Effect of gestational Mediterranean diet intervention on newborn fat mass and cord blood leptin level

Maternal nutritional status is an important determinant of intrauterine growth and neonatal size. No published surveys exist on maternal Mediterranean diet intakes and newborn adiposity. The aim of the study was to evaluate the impact of the individual maternal Mediterranean diet on the in-utero body fat formation and cord leptin level in newborns. Pregnant women with a pre-pregnancy body mass index (BMI) between 30 and 35 kg/m2 (n = 118) were assisted for individual dietary counseling based on the Mediterranean diet healthy eating. According to diet adherence, participants (paired mother and newborn) were divided into an intervention group (n = 57) and a control group (n = 61). We examined the association between diet modification and gestational weight gain, maternal, and cord leptin level together with newborn anthropometry (weight and fat mass %). Gestational weight gain, newborn birth weight, fat mass %, and cord leptin level lower in the intervention (12.22 ± 1.8 kg, 3.57 ± 0.35 kg, 9.27 ± 2.16 %, 11.78 ± 3.63 ng/ml, p = 000, respectively) than in control group (18.03 ± 3.25 kg, 4.02 ± 0.32 kg, 11.85 ± 2.30 %, 35.37 ± 11.14 ng/ml, respectively. Umbilical cord leptin levels strongly correlated with neonatal fat mass percent in both groups. However, maternal serum leptin did not correlate with the newborn parameters in the intervention group. Maternal energy intake from healthy fat and diet intervention is probably associated with decreased fat mass and leptin levels in neonates.


Background
Maternal nutritional and metabolic conditions determine the environment in which the fetus develops. The intrauterine environment can permanently change and mark the baby's physiology and metabolism. This process is also known as fetal programming or fetal origin hypothesis [1]. Fetal metabolic programming was initially studied in the context of maternal undernutrition and/or reduced intrauterine growth. There is an emerging need to evaluate the effect of maternal adiposity on offspring. Adverse maternal environments during the pregnancy, such as those seen with greater maternal body mass index (BMI) at the start of pregnancy and greater gestational weight gain (GWG) have been found to be associated with increased lifelong risks of offspring obesity and metabolic and cardiovascular dysfunction in adulthood [2]. However, little is known about metabolic adaptations in the offspring that occur independently of postnatal growth and nutrition. These metabolic diseases and conditions are now being diagnosed in children and adolescents at alarming rates, mirroring the rising rates of juvenile obesity [3].
The Mediterranean-style diet has been associated with reduced risk of cardiovascular disease, cancer, obesity, and metabolic syndrome [4,5]. Taking this a step further, research is now pointing to the benefits that adherence to a Mediterranean diet could have while pregnant [6].
Virtually, any nutritional or other factors on the maternal side must pass through the syncytioendothelial membrane to reach the fetal blood. In addition to mediating the transport of nutritional and metabolic compounds, the syncytio-trophoblast works as an endocrine organ that releases several hormones and growth factors to the maternal circulation. These factors effectuate profound metabolic and vascular alterations in the maternal body securing adequate conditions for fetal development [7].
Leptin is a polypeptide hormone that aids in the regulation of body weight and energy homeostasis and is linked to a variety of reproductive processes in both animals and humans. Fat mass is the major determinant of circulating leptin levels in children and adults, and cord blood leptin has been found to reflect neonatal fetal mass [8]. In the present study, we hypothesized that gestational adiposity may affect circulating levels of leptin and affect the fetal growth and body composition. Therefore, our aim was to evaluate the impact of individual maternal dietary counseling on the in-utero body fat formation and cord leptin level in newborns.

Methods
This was a cross-sectional study conducted at the antenatal clinic as a part of the gestational nutritional counseling program and neonatal intensive care unit of Ain Shams University Hospital for one and a half years starting June 2016. An informed consent was obtained from pregnant women to be enrolled in the study.
Eligibility included obese women with singleton uncomplicated pregnancies and BMI between 30 and 35 kg/m 2 , attending the obstetric clinic of Ain Shams University Hospital, who continued the interventional nutritional counseling and gave birth at the hospital.
Exclusion criteria: multiple pregnancies, known women with pre-gestational or gestational diabetes and with severe chronic diseases (pulmonary, cardiac, or renal), and any discovered fetal malformation during routine follow-up or emerging diseases that could affect fetal growth and liver functions.
Eligible pregnant women were assisted to a preliminary nutritional education session and lifestyle changes. The interventional diet modification was based on the Mediterranean diet. The scheduled individualized session for nutritional counseling was performed every 4 weeks focusing on the nutritional requirements during pregnancy and the healthy balanced diet modification. A printed handout was given to each participant of the diet regimen required. Each pregnant woman had at least 3 recorded nutritional assessment visits throughout the pregnancy period.
The traditional Mediterranean diet is characterized by a high intake of olive oil, fruits, vegetables, legumes, nuts, and whole-grain products; a moderate intake of fish; and only small amounts of red and processed meat. This dietary pattern is low in saturated fat intake and high in monounsaturated fat intake from olive oil. Total fat in a diet roughly provides 25 to 35% of calories, with saturated fat providing 8% or less of calories). It is rich in fiber and glutathione, provides a balanced ratio of n-6/n-3 essential fatty acids, and high amounts of antioxidants (especially polyphenols from olive oil, vitamins E, and C [9]. Clinical data and routine laboratory blood samples were collected at each visit. Each recorded visit, the mother was asked to answer a simple 14 items Mediterranean diet adherence screener questionnaire [10] to evaluate her diet compliance. A score of 9 of the 14 was the cut off point for discriminating between case and control [11] (Table 1).
According to Mediterranean diet adherence, participant pregnant women were divided into two groups: Obese intervention group: Pregnant women who were compliant and adherent to diet. It included 57 participants (paired mother and newborn). Obese control group: pregnant women who were not adherent to diet instructions. It included 61 participants (paired mother and newborn) (Fig. 1).
3) Newborn length in centimeter. 4) Body mass index was calculated using the following formula: weight (kg)/length (m 2 ). 5) Skinfold thickness (SFT) measurements in millimeter taken from four different sites (tricepsbiceps-quadriceps-subscapular) using slim guide skinfold calipe0072 and their mean were calculated. 6) Body fat mass percentage using the following equation [12]:

Blood sampling
Three millimeters of maternal serum and cord blood was withdrawn from each participant (paired mother and newborn). Samples were collected under complete aseptic conditions in clean glass tubes without anticoagulant. After coagulation, serum was separated, aliquoted, and stored at -20°C for the assay of leptin.

Analytical method
Leptin assay was carried out using a commercially available product of DSL-10-23100 Active [Diagnostic . This assay is an enzymatically amplified two-step sandwich-type immunoassay performed in microtitration wells, which have been coated with antihuman leptin antibody. After incubation of serum and washing off the unbound leptin, the wells were treated with another antihuman leptin detection antibody labeled with the enzyme horseradish peroxidase (HRP). After a second incubation and washing step, the wells were incubated with the substrate tetramethylbenzidine (TMB). An acidic stopping solution was then added, and the degree of the enzymatic turnover of the substrate was determined by dual-wavelength absorbance measurement at 405 and 620 nm. The absorbance measured is directly proportional to the concentration of human leptin present. A set of human leptin standards was used to plot a standard curve of absorbance versus human leptin conc. from which the human leptin concentration in subjects' sera was calculated using a linear curve-fit.

Statistical methods
Data were collected and analyzed using statistical package for social science (SPSS) version for windows (version 15.0.1). All data were expressed in terms of mean values ± SD. Comparisons of parameters among groups were made using student t test after testing and verification of normal distribution. Comparisons between two qualitative variables were performed using chi-square and fisher's exact tests. A p value ≤ 0.05 was significant. Pearson's correlation coefficient (r) test was used for correlating data.

Results
The mean age of pregnant women included was 26 ± 5.4 years, and the neonatal mean gestational age was 38 ± 1.3 weeks. Anthropometric measures of the studied groups and leptin levels (maternal serum and cord blood) are shown in (Table 2). The interventional Mediterranean diet had a negative effect on the gestational weight gain where the mother in the intervention group had significantly lower gestational weight gain as compared to the control (12.22 ± 1.81 kg vs 18.03 ± 3.25 kg, p = 0). The mean birthweight and BMI of newborn of the intervention group were significantly lower than that of the control group (3.57 ± 0.35 vs 4.02 ± 0.32 kg [p = 0] and 15.69 ± 2.24 vs 17.45 ± 2.36 [p = 0.001], respectively). Meanwhile, there were no significant differences in the mean gestational age or length between newborns of both groups.
Maternal serum leptin positively correlated with the gestational weight gain, fat mass %, and cord leptin of the control group (r = 0.882, p = 0.000; r 0.642, p = 0.00; and r 0.977, p = 0.000, respectively), but it did not correlate significantly with that of the intervention group; cord leptin, meanwhile, had a significant positive correlation with gestational weight gain and fat mass % in the intervention group of the newborn (r = 0.379, p value = 0.023 and r = 0.556, p value = < 0.001, respectively), and in the control group also (r = 0.921, p value < 0.001 and r = 0.645, p value < 0.001, respectively) ( Table 3).

Discussion
The dietary guidelines suggest the adoption of healthy eating patterns, which are characterized by higher consumption of fruits, vegetables, and whole grains and lower intake of calories, saturated fat, sodium, refined grains, and added sugars. One such healthy eating pattern contributing to overall health is the Mediterranean diet [13].  In this study, it was hypothesized that dietetic counseling based on the Mediterranean diet (MD) might have a good impact on the appropriate intrauterine environment with neonatal influence on fat development and leptin level. To the best of our knowledge, it is the first study to assess the effect of the Mediterranean diet during pregnancy on cord leptin values and correlate it to infant fat mass percent.
The results of the present study have revealed that gestational weight gain, newborn birth weight, and cord leptin level were lower in the Mediterranean diet intervention group than the control group, with no significant differences in either maternal age or neonatal gestational age between both groups. Our findings are consistent with previous studies which suggested that both gestational weight gain and maternal adiposity have a main role in the determination of infant birth weight and macrosomia. In the study of Sommer et al. [14], gestational weight gain was the strongest independent predictor of both birth weight and neonatal sum of skinfolds, with a 0.21 kg/week increased weight gain giving a 110.7 (95% confidence interval 76.6-144.9) g heavier neonate, and with 0.72 (0.38-1.06) mm larger sum of skinfolds.
Similarly, Hull et al. [15] reported that for women within the appropriate gestational weight gain category, infants born to obese mothers had greater % fat (14.6%) than infants born to normal-weight (11.2%, P .014) and overweight mothers (9.2%, P .002). For women within the excessive gestational weight gain category, infants born to normal-weight mothers had lower %fat (11.8%) than infants born to overweight mothers (13.7%, P .019) and obese mothers (14.2%, P .011). Moreover, gestational weight gain above the figures of the Institute of Medicine (IOM) guidelines was found to be associated with both macrosomia, and neonatal hypoglycemia in the study of Kominiarek et al. [16].
In the present study, the results revealed that skinfold thickness measurements and neonatal fat mass percent values were significantly lower in the intervention group than in the control group. This difference could be explained by the change in monounsaturated fat consumption and less total fat consumed during pregnancy and its positive correlation with the fetal adiposity and leptin level. The intervention group consumed mainly olive oil rich in useful fatty acids, polyunsaturated compounds, and also fat-soluble vitamins (A, E, D, and K). . This finding consolidates the assumption that maternal nutritional status is an important determinant of intrauterine growth and neonatal size. Several studies have highlighted the benefits that adherence to the Mediterranean diet during pregnancy could have on the offsprings [17]. In the study of Lee et al. [18], supplementation of fish oils rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to overweight women during gestation has shown a protective effect to fetal growth and development. These protective effects of fish oils were associated with increased DNA methylation of insulin growth factor 2 (IGF2), which controls intrauterine and postnatal growth.
Therefore, it goes with our hypothesis that fetal leptin levels may be involved in fetal development during late pregnancy.
Our findings also revealed that the cord leptin levels strongly correlated with the average gestational weight gain and neonatal fat mass percent in both study groups. Maternal serum leptin was found to be associated with the fat mass percent and cord leptin levels in the control group. Therefore, our findings support the hypothesis that fetal leptin levels may be involved in fetal development during late pregnancy. In a cohort study done on 638 motherchild pairs providing cord blood serum samples for leptin analysis and complete data on birth outcomes, it was found that both an increased birth size and longer gestational age are associated with higher cord blood leptin levels and that maternal pre-pregnancy BMI and weight gain during pregnancy represented significant indicators of cord blood leptin [19]. These findings are in agreement with our results. Another study conducted on 39 women and their babies to assess the link between leptin level and neonatal adiposity revealed a strong association between maternal leptin and maternal adiposity in pregnancy, and also between cord leptin at delivery and birthweight, as well as other anthropometric markers of fetal growth [20]. Similar results had been also previously obtained by others [21]. It was reported that the synthesis of leptin is in the placenta and that leptin concentration in the cord blood correlates with newborn anthropometry. In addition, umbilical serum leptin levels were found to correlate positively with neonatal birth weights [22]. Several physiological roles for leptin in pregnancy have been proposed, including the regulation of fetal growth and development, fetal/placental angiogenesis, embryonic hematopoiesis, and hormone biosynthesis within the maternal-fetoplacental unit. The specific localization of both leptin and its receptor in the syncytiotrophoblast implies autocrine and/or paracrine relationships in this endocrinologically active tissue [23].

Limitations
The limitations of the study are the focus on superficial adiposity and no access to the visceral and intraabdominal fat.

Conclusion
We found that the energy intake from healthy fat and diet was associated with decreased fat mass and leptin level in neonates. These findings indicate that the composition of the maternal diet is important for birth outcomes. An association between fat intake and birth outcomes should be more investigated in the future studies.