Effects of prenatal maternal smoking on placental IGF-1, leptin, and HPL expression and breastfeeding practices

TL;DR

Prenatal smoking reduces placental hormone levels and may affect breastfeeding practices and fetal development.

Contribution

This study reveals new associations between maternal smoking, placental hormone expression, and breastfeeding outcomes.

Findings

Smoking during pregnancy lowers placental IGF-1, leptin, and HPL expression.

Maternal smoking is linked to altered breastfeeding practices.

Reduced hormone levels may impact fetal development and placental function.

Abstract

Smoking during pregnancy has many negative effects, such as growth retardation, premature birth, and placental detachment. However, although it is known to have negative effects during breastfeeding, it has not been examined in detail as the pregnancy period. This study aimed to investigate the effects of smoking during pregnancy on the placenta and its relationship with breastfeeding practices and placental hormone levels. The study included 70 women who underwent spontaneous vaginal delivery. The participants were divided into two groups: smokers (n = 35) and non-smokers (n = 35). The breastfeeding conditions of both groups were evaluated prior to discharge and on the 10th day, postpartum. Cross-sectional samples of placental tissues were collected, and their IGF-1, leptin, and HPL immunoreactivities were analyzed. A significant difference was found between the smoking status of the mothers and the immunoreactivity of IGF-1, leptin, and HPL (p < 0.05). Pregnant mothers who smoked showed lower expression of IGF-1, leptin, and HPL than non-smoker pregnant mothers. These results indicate that maternal smoking during pregnancy has adverse effects on the placenta and cord blood and may affect fetal development. Moreover, prenatal maternal smoking is associated with changes in breastfeeding practices.

Figures

IGF-1 immunostainings in the placenta villi of smoker and nonsmoker groups. Nonsmoker A, Smoker B. ➠:Villous syncytiotrophoblast cells, →: villous stromal cells. (A1,B1) X100, (A2,B2) X400

Leptin immunostainings in the term placenta villi of smoker and nonsmoker groups. Nonsmoker A, Smoker B. ➠:Villous syncytiotrophoblast cells, →: villous stromal cell. (A1,B1) X100, (A2,B2) X400

HPL immunostainings in the term placenta villi of Nonsmoker and smoker groups. Nonsmoker A, Smoker B. ➠:Villous syncytiotrophoblast cells, →: villous stromal cell. (A1,B1) X100, (A2,B2) X400

H-SCORE statistical analysis of IGF-1, leptin, HPL, immunoreactivity intensities for nonsmoker and smoker pregnant women. Values have been presented as mean ± SD

Full text

Introduction

Maternal smoking remains a major public health problem worldwide and continues to affect a substantial proportion of women of reproductive age [1–4]. Although global smoking prevalence has declined over recent decades, tobacco use among pregnant women persists, particularly in certain regions, and is associated with adverse perinatal outcomes [5–9]. One of the most consistently reported fetal consequences of prenatal smoking exposure is impaired fetal growth, including intrauterine growth restriction and an increased risk of small for gestational-age birth, largely mediated by chronic hypoxia, carbon monoxide exposure, and placental dysfunction [9, 10]. In addition, maternal smoking has been linked to reduced exclusive breastfeeding rates and delayed initiation of lactation, potentially through both behavioral and biological mechanisms [11–19]. Importantly, accumulating evidence suggests that some of the biological effects of smoking on both fetal growth and early postnatal feeding may be mediated through alterations in placental endocrine function. However, the biological pathways connecting prenatal smoking, placental hormonal regulation, and early postnatal feeding outcomes remain incompletely understood.

The placenta plays a central role in regulating fetal growth and maternal metabolic adaptation through the synthesis of key hormones, including insulin-like growth factor-1 (IGF-1), leptin, and human placental lactogen (HPL) [20–26]. IGF-1 is a critical mediator of cellular proliferation, placental blood flow, and fetal tissue development [20, 21]. Placental leptin contributes to energy homeostasis and trophoblast function and may reflect adaptive responses to placental stress in growth restricted pregnancies [23, 24]. HPL, a growth hormone and prolactin-like peptide, increases progressively during gestation in proportion to placental mass and supports fetal growth by modulating maternal glucose and lipid metabolism [26]. Beyond their roles in fetal growth, these hormones are also involved in maternal metabolic and neuroendocrine adaptations that are essential for the initiation and maintenance of lactation. Although maternal smoking is known to impair placental vascularization and endocrine activity, its specific effects on placental IGF-1, leptin, and HPL expression and their potential downstream implications for both fetal development and lactogenesis have not been fully elucidated.

In parallel with its effects on fetal growth, maternal smoking has been consistently associated with suboptimal breastfeeding practices, including delayed initiation and reduced exclusivity, potentially mediated by neuroendocrine and metabolic alterations affecting lactogenesis and milk composition [10–19]. However, whether smoking-related alterations in placental endocrine function particularly in hormones involved in both fetal growth and lactogenesis coexist with differences in early breastfeeding behavior has not been systematically investigated.

Therefore, the aim of the present study was to evaluate the association between maternal smoking during pregnancy and placental as well as umbilical cord blood levels of IGF-1, leptin, and HPL, and to examine whether maternal smoking is related to differences in early breastfeeding practices during the immediate postpartum period.

Materials and methods

Ethical approval

This study was approved by the Manisa Celal Bayar University Health Sciences Ethics Committee (Approval date: 02 August 2017; Decision No: 20.478.486).

Study design and participants

This prospective case–control study included 70 postpartum women (35 smokers and 35 non-smokers) who delivered via spontaneous vaginal birth at Merkezefendi State Hospital between October 2017 and April 2018 [27]. Written informed consent was obtained from all participants before enrollment. Participants completed a questionnaire related to demographics and breastfeeding.

The participants were categorized into two groups:

• Group 1 (smokers): Women who smoked at least 100 cigarettes in their lifetime and continued smoking before and during pregnancy (n = 35).
• Group 2 (non-smokers): Women who had never smoked in their lifetime and did not smoke during pregnancy (n = 35).

Breastfeeding status was assessed prior to hospital discharge and on postpartum day 10.

Inclusion and exclusion criteria for research

Pregnant mothers who had smoked at least 100 cigarettes during their lifetime, smoked during pregnancy, gave birth spontaneously, had no systemic diseases, and did not have any health condition that could prevent breastfeeding were included in the study group. The study also included the use of e-cigarettes and vapes by the participants.

Those who had never smoked, had spontaneous vaginal birth, had no systemic disease (diabetes mellitus, hypertension, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, autoimmune diseases in general, systemic infections such as sepsis, and chronic cardiovascular conditions such as heart failure), and did not have any health condition that could interfere with breastfeeding were included in the control group.

Pregnant mothers with multiple pregnancies, preeclampsia, pregestational or gestational diabetes, thyroid dysfunction, preterm premature rupture of membranes (PPROM), or foreign nationality were excluded from the study.

Placental tissue collection and processing

Immediately after delivery, 35 placental tissue samples per group were collected. The placentas were rinsed with phosphate-buffered saline (PBS) to remove excess blood. Tissue samples (~ 1 cm³) were collected from standardized locations on both the maternal and fetal surfaces near the peripheral margin. Samples were fixed in 10% neutral-buffered formalin for 24–48 h, dehydrated through graded ethanol, and embedded in paraffin blocks. Sections of 5 μm thickness were cut using a microtome (Leica RM 2135, Germany) for immunohistochemical analyses.

Immunohistochemical staining on placenta

A 1 cm³ tissue sample was obtained from both the fetal and maternal surfaces of the placenta, specifically from the peripheral margin, and was then placed in 10% formaldehyde and fixed for 24 h. Samples were collected from the same region of the placenta. The samples were then subjected to dehydration and embedded in paraffin blocks. The embedded tissues were transferred as 5 μm sections onto a microscope slide using a microtome (Leica RM 2135; Bensheim, Germany). For immunohistochemical staining, the sections were first incubated at 60 °C overnight and 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 incubated in 2% trypsin in Tris buffer at 37 °C for 15 min and washed with PBS three times for 5 min. The sections were incubated in a 3% H_2_O_2_ solution for 15 min to inhibit endogenous peroxidase activity. The sections were then washed with PBS and incubated with the 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 (1:100, mouse monoclonal, Thermo Fisher, USA), leptin (1:100, Bioss, Woburn, Massachusetts, USA), IGF-1 (1:100, Bioss, Woburn, Massachusetts, USA). The cells were then washed three times for 5 min each with PBS, followed by incubation with biotinylated IgG and streptavidin–peroxidase conjugate (Thermo Fisher, CA, USA). After washing three times for 5 min with PBS, the sections were incubated with diaminobenzidine (DAB) substrate (Thermo Fisher, CA, USA) for 5 min to detect immunoreactivity, followed by Mayer’s hematoxylin. The sections were covered with a mounting medium and analyzed under a light microscope (CX 31; Olympus, Tokyo, Japan). Negative control samples were processed in an identical manner, but without the primary antibody step. All of the immunostained sections were reviewed by two histologists, who had no knowledge of the groups.The slides were first examined under low magnification (x10 objective) to identify regions with HPL, leptin and IGF-1 staining. Ten random areas were selected and scored for each section. In sections where staining was particularly intense, a single random field was chosen for detailed analysis. The proportion of placenta in each selected field was determined by counting at high magnification. At least 100 cells were scored per field at × 40 magnification for each group. All sections were scored semi-quantitatively, considering both the intensity and percentage of cells stained 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 positively stained cells. For each slide, a value designated as H-SCORE was obtained by applying the following algorithm: H-SCORE = Σ Pi.(i + 1), where i and Pi represent the intensity and percentage of cells stained at each intensity. 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 [28].

Umbilical cord blood collection and biochemical analysis

Whole blood collected from the umbilical cord vein was drawn into 5.5 mL serum separator tubes with yellow caps (Product No: #30011181, Hawach Scientific) for biomarker analysis. The samples were kept at room temperature for at least 30 min to allow coagulation. The tubes were then centrifuged at 1000 x g for 15 min at + 4 °C, and the serum was separated. The obtained serum samples were aliquoted into 0.5 mL polypropylene Eppendorf tubes, one for each biomarker. Samples were stabilized at − 80 °C and stored in a deep freezer until the day of analysis. Once the planned number of patients was reached, the samples were thawed homogeneously by placing them at 4 °C overnight. Prior to the analyses, leptin and human placental lactogen (HPL) levels were measured using the sandwich ELISA method described above. The analysis was performed according to the manufacturer’s protocol using a commercially available ELISA kit (Elabscience; catalog number: E-EL-H6017). Each sample was analyzed in duplicate. Standards and samples were added to 96-well plates and incubated at room temperature for 2 h. The wells were washed with PBS containing 0.05% Tween-20, followed by the addition of a biotin-labeled secondary antibody and incubation for 1 h. After washing, streptavidin-HRP conjugate was added and incubated for 30 min. Color development was achieved by adding 3, 3 ′, 5, 5′-tetramethylbenzidine (TMB) substrate and incubating in the dark for 15 min. The reaction was stopped using 2 N sulfuric acid, and the optical density was measured at 450 nm using a microplate reader (BioTek ELx800). The results were quantified using a standard curve based on a logarithmic regression analysis. Each plate included internal positive and negative control. All samples were analyzed simultaneously under the same conditions to ensure that the half-lives of the biomarkers were unaffected. The coefficient of variation- CV (%) for the replicates was maintained below 10% to ensure measurement reliability [29]. Due to budgetary constraints, the IGF ELISA kit could not be procured; thus, IGF analysis was not performed.

Statistics

A priori sample size estimation was performed using G*Power version 3.1.9.7 for a two-sided comparison of two independent means with equal group allocation. The primary outcome used for the calculation was the difference in placental hormone expression levels between smoking and non-smoking mothers.

Based on previous lactation- and placenta-related studies reporting large differences in hormone concentrations between smokers and non-smokers, a large effect size was assumed (Cohen’s d = 1.15). This value reflects a conservative representation of large effects described in the literature and was not derived post hoc from the present dataset. Using this assumed effect size with an alpha level of 0.05 and 95% power, the minimum required sample size was approximately 20 participants per group [30, 31]. Therefore, enrolling 35 participants per group provided sufficient statistical power to detect large between-group differences.

All statistical analyses were performed using SPSS version 25.0 (SPSS Inc., Chicago, IL, USA). Continuous variables were expressed as mean ± standard deviation. Group comparisons were conducted using independent samples t-tests or one-way ANOVA, categorical variables were analyzed using chi-square tests, and linear regression analyses were applied where appropriate. Statistical significance was defined as p < 0.05.

Results

Study population

A total of 70 women (35 smokers and 35 non-smokers) who delivered via spontaneous vaginal birth were included in this study. Demographic and breastfeeding-related characteristics, placental immunohistochemical markers (IGF-1, leptin, and HPL), and umbilical cord blood biochemical levels of leptin and HPL were evaluated for each participant. All collected placental tissue samples (n = 70) were adequate for histological and immunohistochemical assessment, and all cord blood samples were suitable for biochemical analyses.

Demographic characteristics

The sociodemographic characteristics of the participants are presented in Table 1. Birth weight percentiles were classified as ≤ 10th percentile (small for gestational age, SGA) or > 10th percentile (non-SGA). SGA was significantly more common among infants born to mothers who smoked (75%, n = 18) than among those born to non-smokers (25%, n = 6) (χ² = 9.13, p = 0.003). No statistically significant differences were found between the groups with respect to maternal age, marital status, education level, parity, abortion history, number of living children, infant sex, placental expulsion time (min), or placental weight (g) (p > 0.05).

Table 1. Sociodemographic and obstetric variables of the study population (n = 70)VariableSmokers (n = 35)Nonsmokers (n = 35)x^2^/pFrequency (n)%Frequency (n)%Age (years) < 262057.114402.05/0.15 ≥ 261542.92160Marital status Married3394.335100-/- Single25.700Level of education No formal education617,138.63.77/0.15 Formal education1542.91028.6 Primary school and above14402262.9National Public Health Insurance Coverage Yes1131.42365.78.23/ 0.004* No2468.61234.3Parity None1028.61028.62.88/0.23 11028.61645.7 2 and above1542.9925.7Abortions None2777.13085.7-/- 1617.138.6 2 or more25.725.7Living children None1131.41131.41.34/0.51 11131.41542.9 2 and above1337.1925.7Infant sex Girl925.81542.91.585/0.208 Boy2674.22057.1Placental expulsion time (min) ≤ 15 min3451.53248.51.061/0.303 ≤30 min125375Placental weight (g) 400–500 g562.5337.56.976/0.073 501–600 g1866.7933.3 601–700 g733.31466.7 ≥ 701535.7964.3Birth weight: SGA: ≤10thP18756259.130/ 0.003Non-SGA: >10thP17372963χ²: Chi-square test. Birth weight percentiles were calculated using the Fetal Biometry 3.0 calculator (Perinatology.com). Infants with birth weight ≤ 10th percentile for gestational age were classified as small for gestational age (SGA), and those with birth weight > 10th percentile as non-SGAp < 0.05

Table 2. Comparison of gestational age at delivery between smoking and non-smoking mothers (n = 70)Independent T- testGroups N MeanStd. Deviationtdf p Gestational Age at DeliverySmokers3536.22.5−2.58768 0.012** NonSmokers3537.73Independent T test(*) Indicates significant differences between groups^**^p ≤ 0.05

Comparison of gestational age at delivery revealed a statistically significant difference between smoking and non-smoking mothers (Table 2). The mean gestational age was significantly lower in the smoking group compared with the non-smoking group (36.2 ± 2.5 vs. 37.7 ± 3.0 weeks, respectively; t = − 2.587, df = 68, p = 0.012).

Table 3. Mean ± SD values of term placenta IGF-1, leptin, and HPL immunohistochemistry scores results between birth weight groups (SGA ≤ 10th percentile; Non-SGA > 10th percentile) (n = 70)Independent T- testGroups N MeanStd. Deviationtdf p IGF − 1SGA:≤10thP24103.572.8−2.87468 0.005** Non-SGA: >10thP46159.379.3LeptinSGA:≤10thP24283.193.83.21168 0.002** Non-SGA: >10thP46202.9101.9HPLSGA:≤10thP24149.272.8−2.86668 0.006** Non-SGA: >10thP46218.679.2Independent T test(*) Indicates significant differences between groups^**^p ≤ 0.05

Birth weight percentiles were calculated using gestational age–adjusted reference standards, and infants were categorized as SGA (≤ 10th percentile) or non-SGA (> 10th percentile).

When infants were classified according to birth weight percentiles, significant differences in placental hormone expression were observed between the SGA (≤ 10th percentile) and non-SGA (> 10th percentile) groups. Placental IGF-1 immunohistochemical scores were significantly lower in the SGA group compared to the non-SGA group (103.5 ± 72.8 vs. 159.3 ± 79.3; t = − 2.874, p = 0.005). Similarly, placental leptin expression differed significantly between groups, with higher scores observed in the SGA group than in the non-SGA group (283.1 ± 93.8 vs. 202.9 ± 101.9; t = 3.211, p = 0.002). In addition, placental HPL expression was significantly reduced in the SGA group compared to the non-SGA group (149.2 ± 72.8 vs. 218.6 ± 79.2; t = − 2.866, p = 0.006) (Table 3).

To further investigate the combined effects of maternal smoking status and fetal growth status on placental hormone expression, participants were stratified into four groups based on smoking status (smoker vs. non-smoker) and birth weight percentile: smoker–SGA (≤ 10th percentile), smoker–non-SGA (> 10th percentile), non-smoker–SGA, and non-smoker–non-SGA. This stratification allowed for the assessment of both independent and combined effects of smoking exposure and fetal growth status on placental IGF-1, leptin, and HPL expression. One-way ANOVA demonstrated significant differences in placental IGF-1, leptin, and HPL immunohistochemical scores among the four study groups (p < 0.001 for all markers) (Table 4).

For all three placental hormones, the lowest expression levels were observed in the smoker–SGA group, whereas the highest levels were detected in the non-smoker–non-SGA group.

Table 4. Comparison of placental IGF-1, leptin, and HPL immunohistochemical scores among combined smoking status and fetal growth groups (SGA ≤ 10th percentile; non-SGA > 10th percentile) (n = 70)AnovaGroups N MeanStd. DeviationF p IGF − 1Smoker-SGA1864.824.7183.07 0.000* Smoker-non-SGA1763.319.1Non-Smoker-SGA6219.527.7Non-Smoker-non-SGA29215.631.9LeptinSmoker-SGA1833049.3153.33 0.000* Smoker-non-SGA17326.947.5Non-Smoker-SGA6142.524.5Non-Smoker-non-SGA29130.124.1HPLSmoker-SGA18100.617.1188.33 0.000* Smoker-non-SGA1799.222.2Non-Smoker-SGA629561.5Non-Smoker-non-SGA29288.638.6Anova test(*) Indicates significant differences between groups^**^p ≤ 0.05

Post hoc Tukey multiple comparison analysis revealed that both smoker–SGA and smoker–non-SGA groups had significantly lower IGF-1, leptin, and HPL scores compared with their respective non-smoker counterparts (p < 0.001 for all comparisons) (Table 5).

Table 5. Post hoc multiple comparisons of IGF-1, leptin, and HPL immunohistochemical scores across groups (n = 70)Multiple comparisons between groupsP ValuePosthoc/TukeyIGF − 1LeptinHPLSmoker-SGA vs. Non-Smoker-SGA 0.000*

0.000*

0.000* Smoker-SGA vs. Non-Smoker-non-SGA 0.000*

0.000*

0.000* Smoker-non-SGA vs. Non-Smoker-SGA 0.000*

0.000*

0.000* Smoker- non-SGA vs. Non-Smoker- non-SGA 0.000*

0.000*

0.000* Smoker-SGA vs. Smoker- non-SGA0.9980.9950.999Non-Smoker- SGA vs. Non-Smoker- non-SGA0.9890.8890.974() Indicates significant differences between groups**p ≤ 0.05

One-way ANOVA revealed statistically significant differences in placental IGF-1, leptin, and human placental lactogen (HPL) immunohistochemical scores among groups stratified by combined maternal smoking status and fetal growth categories (SGA ≤ 10th percentile and non-SGA > 10th percentile) (Table 4). Significant group effects were observed for all three markers (IGF-1: F = 183.07, p < 0.001; leptin: F = 153.33, p < 0.001; HPL: F = 188.33, p < 0.001).

Placental IGF-1 immunoreactivity was markedly reduced in both the smoker–SGA and smoker–non-SGA groups compared with their non-smoker counterparts, whereas substantially higher IGF-1 scores were observed in the non-smoker–SGA and non-smoker–non-SGA groups. These findings indicate a strong suppressive effect of maternal smoking on placental IGF-1 expression, which is largely independent of fetal growth category.

In contrast, leptin immunoreactivity was significantly elevated in the placentas of mothers who smoked in both the SGA and non-SGA groups, while the non-smoker groups exhibited considerably lower leptin scores. This pattern suggests that maternal smoking is associated with the upregulation of placental leptin expression, potentially reflecting a compensatory response to smoking-induced placental stress or hypoxia.

Similarly, HPL immunoreactivity was significantly lower in smokers regardless of fetal growth status, whereas non-smokers, particularly the non-smoker–non-SGA group, demonstrated markedly higher HPL expression. These results imply that maternal smoking adversely affects the placental endocrine capacity and fetal metabolic support mechanisms.

Post hoc multiple comparisons using the Tukey test confirmed that the differences in IGF-1, leptin, and HPL immunohistochemical scores between the smoker and non-smoker groups were statistically significant (p < 0.001 for all comparisons) (Table 5). Significant differences were consistently observed between the smoker–SGA and non-smoker–SGA, smoker–SGA and non-smoker–non-SGA, smoker–non-SGA and non-smoker–SGA, and smoker–non-SGA and non-smoker–non-SGA groups. Collectively, these findings demonstrate that maternal smoking status is the predominant determinant of placental IGF-1, leptin, and HPL expression, exceeding the effect of fetal growth classification.

Linear regression analyses

Linear regression models indicated that maternal smoking had a significant negative effect on birth weight, with infants of smoking mothers weighing, on average, 486 g less, explaining 20.8% of the variance (B = − 0.486, p < 0.001). In contrast, maternal age did not have a significant effect on birth weight (p = 0.463) and explained only 0.8% of the variance. A positive association was observed between maternal education level and birth weight, suggesting an estimated increase of approximately 437 g with higher education levels; however, this relationship was of borderline statistical significance (B = 0.437, p = 0.054). Although social security status showed a positive association with birth weight, this effect did not reach statistical significance (B = 0.163, p = 0.151).

A strong and significant association was also identified between gestational age and birth weight, with longer gestational duration being associated with higher birth weight (B = 0.729, p = 0.020), accounting for 13.2% of the variance. Collectively, these findings demonstrate that maternal smoking is the most influential determinant of reduced birth weight in this cohort, exceeding the effects of maternal age, education, and socioeconomic indicators, and underscore the critical role of gestational duration in fetal growth and perinatal health outcomes (Table 1).

Breastfeeding practices

When participants were contacted by phone on the 10th day postpartum to assess characteristics related to breastfeeding practices, it was found that the initiation of breastfeeding within the first hour after birth was lower in smoking mothers (57.1%, n = 20) compared to non-smoking mothers (94.3%, n = 33). Furthermore, the breastfeeding rate was lower in smoking mothers (62.9%, n = 22), while the rate of mothers feeding their babies with both breast milk and formula was higher (37.1%, n = 13). Additionally, 91.4% (n = 64) had initially given colostrum, 75.7% (n = 53) had breastfed within the first hour, 77.1% (n = 54) were currently exclusively breastfeeding, and among those who provided supplementary feeding in addition to breast milk, 12.9% (n = 9) reported doing so because of infant-related problems (Table 6).

Table 6. Breastfeeding practices process -related characteristics/variables of participants (n = 70)VariableSmokers (n = 35)Nonsmokers (n = 35)Totalx^2^/pFrequency (n)%Frequency (n)%Frequency (n)%The first food you give to your child orally Colostrum3188.63394.36491.4-/- Formula milk411.425.768.6Time to initiate feeding after birth Within the first hour2057.13394.35375.7 13.130/0.000* Within the first two hours1542.925.71724.3The breastfeeding status of the baby Only breast milk2262.93291.45477.1 8.102/0.004* Breast milk and formula1337.138.61622.9Reasons for Supplementary Feeding (n = 16) Insufficient milk supply38.60034.3-/- Baby refuses to breastfeed38.60034.3 Maternal health issues12.90011.4 Infant health issues617.138.6912.9**p* < 0.05 × 2: Chi-square test

Immunohistochemical findings

In the smoking group, weak IGF-1 immunoreactivity (Fig. 1B1, B2), moderate leptin immunoreactivity (Fig. 2B1, B2), and weak HPL immunoreactivity (Fig. 3B1, B2) were observed in both villous stromal cells and syncytiotrophoblasts.

Fig. 1IGF-1 immunostainings in the placenta villi of smoker and nonsmoker groups. Nonsmoker A, Smoker B. ➠:Villous syncytiotrophoblast cells, →: villous stromal cells. (A1,B1) X100, (A2,B2) X400

Fig. 2. Leptin immunostainings in the term placenta villi of smoker and nonsmoker groups. Nonsmoker A, Smoker B. ➠:Villous syncytiotrophoblast cells, →: villous stromal cell. (A1,B1) X100, (A2,B2) X400

Fig. 3HPL immunostainings in the term placenta villi of Nonsmoker and smoker groups. Nonsmoker A, Smoker B. ➠:Villous syncytiotrophoblast cells, →: villous stromal cell. (A1,B1) X100, (A2,B2) X400

In the non-smoking group, moderate to strong IGF-1 immunoreactivity was observed in villous stromal cells, whereas weak to moderate IGF-1 immunoreactivity was observed in syncytiotrophoblast cells (Fig. 1A1, A2). Leptin immunoreactivity was strong in villous stromal cells and moderate in syncytiotrophoblasts (Fig. 2A1, A2). HPL immunostaining was strong in syncytiotrophoblast cells but weak in villous stromal cells (Fig. 3A1, A2).

The staining intensity of IGF-1, leptin, and HPL was significantly higher in the non-smoking group than in the smoking group (p < 0.05) (Table 7; Fig. 4).

Table 7. Mean ± SD values of term placenta IGF-1, leptin, and HPL immunohistochemistry staining results between smoker and non-smoker pregnant mothers (n = 70)Independent T- testHScoreGroups N MeanStd. Deviationtdf p IGF − 1Smokers3564.121.87−24.08768*< 0.001Nonsmokers35216.230.9LeptinSmokers35132.224.221.66368< 0.001Nonsmokers35328.547.7HPLSmokers3599.919.5−23.76368< 0.001Nonsmokers35289.742.3Independent T test() Indicates significant differences between groups^^p < 0.05

Fig. 4H-SCORE statistical analysis of IGF-1, leptin, HPL, immunoreactivity intensities for nonsmoker and smoker pregnant women. Values have been presented as mean ± SD

Comparison of placental immunohistochemical staining scores demonstrated significant differences in IGF-1, leptin, and human placental lactogen (HPL) expression between smoking and non-smoking pregnant mothers (Table 7).

Placental IGF-1 immunoreactivity was significantly lower in the smoking group compared with the non-smoking group (64.1 ± 21.87 vs. 216.2 ± 30.9, respectively; t = − 24.087, df = 68, p < 0.001).

Similarly, placental leptin immunohistochemical scores were significantly reduced in smokers compared with non-smokers (132.2 ± 24.2 vs. 328.5 ± 47.7, respectively; t = 21.663, df = 68, p < 0.001).

In addition, placental HPL immunoreactivity was markedly lower in the smoking group than in the non-smoking group (99.9 ± 19.5 vs. 289.7 ± 42.3, respectively; t = − 23.763, df = 68, p < 0.001).

Umbilical cord blood biochemistry findings

A statistically significant difference was identified between the smoking status of pregnant mothers and their mean ± SD for leptin and HPL biochemistry findings. Umbilical cord blood analysis demonstrated significantly lower leptin and HPL levels in infants of smoking mothers than in those of non-smokers (p < 0.001 for both) (Table 8).

Table 8. Leptin and HPL biochemistry findings of smoker and nonsmoker pregnant mothers (n = 70)Biochemistry FindingsGroups N MeanStd. Deviationtdf p Leptin(pg/ml)Smokers3554.611.526.76636.518*< 0.001***Nonsmokers35107.82.2HPL(pg/ml)Smokers350.90.6−24.63368 < 0.001

---

Nonsmokers355.80.9Independent T test(*) Indicates significant differences between groups^**^p < 0.05

Umbilical cord blood biochemical analysis revealed significant differences in leptin and human placental lactogen (HPL) levels between infants born to smoking and non-smoking mothers (Table 8). Mean umbilical cord blood leptin levels were significantly lower in the smoking group compared with the non-smoking group (54.6 ± 11.5 pg/mL vs. 107.8 ± 2.2 pg/mL, respectively; t = 26.766, df = 68, p < 0.001).

Similarly, umbilical cord blood HPL levels were markedly reduced in the smoking group compared with the non-smoking group (0.9 ± 0.6 pg/mL vs. 5.8 ± 0.9 pg/mL, respectively; t = − 24.633, df = 68, p < 0.001).

These findings indicate that maternal smoking during pregnancy is associated with significantly lower leptin and HPL levels in umbilical cord blood.

Discussion

In the present study, maternal smoking during pregnancy was found to be strongly associated with adverse fetal growth outcomes and altered placental endocrine function. Infants of mothers who smoked were considerably more likely to be classified as small for gestational age (SGA, ≤ 10th percentile) than those born to non-smoking mothers, underscoring the well-established link between prenatal smoking and impaired fetal growth. Importantly, by stratifying participants according to both smoking status and gestational age–adjusted birth weight percentiles, and by concurrently evaluating early breastfeeding practices, this study provided a more nuanced understanding of how smoking-related placental hormone dysregulation interacts with fetal growth status and postnatal feeding outcomes.

Birth weight is considered one of the primary indicators of fetal health and intrauterine development and is closely associated with placental hormone levels. Hormones produced by the placenta, such as insulin-like growth factor-1 (IGF-1), leptin, and human placental lactogen (HPL), have been shown in numerous studies to play key regulatory roles in fetal growth. Leptin contributes to fetal energy balance and growth, whereas IGF-1 supports tissue development by promoting cellular proliferation and differentiation. In contrast, HPL enhances fetal growth by modulating maternal metabolism to increase glucose availability to the fetus and by exerting prolactin-like effects. Alterations in the levels of these hormones have been consistently associated with fetal growth restriction [32]. Therefore, assessment of placental hormone expression is essential for understanding the biological mechanisms underlying impaired fetal growth.

By stratifying participants according to both maternal smoking status and gestational age–adjusted birth weight percentiles, the present study provides further insight into the interaction between prenatal smoking exposure and fetal growth in relation to placental endocrine function. The markedly reduced placental IGF-1 and HPL expression observed in the smoker–SGA group, together with the concomitant increase in placental leptin expression, suggests that maternal smoking may exacerbate placental endocrine dysfunction, particularly in pregnancies complicated by impaired fetal growth. The finding that lower placental IGF-1 expression is associated with SGA status is consistent with previous reports demonstrating reduced circulating IGF-I concentrations in neonates born to mothers who smoked during pregnancy and a positive association between IGF-I levels, birth weight, and neonatal length [33]. Given the central role of IGF-1 in placental nutrient transfer and fetal growth regulation, smoking-related suppression of IGF-1 may represent a key mechanistic pathway linking maternal smoking to fetal growth restriction. The observed alterations in placental leptin expression according to fetal growth status are also in agreement with existing evidence indicating that leptin is actively synthesized by trophoblast cells and exerts important endocrine and paracrine functions within the placenta. Increased placental leptin expression in growth-restricted pregnancies has been proposed to reflect a compensatory response to placental stress, hypoxia, or impaired nutrient transport, rather than an indicator of enhanced fetal growth potential [23].

In line with this, increased placental leptin transcript abundance has been reported in pregnancies complicated by intrauterine growth restriction (IUGR) and small for gestational age (SGA), supporting the association between leptin dysregulation and impaired fetal growth.

Similarly, human placental lactogen (HPL) is a key placental hormone whose secretion reflects placental mass and metabolic adaptation during gestation. Although the relationship between HPL and birth weight is complex, several studies have demonstrated significant associations between HPL levels, placental size, and gestational age–adjusted fetal growth, supporting the biological plausibility of HPL involvement in the regulation of fetal growth and nutrient supply [34, 35]. The reduced placental HPL expression observed in smoking mothers in the present study may therefore contribute to impaired fetal growth and adverse neonatal outcomes.

Most studies have focused on the protective effects of breast milk, and it is well established that these effects are modulated by environmental, cultural, and socioeconomic factors. The protective impact of breastfeeding is particularly pronounced in populations characterized by high infant mortality, poor sanitation, inadequate nutrition, and a high prevalence of tobacco exposure [36]. However, maternal tobacco smoking has been shown to adversely affect breast milk quality and composition, including alterations in taste, antioxidant capacity, long-chain polyunsaturated fatty acids, cytokine profiles, immunoglobulin content, and increased exposure to heavy metals [37]. Previous studies have demonstrated that exposure to tobacco smoke constituents through breast milk may negatively influence infant development and increase susceptibility to adverse health outcomes [38–40], including an association between heavy maternal smoking during pregnancy and lactation and an elevated risk of early-onset leukemia [41]. In addition, infants breastfed by smoking mothers have been reported to have a higher risk of colic [42], respiratory infections, and other health complications, potentially due to the transfer of nicotine and other toxic substances into colostrum and breast milk during critical periods of immune system maturation [43–49]. Smoking has also been associated with delayed lactogenesis II, which may interfere with early breastfeeding initiation and adequate colostrum intake [42]. Consistent with these observations, our study found that colostrum feeding rates were lower among smoking mothers (88.6%, n = 31) compared with non-smoking mothers (94.3%, n = 33), whereas formula supplementation was more frequent in the smoking group (11.4% vs. 5.7%). These findings suggest that maternal smoking may impair early lactational performance, potentially through reduced milk secretion and altered milk composition, thereby contributing to suboptimal breastfeeding practices during the early postnatal period and further compounding the risk of adverse outcomes in growth-restricted (SGA) infants.

When reviewing the literature on the timing of breastfeeding initiation, the World Health Organization (WHO) and UNICEF recommend initiating breastfeeding within the first hour after birth [50]. Early initiation of breastfeeding has been shown to play a critical role in reducing the risk of neonatal hypothermia during the immediate postnatal period [51], and breastfeeding support interventions have been demonstrated to positively influence the timing of initiation [52]. Recent studies, including a 2024 investigation in preterm infants, have further reported that early provision of mothers’ milk shortens the time to enteral feeding initiation and improves feeding-related outcomes [53, 54]. Consistent with these recommendations, our study demonstrated a marked delay in breastfeeding initiation among smoking mothers. Initiation of breastfeeding within the first hour postpartum was observed in only 57.1% (n = 20) of smokers, whereas 42.9% (n = 15) initiated breastfeeding during the second hour. In contrast, 94.3% (n = 33) of non-smoking mothers initiated breastfeeding within the first hour, and only 5.7% (n = 2) during the second hour. These findings indicate that maternal smoking during pregnancy is associated with delayed initiation of breastfeeding in the early postnatal period. Taken together, the delayed onset of breastfeeding observed in smoking mothers may be partly explained by smoking-related placental endocrine dysfunction, particularly the reduced expression of HPL and leptin, hormones that are known to influence maternal metabolic adaptation, lactogenesis, and the transition to effective milk secretion in the early postpartum period. Such endocrine disturbances may further compromise postnatal adaptation, especially in infants with impaired fetal growth (SGA), thereby linking prenatal smoking exposure, placental dysfunction, and suboptimal early feeding practices within a unified biological framework.

IGF-1 affects tissue growth and metabolism and plays a critical role in fetal growth and development [21, 55]. In a study conducted by Fleisch et al. in 2017, umbilical cord blood IGF-1 levels were compared among pregnant women who continued smoking during pregnancy, those with a history of smoking, and never-smokers. The lowest IGF-1 concentrations in cord blood were observed in women who continued to smoke throughout pregnancy [31]. Consistent with these findings, the present study demonstrated significantly reduced placental IGF-1 immunoreactivity in mothers who smoked during pregnancy.

Human placental lactogen (HPL) is exclusively produced during pregnancy, and its concentration in maternal serum increases in parallel with fetal and placental development. Although reduced HPL levels have been observed in women with higher body mass index (BMI), the extent to which prenatal health behaviors, including smoking, modulate HPL levels and influence infant birth weight remains incompletely understood [32]. Previous studies have reported an association between maternal smoking and reduced HPL levels. Carl et al. evaluated serial maternal serum HPL measurements during pregnancy and demonstrated that impaired fetal growth was associated with altered HPL trajectories, highlighting the relevance of HPL as a marker of placental and fetal development [56]. Similarly, Spira et al. reported that maternal smoking was associated with both reduced fetal weight and lower HPL levels [57]. Mochizuki et al. further demonstrated that heavy maternal smoking was linked to growth deficiency and decreased urinary estriol and HPL levels compared with non-smokers [58]. Consistent with these findings, the present study demonstrated significantly reduced placental HPL immunoreactivity and lower umbilical cord blood HPL levels in smoking mothers compared with non-smoking mothers, supporting the concept that prenatal tobacco exposure adversely affects placental endocrine function and compromises metabolic and hormonal support to the fetus, thereby contributing to impaired fetal growth and an increased risk of small-for-gestational-age (SGA) outcomes.

Leptin is a peptide hormone produced primarily by white adipose tissue and the placenta and plays a central role in the regulation of energy homeostasis, fetal growth, and metabolic adaptation during pregnancy [59–61]. Beyond its central effects on appetite and energy expenditure, leptin synthesized in the placenta exerts important endocrine and paracrine functions by supporting trophoblast proliferation, survival, and nutrient transport through the activation of intracellular signaling pathways such as JAK/STAT, MAPK, and PI3K [62–67]. Several studies have highlighted the association between placental leptin expression and fetal growth. Placental leptin has been proposed to reflect placental stress or adaptive responses in pregnancies complicated by impaired fetal growth, rather than directly promoting increased fetal size [68]. Pardo et al. reported that cord blood leptin levels were primarily related to gestational age and were not significantly altered by maternal smoking in term AGA newborns [68]. In contrast, Mantzoros et al. suggested that altered leptin concentrations in smoking pregnancies may be associated with reduced fetal growth [69]. In the present study, both placental leptin immunoreactivity and umbilical cord blood leptin levels were significantly lower in smoking mothers compared with non-smoking mothers. Importantly, when stratified according to gestational age–adjusted birth weight percentiles, leptin expression was closely associated with fetal growth category, supporting a link between smoking-related placental leptin dysregulation and small-for-gestational-age (SGA) status. The higher placental and cord blood leptin levels observed in non-smoking mothers are consistent with more favorable fetal growth patterns, whereas reduced leptin expression in smoking mothers likely reflects compromised placental endocrine adaptation in growth-restricted (SGA) pregnancies.

Taken together, these findings suggest that smoking-related alterations in placental leptin expression are more closely related to fetal growth status than to isolated changes in neonatal adiposity, and may represent a compensatory yet insufficient placental response to an adverse intrauterine environment.

One of the main limitations of this study is the lack of biochemical assessment of the quantity and composition of breast milk in smoking mothers. In addition, the relatively small sample size and the absence of direct measurements of nicotine and its metabolites in breast milk limit the interpretation and generalizability of the findings. As this study was conducted at a single center, future multicenter investigations with larger cohorts are warranted to improve external validity. Furthermore, although it is well established that female infants tend to have slightly lower birth weights than male infants, sex-specific differences in gestational age–adjusted fetal growth (SGA vs. non-SGA) were not specifically examined and should be addressed in future studies.

Another important limitation is the lack of pregnancy-specific tobacco use data in the 2022 statistics published by the Turkish Statistical Institute (TURKSTAT), despite strong evidence demonstrating the adverse effects of smoking during pregnancy and lactation. This gap underscores the need for more comprehensive, large-scale epidemiological surveillance of maternal smoking. Finally, experimental studies, including animal models and detailed prenatal histopathological and biochemical evaluation of placental and mammary tissues, may further elucidate the mechanistic pathways linking maternal smoking, placental endocrine dysfunction, impaired lactogenesis, and fetal growth restriction.

Conclusion

Antenatal smoking is inversely associated with fetal growth and is known to exert detrimental effects on placental structure and endocrine function. In the present study, lower levels of IGF-1, leptin, and human placental lactogen (HPL) were observed in both placental tissue and umbilical cord blood of mothers who smoked during pregnancy compared with non-smoking mothers. In addition, placental immunostaining intensities of IGF-1, leptin, and HPL were significantly higher in the non-smoking group, indicating a more favorable placental endocrine profile.

With respect to early feeding outcomes, smoking mothers exhibited lower rates of breastfeeding initiation within the first hour after birth, reduced exclusive breastfeeding rates, and a higher prevalence of mixed feeding practices. Although direct causal pathways between placental hormone alterations and breastfeeding behavior were not specifically examined, the coexistence of smoking-related placental endocrine dysfunction and suboptimal early feeding patterns suggests that prenatal tobacco exposure may adversely affect both intrauterine adaptation and early postnatal metabolic and lactational processes.

Overall, these findings indicate that maternal smoking during pregnancy is associated with impaired placental endocrine function, an increased risk of small-for-gestational-age (SGA) status, and less favorable early breastfeeding practices. Together, these results underscore the critical importance of effective smoking cessation interventions during pregnancy, not only to improve fetal growth and placental function but also to promote optimal early postnatal nutrition and long-term infant health outcomes.