Free Leptin Index is Elevated in Preeclamptic but not Healthy Women throughout Gestation. A Prospective Cohort Study

The ratio leptin/soluble leptin receptor (sOB-r), free leptin index (FLI), is used as a marker of leptin sensitivity/resistance in different pathologies. The aim of this study was to evaluate FLI in healthy non-pregnant, healthy pregnant and mild preeclamptic women during pregnancy. We conducted a nested case-control study within a longitudinal observational prospective cohort study. Serum leptin (p=0.0001) and sOB-r (p=0.0000) levels rose signicantly throughout pregnancy in healthy pregnant and preeclamptic women [leptin (p=0.0000); sOB-r (p=0.0380)]. Serum leptin levels were signicantly higher in preeclamptic compared to healthy pregnant women at 2 nd (p=0.0245) and 3 rd trimesters of pregnancy (p=0.0016). Additionally, serum sOB-r levels were signicantly lower in preeclamptic women during the 2 nd (p=0.0236) and 3 rd trimester (p=0.0024) of pregnancy compared to healthy pregnant women. Moreover, we found that FLI did not vary signicantly during any of the three periods studied in healthy pregnant women (p=0.7640), whereas, increased throughout preeclamptic pregnancy (p=0.0037). Indeed, FLI was signicantly higher at 2 nd (p=0.0053) and 3 rd (p=0.0003) trimesters of pregnancy in preeclamptic compared to healthy pregnant women. Additionally, FLI was signicantly higher during luteal phase compared to the follicular phase (p=0.0039). These results demonstrate that FLI increases signicantly in preeclamptic pregnant women towards the end of pregnancy. FLI is increased in preeclamptic compared with healthy pregnant women 35–37 FLI been evaluated longitudinally in mild preeclamptic pregnant women. Therefore, the current study aims to investigate this index throughout pregnancy in healthy and mild preeclamptic pregnant women in a case-control study nested within a longitudinal prospective cohort. serum samples obtained the follicular and luteal phases serum leptin (Invitrogen, KAC2281) R&D available range levels could contribute to the pathophysiology and underlying mechanisms of hypertensive disorders during pregnancy. Consequently, this index might be helpful as an early predictive biomarker for this hypertensive disorder during pregnancy. Moreover, our study reveals that elevated serum leptin levels and a slightly increased concentration of sOB-r exist in mild preeclamptic women when compared with normotensive pregnant women in the second and third trimesters of pregnancy. These results are consistent with ndings reported in patients with primary hypertension, and support the hypothesis that there is a strong relationship between hypertension, leptin and its sOB-r 50 . Of note, our present results compare favorably and extend those of a previous report showing that hyperleptinemia may precede and contribute to the development of hypertension, rather than being a major cause of it, inasmuch as, in the present study, hyperleptinemia occurs in preeclamptic pregnant women from the second trimester of gestation 33 . Also, FLI was more strongly related with adverse clinical outcomes and associated with masked hypertension than leptin or sOB-r alone; suggesting that leptin and its receptor acting conjointly may be involved in the hypertensive disorders of pregnancy. in pregnant higher leptin concentration and important role in the pathophysiology of preeclampsia, and FLI be useful in predicting


Introduction
Leptin is an adipokine predominantly synthetized and secreted by adipocytes, but it is also produced at lower levels in other tissues, including the placenta, therefore playing pleiotropic roles in the control of energy metabolism, reproductive function, immunity, and bone metabolism, where it exerts these actions by binding to speci c cell surface receptor 1 . Leptin receptors belong to the class I cytokine receptor family, and six splice variants with identical ligand-binding domains and alternative or truncate cytoplasmic domains have been described (LepRa, LepRb, LepRc, LepRd, LepRe and LepRf) 1,2 . Furthermore, the soluble leptin receptor (Ob-Re or sOB-r) represents the major leptin binding protein in blood, and plays an important role in leptin signaling [3][4][5][6][7][8][9] .
Previous studies have demonstrated that circulating leptin levels increase signi cantly during normal pregnancy, reaching a nadir in the third trimester and returning to preconception values in the postpartum period 10 . Additionally, some studies have shown that chronically higher plasma leptin levels are associated with obesity, metabolic syndrome and gestational diabetes mellitus 7,11−16 . Leptin also affects blood pressure and contribute to hypertension through sympathetic nervous system activation on both vasculature and the kidney 17,18 . During normal pregnancy basal sympathetic nerve activity increases 19 and deregulation of leptin levels has been correlated with the pathogenesis of preeclampsia 10,20−23 . For instance, leptin expression is increased in preeclamptic placentas and leptin concentrations are higher in preeclamptic women compared with normotensive pregnant women [24][25][26] .
The rate between circulating leptin and sOB-r, known as free leptin index (FLI = leptin/sOB-r), has been widely used to study leptin sensitivity/resistance 26,27 .
FLI is signi cantly increased in obese patients, due to higher circulating levels of leptin and lower levels of sOB-r compared to healthy normal weight control or obese subjects undergoing a weight reduction diet [28][29][30] . Thus, high FLI values have been associated with progressive and chronic diseases such as obesity, type 2 diabetes (T2D), reproductive diseases and nonalcoholic fatty liver disease (NAFLD) 13,31−34 . In this way, FLI has been associated with a number of pathological pregnancy states, including gestational diabetes mellitus (GDM) and preeclampsia 7,13,35 .
To date, some cross-sectional studies have reported that FLI is increased in preeclamptic compared with healthy pregnant women [35][36][37] . However, FLI has not been evaluated longitudinally in mild preeclamptic pregnant women. Therefore, the current study aims to investigate this index throughout pregnancy in healthy and mild preeclamptic pregnant women in a case-control study nested within a longitudinal prospective cohort.

Study Design and Participants
A nested case-control study within a longitudinal observational prospective cohort study (n = 465) was carried out to compare maternal FLI in healthy and preeclamptic pregnant women, across the three trimesters of pregnancy and three months postpartum. Study participants were recruited among pregnant women attending the obstetrics and gynecology health promotion and disease prevention program at the Engativa Hospital -Bogota. Pregnant women were recruited at 1st trimester (11-13 weeks) of pregnancy and followed until delivery and up to three months postpartum. Gestational age was calculated according to the last menstrual period or ultrasound examination in the rst trimester. Study subjects included healthy pregnant women (n = 43) and woman diagnosed with mild preeclampsia (n = 20) randomly selected from the original cohort (n = 465). Furthermore, twenty healthy non-pregnant women with regular menstrual cycles were included in the study during the follicular and luteal phases of the menstrual cycle.
Demographic, medical and reproductive clinical history was obtained by verbal interview. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were determined and mean arterial pressure (MAP) was calculated. Additionally, follow-up assessments of mothers as part of routine antenatal care visit included anthropometric, biochemical and hormonal determinations. Women with mild preeclampsia and late-onset preeclampsia, were delivered at ≥ 34 weeks' gestation and diagnosed and classi ed according to the ACOG guidelines and elsewhere [Systolic blood pressure (BP) ≥ 140 mmHg and/or diastolic preeclampsia with clinical manifestation occurring after 34 weeks were detected through routine prenatal screening and diagnosed with systolic blood pressure between 140 to 159 mmHg or diastolic blood pressure measures between 90 to 109 mmHg, non-elevated liver enzymes, absence of renal insu ciency, pulmonary edema, cyanosis, new-onset headaches or visual disturbances, and/or right upper quadrant or epigastric pain 40 . Data about maternal health complications, time and type of delivery, and neonatal characteristics at birth were retrieved from medical records.
Additionally, for the present study we excluded women with multiple gestations or the development of any complications during pregnancy, including prepregnancy hypertension, gestational diabetes mellitus, autoimmune and metabolic disorder, thyroid disease, liver and renal disease, acute and chronic infections, and diseases of the hematopoietic system, as well as women who were taking medications that affected metabolism.

Biochemical analysis
All biochemical and hormonal laboratory measurements were performed in the morning hours (07:00-08:00 hours) following an overnight fast (10:00-12:00 hours). Blood samples from the pregnant and non -pregnant women were collected at each visit into BD -Vacutainer® tubes from veins in the antecubital area. Serum glucose, total serum cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-c) and high density lipoprotein cholesterol (HDL-c) levels were measured by enzymatic methods (Labkit Kits, Spain).
Additionally, serum insulin levels were measured by electrochemiluminescence immunoassay using Roche Modular detection system (Roche, Basel, Switzerland) and serum C-reactive protein (CRP) were measured by colorimetric enzyme-linked immunosorbent assay ( Human serum leptin (Invitrogen, KAC2281) and sOB-r (DOBR00, R&D Systems) were determined in duplicate using the commercially available ELISA kits and assays were performed as described by the manufacturer. For serum human leptin, the analytical sensitivity was < 3.5 pg/mL, the assay range 15.6-1000 pg/mL and the intra-and inter-assay coe cient of variation (CV) were 4.6% and 3.6%, respectively. Additionally, for human serum sOB-r ELISA, the analytical sensitivity was < 0.128 ng/mL, the assay range 0.3-20 ng / mL and the intra-and inter-assay coe cient of variation (CV) were 5.5 % and 5.5 %, respectively.
The ratio between circulating leptin to sOB-r levels (leptin/sOB-r) (FLI) was determined as described elsewhere 26,27 .

Statistical analyses
Variables were expressed as means ± standard deviation (SD) or median and interquartile range (IQR) if they were parametric or not parametric. Parametric variables were compared using the Student t-test and one-way analysis of variance (ANOVA). We used the Bonferroni test, if the results were statistically signi cant. Non-parametric variables were evaluated with the Mann-Whitney test. For the longitudinal study, where the data were normally distributed, a repeated measures ANOVA was used with a Bonferroni post hoc test. For non-parametric longitudinal data, a Kruskal Wallis test was used with a Dunn's post hoc test. A p-value < 0.05 was considered statistically signi cant in all analyses, with a 95% con dence interval (CI). We used STATA 15-IC® version for statistical analyses.  Table 1). In healthy pregnant women, circulating leptin levels increased signi cantly throughout gestation (p = 0.0000). This increment was statistically different between rst and second trimester of pregnancy, whereas no signi cant differences were found between second and third trimester ( Fig. 1 and Supplementary Table 1). After delivery, leptin levels markedly dropped ( Fig. 1 and Supplementary Table 1).  Demographic, clinical and biochemical parameters of preeclamptic women. Normal distribution was indicated as mean (SD) and non-normal parameters were indicated as median (IQR). BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure; MBP: mean blood pressure; HOMA-IR: homeostasis model assessment-estimated insulin resistance; FLI: free leptin index. A p-value < 0.05 was considered statistically signi cant.-In preeclamptic women, leptin levels are also signi cantly elevated throughout gestation (p = 0.0000) ( Fig. 1 and table 2). When second (p = 0.0245 and third (p = 0.0016) trimester of preeclamptic women are compared to second and third trimester of healthy pregnant women, a statistically signi cant difference is found in serum leptin levels ( Fig. 1 and Supplementary Table 2).

Serum sOB-r levels are increased during pregnancy in healthy and preeclamptic women
On the other hand, serum sOB-r concentrations were not statistically different between follicular and luteal phases of the menstrual cycle in the non-pregnant women (p = 0.3969) ( Fig. 2 and Supplementary Table 3). In healthy pregnant women, a signi cant increase was observed in serum sOB-r concentrations from rst to second and third trimesters of pregnancy (p = 0.0000) and there were no statistically signi cant differences between second and third trimesters (p = 0.3999) ( Fig. 2 and Supplementary Table 3). Serum sOB-r levels were signi cantly decreased after delivery (Fig. 2 and Supplementary Table 3).
Circulating sOB-r was also signi cantly elevated throughout gestation in preeclamptic women (p = 0.0174) (Fig. 2), where serum sOB-r levels during the second (p = 0.0236) and third trimester (p = 0.0024) were higher than in the rst trimester. In addition, serum sOB-r levels were similar between healthy pregnancy and preeclamptic women ( Fig. 2 and Supplementary Table 4).
3.3. Free leptin index is increased throughout pregnancy in preeclamptic, but not in healthy women The FLI was lower in the follicular than in luteal phase of menstrual cycle of non-pregnant women ( Fig. 3 and Supplementary Table 5) (p = 0.0039). FLI was not signi cantly different at any trimester of pregnancy in healthy pregnant women (p = 0.7640) (Fig. 3). Conversely, FLI increased signi cantly from the rst to third trimesters of pregnancy in preeclamptic pregnant women (Fig. 3) (p = 0.0037). The FLI was signi cantly elevated during the second (p = 0.0053) and third trimester (p = 0.0003) of gestation in preeclamptic women when compared to healthy pregnant women ( Fig. 3 and Supplementary table 6).

Discussion
The present study demonstrates, for the rst time that, at variance to pregnant healthy women, FLI is signi cantly elevated during pregnancy in mild preeclamptic women. The FLI in the second and third trimesters of gestation in mild preeclamptic women was higher than in healthy pregnant women at the same periods, in a nested case-control study within a prospective cohort study. This signi cant increase occurs particularly in the second and third trimesters of pregnancy as result of the notable increased circulating levels of leptin and the moderated elevation in serum levels of sOB-r in preeclamptic women. These results are consistent with the previous ndings reported by Andersson-Hall et al. in healthy pregnant women in a longitudinal prospective cohort study 13 .
Additionally, it is important to note that FLI was signi cantly higher in the luteal versus follicular phase of the menstrual cycle, in response to higher serum leptin concentrations in the luteal phase.
Preeclampsia is a multisystem hypertensive disorder of pregnancy and one of the major causes of maternal and fetal morbidity and mortality worldwide 42 . In addition, it has been associated with endothelial dysfunction, coagulopathies, imbalance between angiogenic and anti-angiogenic factors, acute kidney injury, edema, and development of cardiovascular diseases, systemic in ammatory response and oxidative stress 43 . Furthermore, preeclampsia has been associated with a dysregulated secretion pro le of maternal and placental circulating factor, including growth factors, hormones and some adipokines such as leptin 44 .
Leptin is an adipokine produced and secreted primarily by white adipose tissue, which crosses the blood-brain barrier through a saturable transport system to reach the hypothalamus and activate the sympathetic nervous system that can lead to hypertension [45][46][47] . Previous studies have shown that high-circulating leptin levels are present in animals and humans with hypertension [45][46][47][48][49] . Here, we found that circulating levels of leptin are signi cantly elevated during the second and third trimesters of gestation in both healthy and preeclamptic women. However, this increase is more remarkable in preeclamptic pregnant women, suggesting that high leptin levels could contribute to the pathophysiology and underlying mechanisms of hypertensive disorders during pregnancy.
Consequently, this index might be helpful as an early predictive biomarker for this hypertensive disorder during pregnancy. Moreover, our study reveals that elevated serum leptin levels and a slightly increased concentration of sOB-r exist in mild preeclamptic women when compared with normotensive pregnant women in the second and third trimesters of pregnancy. These results are consistent with ndings reported in patients with primary hypertension, and support the hypothesis that there is a strong relationship between hypertension, leptin and its sOB-r 50 . Of note, our present results compare favorably and extend those of a previous report showing that hyperleptinemia may precede and contribute to the development of hypertension, rather than being a major cause of it, inasmuch as, in the present study, hyperleptinemia occurs in preeclamptic pregnant women from the second trimester of gestation 33 . Also, FLI was more strongly related with adverse clinical outcomes and associated with masked hypertension than leptin or sOB-r alone; suggesting that leptin and its receptor acting conjointly may be involved in the hypertensive disorders of pregnancy.

Conclusions
This longitudinal study indicates that circulating levels of leptin and sOB-r could be considered an independent risk factor for hypertensive disorders that occur in pregnant women. We suggest that higher leptin concentration might play and important role in the pathophysiology of preeclampsia, and FLI could be useful in predicting the onset of hypertensive disorders during pregnancy.