly in especially the villous stromal cells of the nonsmoker group, it was observed to be stained at a moderate intensity in syncytiotrophoblast cells (Figure 2 A1, A2). Whereas in the smoker group, it was striking to see that leptin immunoreaction was stained moderate in both villous stromal and syncytiotrophoblast cells (Figure 2 B1, B2). It was observed that HPL immunostaining was quite strong in especially the syncytiotrophoblast cells in the placenta villi of the nonsmoker group, while weak staining attracted attention in villous stromal cells (Figure 3 A1, A2). It attracted attention that the HPL immunoreaction reduced significantly in the syncytiotrophoblast cells of the smoker group. There was again weak expression in the villous stromal cells (Figure 3 B1,B2). A statistically significant difference was observed between the smoking status of the pregnant women and the median scores of IGF-1, Leptin and HPL histopathological findings (p<0,05). It was illustrated that the IGF-1, Leptin and HPL median scores of smoker pregnant women were lower compared with those of the nonsmokers (Figure 4, Table 2). A statistically significant difference was identified between the smoking status of pregnant women and their median scores for IGF-1, Leptin and HPL histopathological findings (p<0,05). It was observed that the IGF-1, Leptin and HPL median scores of smoker pregnant women were lower compared with those of nonsmokers (Table 2, Figure 4). Table 2 . Comparison of the Placenta Indirect Immunoperoxidase Hscore Findings of Smoker and Nonsmoker Pregnant Women (n=70) Histsopathological Findings Smokers (n=35) Nonsmokers (n=35) Med ± sd Median ( IQR *) Med ± sd Median ( IQR *) z/p IGF -1 64.1±21.87 63.0 (45.0) 216.2±30.9 216.0 (54.0) -7.19/0.00** Leptin 132.2±24.2 129.0 (39.0) 328.5±47.7 340.0 (96.0) -7.19/0.00** HPL 99.9±19.5 99.0 (27.0) 289.7±42.3 300.0 (72.0) -7.19/0.00** * ( IQR *) : Interquartile range. **p<0.05. z: Mann Whitney U test Umbilical cord blood biochemistry Findings A statistically significant difference was identified between the smoking status of pregnant women and their median scores for Leptin and HPL biochemistry findings (p<0,05). It was observed that the Leptin and HPL mean scores of smoker pregnant women were lower compared with those of nonsmokers(Table 3). Table 3 . Leptin and HPL Biochemistry Findings of Smoker and Nonsmoker Pregnant Women (n=70) Biochemistry Findings Groups N Mean Std. Deviation F P Leptin (pg/ml) Smokers 35 54.6 11.5 39.59 <0.001 ** Nonsmokers 35 107.8 2.2 HPL Smokers 35 0.9 0.6 1.98 <0.001 ** Nonsmokers 35 5.8 0.9 **p<0.05. Independent T test Discussion The impact of smoking on female reproductive functions has become a significant concern due to the rising number of young female smokers. Since Simpson's 1957 report, which highlighted a higher incidence of premature births among pregnant women who smoked and showed that this rate increased with the amount of smoking, numerous studies have examined the effects of maternal smoking on the fetüs.[22] Many of these studies have found that babies born to mothers who smoke tend to have lower birth weights compared to those born to non-smokers. In fact, this study aimed to investigate the impact of smoking on breastfeeding. By analyzing the levels of specific biomarkers in postpartum placenta tissue and umbilical cord blood, we gathered information about the subsequent stage: the breastfeeding process. In our study, expression of IGF, Leptin and HPL median scores expressed in the placentas of smoker pregnant women which were lower compared with those of nonsmoker pregnant women. And Leptin and HPL biochemistry findings of smoker pregnant women which were lower compared with those of nonsmoker pregnant women. Fleisch et al. conducted a study in 2017 during which they examined the umbilical cord blood hormones for mothers who smoked during pregnancy and classified the women into 3 groups as those who smoked during their pregnancy, who smoked prior to pregnancy and continued to do so and those who never smoked.[23] Hormones such as IGF-1, IGF-2, IGFB-3, leptin were examined in the cord blood as a result of which it was observed that IGF-1 values were lower for those who smoked during pregnancy.[23] IGF-1 immunoreactivity was examined in the placenta during the present study in which it was determined similar to other studies that it was lower for the group of women who smoked during pregnancy. IGF-1 immunoreactivity was observed to be lower in the placenta tissues of nonsmoker mothers. Correlatively with the fact that babies of smoker mothers in our study have lower head circumference and height values and the fact that babies of smoker women had lower birth weights in our previous study.[24] it was concluded that the IGF-1 and Leptin values expressed from the placenta may have a primary impact on the development of the baby. Leptin is generated at the syncytiotrophoblast cells by adipocyte cells and it is thought to play an impact on fetal development through currently unknown mechanisms. Ypsilantis P et al. carried out a study in 2013, during which it was emphasized that smoking plays a critical role in bodyweight loss and appetite reduction and that the leptin and ghrelin hormones related with appetite control are not clear.[25] For this reason, they used 8 week old male rats in smoker and nonsmoker groups as a result of which it was observed that a significant loss of bodyweight took place in in 4 weeks in the group subject to smoking while on the other hand weight was regained 1 week after the end of exposure. The leptin level decreased during the first 4 weeks in the rat group subject to exposure for 8 weeks only to return to the normal level on the 7 th week after exposure. Based on the results of this study, the leptin level in circulation during the first few weeks of exposure in rats cannot be clearly explained with body weight. [25] The serum leptin level was observed to be much lower compared with cigarette exposure in the study by Ypsilantis et al. and similarly, placenta leptin immunoreactivity was observed to be low in smokers in the present study as well. Similarly, Pardo et al. published a study in 2004 entitled “Does maternal smoking influence leptin levels in term, appropriate-for-gestational-age newborns?” In this study, it was emphasized that leptin is a hormone generated in the placenta and adipose tissues and that it is correlated with neonatal development. 19 smoker and 91 nonsmoker pregnant women were classified in this study. Neonatal anthropometric measurements were taken and leptin levels were measured via radioimmunoassay. Leptin concentrations were observed to be similar between the groups and leptin levels were correlated with ponderal index and similarly with the gestational ages of the newborns. This study presented that maternal smoking during pregnancy does not have an impact on the cord blood leptin levels but that it is in accordance with gestational age.[26] Placenta Leptin immunoreactivity in our study was observed to be lower in smoker pregnant women compared with nonsmokers. Ratio of those that were previously smoking among pregnant women was 26 %, whereas the ratio of current smokers was 16.5 %, age of starting smoking was 18 and the number of cigarettes smoked per day was eight. TNSA-2008 results along with the results of studies carried out in both our country and around the world indicate that smoking is a critical issue among both pregnant, nursing and other women.[3, 27] The HPL median scores of smoker pregnant women were observed to be lower compared with those of nonsmoker pregnant women. There were no previous studies in literature on the correlation between smoking, breastfeeding and placental HPL. Human Prolactin Lactogen (HPL) is a single chain polypeptide molecule produced by syncytiotrophoblasts in the placenta with its major biological impact due to its resemblance of the growth hormone and prolactin. HPL secretion acts as a growth hormone thus supplementing fetal growth.[15] It was determined statistically in our study that the HPL expressions of smoker pregnant women are lower compared with nonsmoker pregnant women. While based on our immunohistochemical findings, HPL immunostaining is quite strong in the placenta villi of the nonsmoker group, HPL immune reaction was observed to decrease distinctively in syncytiotrophoblast cells and villous stromal cells in the smoker group. Carl J et al. published a study in 1991 in which 75 women were included in human placental lactogen (hPL) for normal pregnancy. HPL levels were recorded during the period starting from 26 th week until birth. It was observed as a result of the measurement conducted via Modifiye Gompertz equation method that development retardation continued until the 36 th week.[15] In another study, smoking was not shown to affect placenta size and it was emphasized that smoking caused the fetus weight and HPL levels to be lower.[28] It was also observed in our study that the HPL levels were lower among smoker pregnant women compared with nonsmokers. Mochizuki et al. carried out a study in 1984 during which it was emphasized that maternal smoking results in growth deficiency. Based on the findings of this study, the urinary estriol and hPL levels of heavy smoker women are lower compared with those of the nonsmokers.[29] Similar to these studies, the HPL amount expressed from the placenta was observed to be lower for smokers in our study, however different from the study by Mochizuki et al., we showed only the immunoreactivity of HPL in the placenta. However, we also examined leptin and IGF-1 expressions in addition to HPL. Conclusion Although our sample is similar to other histopathological studies in the literature, the fact that our study was conducted at a single center may not reflect the results of the general population. This constitutes the main limitation of our study. It is known that there is a statistically significant correlation between prematernal and maternal smoking and the birth weight of the newborn and that smoking reduces birth weight and placenta weight in addition to making adverse impacts on placenta histology. Low IGF-1 Leptin and HPL levels expressed in the placenta among maternal smoker women were observed. Lower HPL expression values of the placentas of smoker mothers led us to think that it is related with insufficient mother’s milk during the postpartum nursing period. Declarations Author contributions All authors participated in the design, interpretation of the studies and analysis of the data and review of the manuscript; HYS,KS and SGG Conception or design of the work, HYS data collection, HYS and SGG histology analysis and HYS,KS drafting the article, OS biochemical analysis, SGG,KS and HYS final approval of the version to be published. Ethical approval This study was approved by Manisa Celal Bayar University Health Science Ethics Committee on 02/ 08/ 2017 with the number 20.478.486. Informed consent Informed consent was obtained from all individual participants included in the study. Declaration of Helsinki The study was conducted according to the principles of Helsinki Declaration. Availability of research data Authors are available and ready to supply the data upon any requests through the corresponding author. Funding This study was supported by a grant from the Manisa Celal Bayar University Research Fund, Manisa, Turkey. [Project no: 2017-216]. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgement This study was registered at www.clinicaltrials.gov. Trial registration number is NCT06456645. References Higgins, S., Smoking in pregnancy. J Current Opinion in Obstetrics Gynecology, 2002. 14 (2): p. 145-151. Andres, R.L. and M.-C. Day. Perinatal complications associated with maternal tobacco use . in Seminars in Neonatology . 2000. Elsevier. Shiverick, K. and C.J.P. Salafia, Cigarette smoking and pregnancy I: ovarian, uterine and placental effects. 1999. 20 (4): p. 265-272. Fuglsang, J., P.J.G.H. Ovesen, and I. Research, Aspects of placental growth hormone physiology. 2006. 16 (2): p. 67-85. Koutsaki, M., et al., Decreased placental expression of hPGH, IGF-I and IGFBP-1 in pregnancies complicated by fetal growth restriction. 2011. 21 (1): p. 31-36. Sferruzzi‐Perri, A., et al., The neglected role of insulin‐like growth factors in the maternal circulation regulating fetal growth. 2011. 589 (1): p. 7-20. Hassink, S.G., et al., Placental leptin: an important new growth factor in intrauterine and neonatal development? 1997. 100 (1): p. e1-e1. Saad, M.F., et al., Diurnal and ultradian rhythmicity of plasma leptin: effects of gender and adiposity. 1998. 83 (2): p. 453-459. Haschke, F., M.A.J.J.o.p.g. van't Hof, and nutrition, Euro-Growth references for breast-fed boys and girls: influence of breast-feeding and solids on growth until 36 months of age. 2000. 31 (1): p. S60-S71. Ong, K.K., et al., Size at birth and early childhood growth in relation to maternal smoking, parity and infant breast-feeding: longitudinal birth cohort study and analysis. 2002. 52 (6): p. 863-867. Butler, N., H. Goldstein, and E.J.B.M.J. Ross, Cigarette smoking in pregnancy: its influence on birth weight and perinatal mortality. 1972. 2 (5806): p. 127-130. Murphy, J., et al., The effect of maternal cigarette smoking on fetal birth weight and on growth of the fetal biparietal diameter. 1980. 87 (6): p. 462-466. Mantzoros, C.S., et al., Effect of birth weight and maternal smoking on cord blood leptin concentrations of full-term and preterm newborns. 1997. 82 (9): p. 2856-2861. Beck, T., G. Schweikhart, and E.J.A.o.g. Stolz, Immunohistochemical location of HPL, SP1 and β-HCG in normal placentas of varying gestational age. 1986. 239 (2): p. 63-74. Sørensen, S., et al., Serial measurements of serum human placental lactogen (hPL) and serial ultrasound examinations in the evaluation of fetal growth. 2000. 60 (1): p. 25-34. Rodríguez, A., Novel molecular aspects of ghrelin and leptin in the control of adipobiology and the cardiovascular system. Obes Facts, 2014. 7 (2): p. 82-95. Strazhesko, I.D., et al., Growth Hormone, Insulin-Like Growth Factor-1, Insulin Resistance, and Leukocyte Telomere Length as Determinants of Arterial Aging in Subjects Free of Cardiovascular Diseases. Front Genet, 2017. 8 : p. 198. Spellacy, W.N., W.C. Buhi, and S.A. Birk, The effect of smoking on serum human placental lactogen levels. Am J Obstet Gynecol, 1977. 127 (3): p. 232-4. https://www.cdc.gov/nchs/nhis/tobacco/tobacco_glossary.htm. Yeşil Sarsmaz, H., et al., The relationship between oxidative stress and apoptosis of histopathological changes in the ovary made by mad honey containing grayanotoxin. Food Chem Toxicol, 2024. 187 : p. 114634. Kharb, S. and S. Nanda, Patterns of Biomarkers in Cord Blood During Pregnancy and Preeclampsia. Curr Hypertens Rev, 2017. 13 (1): p. 57-64. Simpson, W.J., A preliminary report on cigarette smoking and the incidence of prematurity. Am J Obstet Gynecol, 1957. 73 (4): p. 807-15. Fleisch, A.F., et al., Associations of maternal prenatal smoking with umbilical cord blood hormones: the Project Viva cohort. 2017. 72 : p. 18-26. Balcıoğlu, E., H.Y. Sarsmaz, and G.S.M.D. Gürgen, An Examınatıon Of The Relatıonshıp Between Smokıng Durıng-Before Pregnancy And Newborn Bırth Weıght/Placenta Weıght And Its Hıstopathologıcal Impacts On The Placenta Tıssue. 2020: p. 115. Ypsilantis, P., et al., Effects of Cigarette Smoke Exposure and Its Cessation on Body Weight, Food Intake and Circulating Leptin, and Ghrelin Levels in the Rat. Nicotine & Tobacco Research, 2013. 15 (1): p. 206-212. Pardo, I., et al., Does maternal smoking influence leptin levels in term, appropriate-for-gestational-age newborns? 2004. 15 (6): p. 408-410. Ladomenou, F., A. Kafatos, and E.J.A.P. Galanakis, Risk factors related to intention to breastfeed, early weaning and suboptimal duration of breastfeeding. 2007. 96 (10): p. 1441-1444. Spira, A., et al., Smokıng Durıng Pregnancy And Placental Pathology. Biomedicine Express, 1977. 27 (7): p. 266-270. Mochizuki, M., et al., Effects of smoking on fetoplacental-maternal system during pregnancy. 1984. 149 (4): p. 413-420. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6139249","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":424337128,"identity":"2d55320a-160a-492f-8ab7-744c0f0ed686","order_by":0,"name":"Hayrunnisa Yeşil Sarsmaz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYDADNgYGxgdAmoePFC3MBiAtbCRZJAHVix/It599/OlGTV0eH//hZ5Vfc+xk2BiYHz66gUeLwZl0M+mcY2zFbBJpZrdltyUDHcZmbJyDTwtDGhtzDhtPYpsEg9ltyW3MQC08bNL4tMj3P2P+nPNPIrGN//i3Yslt9YS1MNxIY5DObTNIbGPIMWP8uO0wYS0GN56xSef2JQAdllMszbjtOA8bMwG/yPenAR32rS5xfv/xjR9/bqu252dvfvgYr8OQATMPmCRWOQgw/iBF9SgYBaNgFIwYAACPvz+ItMG0NgAAAABJRU5ErkJggg==","orcid":"","institution":"Manisa Celal Bayar University","correspondingAuthor":true,"prefix":"","firstName":"Hayrunnisa","middleName":"Yeşil","lastName":"Sarsmaz","suffix":""},{"id":424337129,"identity":"88ab7a6d-448b-49af-8868-a4a77f3c4f0f","order_by":1,"name":"Seren Gülşen Gürgen","email":"","orcid":"","institution":"Manisa Celal Bayar University","correspondingAuthor":false,"prefix":"","firstName":"Seren","middleName":"Gülşen","lastName":"Gürgen","suffix":""},{"id":424337130,"identity":"1309dab8-74bd-4176-bb5a-4e9bca0d4674","order_by":2,"name":"Kemal Sarsmaz","email":"","orcid":"","institution":"Manisa Celal Bayar University","correspondingAuthor":false,"prefix":"","firstName":"Kemal","middleName":"","lastName":"Sarsmaz","suffix":""},{"id":424337131,"identity":"ec535070-c95f-4ce7-ac8d-5086ad162d85","order_by":3,"name":"Oya Sayın","email":"","orcid":"","institution":"Dokuz Eylül University","correspondingAuthor":false,"prefix":"","firstName":"Oya","middleName":"","lastName":"Sayın","suffix":""}],"badges":[],"createdAt":"2025-03-02 12:08:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6139249/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6139249/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77781882,"identity":"0e81ef06-0d69-4cf2-a029-356f400153ba","added_by":"auto","created_at":"2025-03-05 12:56:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1565297,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figuresnigerian1.png","url":"https://assets-eu.researchsquare.com/files/rs-6139249/v1/7f830f059b149db0b13da85f.png"},{"id":77782121,"identity":"548687db-3de2-4f9a-90a1-466767c092f5","added_by":"auto","created_at":"2025-03-05 13:04:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1772915,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figuresnigerian2.png","url":"https://assets-eu.researchsquare.com/files/rs-6139249/v1/d1d7b8f3cf7b8af419e8e48c.png"},{"id":77781883,"identity":"0b6f4dcb-88f3-4753-9bb0-9dfc2800a70a","added_by":"auto","created_at":"2025-03-05 12:56:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1635679,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figuresnigerian3.png","url":"https://assets-eu.researchsquare.com/files/rs-6139249/v1/888cdd79134d1efb09860e10.png"},{"id":77781887,"identity":"7633120e-7b08-4df5-a462-869fc5860b3b","added_by":"auto","created_at":"2025-03-05 12:56:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":30274,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6139249/v1/620cc75e15cbd4a9c3170398.png"},{"id":78164303,"identity":"747c8cc5-1419-4c78-906f-15ade0f92886","added_by":"auto","created_at":"2025-03-10 13:47:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6486776,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6139249/v1/dfd96c64-1fe1-4520-8c8d-d9f2f2729abc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of HPL, Leptin, and IGF-1 in Placental Tissues of Maternal Smoking During Pregnancy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIt is known that 20-50 % of women in developed countries smoke starting from the onset of pregnancy. [1] Intrauterine growth restriction (IUGR) caused on the fetus by the cigarettes smoked by the mother during pregnancy is the most frequently encountered complication. [1-3] Chronic and acute hypoxia along with the presence of carbon monoxide in maternal circulation leads to changes in the transmission of oxygen to the fetus and hence lower birth weights.[2] Even though this impact on fetal growth takes place subject to dose during the third trimester of pregnancy, the specific impact of maternal smoking on the placenta and thus on the fetus along with the correlations between the various factors expressed by the placenta have not yet been properly understood. [2, 3]\u003c/p\u003e\n\u003cp\u003eInsulin Growth Factor (IGF-1) has a polypeptide structure and it is a hormone comprised of 70 amino acids that structurally resembles proinsulin. The primary synthesis location of IGFs that are synthesized in almost all tissues is known as the liver, it is important for cell growth and differentiation and is included in the functioning of many organs[4]. Maternal tissue development and placental blood flow are induced by the combination of steroids, eritropoetin, renin, aldosteron and IGF-1. Nutritional support for fetal and neonatal development is provided through uteroplacental blood flow and postpartum milk release.[5] Insulin resembling growth factors make an impact on maternal tissue growth and metabolism thus playing a critical role in fetal growth and development. Maternal IGF that regulates placenta formation, substrate transportation and hormone release affects the usability of maternal substrate or the provision of nutrition to the fetus thus playing a role in fetal development. [6]\u003c/p\u003e\n\u003cp\u003eLeptin is a hormone that is primarily synthesized in adipose tissue and less in placental tissue.[7] It is thought to play a role in eating habits and the controlling of the energy balance through its impacts on the central nervous system. It reflects the fat reserve in the body and is synthesized from white and brown fat tissue. Leptin overcomes the blood-brain barrier through its special receptors after mixing in with the blood thus affecting the central nervous system to reduce nutrition intake while increasing energy consumption. All adipocytes have leptin release ability, however large adipocytes release more hormones compared with the smaller ones. [8] Placenta is in effect during the fetal period as well as a source of leptin.[9] Following its breaking off from the placenta after birth, milk releasing breast epithelium cells become the leptin source and thus mother\u0026rsquo;s milk that is the source of leptin during postpartum period for the newborn enables a significant amount of leptin intake.[10] It is known based on previously conducted studies that smoker women give birth to babies with less birthweight compared with nonsmoker women.[11, 12] Mantzoros et al. put forth as a result of the comparison between women who smoked during their pregnancy and those who did not that the varying leptin concentrations may be related with the decrease in fetal growth. [13]\u003c/p\u003e\n\u003cp\u003eHuman Prolactin Lactogen (HPL) is a single chain polypeptide molecule generated in the placenta by syncytiotrophoblasts, with a short half-life but even so with a high level in circulation. [14] It appears in the mother\u0026rsquo;s serum during the 6\u003csup\u003eth\u003c/sup\u003e-7\u003csup\u003eth\u003c/sup\u003e week of pregnancy. The HPL level in maternal circulation is related with fetal and placental weight and slowly increases until the last 4 weeks of pregnancy. It mixes in with the fetal circulation in a very low amount. Placental lactogen and HGH hormones display molecular similarities. The major biological impact of HPL is due to the growth hormone and prolactin similarity. HPL release acts as the growth hormone thus supporting fetal growth. \u0026nbsp;[15]\u003c/p\u003e\n\u003cp\u003eSome studies in the literature have shown that IGF-1 deficiency significantly increases the risk of cardiovascular diseases, and leptin reduces vasodilation through the expression of inducible NO synthase (iNOS) in the vascular wall. [16, 17] No study was observed in literature that has previously examined the correlation between IGF-1, Leptin and HPL expressions in the placentas of smoker and nonsmoker pregnant women. Thus, the current study aimed to examine the effects of IGF-1, Leptin and HPL expressions in the placental tissues of smoker and nonsmoker pregnant women.\u0026nbsp;\u003c/p\u003e"},{"header":"Materials and Method","content":"\u003cp\u003eThis study was approved by Manisa Celal Bayar University Health Science Ethics Committee on 02/ 08/ 2017 with the number 20.478.486.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParticipants\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur study was conducted with 70 women[18] (case: 35 smokers, control: 35 nonsmokers) [18]who applied to the T.R. Ministry of Health Manisa Merkezefendi State Hospital Delivery Room Unit during the dates of March-May and who gave birth through spontaneous vaginal birth. In this investigation was received a statement that the subject gave \u0026ldquo;informed consent\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eInclusion criteria for research\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePregnant women who have started smoking at least 100 cigarettes in her lifetime and who now smokes every day[19], pregnant women who have never smoked throughout their life, including pregnancy[19],pregnant women who gave birth at term, multiple pregnancy, preeclampsia, gestational diabetes, thyroid dysfunction, those who do not have a systemic disease and due to systemic disease during pregnancy, those who do not use, pregnant women who had spontaneous vaginal delivery, in order not to encounter communication barriers and because cultural differences may vary, the pregnant woman is not a foreign national, pregnant women who do not have any barriers to communication by phone, those who do not have any health problems that prevent breastfeeding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eExclusion\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eCriteria for research\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMultiple pregnancy, insulin dependent diabetes, pregnant women who have had a cesarean section, preeclampsia, thyroid disease.\u003c/p\u003e\n\u003cp\u003eGroups were classified as Group 1(case): Smokers \u0026ldquo;before and during pregnancy\u0026rdquo; (n=35), Group 2(control): Non-smokers \u0026ldquo;lifetime and during pregnancy\u0026rdquo; ( n=35).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlacenta Tissue Selection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study was carried out at the Manisa Merkez Efendi State Hospital during October 2017-April 2018 for a period of about 6 months by taking the necessary ethical and official permissions. It was aimed to relationship between \u0026nbsp; histopathological changes \u0026nbsp;of the placenta tissues acquired from \u0026ldquo;pregnant women who have started smoking at least 100 cigarettes in her lifetime and who now smokes every day\u0026rdquo; and from \u0026ldquo;pregnant women who have not smoked and non exposed second-hand smoke\u0026rdquo; at all in addition to examining umbilical cord bloods[19]. It took about 6 months between October 2017-April 2018 to collect the placentas.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical Staining on placenta\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe placenta tissues separated from the mothers were weighed rapidly. Following these processes, a sample tissue with dimensions of 1 cm3 was taken from the larger placenta tissue which was then placed in 10% formaldehyde and fixed for 24 hours. They were then subject to dehydration processes and embedded in paraffin blocks. The embedded tissues were transferred as 5 \u0026mu; sections onto a microscope slide via a microtome (Leica RM 2135; Bensheim, Germany). For immunohistochemical staining, the sections were first incubated at 60 \u0026ordm;C overnight and were then incubated in xylene for 30 min. After washing with serial concentrations of ethanol (95, 80, 70, and 60%), the sections were washed with distilled water and phosphate-buffered saline (PBS) for 10 min. They were then held in 2% trypsin in Tris buffer at 37 C for 15 min and were washed with PBS three times for 5 min. The sections were incubated in a solution of 3% H2O2 for 15 min to inhibit endogenous peroxidase activity. After that, the sections were washed with PBS and were incubated with blocking solution, Large Volume Ultra V Block (Thermo Fisher, USA) for 1 h. The sections were then incubated at 4 C with primary antibodies overnight: HPL (Mouse monoclonal, Thermo Fisher, USA), Leptin (Bioss, Woburn, \u0026nbsp;Massachusetts, USA), IGF-1 (Bioss, Woburn, Massachusetts- USA) Then, they were washed three times for 5 min, each with PBS, followed by incubation with biotinylated IgG and then with streptavidin\u0026ndash;peroxidase conjugate (Thermo Fisher, CA, USA). After washing three times for 5 min with PBS, the sections were incubated with DAB substrate containing diaminobenzidine (Thermo Fisher, CA, USA) for 5 min to detect immunoreactivity and then with Mayer\u0026rsquo;s hematoxylin. The sections were covered with mounting medium and were analyzed on a light microscope with a CX 31 microscope (Olympus, Tokyo, Japan). Control samples were processed in an identical manner, but in the\u003c/p\u003e\n\u003cp\u003eabsence of primary antibody step. \u0026nbsp;All of the immunostained sections were reviewed by two histologists, who had no knowledge of the groups. Slides were examined under low power (X10 objective) to identify the regions containing HPL, Leptin and, IGF-1 staining ten areas selected at random were scored and in sections, where all of the staining appeared intense, one random field was selected. The proportion of placenta \u0026nbsp;in each selected field was determined by counting at high magnification. At least 100 cells were scored per X 40 field for each group. All the sections were scored in a semiquantitative manner, which considers both the intensity and the percentage of cells, staining at each intensity. The intensities were classified as 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining), whereas 10% groupings were used for the percentage of cells that stained positive. For each slide, a value designated H-SCORE was obtained by the application of the algorithm: H-SCORE: \u0026Sigma; Pi.(i+1), where I and PC represent the intensity and the percentage of cells that stain at each intensity, respectively, and the corresponding H-SCOREs were calculated separately. Numbers were calculated on 10 randomly selected areas using the image analysis system (Leica Q Win V3 Plus Image), and the mean values were employed for analysis.[20]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUmbilical cord blood biochemistry analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole blood taken from the umblical cord was collected into 5.5 ml tubes with yellow caps and serum separator gel for biomarker analyses. After waiting at least 30 minutes for the coagulation reaction, the tubes were centrifuged at 1000g x 15 min at +4\u0026deg;C. The serum samples obtained will be portioned separately into polypropylene 0.5 ml eppendorf tubes for each biomarker. Samples were stabilized at -80 and stored in a deep freezer until the working day. When the planned number of patients is reached, serum samples will be raised to +4\u0026deg;C the night before the working day and all samples will be thawed homogeneously.[21] Analyzes of Leptin and HPL \u0026nbsp;biomarkers were performed by ELISA method. When the number of all blood samples reaches 35 patients in the case group and 35 patients in the control group, all of them were studied at the same time to be statistically significant. Therefore, the half-lives of the biomarkers weren\u0026rsquo;t affected.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSPSS 25.0(SPSS Inc.; Chicago, IL, USA)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e software was used for data evaluation. Statistically significant results were defined as those with a P value less than 0.05. The data analysis focused on the \u0026quot;mean \u0026plusmn;SD\u0026quot; values. Independent t test \u0026nbsp;and Mann Whitney U test were used for this study.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 70 parturients who presented to the labor ward of the one study centers participated in the study and all the samples taken (100%) were adequate and analyzed. The study found that 57.1% of pregnant women who smoke are aged 25 or younger, 94.3% are married, 42.9% are literate, 80% are housewives, 91.4% have a similar income status, 68.6% do not have social security, 74.3% have had multiple pregnancies, 42.9% have given birth to two or more children, 77.1% have not had a miscarriage, and 37.1% have two or more living children. The study found that 60% of pregnant women who do not smoke are over the age of 26, 100% are married, 62.9% have an education level of primary school or higher, 97.1% are housewives, 94.3% have a similar income status, 65.7% have social security, 74.3% have had multiple pregnancies, 45.7% have had one birth, 85.7% have not had a miscarriage, and 42.9% have one living child (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1:\u0026nbsp;\u003c/strong\u003eSociodemographic characteristics/variable of participants (n=70)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"565\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eVariable\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSmokers (n=35)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNonsmokers (n=35)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;(n=70)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ex\u003csup\u003e2\u003c/sup\u003e/p\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFrequency (n)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFrequency (n)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFrequency (n)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge (years)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026lt;26\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026ge;26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e57.1\u003c/p\u003e\n \u003cp\u003e42.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e48.6\u003c/p\u003e\n \u003cp\u003e51.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2.05/0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMarital status\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eMarried\u003c/p\u003e\n \u003cp\u003eSingle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e94.3\u003c/p\u003e\n \u003cp\u003e5.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e68\u003c/p\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e97.1\u003c/p\u003e\n \u003cp\u003e2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e-/-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLevel of education\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNo formal education\u003c/p\u003e\n \u003cp\u003eFormal education\u003c/p\u003e\n \u003cp\u003ePrimary school and above\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e17,1\u003c/p\u003e\n \u003cp\u003e42.9\u003c/p\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e8.6\u003c/p\u003e\n \u003cp\u003e28.6\u003c/p\u003e\n \u003cp\u003e62.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e12,9\u003c/p\u003e\n \u003cp\u003e35.7\u003c/p\u003e\n \u003cp\u003e51.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3.77/0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSocial Security\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e31.4\u003c/p\u003e\n \u003cp\u003e68.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e65.7\u003c/p\u003e\n \u003cp\u003e34.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e48.6\u003c/p\u003e\n \u003cp\u003e51.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e8.23/0.004*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParity\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003ePrimipar\u003c/p\u003e\n \u003cp\u003eMultipar\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e25.7\u003c/p\u003e\n \u003cp\u003e74.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e25.7\u003c/p\u003e\n \u003cp\u003e74.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e25.7\u003c/p\u003e\n \u003cp\u003e74.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.00/1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of births\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003cp\u003e2 and above\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e28.6\u003c/p\u003e\n \u003cp\u003e28.6\u003c/p\u003e\n \u003cp\u003e42.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e28.6\u003c/p\u003e\n \u003cp\u003e45.7\u003c/p\u003e\n \u003cp\u003e25.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e28.6\u003c/p\u003e\n \u003cp\u003e37.1\u003c/p\u003e\n \u003cp\u003e34.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2.88/0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of abortions\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNone\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003cp\u003e2 or more\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e77.1\u003c/p\u003e\n \u003cp\u003e17.1\u003c/p\u003e\n \u003cp\u003e5.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e85.7\u003c/p\u003e\n \u003cp\u003e8.6\u003c/p\u003e\n \u003cp\u003e5.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e81.4\u003c/p\u003e\n \u003cp\u003e12.9\u003c/p\u003e\n \u003cp\u003e5.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e-/-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of living children\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003cp\u003e2 and above\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e31.4\u003c/p\u003e\n \u003cp\u003e31.4\u003c/p\u003e\n \u003cp\u003e37.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e31.4\u003c/p\u003e\n \u003cp\u003e42.9\u003c/p\u003e\n \u003cp\u003e25.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e31.4\u003c/p\u003e\n \u003cp\u003e37.1\u003c/p\u003e\n \u003cp\u003e31.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.34/0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*p\u0026lt;0,05 x2: Chi-square test\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical Findings\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhereas IGF-1 immunoreaction was stained at intensities ranging from moderate to strong in the villous stromal cells of the nonsmoker group, it was at intensities ranging from weak to moderate in syncytiotrophoblast cells (Figure 1 A1,A2). Whereas weak IGF-1 reaction was observed in both villous stromal cells and syncytiotrophoblast cells in the smoker group (Figure 1 B1, B2). While leptin immunoreaction was stained strongly in especially the villous stromal cells of the nonsmoker group, it was observed to be stained at a moderate intensity in syncytiotrophoblast cells (Figure 2 A1, A2). Whereas in the smoker group, it was striking to see that leptin immunoreaction was stained moderate in both villous stromal and syncytiotrophoblast cells (Figure 2 B1, B2). It was observed that HPL immunostaining was quite strong in especially the syncytiotrophoblast cells in the placenta villi of the nonsmoker group, while weak staining attracted attention in villous stromal cells (Figure 3 A1, A2). It attracted attention that the HPL immunoreaction reduced significantly in the syncytiotrophoblast cells of the smoker group. There was again weak expression in the villous stromal cells (Figure 3 B1,B2). A statistically significant difference was observed between the smoking status of the pregnant women and the median scores of IGF-1, Leptin and HPL histopathological findings (p\u0026lt;0,05). It was illustrated that the IGF-1, Leptin and HPL median scores of smoker pregnant women were lower compared with those of the nonsmokers (Figure 4, Table 2). A statistically significant difference was identified between the smoking status of pregnant women and their median scores for IGF-1, Leptin and HPL histopathological findings (p\u0026lt;0,05). It was observed that the IGF-1, Leptin and HPL median scores of smoker pregnant women were lower compared with those of nonsmokers (Table 2,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFigure 4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e. Comparison of the Placenta Indirect Immunoperoxidase Hscore Findings of Smoker and Nonsmoker Pregnant Women (n=70)\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"631\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHistsopathological Findings\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 172px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSmokers (n=35)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 206px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNonsmokers (n=35)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMed\u003c/strong\u003e\u003cstrong\u003e\u0026plusmn;\u003c/strong\u003e\u003cstrong\u003esd\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eIQR\u003c/strong\u003e\u003cstrong\u003e*)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMed\u003c/strong\u003e\u003cstrong\u003e\u0026plusmn;\u003c/strong\u003e\u003cstrong\u003esd\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eIQR\u003c/strong\u003e\u003cstrong\u003e*)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ez/p\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIGF -1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e64.1\u0026plusmn;21.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e63.0 (45.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e216.2\u0026plusmn;30.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003e216.0 (54.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e-7.19/0.00**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLeptin\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e132.2\u0026plusmn;24.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e129.0 (39.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e328.5\u0026plusmn;47.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003e340.0 (96.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e-7.19/0.00**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHPL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e99.9\u0026plusmn;19.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e99.0 (27.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e289.7\u0026plusmn;42.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003e300.0 (72.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e-7.19/0.00**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eIQR\u003c/strong\u003e\u003cstrong\u003e*)\u003c/strong\u003e: Interquartile range. **p\u0026lt;0.05. z: Mann Whitney U test\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUmbilical cord blood biochemistry Findings\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA statistically significant difference was identified between the smoking status of pregnant women and their median scores for Leptin and HPL biochemistry findings (p\u0026lt;0,05). It was observed that the Leptin and HPL mean scores of smoker pregnant women were lower compared with those of nonsmokers(Table 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e. Leptin and HPL Biochemistry Findings of Smoker and Nonsmoker Pregnant Women (n=70)\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBiochemistry Findings\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroups\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStd. Deviation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLeptin\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(pg/ml)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eSmokers\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e54.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e11.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e39.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026lt;0.001\u003c/em\u003e\u003c/strong\u003e**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eNonsmokers\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e107.8\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHPL\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eSmokers\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e1.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026lt;0.001\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e**\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eNonsmokers\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e5.8\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e**p\u0026lt;0.05. Independent T test\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe impact of smoking on female reproductive functions has become a significant concern due to the rising number of young female smokers. Since Simpson\u0026apos;s 1957 report, which highlighted a higher incidence of premature births among pregnant women who smoked and showed that this rate increased with the amount of smoking, numerous studies have examined the effects of maternal smoking on the fet\u0026uuml;s.[22] Many of these studies have found that babies born to mothers who smoke tend to have lower birth weights compared to those born to non-smokers. In fact, this study aimed to investigate the impact of smoking on breastfeeding. By analyzing the levels of specific biomarkers in postpartum placenta tissue and umbilical cord blood, we gathered information about the subsequent stage: the breastfeeding process.\u0026nbsp;In our study, expression of IGF, Leptin and HPL median scores expressed in the placentas of smoker pregnant women which were lower compared with those of nonsmoker pregnant women. And\u0026nbsp;Leptin and HPL biochemistry findings of smoker pregnant women which were lower compared with those of nonsmoker pregnant women.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFleisch et al. conducted a study in 2017 during which they examined the umbilical cord blood hormones for mothers who smoked during pregnancy and classified the women into 3 groups as those who smoked during their pregnancy, who smoked prior to pregnancy and continued to do so and those who never smoked.[23] Hormones such as IGF-1, IGF-2, IGFB-3, leptin were examined in the cord blood as a result of which it was observed that IGF-1 values were lower for those who smoked during pregnancy.[23] IGF-1 immunoreactivity was examined in the placenta during the present study in which it was determined similar to other studies that it was lower for the group of women who smoked during pregnancy. IGF-1 immunoreactivity was observed to be lower in the placenta tissues of nonsmoker mothers. Correlatively with the fact that babies of smoker mothers in our study have lower head circumference and height values and the fact that babies of smoker women had lower birth weights in our previous study.[24] it was concluded that the IGF-1 and Leptin values expressed from the placenta may have a primary impact on the development of the baby.\u003c/p\u003e\n\u003cp\u003eLeptin is generated at the syncytiotrophoblast cells by adipocyte cells and it is thought to play an impact on fetal development through currently unknown mechanisms. Ypsilantis P et al. carried out a study in 2013, during which it was emphasized that smoking plays a critical role in bodyweight loss and appetite reduction and that the leptin and ghrelin hormones related with appetite control are not clear.[25] For this reason, they used 8 week old male rats in smoker and nonsmoker groups as a result of which it was observed that a significant loss of bodyweight took place in in 4 weeks in the group subject to smoking while on the other hand weight was regained 1 week after the end of exposure. The leptin level decreased during the first 4 weeks in the rat group subject to exposure for 8 weeks only to return to the normal level on the 7\u003csup\u003eth\u003c/sup\u003e week after exposure. Based on the results of this study, the leptin level in circulation during the first few weeks of exposure in rats cannot be clearly explained with body weight. [25] The serum leptin level was observed to be much lower compared with cigarette exposure in the study by Ypsilantis et al. and similarly, placenta leptin immunoreactivity was observed to be low in smokers in the present study as well. Similarly, Pardo et al. published a study in 2004 entitled \u0026ldquo;Does maternal smoking influence leptin levels in term, appropriate-for-gestational-age newborns?\u0026rdquo; In this study, it was emphasized that leptin is a hormone generated in the placenta and adipose tissues and that it is correlated with neonatal development. 19 smoker and 91 nonsmoker pregnant women were classified in this study. Neonatal anthropometric measurements were taken and leptin levels were measured via radioimmunoassay. Leptin concentrations were observed to be similar between the groups and leptin levels were correlated with ponderal index and similarly with the gestational ages of the newborns. This study presented that maternal smoking during pregnancy does not have an impact on the cord blood leptin levels but that it is in accordance with gestational age.[26] Placenta Leptin immunoreactivity in our study was observed to be lower in smoker pregnant women compared with nonsmokers.\u003c/p\u003e\n\u003cp\u003eRatio of those that were previously smoking among pregnant women was 26 %, whereas the ratio of current smokers was 16.5 %, age of starting smoking was 18 and the number of cigarettes smoked per day was eight. TNSA-2008 results along with the results of studies carried out in both our country and around the world indicate that smoking is a critical issue among both pregnant, nursing and other women.[3, 27] The HPL median scores of smoker pregnant women were observed to be lower compared with those of nonsmoker pregnant women. There were no previous studies in literature on the correlation between smoking, breastfeeding and placental HPL.\u0026nbsp;Human Prolactin Lactogen (HPL) is a single chain polypeptide molecule produced by syncytiotrophoblasts in the placenta with its major biological impact due to its resemblance of the growth hormone and prolactin. HPL secretion acts as a growth hormone thus supplementing fetal growth.[15] It was determined statistically in our study that the HPL expressions of smoker pregnant women are lower compared with nonsmoker pregnant women. While based on our immunohistochemical findings, HPL immunostaining is quite strong in the placenta villi of the nonsmoker group, HPL immune reaction was observed to decrease distinctively in syncytiotrophoblast cells and villous stromal cells in the smoker group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCarl J et al. published a study in 1991 in which 75 women were included in human placental lactogen (hPL) for normal pregnancy. HPL levels were recorded during the period starting from 26\u003csup\u003eth\u003c/sup\u003e week until birth. It was observed as a result of the measurement conducted via Modifiye Gompertz equation method that development retardation continued until the 36\u003csup\u003eth\u003c/sup\u003e week.[15] In another study, smoking was not shown to affect placenta size and it was emphasized that smoking caused the fetus weight and HPL levels to be lower.[28] It was also observed in our study that the HPL levels were lower among smoker pregnant women compared with nonsmokers. Mochizuki et al. carried out a study in 1984 during which it was emphasized that maternal smoking results in growth deficiency. Based on the findings of this study, the urinary estriol and hPL levels of heavy smoker women are lower compared with those of the nonsmokers.[29] Similar to these studies, the HPL amount expressed from the placenta was observed to be lower for smokers in our study, however different from the study by Mochizuki et al., we showed only the immunoreactivity of HPL in the placenta. However, we also examined leptin and IGF-1 expressions in addition to HPL.\u003c/p\u003e"},{"header":"Conclusion","content":"Although our sample is similar to other histopathological studies in the literature, the fact that our study was conducted at a single center may not reflect the results of the general population. This constitutes the main limitation of our study. It is known that there is a statistically significant correlation between prematernal and maternal smoking and the birth weight of the newborn and that smoking reduces birth weight and placenta weight in addition to making adverse impacts on placenta histology. Low IGF-1 Leptin and HPL levels expressed in the placenta among maternal smoker women were observed. Lower HPL expression values of the placentas of smoker mothers led us to think that it is related with insufficient mother’s milk during the postpartum nursing period."},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors participated in the design, interpretation of the studies and analysis of the data and review of the manuscript; HYS,KS and SGG Conception or design of the work, HYS data collection, HYS and SGG histology analysis and HYS,KS drafting the article, OS biochemical analysis, SGG,KS and HYS final approval of the version to be published.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by Manisa Celal Bayar University Health Science Ethics Committee on 02/ 08/ 2017 with the number 20.478.486.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Helsinki\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The study was conducted according to the principles of Helsinki Declaration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of research data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Authors are available and ready to supply the data upon any requests through the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by a grant from the Manisa Celal Bayar University Research Fund, Manisa, Turkey. [Project no: 2017-216].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was registered at www.clinicaltrials.gov. Trial registration number is NCT06456645.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eHiggins, S., \u003cem\u003eSmoking in pregnancy.\u003c/em\u003e J Current Opinion in Obstetrics Gynecology, 2002. \u003cstrong\u003e14\u003c/strong\u003e(2): p. 145-151.\u003c/li\u003e\n \u003cli\u003eAndres, R.L. and M.-C. Day. \u003cem\u003ePerinatal complications associated with maternal tobacco use\u003c/em\u003e. in \u003cem\u003eSeminars in Neonatology\u003c/em\u003e. 2000. Elsevier.\u003c/li\u003e\n \u003cli\u003eShiverick, K. and C.J.P. 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Galanakis, \u003cem\u003eRisk factors related to intention to breastfeed, early weaning and suboptimal duration of breastfeeding.\u003c/em\u003e 2007. \u003cstrong\u003e96\u003c/strong\u003e(10): p. 1441-1444.\u003c/li\u003e\n \u003cli\u003eSpira, A., et al., \u003cem\u003eSmokıng Durıng Pregnancy And Placental Pathology.\u003c/em\u003e Biomedicine Express, 1977. \u003cstrong\u003e27\u003c/strong\u003e(7): p. 266-270.\u003c/li\u003e\n \u003cli\u003eMochizuki, M., et al., \u003cem\u003eEffects of smoking on fetoplacental-maternal system during pregnancy.\u003c/em\u003e 1984. \u003cstrong\u003e149\u003c/strong\u003e(4): p. 413-420.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Smoking cigarette, placenta, IGF-1, Leptin, HPL, breastfeeding","lastPublishedDoi":"10.21203/rs.3.rs-6139249/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6139249/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Maternal smoking during pregnancy although it is known to be associated with fetal growth restriction, it’s effect on postpartum breastfeeding is not yet clear. The aim of the present study was to examine the impacts of smoking in pregnancy on breastfeeding as well as its impacts on placental immunoreactivity. The study was conducted on 70 women who gave birth via spontaneous vaginal delivery. Groups were classified as Smokers (n=35) and Non-smokers (n=35). Cross-sections were taken from the placenta tissues after which their IGF-1, Leptin, HPL immunoreactivities were examined. A significant difference was identified between the smoking status of the pregnant women and findings on their IGF-1, Leptin and HPL immunoreactivities (p\u003c0,05). Lower IGF-1, Leptin and HPL median scores were observed for smoker pregnant women compared with non-smoker pregnant women. Lower placental IGF-1 Leptin and HPL expressions in smoker groups were found to be correlated with the lower values related with the baby and breastfeeding. Lower HPL expression values of the placentas of smoker mothers led us to think that it is related with insufficient mother’s milk during the postpartum nursing period.","manuscriptTitle":"Evaluation of HPL, Leptin, and IGF-1 in Placental Tissues of Maternal Smoking During Pregnancy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-05 12:56:44","doi":"10.21203/rs.3.rs-6139249/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4ba34a3f-766d-4302-bcee-d4ba67159485","owner":[],"postedDate":"March 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-10T13:39:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-05 12:56:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6139249","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6139249","identity":"rs-6139249","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}