Body weight gain and serum leptin levels of non-overweight and overweight/obese pregnant women

Background

Obesity is currently considered to be one of the major public health problems worldwide [1]; consequently, the number of obese women who become pregnant increases each year [2]. This condition is related with several diseases, such as hypertension, type-2 diabetes mellitus, dyslipidemia, metabolic syndrome, cardiovascular disease, and hepatic steatosis [3–5].

Obesity is also associated with adverse pregnancy outcomes such as pre-eclampsia, gestational diabetes mellitus (GDM), caesarean delivery, macrosomia, neural tube defect, thromboembolism, postpartum hemorrhage, puerperal infection, and increased risk of maternal and perinatal mortality [6–15]. According to the National Health and Nutrition Examination Survey, in the United States, more than half of pregnant women are overweight or obese [16]. The risks of most of these complications are amplified by excessive weight gain during pregnancy, which proportionally increases the degree of obesity [12].

Normal pregnancy has been characterized as a “diabetogenic” state [17]. Changes in the metabolism of carbohydrates and lipids occur during normal pregnancy to ensure a continuous supply of nutrients to the fetus despite non-continuous food intake by pregnant women [17]. These changes are progressive and promote the growth of adipose tissue during early pregnancy, which is followed by the induction of insulin resistance (IR) and lipolysis during late pregnancy [17–19].

In humans, leptin is the protein product of the ob gene and is primarily synthesized in adipose tissue. It sends an afferent signal to the central nervous system, where it acts to control food intake, insulin secretion, glucose utilization, glycogen synthesis, fatty acid metabolism, and in the regulation of adipose tissue, energy expenditure, body weight, and appetite [20,21]. During pregnancy, leptin is also synthesized by the placenta [22] and plays an important role in regulating maternal energy metabolism and the induction of physiological insulin resistance [17,22,23]. Elevated levels of this adipocytokine are also associated with GDM [22,24–26], pre-eclampsia [11,22,27], and intrauterine growth restriction (IUGR) [28,29]. Leptin levels increase progressively during pregnancy, and this increase is related to body fat and to the regulation of placental growth, transfer of nutrients, angiogenesis, and trophoblastic invasion [30]. Elevated leptin serum levels have been associated with maternal BMI [17,25]. However, these relationships are complex and have yet to be fully clarified [22,25].

In this study, we evaluated changes in serum leptin levels during pregnancy in overweight/obese and normal-weight pregnant women. In addition, we evaluated the percent and total body weight gain as possible factors influencing the levels of this adipocytokine in obese and non-obese pregnant women.

Material and Methods

ANTHROPOMETRIC EVALUATION:

The anthropometric characteristics collected for the study group included the patients’ weights and heights. Weight was measured in kilograms (within 100 grams) using a Filizola calibrated electronic balance (Personal Line 200; Filizola S.A., São Paulo, Brazil). Height was measured in meters (to 2 decimal places). Pre-pregnancy weight was obtained by patient records, and was collected during the first prenatal visit. Pre-pregnancy BMI was calculated from the ratio of body mass (kg) to squared height (m) and classified into 2 groups – non-overweight (BMI <25 kg/m2) and overweight/obese (BMI ≥25kg/m2) – based on criteria from the World Health Organization [31]. Gestational ages were verified using the parameters collected from ultrasounds performed between weeks 11 and 13 of pregnancy. If necessary, the results were used to retrospectively correct the calculations made on the first visit, which were based on last menstrual period as determined by the patient. Total weight gain was calculated by subtracting the weight measured at the last prenatal visit before birth, performed between weeks 37 and 40, from the pre-pregnancy weight reported at the first visit. Based on the total weight gain and pre-pregnancy weight of the patient, we calculated the percent weight gain.

LEPTIN MEASUREMENT:

During each study visit for prenatal care, we obtained a maternal serum sample, which was preserved in triplicate in labeled 2-ml cryogenic tubes (CRAL, SP, Brazil) and stored in a Thermo Scientific Freezer (model 902; Thermo Electric Scientific, Winchester, VA, USA) at −80°C for subsequent analysis. Serum leptin levels (ng/ml) were analyzed with enzyme-linked immune sorbent assays (ELISA) using a human leptin ELISA kit (KAC2281; Invitrogen Corporation, Carlsbad, CA, USA) according to the manufacturer’s instructions. The detection limit of this assay is 3.5 pg/ml. The inter-assay coefficient of variation was 3.9% for 150.6 pg/ml and 5.3% for 240.7 pg/ml. The readings were taken using an ELISA microplate reader (Expert Plus; Asys Hitech, Eugendorf, Austria) at 450 nm. These measurements were performed by researchers in the Clinical Immunology and Immunopathology Laboratory at the Center for Reproductive Biology, Federal University of Juiz de Fora.

STATISTICAL ANALYSES:

For the statistical analyses, pregnant women were divided into 2 groups according to their BMIs (<25 kg/m² or ≥25 kg/m²). Next, linear regression analyses were performed using maternal weight vs. gestational age, and leptin levels vs. gestational age. To compare leptin levels across the 3 time periods defined in this study, a 1-sided ANOVA was performed on the 2 groups, which was followed by a post hoc Bonferroni test. Subsequently, we used the t test to compare absolute weight gain and percent weight gain between the 2 groups. All of the statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). We adopted a level of significance of P<0.05.

Results

The socio-demographic and clinical/obstetric characteristics of the cohort are described in Table 1. We found that 95.2% (40/42) of the women were between 20 and 30 years of age, 66.7% (28/42) were nulliparous, 95.2% (40/42) had no history of pre-eclampsia during previous pregnancies, and 100% had no previous history of GDM, macrosomia, or stillbirths. As for family history (FH), 83.3% (35/42) did not have a FH of pre-eclampsia, 76.2% (32/42) did not have a FH of dyslipidemia, and 78.6% (33/42) did not have a FH of obesity.

Figure 1 shows that, in the course of gestation, there was a progressive increase in maternal weight gain in both the non-overweight (BMI <25 kg/m2) and the overweight/obese (BMI ≥25 kg/m2) groups of pregnant women. There was a progressive increase in maternal serum levels of this adipocytokine in both groups. However, this increase was significantly greater in the non-overweight group (BMI <25 kg/m2) (Figure 2). Additionally, when the mean serum leptin levels were compared during the first (T1), second (T2), and third (T3) trimesters of pregnancy, we concluded that there were significant increases (T1 vs. T2, P<0.01 and T1 vs. T3, P<0.001) in leptin levels in Group 1 (non-overweight or G1), as seen in Figure 3. However, we did not observe a significant increase in leptin levels (P>0.05) during the 3 trimesters in Group 2 (overweight/obese or G2).

When we analyzed the total weight gain during pregnancy in the 2 groups, we found that G1 had a greater total weight gain than G2, although the difference between the 2 groups was not significant (P>0.05) (Figure 4A). However, when we compared the percent weight gain during pregnancy between G1 and G2, there was a significantly higher percent weight gain in G1 compared to G2 (P<0.001) (Figure 4B).

Discussion

This study contributes to the body of knowledge available on the topic of evaluation of obesity in pregnancy and its association with serum leptin levels. Recently, obesity and leptin have been the subject of many studies [30–32], but there is still a significant gap in knowledge about the behavior of this hormone in women who were not overweight and those who were overweight/obese during pregnancy. From a methodological standpoint, this study provides adequate control of many intervening variables, such as the monitoring of all volunteers by a single physician, which minimizes the possible variations in data collection.

The changes in serum leptin levels during pregnancy have been well established through many studies for nearly 2 decades [32–34]. However, there are still many gaps in knowledge about the levels of this adipocytokine in pregnant women in relation to BMI and gestational weight gain.

We found that serum leptin levels in non-overweight (BMI <25 kg/m2) and overweight/obese (BMI ≥25 kg/m2) pregnant women increased progressively throughout pregnancy and the mean leptin concentrations were significantly higher in overweight/obese compared to normal-weight pregnant women. Similar findings were also reported by some recent studies that directly related leptin levels with groups of obese and non-obese pregnant women [25,35–38].

Our results also suggest that the leptin levels in non-overweight women were significantly higher (P<0.001) during pregnancy compared to overweight/obese women (Figures 2 and 3). This seems paradoxical, considering that probably the largest increase in leptin should have been detected in patients with overweight/obesity. Similar data were also observed by Misra [25] in the analysis of the serum leptin levels in non-overweight and in overweight/obese pregnant women. The authors concluded that the overweight/obese group had significantly higher serum leptin concentrations than the non-overweight group during pregnancy (P<0.01) and, although these concentrations increased significantly across gestation for both groups, the rate of increase was significantly smaller in overweight/obese women (P<0.05). They also concluded that factors other than fat mass alone can influence leptin concentrations in overweight/obese women compared to non-overweight women during pregnancy. This study suggests a hypothesis to explain this increase in leptin levels in the non-overweight group.

Analyzing the total weight gain and percent weight gain in both groups of pregnant women in our cohort, the results suggest that G1 had a greater total weight gain than G2, although the difference between the 2 groups was not significant (P>0.05) (Figure 4A). Furthermore, when we analyzed both groups for their percent weight gain during pregnancy compared to their pre-pregnancy weight, the non-overweight patients had a significantly greater percent weight gain (P<0.001) compared to the overweight/obese women (Figure 4B). Our finding suggests that the adipose tissue in women with a BMI <25 kg/m2 exhibits a higher percent increase during pregnancy compared to women with a BMI ≥25 kg/m2. This increase in adipose tissue can directly influence the production of adipocytokines and other inflammatory mediators by adipocytes. These findings suggest a hypothesis to explain the increase in leptin levels in non-overweight group

A limitation of this study was that the pre-pregnancy BMI was calculated using the pregnant woman’s self-reported weight at the first prenatal visit, and this information was subjective and prone to error. Another limitation is the small sample size, which may reduce the external validity of the research.

Conclusions

In summary, our results indicate that the significantly greater increase in serum leptin levels in non-overweight pregnant women may be explained by the significantly higher percent weight gain in this group compared to overweight/obese women. This study suggests that controlling both weight gain and the percent weight gain during the prenatal period may be an important preventive measure to control leptin levels during pregnancy and its subsequent medical complications.