Molecular Human Reproduction, Vol. 6, No. 8, 763-769,
August 2000
© 2000 European Society of Human Reproduction and Embryology
Pregnancy |
Placental leptin in normal, diabetic and fetal growth-retarded pregnancies
1 Division of Nutrition and Development, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, UK and 2 Laboratory for Pregnancy and Newborn Research, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA
Abstract
Leptin expression in third trimester placenta (p) and leptin concentrations in umbilical cord blood (cb) were investigated in normal pregnancies [n = 10 (p), 31 (cb)] and abnormal pregnancies complicated with (i) maternal insulin-dependent diabetes [IDDM: n = 3 (p), 13 (cb)], (ii) gestational diabetes [GD: n = 2 (p), 10 (cb)] and (iii) fetal growth retardation [FGR: n = 5 (p), 5 (cb)]. By in-situ hybridization and immunohistochemistry, placental leptin mRNA and protein were co-localized to the syncytiotrophoblast and villous vascular endothelial cells. Leptin receptor was immunolocalized to the syncytiotrophoblast. Relative to controls, the FGR group was characterized by low concentrations of placental and cord blood leptin. In a twin pregnancy, the normal-sized infant exhibited more placental and cord blood leptin than its growth-retarded twin. In contrast, both diabetic groups exhibited high concentrations of placental leptin mRNA and protein. The IDDM group exhibited the highest concentrations of leptin in cord blood. No change was observed in the expression of the leptin receptor in either the growth-retarded or diabetic pregnancies. In conclusion, the localization of placental leptin suggests that it may be released into both maternal and fetal blood. Furthermore, in fetal growth-retarded and diabetic pregnancies, the changes in leptin expression in the placenta and in leptin concentrations in umbilical cord blood appear to be related.
diabetes/fetal/leptin/placenta/trophoblast
Introduction
Leptin, the protein product of the ob (obese) gene, is a 16 kDa hormone, whose principle site of synthesis is white adipose tissue, although expression of leptin now appears to be much more widespread. Leptin is considered to be a signalling molecule to the brain, where the leptin receptor has been localized, for the regulation of whole-body energy balance. The leptin receptor gene has been shown to have several splice variants (Cioffi et al., 1996
; Lee et al., 1996). The ob-Rb variant encodes a receptor with a long intracellular domain that is thought to be essential for intracellular signal transduction (Tartaglia et al., 1995). It is increasingly recognized that leptin is also an important metabolic hormone influencing processes such as insulin secretion, glucose utilization, glycogen synthesis and fatty acid metabolism (Pi-Sunyer et al., 1999; Trayhurn et al., 1999).
Recent studies suggest that leptin may play an important role during pregnancy. In humans and rodents, maternal circulating leptin concentrations rise towards the end of pregnancy and fall to below pre-pregnancy concentrations at around birth (Chien et al., 1997; Hardie et al., 1997; Tomimatsu et al., 1997). The source of this peak in leptin in late pregnancy remains to be established, particularly as there is no positive correlation between leptin and body mass index (BMI) in pregnant women.
One possible explanation for the increase in leptin towards the end of pregnancy is synthesis by the placenta. Both the human (Green et al., 1995; Masuzaki et al., 1997) and murine placenta (Hoggard et al., 1997a) have been shown to express both leptin and the leptin receptor. It is interesting to note that the human leptin gene has a placental specific upstream enhancer (Bi et al., 1997). This implies that in the human, placental leptin is differentially regulated to leptin of adipose origin. Several recent studies have also demonstrated a positive correlation between leptin concentrations in the umbilical cord blood and body birthweight, suggesting that placental leptin may also have a role in fetal development (Hassink et al., 1997; Schubring et al., 1997; Gross et al., 1998; Marchini et al., 1998; Shekhawat et al., 1998; Ertl et al., 1999; Gomez et al., 1999; Ong et al., 1999; Shaarawy and El-Maliah, 1999).
The purpose of this study was to determine how retarded fetal growth and maternal diabetes may affect placental leptin expression and concentrations of leptin in cord blood. Normal third trimester pregnancies were compared to those complicated with retarded fetal growth and maternal diabetes in terms of (i) placental leptin expression (mRNA, protein), (ii) leptin receptor expression (protein) and (iii) cord blood leptin concentrations. In addition, normal first and third trimester placentae were compared in terms of the precise anatomical location of leptin and the leptin receptor.
Materials and methods
Patients
Third trimester
A placental biopsy and venous cord blood sample were collected at the time of pre-labour Caesarean section from four groups of subjects: (i) A control group of uncomplicated singleton term pregnancies (37 completed weeks by early ultrasound dating) undergoing Caesarean section because of primigravid breech presentation or previous Caesarean section. (ii) A group of insulin-dependent diabetic (IDDM) women ranging from 35 to 41 weeks gestation. The indications for term Caesarean section were identical to that for the control group. Premature delivery was indicated where there was evidence of significant fetal compromise (reduced growth, reduced liquor volume and raised Doppler umbilical artery resistance index) or of worsening proteinuric hypertension. All women in this group were using injectable insulin therapy and self-monitored their blood glucose. (iii) A group of women with gestational diabetes requiring injectable insulin to maintain blood glucose in the normal range. This group consisted of women newly presenting in the current pregnancy and diagnosed with gestational diabetes on the basis of a 75 g oral glucose tolerance test. In this group, six were diagnosed before 12 weeks and four were diagnosed between 20 and 34 weeks. The indications for delivery in this group were similar to women in the IDDM group. For both the IDDM and gestational diabetic subjects, self-recorded blood glucose monitoring and glycosylated haemoglobin concentrations suggested that maternal blood glucose had been maintained in the normal range for the fortnight prior to delivery. (iv) A group of women delivered prematurely because of significant worsening proteinuric hypertension or fetal compromise (absent end diastolic flow on umbilical Doppler studies, reduced growth and liquor volumes). Antenatal dexamethasone (two doses of 12 mg 24 h apart) were administered prior to delivery where the gestation was less than 32 weeks (n = 1: 27 weeks). In total, three of five growth-retarded pregnancies were premature. The demographic details of all four groups of third trimester pregnancies are summarized in Table I.
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All women fasted overnight prior to Caesarean section which was carried out under spinal anaesthesia. For the diabetic patients an insulin and dextrose infusion, adjusted to maintain blood glucose at ~4 mmol, was started at about 0700 h, some 2–3 h before planned delivery. The study was approved by the local ethics committee and all women gave informed consent prior to participation in the study.
First trimester
Placental tissue was taken from women undergoing surgical termination of normally progressing pregnancies (n = 3).
Sample collection and preparation
Venous cord blood was collected into lithium heparinized tubes at Caesarean section after the umbilical cord had been clamped. The sample was immediately centrifuged and separated and the plasma stored in duplicate aliquots at –20°C until assay. The placenta was then delivered onto ice and a full-thickness sample of ~2 cm square was taken. The placental biopsy was taken at random with the aid of a 20x20 cm grid divided into 2 cm squares placed over the placenta, using randomly selected coordinates. In some cases the random co-ordinates specified an area of the grid outside the placenta, and in this case new coordinates were selected.
The placental biopsy was fixed for 24 h in 4% paraformaldehyde and stored in 70% ethanol until routinely embedded in wax. Sections 5 µm thick were cut for the immunohistochemical localization of (i) leptin protein and (ii) leptin receptor. For the detection of leptin mRNA by in-situ hybridization, 5 µm sections were cut over DEPC (diethylpyrocarbonate; Sigma, Poole, Dorset, UK)-treated water and taken onto baked 3-aminopropyltriethoxysilane (`Tespa'; Sigma)-coated slides.
Cloning of leptin cDNA
Total RNA was extracted from human normal term placental tissues using RNAzol (BRL) and cDNA were generated by reverse transcription using the Superscript Pre-amplification system (Gibco BRL) as previously described (Hoggard et al., 1997b). The cDNA primers to the human leptin gene were 5'-GTCCAAGCTGTGCCCATC-3' and 5'-ATCCCGGAGGTTCTCCAG-3'; Genbank U43653; a 256 bp fragment was generated.
Polymerase chain reaction (PCR) was performed on a Hybaid Touchdown thermal cycler as previously described (Hoggard et al., 1997b) using the following amplification conditions [94°C (4 min), one cycle; 94°C (1 min), 55°C (1 min), 72°C (1 min), 35 cycles; 72°C (10 min), one cycle]. Agarose gel electrophoresis (2%) in the presence of ethidium bromide confirmed the presence of a single band of the expected size. The PCR product was purified using Wizard PCR preps (Promega) and cloned directly into pGEM-T (Promega). The sequence and orientation of the insert was confirmed by automated sequencing. Plasmids were linearized with Sac I or Apa I for transcription with T7 or SP6 RNA polymerase to generate antisense and sense riboprobes.
In-situ hybridization
In-situ hybridization was carried out as previously described with a few minor modifications (Hoggard et al., 1997a). Briefly, 5 µm paraffin wax sections were dewaxed through xylene and rehydrated through a decreasing ethanol series. Slides were then washed in DEPC-treated water (5 min), immersed in 0.1 N HCl for 20 min followed by 2x standard saline citrate (SSC) for 30 min at room temperature. Sections were then exposed to 2.0 µg/ml proteinase K in 0.2 mol/l Tris–HCl pH 7.6, 0.05 mol/l EDTA at 37°C for 20 min and post fixed in 0.4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at 4°C. Following two further 5 min washes in PBS, sections were acetylated by emersion in 0.25% acetic anhydride in 0.1 mol/l triethanolamine for 10 min, washed in 2x SSC, dehydrated through an ascending ethanol series and left to air dry.
Hybridization was carried out using 35S-labelled cRNA probes and 1x106 c.p.m. was applied to each section. The probes were prepared in 50% formamide, 0.3 mol/l NaCl, 1xDenhardt's, 10 mmol/l Tris (pH 8.0), 1.0 mmol/l EDTA, 1 mg/ml tRNA, 10 mmol/l dithiothreitol, 20% Dextran sulphate. Probe containing hybridization mix (60 µl) was sealed under sterile coverslips using DPX mountant and hybridization was carried out at 58°C overnight. Following hybridization, the DPX was pealed away and the cover slips were soaked off in 4xSSC. Sections were further washed in fresh 4xSSC and treated with RNase A (20 µg/ml) in 0.5 mol/l NaCl, 10 mmol/l Tris, 1 mmol/l EDTA at 37°C for 30 min. All sections were desalted by emersion in decreasing SSC solutions with a final high stringency wash in 0.1xSSC at 60°C, dehydrated and air-dried. Finally the slides were dipped in K5 autoradiography emulsion (Ilford Ltd, Mobberely, Cheshire) diluted with an equal volume of distilled water, dried overnight and transferred to a light sealed box for 14 days at 4°C. After developing with Kodak D-19 developer, the slides were counterstained with haematoxylin and examined by light microscopy.
Antisera
The human leptin antiserum is a rabbit anti-leptin antiserum that was raised at the Rowett Research Institute against recombinant human leptin (Peprotech). This antiserum was used for immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). The anti-leptin receptor antiserum is an affinity-purified goat polyclonal antibody raised against a peptide corresponding to an amino sequence mapping at the carboxy terminus of mouse ObR (Santa Cruz Biotechnology, Heidelberg, Germany). This antiserum detects all splice variants of the leptin receptor and was used for immunohistochemistry.
Immunohistochemistry
Immunolocalization of leptin
Tissue sections were dewaxed in histoclear, rehydrated through a descending ethanol series and washed in 0.1 mol/l PBS for 2x5 min. Non-specific endogenous peroxidase activity was blocked by treatment with 3.0% hydrogen peroxide in water for 5 min at room temperature and the leptin epitope was exposed by microwaving for 2x5 min in 0.1 mol/l citrate buffer. Tissue sections were then washed with PBS for 2x5 min and exposed to a non-immune block with goat serum (150 µl in 10 ml) for 20 min at room temperature. Rabbit anti-human leptin polyclonal antibody (1mg/ml) was then applied at a dilution of 1 in 50 overnight at 4°C and this was followed with biotinylated goat anti-rabbit antibody for 30 min at room temperature. An avidin–biotin–peroxidase detection system (Vector stain; Vector Laboratories, Peterborough, UK) was then applied for a further 30 min (room temperature). Finally the sections were developed with diaminobenzidine (DAB kit; Vector Laboratories) for 10 min to generate a brown-coloured product and lightly counterstained with haematoxylin prior to mounting. Negative controls were performed by replacing the primary antibody with rabbit immunoglobulin at the same concentration as the primary antibody. The anti-human leptin antibody was also incubated with excess recombinant human leptin and the absorbed antibody was used as a further negative control in some experiments.
Immunolocalization of leptin receptor
The leptin receptor epitope was exposed by incubation with 0.1% trypsin in 0.1% calcium chloride (pH 7.8) for 10 min at 37°C. Tissue sections were incubated with polyclonal goat anti-human leptin receptor antibody at a dilution of 1 in 25 for 60 min at room temperature and developed as above. Negative controls were performed by replacing the primary antibody with rabbit immunoglobulin at the same concentration as the primary antibody. Rat kidney was used as a positive control tissue.
Assessment of immunostaining and in-situ hybridization
Immunoreactivity was assessed using light microscopy. Qualitatively, the localization of leptin and the leptin receptor was noted in each sample. The relative immunostaining intensity for both antibodies was carried out by two blinded observers and visually assessed on the intensity of staining, i.e. tissues were assessed as being very positive, positive or negative. Reproducibility was demonstrated by three separate experiments in which all samples were analysed together. The in-situ hybridization results were also assessed qualitatively as described above. This approach allowed gross differences between groups to be assessed.
ELISA of leptin in cord blood
Leptin concentrations of the samples were determined in a solid phase sandwich ELISA. Briefly, high-affinity microtitre plates were coated with 1 µg/ml rabbit anti-leptin immunoglobulin G (IgG), washed, blocked with bovine serum albumin, washed and developed as previously described (Hardie et al., 1997). Leptin concentrations were expressed as recombinant leptin equivalents (detection limit 100 pg/ml) by comparison with human recombinant leptin standards.
Assay for insulin in cord blood
Insulin concentrations were determined by radioimmunoassay using a commercially available kit (Pharmacia, UK). The intra-assay coefficient of variation was 6.9%. Briefly, this competition assay used a fixed amount of 125I-labelled insulin and antibody and varying amounts of unlabelled test insulin. In accordance with the Pharmacia kit, separation of insulin-bound antibody was achieved using secondary antibody bound to Amerlex-M magnetic beads, and insulin concentration was calculated by interpolation from a standard curve. Insulin concentration was expressed in microunits/ml.
Statistical analysis
For statistical analysis, a normal distribution of the leptin and insulin assay data was achieved by log transformation and the data were assessed by analysis of variance (ANOVA). Significant differences between groups were tested by calculation of Bonferroni intervals. Regression analysis was used to assess the effects of gestation on log insulin and log leptin concentrations. To investigate linear relationships, correlations were calculated and P values determined where appropriate.
Results
Leptin and leptin receptor expression in first and third trimester placenta
In all first trimester placental samples examined, leptin mRNA and protein were co-localized to the syncytiotrophoblast and cytotrophoblast lineages (Figure 1a,b,e,f). In normal third trimester placental tissue, when cytotrophoblast cells are sparse, leptin mRNA and protein remained co-localized to the syncytiotrophoblast (Figure 1c,d,g,h). Leptin protein was clearly localized to villous vascular endothelial cells in both the first and third trimester (Figure 1e,g). In addition, leptin mRNA was co-localized to this cell type and this was most evident in third timester placenta (Figure 2i, parts c,d: mRNA, protein). The leptin receptor was immunolocalized to only the syncytiotrophoblast in both first and third trimester placenta (Figure 1i–l). All IgG and absorbed antibody controls were negative. The rat kidney positive controls exhibited positive leptin receptor immunostaining in the distal tubules (data not shown).
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Placental leptin expression in growth-retarded and diabetic pregnancies
Placental leptin mRNA and protein in placentae associated with growth-retarded infants exhibited less hybridization and lower intensity immunostaining than normal placentae [Figure 2i
, parts a–d]. This observation was consistent when each of the five samples from growth-retarded pregnancies were compared to the controls (n = 10). In contrast, placentae from diabetic pregnancies exhibited more intense hybridization and immunostaining for leptin than controls [Figure 2i parts c–f]. Again, this observation was consistent when each of the five samples from diabetic pregnancies was compared to the controls (n = 10). No changes in leptin receptor expression were observed (results not shown) in any of the samples from the different types of pregnancies.
In a twin pregnancy where one baby was normal-sized and the other growth-retarded, less intense placental hybridization and immunostaining for leptin was observed in association with the growth-retarded infant [Figure 2ii parts a–d].
Leptin and insulin concentrations in cord blood
Leptin and insulin concentrations in cord blood samples collected from control, diabetic and growth-retarded pregnancies are summarized in Table II. The range of gestational ages for the control and diabetic groups was from 35 to 42 weeks (see Table I). Log (e) transformations were necessary to normalize the data. By regression analysis, there was no significant linear relationship between gestation (length) and log (e) leptin or log (e) insulin. The results were therefore subdivided into established IDDM prior to pregnancy and gestational diabetic subgroups. Only leptin was measured in the samples from pregnancies complicated with fetal growth retardation.
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Insulin concentrations were significantly higher in the IDDM group as compared to the gestational diabetic group and controls (ANOVA: P < 0.001; Bonferroni intervals:
= 0.05). In addition, the highest leptin concentrations were also found in the IDDM group. However, as leptin concentrations exhibited a wide range, the Bonferroni intervals did not reveal any significant differences between the groups. Two out of 13 IDDM cord blood samples had leptin concentrations below the lowest standard used in the ELISA and were given the value of the lowest point of the linear part of the ELISA curve for the analysis. In the growth-retarded group three of the five samples were below the lowest standard used in the ELISA. Since three samples were taken from premature infants, the effects of prematurity on placental leptin could not be ruled out. Consequently no statistical comparisons were made between the growth-retarded group and the controls. However, in the 33 week twin pregnancy (see Figure 2), the cord blood leptin associated with the normal-sized infant was higher than that associated with the growth-retarded infant (0.3644 verses not detectable). A significant positive correlation between log(e) cord blood leptin and fetal weight was observed in the gestational diabetic group (P = 0.0169) but not in the other groups. Discussion
The human placenta has recently been identified as a major source of leptin and the existence of a placental specific upstream enhancer indicates that placental leptin may be regulated differently to that of adipose origin (Green et al., 1995; Bi et al., 1997; Masuzaki et al., 1997). The role of leptin at the maternal–fetal interface, however, remains unclear. Insight into the potential role(s) of leptin at the maternal–fetal interface can be generated by correlating third trimester placental leptin expression and cord blood leptin concentrations to fetal size or abnormal developmental conditions. For the first time, we have shown that both placental expression and cord blood leptin concentrations are lower than normal in pregnancies complicated with fetal growth retardation. In contrast, placental leptin mRNA and protein concentrations are increased in pregnancies complicated with maternal diabetes. Although visually assessed placental immunostaining is only semi-quantitative, the differences between the groups (growth-retarded versus control and diabetic versus control) were striking and clearly followed the changes observed in cord blood leptin. Our localization studies on first and third trimester placentae also show that leptin is expressed by the syncytiotrophoblast in contact with maternal blood and in the vascular endothelial cells in contact with fetal blood. Leptin may therefore be released into both the maternal and the fetal circulation. Interestingly, we could only detect the leptin receptor on the maternal side of the placenta in the syncytiotrophoblast, indicative of a possible autocrine role at the maternal interface.
Previous studies have shown that umbilical cord blood leptin concentrations are lower in small-for-gestational-age infants and that newborns with intrauterine growth retardation have significantly lower serum leptin concentrations than normal growth controls (Jaquet et al., 1998; Marchini et al., 1998; Tamura et al., 1998). Further, concentrations of neonatal serum leptin markedly decrease after birth (Yura et al., 1998). As this fall much exceeded the leptin response to fasting observed in adults, it cannot be associated with nutrient deprivation but rather removal of placental leptin supply. In addition, serum leptin concentrations in newborns were found to be increased more than three fold compared with children after correlation for adiposity (Hassink et al., 1997). This would suggest that the placenta is an important source of leptin in the fetal circulation. In support of this, venous cord blood leptin concentrations are reported to be higher than arterial concentrations and we have shown that leptin is expressed by villous vascular endothelial cells in contact with fetal blood (Yura et al., 1998; Gomez et al., 1999). These findings, in combination with a correlation of cord blood leptin concentrations with birthweight, suggest that placental leptin may function as a fetal growth factor. In support of this, our studies of a 33 week twin pregnancy have shown that a growth-retarded twin had markedly lower concentrations of placental and cord blood leptin than its normal-sized twin. Further, although three of our fetal growth-retarded group were premature, our studies indicate that low concentrations of cord blood leptin which characterize low birthweight infants may reflect low concentrations of leptin in the placenta. However, a contribution from fetal adipose tissue cannot be ruled out (Bernard et al., 1999; Yuen et al., 1999).
Placental and cord blood leptin concentrations are also affected by maternal diabetes and this has also been linked with fetal growth. Indeed, in pregnant women with IDDM, Northern analysis studies have shown that a 3–5-fold increase in placental leptin mRNA is associated with elevated concentrations of leptin and insulin in venous cord blood (Gross et al., 1998; Lepercq et al., 1998). Poorly controlled maternal diabetes is associated with neonatal macrosomia and this is thought to be due to fetal hyperinsulinaemia resulting from maternal hyperglycaemia (Pederson, 1954). It is possible therefore that fetal insulinaemia may induce fetal macrosomia through the induction of placental and fetal `leptinaemia'. Indeed, leptin concentrations in cord blood have been reported to correlate with both birthweight and placental weight whereas maternal leptin concentrations do not (Hassink et al., 1997; Schubring et al., 1997; Gross et al., 1998; Marchini et al., 1998; Shekhawat et al., 1998; Ertl et al., 1999; Gomez et al., 1999; Ong et al., 1999; Shaarawy et al., 1999). In the present study, the highest cord blood leptin concentrations were found in women with IDDM and this group also exhibited significantly higher insulin concentrations. The increased cord blood leptin concentrations were also associated with an increased expression of leptin in the placenta. Although the limited number of placental tissue samples available from diabetic pregnancies (n = 5) precluded further subdivision of the in-situ hybridization and immunostaining data into established and gestational diabetic, elevated leptin expression was observed in each sample. Interestingly, in the IDDM cohort, no increased birthweight was observed. This may reflect a level of metabolic control during pregnancy adequate for normal fetal development (Lepercq et al., 1998). Alternatively, this may reflect saturation of fetal leptin receptor. Further studies are necessary to investigate these possibilities.
The co-localization of leptin and its receptor on the syncytiotrophoblast not only indicates that placental leptin is released into the maternal circulation but may also have an autocrine role on the placenta. In this regard, abnormal trophoblast proliferation or invasion is associated with abnormal placental leptin release, e.g. choriocarcinoma, hydatidiform mole and pre-eclampsia all provide evidence that leptin may be directly involved in the growth of the placenta (Sagawa et al., 1997; Mise et al., 1998; McCarthy et al., 1999). The downstream effects of leptin binding to the placental receptor have not been characterized. However, leptin may play a role in placental angiogenesis (Sierra-Honigmann et al., 1998).
The soluble leptin receptor (OB-Re), which is thought to be shed by the placenta, is reported to increase 1.5 fold in the circulation in IDDM pregnancies (Gavrilova et al., 1997; Lewandowski et al., 1999). However, we observed no change in placental leptin receptor expression in the IDDM or fetal growth-retarded groups. This may reflect our immunolocalization protocol which detected all splice variants of leptin receptor and therefore total leptin receptor expression. Alternatively, the soluble leptin receptor may be expressed at other sites (Bodner et al., 1999).
In conclusion, the localization of placental leptin suggests that it is released into maternal and fetal blood. The localization of the leptin receptor on the maternal side of the placenta suggests that placental leptin may have an autocrine role on the placenta as well as a paracrine/endocrine role in the mother. On the fetal side, leptin release by vascular endothelial cells may act directly on the fetus. In pregnancies complicated with fetal growth retardation and maternal diabetes, changes in placental and cord blood leptin concentrations appear to be related. The implications of these changes in terms of the role of placental leptin at the human maternal–fetal interface require further investigation.
Acknowledgments
This work is supported by the Scottish Agricultural, Environment and Fisheries Department. We are grateful to Dr Grietje Zuur, Biomathematics and Statistics Scotland, for statistical expertise.
Notes
3 To whom correspondence should be addressed at: Maternal-Fetal Physiology Group, Division of Nutrition and Development, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen, AB21 9SB, UK. E-mail: rgl{at}rri.sari.ac.uk 
References
Bernard, S.J., Yuen, I., McMillen, C. et al. (1999) Abundance of leptin mRNA in fetal adipose tissue is related to fetal body weight. J. Endocrinol., 163, 149–157.[Abstract]
Bi, S., Gavrilova, O., Gong, D.W. et al. (1997) Identification of a placental enhancer for the human leptin gene. J. Biol. Chem., 272, 30583–30588.
Bodner, J., Ebenbichler, C.F., Wolf, H.J. et al. (1999) Leptin receptor in human term placenta: in situ hybridization and immunohistochemical localization. Placenta, 20, 677–682.[Web of Science][Medline]
Chien, E.K., Hara, M., Rouard, M. et al. (1997) Increase in serum leptin and uterine leptin receptor messenger RNA levels during pregnancy in rats. Biochem. Biophys. Res. Commun., 237, 476–480.[Web of Science][Medline]
Cioffi, J.A., Shafer, A.W., Zupancic, T.J. et al. (1996) Novel B219/Ob Receptor isoforms — possible role of leptin in hematopoiesis and reproduction. Nature Med., 2, 585–589.[Web of Science][Medline]
Ertl, T., Funke, S., Sarkany, I. et al. (1999) Postnatal changes of leptin levels in full-term and preterm neonates: their relation to intrauterine growth, gender and testosterone. Biol. Neonate, 75, 167–176.[Web of Science][Medline]
Gavrilova, O., Barr, V., Marcus-Samuels, B. et al. (1997) Hyperleptinemia of pregnancy associated with the appearance of a circulating form of the leptin receptor. J. Biol. Chem., 272, 30546–30551.
Gomez, L., Carrascosa, A., Yeste, D. et al. (1999) Leptin values in placental cord blood of human newborns with normal intrauterine growth after 30–42 weeks of gestation. Horm. Res., 51, 10–14[Web of Science][Medline]
Green, E.D., Maffei, M., Braden, V.V. et al. (1995) The human obese (Ob) gene — RNA expression pattern and mapping on the physical, cytogenetic, and genetic maps of chromosome-7. Genome Res., 5, 5–12.
Gross, G.A., Solenberger, T., Philpott, T. et al. (1998) Plasma leptin concentrations in newborns of diabetic and nondiabetic mothers. Am. J. Perinatol., 15, 243–247.[Web of Science][Medline]
Hardie, L., Trayhurn, P., Abramovich, D. et al. (1997) Circulating leptin in women: a longitudinal study in the menstrual cycle and during pregnancy. Clin. Endocrinol., 47, 101–106.[Medline]
Hassink, S.G., de Lancey, E., Shelow, D.V. et al. (1997) Placental leptin: an important new growth factor in intrauterine and neonatal development? Pediatrics, 100, E1NE7.
Hoggard, N., Hunter, L., Duncan, J. et al. (1997a) Leptin and leptin receptor mRNA and protein expression in the murine fetus and placenta. Proc. Natl. Acad Sci. USA, 94, 11073–11078.
Hoggard, N., Mercer, J.G., Rayner, D.V. et al. (1997b) Localization of leptin receptor mRNA splice variants in murine peripheral tissues by RT-PCR and in situ hybridization. Biochem. Biophys. Res. Commun., 232, 383–387.[Web of Science][Medline]
Jaquet, D., Leger, J., Levy-Marchal, C. et al. (1998) Ontogeny of leptin in human fetuses and newborns: effect of intrauterine growth retardation on serum leptin concentrations. J. Clin. Endocrinol. Metab., 83, 1243–1246.
Lee, G.H., Proenca, R., Montez, J.M. et al. (1996) Abnormal splicing of the leptin receptor in diabetic mice. Nature, 379, 632–635.[Medline]
Lepercq, J., Cauzac, M., Lahlou, N. et al. 1998 Overexpression of placental leptin in diabetic pregnancy; a critical role for insulin. Diabetes, 47, 847–850.
Lewandowski, K., Horn, R., O'Callaghan, C.J. et al. (1999) Free leptin, bound leptin, and soluble leptin receptor in normal and diabetic pregnancies. J. Clin. Endocrinol. Metab., 84, 300–306.
Marchini, G., Fried, G., Ostlund, E. et al. (1998) Plasma leptin in infants: relations to birth weight and weight loss. Pediatrics, 101, 429–32.
Masuzaki, H., Ogawa, Y., Sagawa, N. et al. (1997) Non-adipose tissue production of leptin: Leptin as a novel placenta-derived hormone in humans. Nature Med., 3, 1029–1033.[Web of Science][Medline]
McCarthy, J.F., Misra, D.N., Roberts, J.M. (1999) Maternal plasma leptin is increased in pre-eclampsia and positivity correlates with fetal cord concentration. Am. J. Obstet. Gynecol., 180, 731–736.[Web of Science][Medline]
Mise, H., Sagawa, N. and Matsumoto, T. (1998) Augmented placental production of leptin in pre-eclampsia: possible involvement of placental hypoxia. J. Clin. Endocrinol. Metab., 83, 3225–3229.
Ong, K.K., Ahmed, M.L., Sheriff, A. et al. (1999) Cord blood leptin is associated with size at birth and predicts infancy weight gain in humans. ALSPAC study team. Avon longitudinal study of pregnancy and childhood. J. Clin. Endocrinol. Metab., 84, 1145–1148.
Pederson, J. (1954) Weight and length at birth of infants of diabetic mothers. Acta Endocrinol., 16, 330–342.
Pi-Sunyer, F., Laferrere, B., Aronne, L.J. et al. (1999) Therapeutic controversy: obesity — a modern-day epidemic. J. Clin. Endocrinol. Metab., 84, 3–12.
Sagawa, N., Mori, T., Masuzaki, H. et al. (1997) Leptin production by hydatidiform mole. Lancet, 350, 1518–1519.[Medline]
Schubring, C., Kiess, W., Englaro, P. et al. (1997) Levels of leptin in maternal serum, amniotic fluid, and arterial and venous cord blood: relation to neonatal and placental weight. J. Clin. Endocrinol. Metab., 82, 1480–1483.
Shaarawy, M. and El-Mallah, S.Y. (1999) Leptin and gestational weight gain: relation of maternal and cord blood leptin to birth weight. J. Soc. Gynecol. Invest., 6, 70–73.[Web of Science][Medline]
Shekhawat, P.S., Garland, J.S., Shivpuri, C. et al. (1998) Neonatal cord blood leptin: its relationship to birth weight, body mass index, maternal diabetes, and steroids. Pediatr. Res., 43, 338–343[Web of Science][Medline]
Sierra-Honigmann, M.R., Nath, A.K., Murakami, C. et al. (1998) Biological action of leptin as an angiogenic factor. Science, 281, 1683–1686.
Tamura, T., Goldenberg, R.L., Johnston, K.E. et al. (1998) Serum leptin concentrations during pregnancy and their relationship to fetal growth. Obstet. Gynaecol., 91, 389–395.[Web of Science][Medline]
Tartaglia, L.A., Dembski, M., Weng, X. et al. (1995) Identification and expression cloning of a leptin receptor, Ob-R. Cell, 83, 1263–1271.[Web of Science][Medline]
Tomimatsu, T., Yamaguchi, M., Murakami, T. et al. (1997) Increase of mouse leptin production by adipose tissue after midpregnancy: gestational profile of serum leptin concentration. Biochem. Biophys. Res. Commun., 240, 213–215.[Web of Science][Medline]
Trayhurn, P., Hoggard, N., Mercer, J.G. et al. (1999) Leptin: fundamental aspects. Int. J. Obes., 23 (Suppl. 1), 22–28.
Yuen, B.S., McMillen, I.C., Symonds, M.E.. et al. (1999) Abundance of leptin mRNA in fetal adipose tissue is related to fetal body weight. J. Endocrinol., 163, R11–R14.[Abstract]
Yura, S., Sagawa, N., Mise, H. et al. (1998) A positive umbilical venous–arterial difference of leptin level and its rapid decline after birth. Am. J. Obstet. Gynecol., 178, 926–930.[Web of Science][Medline]
Submitted on December 17, 1999; accepted on May 25, 2000.
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