November 26, 2004
Serum Leptin Levels and Uterine Doppler Flow Velocimetry at 20 Weeks’ Gestation As Markers for the Development of Pre-Eclampsia
Key words: LEPTlN, PRE-ECLAMPSIA, DOPPLER FLOW VELOCIMETRY
Leptin is a protein of 167 amino acid residues and is mainly secreted by adipocytes1. It circulates in amounts related to fat mass and acts on the hypothalamic centers regulating body weight homeostasis, signaling the amount of body fat1.
It has been proposed that leptin may regulate reproductive function. Ob/ob mice, a mutant strain lacking leptin, are infertile3. Adminstration of recombinant leptin to these animals corrects the reproductive defect4,5.
Serum leptin levels increase during pregnancy, reaching a maximum at the end of the second trimester and plateauing thereafter. Leptin concentrations return to normal values within 24 h after parturition6. Furthermore, it has been demonstrated that the placenta produces leptin6,7 and that leptin levels are altered in some pathological conditions such as intrauterine growth restriction, both in maternal serum and in cord blood8-10.
Pre-eclampsia is a common pregnancy-associated disorder characterized by clinical features such as hypertension, proteiiiuria and uteroplacental hypoperfusion, with subsequent placental hypoxia, and is frequently accompanied by fetal intrauterine growth restriction11,12. It seems that this condition is favored by an altered placentation process, with defective remodeling of the spiral arteries, reduced differentiation of invasive trophoblastic cells and ischemic damage to the placental tissue13,14.
These histological alterations lead to insufficient changes of the uteroplacental circulation into a low-impedance system. Altered Doppler flow velocimetry of the uterine arteries during the second trimester, with high resistance index (RI) or persistence of an early diastolic notch, has been shown to be correlated with the risk of developing pre-eclampsia later during pregnancy15-18.
Recent studies, aiming at identifying markers for the early detection of women at risk for preeclampsia, suggest that leptin levels in women affected by severe pre-eclampsia are significantly higher than in normal healthy women19,20. Furthermore, hypoxia seems to stimulate leptin production in cultured placental cells6.
The aim of this study was to investigate whether alterations of uterine artery Doppler flow velocimetry during the early second trimester are accompanied by changes in leptin levels, and whether these changes may be an important early risk factor for the development of preeclampsia.
MATERIALS AND METHODS
From January 2000 to March 2003, we collected a blood sample in the morning after an overnight fast, from all the patients undergoing a secondor third-trimester ultrasonographic scan in our department, and determined their body mass index (BMI). The samples were immediately centrifugea for 10 min at 5000 rpm and sera were stored at -80C.
In the same period, we selected, among the patients who delivered in our Obstetric Ward, 50 women affected by pre-eclampsia as defined by the American College of Obstetricians and Gynecologists (systemic blood pressure ≥ 140/90 mmHg; proteinuria ≥ 0.2 g/day) and who had undergone a second-trimester ultrasound examination in our department, showing abnormal Doppler flow velocimetry and of- whom we had sera stored (group A). Inclusion criteria were: presence of preeclampsia developed during the third trimester, pre-pregnancy BMI of ≤ 27 kg/m^sup 2^, abnormal Doppler flow velocimetry at the 20 weeks' gestation ultrasound scan (mean RI ≥ 0.68 and/ or presence of a protodiastolic notch), fetal biometry at the 20 weeks' ultrasound scan corresponding to gestational age, and normal blood count and biochemistry. Exclusion criteria were: any systemic illness excluding pre-eclampsia (in particular, pre-existing chronic hypertension, renal diseases and diabetes), cigarette smoking and fetal malformations.
We also matched 100 controls for age, prepregnancy BMI and gestational age at the secondtrimester ultrasound scan who did not develop preeclampsia. We divided them into two groups: women with normal second-trimester Doppler flow velocimetry (group B, n = 50) and women with abnormal Doppler flow velocimetry (group C, n = 50).
None of these subjects was engaged in any significant physical activity and all women followed a similar diet as determined by a questionnaire completed by the women before the ultrasound scan.
The study was approved by our Institutional Review Board and all the subjects recruited gave their consent to participate in the study.
All women underwent ultrasonographic and Doppler pulsed velocimetry at 20-22 weeks' gestation using a Toshiba Vision 6000 machine with a 5-MHz convex probe (Toshiba Medical System, Rome, Italy). The scans were performed by the same operator (M.P.). Complete biometry of the fetus was performed. During the same scan the uterine arteries were identified by the use of color Doppler and the RI -was measured bilaterally, using a mean RI of 0.68 as a cut- off value to define altered maternal velocimetry. Furthermore, the presence of a protodiastolic notch in the uterine arteries' waveforms was recorded and regarded as a sign of altered velocimetry.
Blood samples were drawn in the morning (08.00-10.00) after an overnight fast. Blood was immediately centrifugea and sera were stored at -80C until testing.
For each subject we recorded the following data: type of delivery, gestational week at delivery, neonatal birth weight, any adverse event, such as intrauterine growth restriction (IUGR), hypertension, edema, proteinuria and development of preeclampsia, according to the criteria of the American College of Obstetricians and Gynecologists21.
Serum leptin levels were determined in duplicate using a human leptin radioimmunoassay (Linco Research, St Charles, MO, USA), with an intraassay coefficient of variation (CV) of 3.4-8.3%, an interassay CV of 3.6-6.2% and a sensitivity of 0.5 ng/ml.
Statistical analysis of the data was performed on an IBM- compatible PC using the Statistical Package for Social Science (SPSS), version 8.0.
Data distribution was assessed using the ShapiroWilk's test. All variables showed a normal distribution. Differences among groups were evaluated using ANOVA followed by Scheffe procedure for post- hoc comparison of means. Correlation between serum leptin levels, UMI and RI of the uterine arteries was assessed using the Pearson product-moment correlation. Significance was set at p
The characteristics of the women studied are reported in Table 1. No differences were observed in age, BMl, parity and gestational age at US scan. The mean neonatal birth weight was significantly lower in group A in comparison with women in groups B and C (2100 450 g vs. 3200 417 g and 3150 300 g, respectively), owing to the development of IUGR in several cases (18/50, 36%).
Table 1 Characteristics of the women studied. Group A: women with altered velocimctry at the second-trimester ultrasonography (US) scan with subsequent pre-eclampsia; group B: women with normal velocimetry at the second-trimester US scan; group C: women with altered velocimetry at the second-trimester US scan with normal outcome of pregnancy
The RI of uterine arteries was significantly higher in groups A and C (p
Serum leptin levels in the second trimester in the three groups evaluated are reported in Figure 1. No significant differences were observed in the three groups (group A: 18.6 4.2 ng/ml; group I: 19.7 3.2 ng/ml; group C: 18.0 4.6). Furthermore, we compared women in group A showing an altered RI but absence of a protodiastolic notch of the waveform and those with a protodiastolic notch, and found no differences (data not shown).
During the third trim\ester, women in group A showed significantly higher serum leptin levels in comparison with women in groups B and C (42.6 15.4 ng/ml vs. 24.4 12.3 ng/ml and 26.1 10.5 ng/ml, respectively; p
Serum leptin levels did not show a significant correlation with BMI either in the second trimester (Pearson p = 0.55, 0.60 and 0.58 in groups A, B and C, respectively) or in the third trimester (Pearson p = 0.52, 0.50 and 0.42 in group A, B and C, respectively) in all groups. Scrum leptin levels did not correlate with values of the RI in any of the groups.
Pre-eclampsia is charaterized by hypertension and proteinuria, probably caused by uteroplacental hypoperfusion and placental hypoxia. These seem to depend on altered trophoblast invasion of the spiral arteries14, leading to high-resistance blood flow to the uteroplacental district and release of a number of factors causing the well-known preeclamptic multisystemic syndrome22.
Figure 1 Maternal serum leptin levels in the second and third trimesters. Values are mean SD. *p
Several studies have demonstrated that Doppler flow velocimetry of the uteroplacental circulation during the second trimester showing high impedance to flow may be predictive of subsequent preeclampsia16,17,23,24 so that indexes of placental resistance during the second trimester can be used as a marker of altered placentation and subsequent development of pre-eclampsia.
Several attempts have been made to identify markers for the early detection of women at risk of developing pre-eclampsia22. Maternal serum leptin concentrations were significantly higher in preeclamptic women in some studies19,20. On the other hand, second- trimester evaluation of this protein in women who subsequently developed pre-eclampsia led to contrasting results25,26.
In this study, maternal serum leptin levels were not different in women with subsequent normal outcome of pregnancy and women who developed severe pre-eclampsia. Furthermore, these concentrations were similar in those women with altered second-trimester uterine artery blood flow who did not develop pre-eclampsia.
These data suggest that leptin levels during the second trimester are not different in subjects who subsequently develop pre- eclampsia in comparison with women who do not develop pre- eclampsia, even in the presence of altered blood flow directed toward the uteroplaccntal district, as diagnosed by Doppler velocimetry.
Recently Anim-Nayme and colleagues25 performed a longitudinal study on leptin levels in preeclamptic women. They found that women who developed pre-eclampsia had significantly higher plasma leptin concentrations, starting before the onset of pre-eclampsia, suggesting that leptin may have a role in the pathogenesis of this condition. Indeed, it has been demonstrated that leptin stimulates central receptors that regulate blood pressure anci/or heart rate27. Nevertheless, leptin levels seem to rise significantly after 32 weeks' gestation, even in normal pregnancy.
In contrast, Clausen and colleagues26 found significantly lower leptin levels at 18 weeks' gestation in women destined to develop preeclampsia. The authors argued that these low leptin levels in the maternal circulation may be due to reduced placental secretion secondary to altered placentation and that, in turn, this reduction may affect the invasive properties of the trophoblast and angiogenesis during placentation before 20 weeks' gestation. The discprepancies with our data may be due to the limited number of subjects included in our study. The limited size of the sample may not allow identifation of small, but significant, differences. Another possible explanation is that placental hypoxia appears after 20 weeks' gestation, inducing an increase of placental leptin secretion. Therefore, maternal serum leptin levels at 20-24 weeks' gestation could be similar to those of women not destined to develop pre-eclampsia.
Since we observed significantly higher leptin levels in patients with pre-eclampsia during the third timester, it is likely that the onset of severe hypoxia is needed to induce placental up-regulation of leptin gene expression28 and, therefore, higher maternal leptin levels. This condition is probably not present before the onset of clinical pre-eclampsia, and placental leptin production probably remains normal.
The nature of the correlation between hypoxia and raised serum leptin levels remains unexplained. Since hypoxia induces the expression of several placental genes19, it is possible that this increase may reflect a general response of the placenta to hypoxia. Indeed, since there seems to be some leptin resistance during pregnancy, it is unlikely that this rise may induce an increase in food intake by the mother in order to compensate fetal growth restriction. Another hypothesis is that the leptin production increase may induce placental proliferation. We previously demonstrated that leptin induced proliferation of a human placental cell line (JAr) modulating the activation of IRSl/MAP kinase signalling . This may counteract the fetal and placenta! growth restriction frequently observed in cases of severe pre-eclampsia.
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G. A. Tommaselli, M. Pighctti, A. Nasti, A. D'Elia, M. Guida, C. Di Carlo, G. Bifulco and C. Nappi
Department of Obstetrics and Gynecology, University of Naples 'Federico II', Naples, Italy
Correspondence: Dr G. A. Tommaselli, Department of Obstetrics and Gynecology, University of Naples 'Federico ?Γ, Via S. Pansini, 5-80131, Naples, Italy Phone: +390817462903; Fax: +390817462905; e- mail: [email protected]
Copyright CRC Press Sep 2004