Leptin and leptin-binding activity in women with recurrent miscarriage: correlation with pregnancy outcome

S.M. Laird1,2,3, N.D. Quinton1, B. Anstie2, T.C. Li2 and A.I.F. Blakemore1,4

1 Division of Biomedical Sciences/Biomedical Research Centre, Sheffield Hallam University, City Campus, Sheffield S1 1WB, 2 Biomedical Research Unit, Jessop Hospital for Women, Leavygreave Road, Sheffield S3 7RE, UK4


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Previous studies in humans and mice have suggested the importance of leptin in fetal growth. Recurrent miscarriage may be a result of abnormal placental and/or fetal development and therefore abnormal leptin levels may be associated with this form of pregnancy loss. METHODS: Leptin and leptin-binding activity (LBA) were measured in blood obtained from women who had a history of recurrent miscarriage (n = 53) during weeks 5–6 and 7–8 of pregnancy, and the concentrations were correlated with subsequent pregnancy outcome. RESULTS: Concentrations of leptin ranged from 1.4–62.8 ng/ml, but there was a strong correlation (r = 0.825, P < 0.001) between leptin values at weeks 5–6 and 7–8 in the same woman. Women who subsequently miscarried had significantly lower plasma leptin concentrations on both weeks 5–6 (13.34 ± 2.1 ng/ml) (P < 0.05) and 7–8 (13.71 ± 2.4 ng/ml) (P < 0.01) of pregnancy, than women who subsequently had a term birth (22.04 ± 2.43 ng/ml week 5–6, 24.76 ± 3.66 ng/ml week 7–8). LBA values ranged from 1–8.5% but there was no significant difference in LBA in blood obtained from women who subsequently miscarried or had a live birth. CONCLUSIONS: The significantly lower concentrations of leptin in women who subsequently miscarried suggest that leptin may play a role in preventing miscarriage. However, as there was a considerable overlap between the values of leptin in women who subsequently miscarried, and those that had a live birth, these measurements are of limited use in the prediction of pregnancy outcome in these women.

Key words: leptin/leptin-binding activity/pregnancy outcome/recurrent miscarriage


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Leptin, the product of the ob gene, is produced by adipocytes and acts as an endocrine signal to the hypothalamus on the body fat mass. It also plays an important role in reproduction. Mice that are deficient in leptin (ob/ob mice) are infertile, but fertility can be restored by injection of recombinant leptin (Chehab et al., 1996Go; Cunningham et al., 1999Go). Several studies have suggested that this effect of leptin on reproduction is due to its interaction with the hypothalamic-pituitary-gonad axis and its ability to modulate LH and follicle stimulating hormone (FSH) production (Barash et al., 1997; Yu et al.,1997Go). In humans, plasma leptin concentrations are higher in females than males (Hickey et al., 1996Go) and are significantly higher during the luteal phase of the menstrual cycle (Hardie et al., 1997Go; Quinton et al., 1999aGo). Other studies, both in the mouse and human, have suggested that leptin may also have a direct effect on the reproductive organs. The presence of leptin receptors in ovarian granulosa and thecal cells has been shown, and leptin decreases steroid production by these cells (Karlsson et al., 1997Go; Spicer and Francisco, 1997Go). Recently the presence of both leptin and its receptor in the endometrium has also been reported (Alfer et al., 2000Go; Gonzalez et al., 2000Go).

Leptin is bound in the circulation by a high affinity binding protein that has been identified as a soluble leptin receptor. Plasma leptin-binding activity has only been measured in a limited number of studies, but has been shown to be lower in obese subjects (Houseknecht et al., 1996Go). In addition a recent study from our laboratory has shown concentrations of leptin-binding activity (LBA) to be low at birth, to increase during the pre-pubertal years and then to fall during puberty and remain stable during adult life. Concentrations of LBA correlated well with Tamer stage of puberty indicating that LBA concentrations may also be important for reproductive function (Quinton et al., 1999bGo).

Leptin is also produced by the placenta (Masuzaki et al., 1997Go) and serum concentrations of leptin are increased during pregnancy, reaching a peak at ~28 weeks gestation (Butte et al., 1997Go; Tamura et al., 1998Go). A number of recent studies have also suggested that low concentrations of leptin during pregnancy are associated with sub-optimal pregnancy outcomes (Lepercq et al., 1998Go; Mise et al., 1998Go) and that fetal birth weight is correlated with neonatal cord blood leptin concentrations (Shekhawat et al., 1998Go; Tamura et al., 1998Go; Schulz et al., 2000Go).

Between 1–2% of fertile women experience recurring pregnancy loss (Li, 1998Go, Brigham et al., 1999Go), which is associated with a number of conditions including parental chromosomal anomalies, various endocrinological abnormalities including polycystic ovarian syndrome (PCOS) and luteal phase deficiency, uterine anomalies, cervical incompetence and anti-phospholipid syndrome (Li, 1998Go). No apparent cause can be found in ~50% of cases (Quenby and Farquharson, 1993Go). In the majority of cases the miscarriage may be associated with abnormal development of the feto-placental unit.

One previous study measured concentrations of leptin in women with a history of recurrent miscarriage and compared them to normal women in the first trimester of pregnancy (Lage et al.1999Go). However in this study leptin was measured in the 24 h subsequent to fetal loss. It is therefore possible that the decreased concentrations seen compared with non-pregnant women and women in the first trimester of pregnancy could be a consequence of the miscarriage itself. In our study plasma leptin and LBA were measured during early pregnancy in women who had a history of at least three miscarriages. The levels were correlated with subsequent pregnancy outcome in these women.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Human subjects
All women included in this study attended the Recurrent Miscarriage Clinic at the Jessop Hospital for Women, Sheffield. They all had a history of three or more consecutive miscarriages. Immediately after the women had conceived they attended the Recurrent Miscarriage Clinic at regular intervals for blood sampling. The plasma samples were stored at –20°C until analysed. They all had pelvic ultrasonography at week 6 to confirm the presence of a pregnancy. Any women with evidence of pregnancy loss at this stage were excluded from the study. All women gave informed consent for the use of their blood in this study.

Leptin and LBA were measured in plasma obtained from 53 recurrent miscarriage women. Twenty-three of these women subsequently miscarried, a further 23 women had a term pregnancy while the other seven women had a pre-term birth. Among the 53 subjects, 28 (53%) had primary recurrent miscarriage (never had a previous live birth), including 13/23 women who miscarried, 12/23 women who had a live birth and 3/7 women who had a pre-term birth. The majority (40/53, 75%) of women suffered from unexplained recurrent miscarriage. In the unexplained group of women 21/40 suffered secondary recurrent miscarriage and 19/40 suffered primary recurrent miscarriage. In women who had a term birth, two had PCOS, one had a uterine abnormality (septate uterus), one had anti-phospholipid syndrome, and one had both anti-phospholipid syndrome and a uterine abnormality. In women who miscarried, three had anti-phospholipid syndrome, two had PCOS, one had a uterine abnormality, one had PCOS and a uterine abnormality and one had PCOS and anti-phospholipid syndrome. In addition one of the women who had a pre-term birth had PCOS. The six women with anti-phospholipid syndrome were undergoing treatment with aspirin (25 mg per day) and clexane (20 mg per day) when the blood samples were taken.

Leptin and LBA were measured in samples obtained at fortnightly intervals either on weeks 5 and 7, or weeks 6 and 8 of pregnancy for all but four women, in whom only one blood sample was obtained. The pregnancies were dated according to the last menstrual period. For analysis, the samples obtained on weeks 5 and 6 and weeks 7 and 8 were considered together. The timing of the miscarriage was defined by the loss of fetal heartbeat.

Leptin measurement
Leptin was measured using a specific Human Leptin ELISA kit (R & D Systems, Abingdon, Oxon, UK) according to the manufacturers instructions. Plasma samples were diluted 1:100 as suggested to ensure that values were within the standard curve. The sensitivity of the assay was 7.8 pg/ml and the inter-assay and intra-assay variations were 3.5–5.4% and 3.0–3.3% respectively.

Leptin-binding activity measurement
LBA was measured using our previously published method (Quinton et al., 1999bGo). Endogenous leptin was removed from the plasma samples using dextran-coated charcoal. 500 µl of pre-chilled Norit A charcoal (2%) and Dextran T70 (0.2%) in assay buffer (0.01 mol/l phosphate buffer (pH 7.4), 0.18 mol/l MgCl2, 1% BSA) was added to 200 µl of plasma at room temperature, vortexed, and immediately placed on ice for 5 min. The sample was centrifuged at 4°C for 12 min at 10 000 g. The supernatant was removed and placed in a new tube and stored at –20°C. Fifty microlitres of stripped plasma was incubated overnight with 150 µl assay buffer, 100 µl [I125] leptin, in the presence (non-specific) or absence (specific) of 1 µg unlabelled leptin (R & D Systems). Bound and unbound leptin was separated by the addition of 1 ml pre-chilled dextran coated charcoal (2% charcoal, 0.2% dextran in assay buffer). After vortexing the samples were incubated at 4°C for 10 min and then centrifuged at 10 000 g for 12 min. One ml of supernatant was aspirated and counted in a gamma counter. The specific binding (total binding minus non-specific binding) was expressed as a percentage of the total [I125] leptin counts and called the LBA. All samples were measured in duplicate.

Statistical analysis
Leptin concentrations and LBA were compared in samples obtained from women who subsequently miscarried and those who had a term birth, and between women who had a pre-term birth and those who had a term birth, using the Student's t-test or the non-parametric Mann–Whitney test as appropriate.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Plasma leptin concentrations varied between 1.4–62.8 ng/ml in this group of recurrent miscarriage women. Despite this wide range in leptin concentrations throughout the whole group, leptin concentrations in blood obtained in week 5–6 and week 7–8 of pregnancy were similar in individual women. There was a strong correlation (r = 0.825; P < 0.001) between concentrations of leptin obtained on both weeks 5–6 and 7–8 of pregnancy in the same women regardless of pregnancy outcome. In addition similar correlations between leptin concentrations obtained on weeks 5–6 and 7–8 were seen in women who subsequently miscarried (r = 0.841; P < 0.001) and those who had a term birth (r = 0.860; P < 0.001). The correlation for the pre-term group was slightly lower (r = 0.604; P < 0.05), but this probably reflects the fact that there were only six patients in this group. The time of miscarriage is known for 19 of the 23 miscarried pregnancies and of these 19, only seven miscarried within a week of the second leptin measurement (week 8). The timing of the other 12 miscarriages was seven at week 9, one at week 10, two at week 12 and two at week 18. Leptin concentrations at both weeks 5–6 and 7–8 showed no correlation with the week of miscarriage.

Table IGo shows concentrations of leptin in the whole group of recurrent miscarriage women divided according to age, number of previous miscarriages, BMI, pregnancy outcome, type of recurrent miscarriage (primary or secondary) and whether the reason for repeated miscarriage is known. Only when patients were divided according to BMI and subsequent pregnancy outcome were any significant differences in leptin concentrations observed. Multivariate analysis of these parameters using either ANOVA or logistic regression analysis suggested that for this population of women only leptin concentrations (P < 0.032 and P < 0.038 for weeks 5–6 and 7–8 respectively) were significantly important in pregnancy outcome. The next most important parameter appeared to be BMI, but this correlation failed to reach statistical significance. Analysis using the Students t-test showed that women who subsequently miscarried had significantly lower plasma leptin concentrations on both weeks 5–6 (P < 0.05) and 7–8 (P < 0.01) of pregnancy than women who subsequently had a term birth. In contrast, there was no significant difference in the plasma leptin concentrations obtained from women who subsequently had a term birth or pre-term delivery at either time period of pregnancy. In addition levels of leptin were lower in women with a BMI of <=25 on both weeks 5–6 (P < 0.01) and weeks 7–8 (P < 0.01) of pregnancy compared to women with a BMI >25.


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Table I. Plasma leptin concentrations (ng/ml) in recurrent miscarriage (RM) women
 
Figure 1Go shows the concentrations of leptin in the plasma of recurrent miscarriage women obtained on weeks 5–6 and 7–8 of pregnancy who subsequently miscarried, had a live birth or had a pre-term delivery. Calculation of the 5th percentile values for plasma leptin for the women who had a term pregnancy gives a value of 6 ng/ml at week 5–6 and 8 ng/ml at week 7–8 of pregnancy. Seven out of 22 women who miscarried (32%) had plasma leptin concentrations below these values both at weeks 5–6 and 7–8. In contrast none of the women who had a pre-term birth had leptin below this value. Table IIGo shows that there were no significant differences in the age, BMI and number of previous miscarriages in the recurrent miscarriage women who subsequently miscarried, had a live birth or a pre-term delivery.



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Figure 1. Concentration of leptin in serum samples from recurrent miscarriage women on week 5–6 and 7–8 of pregnancy who subsequently had a term pregnancy, miscarriage or pre-term delivery. Solid line indicates the mean value. Concentrationswere significantly lower in women who miscarried (P < 0.05 and P < 0.01). Dashed line indicates the 5th percentile values for term pregnancy values at the same time in pregnancy.

 

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Table II. Age, BMI and miscarriage history in women who miscarried, had a term pregnancy or a pre-term birth
 
The LBA values ranged from 1.1–8.5% in this group of women. In contrast to leptin, there was no correlation between levels of LBA in blood from the same women obtained on weeks 5–6 and weeks 7–8 of pregnancy. In addition there was no correlation between LBA and leptin concentrations in the same blood samples and LBA did not correlate with BMI. There was no significant difference in the concentrations of LBA in blood obtained from women who subsequently miscarried (3.79 ± 0.39% at week 5–6, 3.36 ± 0.35% at week 7–8), had a live birth (4.0 ± 3.8% at week 5–6, 4.1 ± 0.45% at week 7–8) or a pre-term delivery (3.72 ± 0.65% at week 5–6, 3.43 ± 0.88% at week 7–8) (Figure 2Go).



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Figure 2. Leptin-binding activity in serum samples from weeks 5–6 and 7–8 of pregnancy from women suffering from recurrent miscarriage who subsequently had a term pregnancy, miscarriage or pre-term delivery. Solid line indicates the mean value. No significant differences were seen between any group of women.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Previous studies have suggested the importance of leptin in reproduction. It has been shown to be produced by trophoblastic placental cells (Masuzaki et al., 1997Go; Yura et al., 1998Go) and concentrations increase during pregnancy to reach a maximum at about week 30 (Lewandowski et al., 1999Go; Stock et al., 1999Go). Low concentrations of maternal plasma leptin are associated with sub-optimal pregnancy outcomes (Mise et al.1998Go; Lage et al., 1999Go; Lea et al., 2000Go) and fetal birth weight is correlated with neonatal cord blood leptin concentrations (Shekhawat et al., 1998Go; Tamura et al., 1998Go). In this study, we measured concentrations of leptin in blood obtained during the first trimester of ongoing pregnancies from women suffering from recurrent miscarriage and correlated it with a number of parameters that are known to be important in determining pregnancy outcome in these women. In contrast to other studies (Lage et al., 1999Go), where only one sample was taken from each woman, in this study we measured leptin in blood samples obtained from each woman at two different time periods in early pregnancy, to increase the accuracy of the results obtained. The fact that the dating of the pregnancy was from the day of the last menstrual period and the desire to obtain two blood samples meant that the only period of time which was suitable for this study were weeks 5–8 of pregnancy. Plasma leptin correlated with pregnancy outcome, with statistically lower concentrations present in women who miscarried, and BMI, but showed no correlation with age, number of previous miscarriages, type of recurrent miscarriage (primary or secondary) or whether the reason for repeated miscarriage was known. However, although serum leptin was statistically lower in women who subsequently miscarried, there was a large overlap in concentrations in the two groups of women. Therefore, on its own, leptin measurement may not be clinically useful in predicting pregnancy outcome.

Previous studies have shown that plasma leptin concentrations in normal fertile women are increased in the first trimester of pregnancy compared with pre-pregnancy (Hardie et al., 1997Go; Lage et al., 1999Go). The concentrations of leptin measured in our study appeared to be slightly lower than those found in normal fertile women in the previous study. This may be due to differences in the assay systems used. However, the concentrations of leptin in women who had a live birth in our study were higher than those found in the luteal phase of the menstrual cycle reported in our previous study using the same method (Quinton et al., 1999aGo).

The aim of this study was to correlate plasma leptin with pregnancy outcome in women who suffer recurrent miscarriage; regardless of the cause of repeated miscarriage. This does however produce a heterogeneous population. Sub-group analysis of the data from only women with unexplained miscarriage (n = 40) produce results similar to the whole group. In addition, the number of recurrent miscarriage women below the 5th percentile value for those who had a live birth was also similar (data not shown). This suggests that in this group of women lowered leptin concentrations are associated with miscarriage whatever the cause.

Leptin is a product of adipose tissue and plasma concentrations are known to correlate with the BMI (Considine et al., 1996Go). However as pregnancy progresses changes in maternal leptin and BMI do not correlate as the placenta begins to contribute to plasma leptin (Ashworth et al., 2000Go). Plasma leptin concentrations correlated well with BMI in this study. There was however no significant difference in the BMI values in women who miscarried, had a live birth or had a pre-term birth. Thus the differences in leptin concentrations in the miscarriage and live birth group of women may reflect an independent association and suggests that, even at this early stage in pregnancy, placental derived leptin contributes to maternal circulating leptin. We, and others, have previously shown that low concentrations of human chorionic gonadotrophin (HCG) in women suffering from recurrent miscarriage are associated with subsequent pregnancy loss (France et al., 1996Go; Li et al., 1998Go). In this study there was no overall correlation between the amounts of HCG and leptin in the same samples (data not shown). This may be due to the fact that HCG is only produced by the placenta, while leptin is also produced by other tissues.

It is possible that the lower leptin values in plasma in weeks 7–8 of pregnancy may be a consequence of an already failing pregnancy. However the similar correlations seen between leptin concentrations on weeks 5–6 and weeks 7–8 in both women who miscarried and women who had a term pregnancy suggest that this is not the case. The fact that concentrations of leptin at weeks 5–6 were significantly lower in the group of women who subsequently miscarried than those who had a live birth, despite the fact that the miscarriage did not occur for at least another 2 weeks, and the lack of correlation of leptin concentrations with time of miscarriage, further supports the view that the decreased leptin in women who miscarried is not due to an already failing pregnancy.

This study has shown decreased concentrations of leptin in women who subsequently miscarry. Previous studies have also shown low concentrations of leptin in growth retarded pregnancies and increased maternal plasma leptin concentrations have also been associated with pregnancies complicated by pre-eclampsia (Mise et al., 1998Go; Anim-Nyame et al., 2000Go) and diabetes (Lepercq et al., 1998Go; Lea et al., 2000Go). Taken together this suggests a role for leptin in the maintenance of healthy pregnancy. All these conditions are thought to be due to an abnormal development of the feto-placental unit, which is under multi-factorial control. Recent studies have shown that the production of leptin by cytotrophoblast cells is modulated by IL1ß and 17ß oestradiol (Chardonnens et al., 1999Go). In other cells leptin is known to interact with cytokine networks (Granowitz, 1997Go; Yamaguchi et al., 1998Go) and therefore leptin may affect pregnancy outcome by affecting the balance of cytokines in the feto-placental unit. In addition leptin has been shown to affect cytotrophoblast cell function. It increases HCG synthesis and increases integrin and metalloproteinase production by cytotrophoblast cells (Gonzalez et al., 2000Go). Finally leptin has angiogenic activity (Sierra-Honigman et al., 1998; Ashworth et al., 2000Go) and therefore could affect placental capillary growth.

In contrast to leptin concentration, plasma LBA was similar in all three groups of women. LBA is postulated to be due mainly to the truncated, soluble form of the leptin receptor. The presence of the leptin receptor in human placenta has been shown (Lea et al., 2000Go; Schulz et al., 2000Go) and one of these studies suggests that the short form of the receptor, of a similar size to the soluble form, is the predominant form found in placental tissue (Schultz et al., 2000). As with other soluble cytokine receptors, the presence of this soluble receptor competitively inhibits the binding of leptin to membrane bound cellular receptors (Liu et al., 1997Go). Another function of the soluble leptin receptor may be to deliver leptin to target tissues (Gavrilova et al., 1997Go) and it is postulated that its role in the placenta is to enhance delivery of leptin to the feto-placental unit (Schulz et al., 2000Go). There are few studies of plasma LBA concentrations during early pregnancy, but concentrations of LBA in plasma of pregnant women obtained during weeks 20–30 of pregnancy appear to be similar to those of non-pregnant women (Lewandowski et al., 1999Go). The LBA concentrations found in this study in blood obtained early in pregnancy were similar to those of non-pregnant adult women published in our previous study (Quinton et al., 1999bGo). Taken together, these studies suggest that in human pregnancy there is no increase in plasma LBA. The lack of correlation between LBA and pregnancy outcome suggests that leptin itself is the most important component of the leptin system in recurrent miscarriage. This agrees with the work of others who have shown changes in leptin, but not its receptor, in the placenta of diabetic and fetal-growth retarded pregnancies (Lea et al., 2000Go).

In summary, this is the first report of the measurement of plasma leptin and LBA in early pregnancy in women who suffer recurrent miscarriage. The results show that leptin concentrations were lower in recurrent miscarriage women who subsequently miscarried compare with those who had a live birth. In contrast, there was no difference in LBA between both groups of women. The significantly lower concentrations of leptin in women who subsequently miscarried suggest that leptin may play a role in preventing miscarriage. However, as there was a considerable overlap between leptin levels in women who subsequently miscarried and had a live birth these measurements, by themselves, are of limited use in the prediction of pregnancy outcome in these women.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors would like to thank the nursing staff of the recurrent miscarriage clinic and Elizabeth Tuckerman for their assistance in the collection of samples for this study and to Dan Meechan for assistance with the LBA assay. The work was supported financially by the Biomedical Research Centre at Sheffield Hallam University and by Knoll Limited (BASF).


    Notes
 
3 To whom correspondence should be addressed at: Division of Biomedical Sciences/Biomedical Research Centre, Sheffield Hallam University, City Campus, Sheffield S1 1WB, UK. E-mail: s.m.laird{at}shu.ac.uk Back

4 Current address: Department of Medical and Community Genetics, Imperial College of Science, Technology and Medicine, Kennedy-Galton Centre, Northwick Park Hospital, Watford Road, Harrow HA1 UJ, UK Back


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 Materials and methods
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 Discussion
 Acknowledgements
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Submitted on March 21, 2001; accepted on June 6, 2001.