Changes in Maternal Serum Leptin Levels during Pregnancy and after Labor in Preeclampsia , and Its Correlation to Neonatal Cord Leptin

Objective: The aim of this study is to determine changes in maternal serum leptin level during pregnancy and after labor in preeclamptic patients compared to healthy pregnant women. Furthermore, to investigate whether maternal serum leptin levels are correlated to the clinical characteristics and laboratory parameters of the study participants, and the possible correlation between maternal and neonatal leptin levels. Subjects and Methods: In this case control study, a total number of fifty five pregnant women in third trimester of pregnancy (≥28 weeks) were recruited. All of them were of the same age, body mass index, and gestational age. After a detailed obstetrical and medical history, they were divided into 2 groups. Group (A) 30 pregnant women with preeclampsia, and Group (B) 25 normotensive pregnant women. Results: During pregnancy, maternal serum leptin levels were significantly higher 41.0 ± 9.78 ng/ml in preeclamptic group compared to control group 24.6 ± 3.64 ng/ml (p = 0.007). After labor, it decreased significantly in both groups to 15.3 ± 3.19, and 11.2 ± 2.68 ng/ml respectively (p = 0.001, 0.002). In group (A) there were significant positive correlations between maternal serum leptin and diastolic blood pressure (rr = 0.419, PP = 0.021), total cholesterol (rr = 0.383, PP = 0.026), and uric acid (rr = 0.424, PP = 0.012) compared to controls, and no significant correlations were found between maternal serum leptin and body mass index, neonatal birth weight or cord leptin level in both groups. Conclusion: Maternal serum leptin is significantly increased in preeclamptic patients compared with normal pregnant women independent of body mass index. There is strong evidence that placenta, rather than maternal adipose tissue is responsible for that. In addition, maternal serum leptin levels were found to correlate positively with diastolic blood pressure, uric acid, and total cholesterol, but not correlated with body mass index, cord blood leptin and birth weight. How to cite this paper: Youssry, M.A., Gabreel, M.A. and Patel, T.A. (2016) Changes in Maternal Serum Leptin Levels during Pregnancy and after Labor in Preeclampsia, and Its Correlation to Neonatal Cord Leptin. Open Journal of Obstetrics and Gynecology, 6, 588-600. http://dx.doi.org/10.4236/ojog.2016.610074 Received: July 30, 2016 Accepted: August 27, 2016 Published: August 30, 2016 Copyright © 2016 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access M. A. Youssry et al.


Introduction
Leptin is a homeostatic hormone, produced by the obesity (Lep°b) gene on chromosome 7, synthesized and secreted by adipocytes [1] [2], and other organs particularly the placenta which may contribute to leptin levels during pregnancy [2] [3].
This anti-obesity hormone mainly acts by binding to specific central and peripheral receptors in the hypothalamus, adipose tissue, liver and pancreatic beta cells.Leptin stimulates a negative energy balance by increasing energy expenditure and reducing food intake thus controlling body weight [4].It also modulates glucose metabolism via increasing insulin sensitivity, activates the sympathetic nervous system, and it has been implicated in the control of the reproductive functions particularly embryonic development [5].Furthermore, leptin is involved in the regulation of immune responses and inflammations [6], since it is considered as a pro-inflammatory cytokine that belongs to the type I cytokine superfamily [7].
However, the regulation of maternal leptin during pregnancy is difficult [8].Serum leptin concentrations double during the course of a normal pregnancy and decrease just before labor [9].Trophoblasts are obligated for the considerably increased leptin levels during the first two trimesters of normal pregnancies [1], subsequently necessary substrates for placental and fetal growth are provided by mobilizing maternal fat stores [8].Fluctuation of leptin levels in pregnancy correlates with estradiol and human chorionic gonadotropin (HCG) levels in maternal circulation.This situation suggests that there is a dynamic relation between fluctuating levels of reproductive hormones oscillated in pregnancy and leptin [9].
Moreover, another important source of leptin could be fetal adiposity, and fetal leptin levels are strongly correlated to birth weight [10].However, leptin receptors possibly are participating in the endocrine control of human pregnancy; these receptors are abundant in the uterine endometrium, trophoblast, and the fetus [11].Leptin levels show further increase in complicated pregnancies as preeclampsia, and intrauterine growth restriction (IUGR) [12] [13].In such conditions there is reduction in placental circulation, therefore leptin acts as a gestational hormone for regulation of placental and fetal growth, and could be a marker for severity of preeclampsia and IUGR [14].
Preeclampsia refers to the new onset of hypertension and either proteinuria or end organ dysfunction after 20 weeks of gestation in a previously healthy woman [15].It is one of the major causes of maternal and perinatal morbidity and mortality [16].The etiology and pathogenesis are not completely understood, but the overproduction of cytokines, and chemokines may trigger a generalized endothelial dysfunction which is characteristic of preeclampsia [17].In addition to angiogenic and anti-angiogenic imbalance which has also been implicated in the development of disease [18].Adipose tissue also acts as endocrine tissue producing a wide range of cytokines and chemokines such as leptin, which play a role in normal pregnancy, as well as in preeclampsia [19].More specifically, it has not yet been established whether leptin levels are simply a marker of fat mobilization, or whether there is a disrupted relationship between the serum leptin and adiposity in preeclampsia [20].
Several studies demonstrated that serum leptin levels increase in preeclamptic patients [19] [21] [22], while others showed unchanged or decreased leptin levels [23] [24].Over expression of leptin gene was demonstrated in preeclamptic women compared with normal pregnancies [25].Although there is placental hypoperfusion in preeclampsia, only one-third of neonates born have IUGR due to increased availability of nutrients in placenta [26].
This aim of this study is to determine changes in maternal serum leptin level during pregnancy and after labor in preeclamptic patients compared to healthy pregnant women.Furthermore, to investigate whether maternal serum leptin levels are correlated to the clinical characteristics and laboratory parameters of the study participants, and the possible correlation between maternal and neonatal leptin levels.

Subjects and Methods
This case control study was conducted at the Department of Obstetrics and Gynecology of Ibn Sina College General Hospital, Saudi Arabia, from November 2014 till April 2016.A total number of fifty five pregnant women attending in the third trimester of pregnancy (≥28 weeks) were recruited in the study.
This study was approved by the Hospital Research Ethics Committee and has been performed in accordance with the ethical standards as in Declaration of Helsinki (1964) and its later amendments, and a written informed consent was obtained from each participant.All participants were of the same age, body mass index, and gestational age.
They were briefed about the nature of the study, and after a detailed obstetrical and medical history, they were divided into 2 groups.Group (A) 30 pregnant women with preeclampsia, and Group (B) 25 normotensive pregnant women.
Preeclampsia refers to the new onset of hypertension (systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg on two occasions four hours apart and proteinuria ≥ 300 mg in a 24 h urine collection or at least on dipstick measurements ≥ +1, or new onset of end organ dysfunction in the absence of proteinuria (American College Of Obstetrics and Gynecology 2013) [15].Blood pressure was measured with the arm at the level of the heart using an appropriately sized cuff (length of 1.5 times the circumference of the arm) with a mercury sphygmomanometer after 10 minutes rest period.

Inclusion criteria
Group (A): Thirty preeclamptic women with singleton pregnancies in their third trimester.
Group (B): Twenty five normal healthy women with singleton pregnancies in their third trimester as controls.

Exclusion criteria
• Pre-existing chronic hypertension.
• Gestational diabetes or Pre-existing diabetes.
• Chronic renal or liver disease.
Maternal fasting blood samples were obtained from the antecubital vein from all subjects at antenatal visit after the preeclampsia diagnosis was confirmed for group (A), and controls for group (B).Complete blood count, complete urine analysis, urea, creatinine, uric acid, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol, triglycerides, and serum leptin were measured.In the post-partum stage, during the first 24 hours after labor another blood sample was taken from all the pregnant women from antecubital region for serum leptin.Umbilical vein samples were taken immediately following clamping of the cord before separation of the placenta, then serum was separated and serum leptin level was measured.In addition to neonatal weights, head circumference, and other measurements.
The blood samples were collected under strict aseptic measures.Each sample was labeled with patient's name and identification number.Serum Leptin levels were measured by enzyme-linked immunosorbent assay (ELISA) method using DRG leptin (Sandwich) ELISA Kit (EIA-2395) purchased from DRG instruments (GmbH, Germany) following the manufacturer's recommendations.

Statistical Analysis
The data was collected and entered into the personal computer.Statistical analysis was done using Statistical Package for Social Sciences (SPSS/version 20) software.Arithmetic mean, standard deviation, for categorized variables Chi-square test was used, while for numerical data t-test was used to compare two groups.The correlation was analyzed by Spearman correlation coefficients.The level of significance was 0.05.
Sample size calculation: A minimum two groups sample sizes of 30 and 25 patients achieve 90% power to detect a difference of 20% between the null hypotheses of both groups.It was calculated by using Med Calc statistical software assuming area under ROC to be 0.80, an alpha of 0.05 and power of study 90.0%.

Results
The participants of this study were attending the obstetrics and gynecology department of our hospital, where an average of a thousand deliveries took place per year.Thirty of them were diagnosed as preeclamptics (group A), and twenty five normal pregnant women (group B) as controls.The demographic characteristics of the study participants are illustrated in (Table 1).There were no statistically significant differences between the two groups for maternal age, parity, gestational age, body mass index (BMI), and mode of delivery.The mean systolic and diastolic blood pressure in preeclamptic women showed a significant increase compared to controls (153.4 ± 10.3, 98.6 ± 7.4 vs 108.6 ± 12.03, 72.3 ± 7.96 respectively, p = 0.001*, 0.001*) (Table 1).
The clinical characteristics of the newborns are described in (Table 3).There were no statistically significant differences between the neonates in the two groups regarding birth weight, sex, length, head circumference, and Apgar score.It is also evident that,     no statistically significant difference was detected in umbilical cord leptin between the two groups (p = 0.078) in (Table 3).In group (A) there were significant positive correlations between maternal serum leptin and diastolic blood pressure (r = 0.419, p = 0.021*), total cholesterol (r = 0.383, p = 0.026*), and uric acid (r = 0.424, p = 0.012*) compared to controls, while no significant correlations were found between maternal serum leptin and BMI, neonatal birth weight, or cord leptin level in both groups (Table 4).

Discussion
Leptin, is mainly secreted by the white adipose tissue, and recently proved that this hormone is also secreted by the placental trophoblast cells into the maternal circulation in considerable amounts [2].Longitudinal studies have documented that leptin levels increase with increasing gestational age [27] [28], or remain stable between the second and third trimester [29].Mise et al. reported for the first time a significant increase in serum leptin levels in preeclampsia particularly in severely preeclampsia.They documented that, increase in placental leptin messenger RNA expression was proportional to increase in serum leptin levels in preeclampsia, and serum leptin decreased following delivery.This refers to the fact that an increase in serum leptin levels in preeclamptic women is related to placental production [3].
In our study, group (A) and (B) matched well regarding maternal age, parity, BMI, and gestational age.The results of our study showed that, serum leptin level increased with increased blood pressure, as there was a significant increase of serum leptin in preeclamptic women compared to normotensive pregnant women (41.0 ± 9.78 vs 24.6 ± 3.64 ng/ml respectively, p = 0.007*), which is in agreement with many other previous studies [19] [21] [22], and maternal leptin had a significant positive correlation with diastolic blood pressure in preeclamptic patients (r = 0.419, p = 0.021*), but not in healthy pregnant women.Placental ischemia explains rapid increase in leptin concentration during third trimester in preeclampsia.Placental hypoperfusion produces local hypoxia that consequently augments leptin gene expression in the placenta [3].Also high leptin levels may be due to the haemoconcentration [30], impaired renal function, and reduced renal clearance in preeclampsia [31].Some of previous studies documented high circulating leptin level even before the clinical onset of the disease [32]- [34], which might have a prognostic role in preeclamsia.Ning et al. stated that preeclampsia risk increased by 30% with every 10 ng/ml increase in leptin concentration [34].On the contrary, other studies showed decreased or unchanged circulating leptin levels in preeclamptic patients compared to controls [23] [24].Furthermore, Masuyama et al. reported significant elevation of leptin in early and late onset preeclampsia compared to controls [22], while Molvarec et al. found no statistically significant difference in serum leptin concentration between late and early onset preeclampsia and among peeclamptic patients with mild and severe form of the disease [35].In the current study, it is also revealed that leptin decreased significantly in the early post partum period in group (A) (15.3 ± 3.19, p = 0.001*) and group (B) (11.2 ± 2.68, p = 0.002*) which is in line with the results published by Mc Carthy et al. [30], and Saylik et al. [36], but we did not detect any significant difference between both groups in postpartum maternal serum leptin levels.
Leptin has been shown to interfere with lipoprotein and is associated with atherosclerosis [37].Our findings clarified that, there was a significant increase in serum levels of triglycerides, and total cholesterol in preeclamptic women compared to controls, and leptin is also positively correlated with total cholesterol in preeclamptic patients (r = 0.383, p = 0.026) but not in controls.These findings are in agreement with results published by Bayhan et al. [38], and Ray et al. [39].
The results of previous studies on correlation between serum leptin and BMI are inconsistent [20] [40] [41], our data showed that, there was no significant correlation between serum leptin and BMI in both groups.This is because in pregnancy the BMI does not accurately show the amount of body fat because the fetus, placenta, amniotic fluid, increased plasma volume and extracellular fluid increase the maternal weight which is prominent in preeclampsia.
Many studies investigated the correlation between maternal serum leptin and baby weight at birth.In our study we did not determine any significant correlation which is in agreement with Tamas et al. [28], and Kolusarı et al. [42], while a significant negative correlation was determined in another study by Saylik et al. [36].Although maternal serum leptin levels in our study were higher than cord blood levels, but there was no significant correlation between them.This contradicts the results published by Mc Carthy et al. as they found a strong correlation between them in preeclamptic women [30].Comparing the cord blood leptin level between both groups, there was no statistically significant difference (10.6 ± 4.22 vs 12.5 ± 5.51, p = 0.078), which was supported previously by Mc Carthy et al. [30].On the other hand Odegard et al. [43], reported higher cord leptin, and Asnafi et al. [44], reported lower cord leptin in preeclamptic group compared to controls.

Study Limitation
Our study had some limitations such as; sample collection for this study requires adequate time to be completed based on inclusion and exclusion criteria.In addition, smaller sample size lacking some parameters hence our findings still need more interpretation for further study.This should be addressed by future prospective studies to verify and clarify the relationship between leptin and other uninvestigated parameters.

Conclusion
In summary, maternal serum leptin is significantly increased in preeclamptic patients compared with normal pregnant women independent of BMI.The exact mechanism is not clear, but there is strong evidence that placenta, rather than maternal adipose tissue is responsible for that.In addition, maternal serum leptin levels were found to correlate positively with diastolic blood pressure, uric acid, and total cholesterol, but not correlated with body mass index, cord blood leptin and birth weight.The results of the current study are consistent with pathophysiological roles played by leptin during pregnancy especially in hypoperfused placentae as in preeclampsia which required further studies to explore.

Figure 1 .
Figure 1.Box and whiskers plot show antepartum maternal serum leptin in group (A), and (B).(Upper whisker: represents the upper 25% of the distribution.interquartile range box: middle 50% of the data.Lower whisker represents the lower 25% of the distribution).

Figure 2 .
Figure 2. Box and whiskers plot show postpartum maternal serum leptin in group (A), and (B).(Upper whisker: represents the upper 25% of the distribution.interquartile range box: middle 50% of the data.Lower whisker represents the lower 25% of the distribution).

Table 1 .
Demographic characteristics of patients in group (A), and (B).
Note: Data presented as mean ± SD; *Significant (p < 0.05); VD: vaginal delivery; CS: cesarean section; a Based on t-test; b Based on Chi square test.

Table 2 .
Biochemical data of patients in group (A), and (B).

Table
Neonatal demographic and biochemical data in group (A), and (B).
Note: Data presented as mean± SD; *Significant (p < 0.05); a Based on t-test; b Based on Chi-square test.

Table 4 .
Correlation between maternal serum leptin and other measured variables in group (A) & (B).