Molecular Human Reproduction, Vol. 6, No. 7, 595-601,
July 2000
© 2000 European Society of Human Reproduction and Embryology
Uterine physiology |
The endometrium as a novel target for leptin: differences in fertility and subfertility
1 Department of Anatomy and Reproductive Biology, Wendlingweg 2, RWTH University of Aachen, 52057 Aachen, 2 Department of Obstetrics and Gynecology, Pauwelsstraße 30, RWTH University of Aachen, 52057 Aachen, and 3 Institute of Reproductive Medicine, 66111 Saarbrücken, Germany
Abstract
Leptin and its receptor are involved in endocrine and paracrine regulation of metabolism, obesity and reproduction. Here, we describe the detection of the functional long isoform receptor of leptin in human endometrium. The leptin receptor protein was shown to be expressed in glandular and luminal epithelium and is periodically regulated throughout the menstrual cycle, demonstrating main expression in follicular and mid-luteal phase. In contrast, leptin receptor mRNA is detectable by reverse transcription–polymerase chain reaction (RT–PCR) as a constitutive component. Since RT–PCR analyses showed that leptin is not expressed in this tissue, the present study suggests that the human endometrium is a novel target for leptin. Therefore, we investigated 11 subfertile patients who underwent two biopsies in one menstrual cycle. The patients presented with a repetitive endometrial maturation defect, but showed adequate serum hormone concentrations and normal steroid hormone receptor expression and down-regulation in the endometrium. These patients were, however, deficient for expression of the functional leptin receptor. These analyses provide evidence that the lack of the leptin receptor in an ovulatory cycle may contribute to subfertility by a yet undefined `endometrial factor'.
endometrium/functional leptin receptor/immunohistochemistry/RT–PCR/subfertility
Introduction
Leptin is produced mainly in adipose tissue (Zhang et al., 1994
) and is rapidly cleared from the circulation by glomerular filtration followed by metabolic degradation in the renal tubules (Cumin et al., 1997). Human plasma leptin concentrations show pulsatile patterns in men and women. Obese patients show higher leptin concentrations, but pulsatility can be preserved (Licino et al., 1997). In menstruating women, plasma leptin increases from the early follicular phase to the mid-luteal phase and returns to the baseline by menstruation. In contrast, leptin concentrations did not change significantly in men or post-menopausal women. The cause of the increase in plasma leptin concentrations during the menstrual cycle has not yet been clarified (Riad-Gabriel et al., 1998). Initially, the leptin receptor was described in mouse choroid plexus (Tartaglia et al., 1995). Since then, several leptin receptor splice variants have been detected in human (Cioffi et al., 1996) and mouse tissues (Lee et al., 1996). There is evidence that leptin influences the hypothalamic–pituitary–adrenal (Glasow et al., 1998; Pralong et al., 1998) and hypothalamic–pituitary–thyroidal axes (Pinkney et al., 1998). The effects of leptin on the human hypothalamus, pituitary and ovary are also well known (Cioffi et al., 1997; Dieterich and Lehnert, 1998; Baskin et al., 1999). It may be that the well-known influence of various leptin concentrations on reproductive target organs, e.g. the effect on steroid production in the ovary (Zachow et al., 1997; Spicer and Francisco, 1998) could have a relevant regulatory capacity also for endometrial transformation and differentiation. The reproductive axis leads to the endometrium, as an important end organ that is necessary for successful reproduction. The fact that the leptin receptor has been identified in several organs of the reproductive axis, led us to search for leptin and its receptor in the endometrium of fertile and subfertile patients.
Materials and methods
Patients and controls
A total of 63 endometrial biopsies from normal cycling women of proven fertility (40.0 ± 4.1 years, mean ± SD) undergoing hysterectomy for benign uterine diseases were investigated. Informed consent was obtained from each woman, according to and by approval of the ethical committee of the Medical School, University of Aachen. All patients investigated had received no hormonal treatments in the last 3 months. Of these patients, 91% achieved pregnancies and births (range: 1–4 children). The biopsies were taken throughout the menstrual cycle: 12 biopsies at the early to mid-follicular phase (days 1–9), 11 biopsies at the late follicular phase (days 10–14), 11 biopsies at the early luteal phase (days 15–19), 18 biopsies at the mid-luteal phase (days 20–24) and 11 biopsies at the late luteal phase (days 25–28). The following parameters were taken into account for dating the endometrial biopsies of fertile and subfertile patients: (i) clinical report; (ii) histological dating (Noyes et al., 1950); (iii) assessment of serum hormone concentrations (progesterone, oestradiol, LH and FSH) on the day of hysterectomy or for the subfertile patients at the time of biopsy sampling (giving results within the normal ranges of all those hormones throughout the cycles; and (iv) in addition, for 30 biopsy samples immunohistochemical detection of progesterone receptors and oestrogen receptors as well as the proliferation marker Ki-67. The combination of these parameters contribute to an optimal dating of the endometrium.
In addition, endometrial biopsies were obtained from 11 subfertile women (32.90 ± 3.03 years, mean ± SD) in the late proliferative and mid-luteal phases of one untreated cycle. Subfertility was defined for a group of patients who did not achieve a pregnancy within 1 year, and who, after clinical examination in the infertility centre, revealed no pathological parameter except an endometrial thickness of <7 mm. This sonographic phenomenon was seen repeatedly in several stimulation cycles, regardless of the routine protocols for follicle stimulation using clomiphene; human menopausal gonadotrophin (HMG); recombinant FSH (rFSH); gonadotrophin-releasing hormone (GnRH) analogue/HMG or rFSH; oestrogen and rFSH. In all of these subfertile patients, the oestradiol and progesterone values in peripheral blood were within the normal physiological range throughout the cycles.
Normally, this group of patients would have been assigned in the category of `idiopathic infertility'. We were, however, studying these patients with particular attention to the specific insufficiency of endometrial function, focussing on the molecular, biological, histological, and immunohistochemical parameters. Our contention was that these patients have a reduced chance to become pregnant because of disturbed endometrial development and differentiation. Subfertile patients usually ovulated and, in particular, exhibited an adequate progesterone serum concentration in the mid-luteal phase.
Tissue samples of first trimester placenta served as a positive control, for immunodetection and reverse transcription–polymerase chain reaction (RT–PCR) analysis of leptin and its receptor (Masuzaki et al., 1997; Bodner et al., 1999).
RT–PCR
RT–PCR primers used to amplify the long isoform leptin receptor were (forward) 5'-GCT ATT TTG GGA AGA TGT-3' and (reverse) 5'-TGC CTG GGC CTC TAT CTC-3'. Annealing temperature was 50°C, 35 cycles. Primers for leptin analysis were (forward) 5'-CCA AAA CCC TCA TCA AGA C-3' and (reverse) 5'-CAC CTC TGT GGA GTA G-3' (Cioffi et al., 1997). Annealing temperature was 52°C, 35 cycles. Primers for the `housekeeping' gene cytochrome oxidase subunit I (CYT) were (forward) 5'-CGT CAC AGC CCA TGC ATT TG-3' and (reverse) 5'-GGT TAG GTC TAC GGA GGC TC-3'. Annealing temperature was 57°C, 20 cycles. Ethidium bromide staining of the agarose gel electrophoresis confirmed the expected 498 bp product for OB-R (B219 long form) and 338 bp product for leptin and a sequence analysis of the PCR products showed 100% homology to leptin and leptin receptor.
Immunohistochemistry
Immunohistochemistry was performed on paraffin sections (4–5 µm) using a streptavidin–biotin amplification. For the negative control, phosphate-buffered saline (PBS; Dulbecco), without Ca2+ and Mg2+/1.5% bovine serum albumin (BSA) replaced the primary antibody. In addition, rabbit immunoglobulin G (IgG; Dako, Hamburg, Germany) and goat IgG (Dianova, Hamburg, Germany) were used at an equal concentration as the primary antibody. We also performed a negative control with blocking peptides against leptin and leptin receptor [blocking peptide Ob (A20)P, blocking peptide Ob-R (C20)P, Santa Cruz Biotechnology, Heidelberg, Germany].
Paraffin-embedded sections for leptin and leptin receptor antibody were deparaffinized, rehydrated in a graded series of ethanol, rehydrated in PBS and pretreated with trypsin (Dako) for 15 min by room temperature for demasking antigens. Normal swine serum (Dako) was diluted in PBS/1.5% BSA (1:20) and incubated on sections for 10 min. The primary antibodies were applied overnight at 4°C [leptin antibody, Ob (A20), dilution 1:40; long isoform leptin receptor antibody, Ob-R (C20), dilution 1:40; Santa Cruz Biotechnology]. The sections were then incubated with a biotinylated second antibody (multi-link, swine anti goat, rabbit, mouse; Dako) for 30 min. Dilution for the second antibody was 1:150 in PBS/1.5% BSA. An incubation with streptavidin–peroxidase conjugate (Dako) with a dilution of 1:333 in PBS/1.5% BSA followed for 10 min. After each incubation step the tissues were washed three times with PBS. Visualising of the specific antigen was performed by peroxidase catalysing the substrate and converting the chromogen aminoethyl carbazole (AEC; Zymed Laboratories Inc, San Francisco, CA, USA) to a red deposit.
Steroid hormone receptors were analysed by immunohistochemistry from tissues of fertile and subfertile patients. We used antibodies from Immunotech (Hamburg, Germany) against the progesterone receptor (10A9, dilution 1:50) and oestrogen receptor 
(1D5, dilution 1:50).
Paraffin-embedded sections for oestrogen and progesterone receptor staining were also deparaffinized and rehydrated. The slides were heated in a citrate buffer by microwave (4x5 min; 600 W) for demasking the hormone receptor antigens. Immunohistochemical staining was performed by a streptavidin–biotin–peroxidase method (Histostain-SP Kit; Zymed Laboratories Inc) at room temperature.
Results
Detection of leptin and leptin receptor mRNA in human endometrium
We performed RT–PCR to identify the mRNA of leptin and the functional long isoform of leptin receptor. The leptin receptor mRNA was detectable as a constitutive component of the endometrial tissue throughout the menstrual cycle. However, RT–PCR revealed no signal for leptin mRNA in the human endometrium, whereas first trimester placenta tissue, serving as positive control, showed a strong signal (Figure 1). Sequence analysis of the amplified RT–PCR products confirmed that we had specifically amplified leptin and leptin receptor mRNA.
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Detection of leptin and leptin receptor protein in human endometrium
Immunohistochemistry revealed clearly positive staining of the long isoform of the leptin receptor and of leptin in glands and luminal epithelium of the endometrium during follicular and mid-luteal phases (Figure 2
). In the early luteal phase, the glands were almost negative, whereas in late luteal phase only the protrusions of these cells demonstrated a remarkable staining for leptin and its receptor. The luminal epithelium is mainly positive during follicular and mid- to late luteal phase.
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The connective tissue, i.e. the so-called stroma of the endometrium, stained quite weakly during the follicular phase, however demonstrated a stronger staining in the functionalis layer during the luteal phase endometrium. The basal layer of the endometrium remained only weakly stained. In some particular cases, we could detect a positive signal from endothelial cells of myometrial and occasionally of endometrial vessels with correlation to days 23–25 of the menstrual cycle. Table I
summarizes the results of leptin and leptin receptor staining in samples from women of known fertility.
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Analysis of endometrium from subfertile patients
In each case of the 11 individual subfertile patients, two biopsies were taken in the same menstrual cycle, one during follicular and one during mid-luteal phase. The biopsies taken during the first half of the patient's cycle demonstrated histologically a follicular phase endometrium. The endometrium at the time of the second biopsy during the mid-luteal phase, however, showed an arrested transformation at the early luteal phase, as has been deduced by the investigations on uterine secretion protein patterns in these patients (Alfer et al., 1998
, 1999a; Beier-Hellwig et al., 1989) although the patients did ovulate and display physiological concentrations of serum hormones. The inability to demonstrate immunohistologically any adequate staining pattern for leptin receptor and leptin in these patients second biopsy (d 23), was in accord with the classical dating of endometrial tissue by Noyes criteria (Noyes et al., 1950) of an histological early luteal phase (days 17–18). The second biopsy was negative in 6 cases (Figure 3) and exhibited positive protrusions in most glands (five cases). At the first biopsy of these patients, however, histologically in phase, the leptin receptor protein could not be identified in the glands (eight cases) (Figure 3) in contrast with fertile women. Three cases demonstrated a weak luminal and atypical staining. Fibroblasts (stroma) stained only weakly. Table II summarizes the results of leptin and lepin receptor staining in samples from subfertile women.
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Detection of steroid hormone receptors
Fertile and subfertile patients exhibited an adequate staining pattern for oestrogen and progesterone receptors in the glands and in the stroma of late follicular phase. Tissues of the mid-luteal phase demonstrated a down-regulation of these receptors until the glands were totally negative. In the stromal compartment the receptors could still be identified. There was no difference in steroid hormone receptor expression and down-regulation between the endometrium of fertile and subfertile patients.
Positive and negative controls
Immunohistological investigation of first trimester placenta revealed a positive signal for leptin and leptin receptor in the cytoplasm of syncytiotrophoblast cells (Figure 4). All controls for the endometrium and placenta, with non-immune IgG instead of the first antibody, were totally negative. The control tissues with blocking peptide added to leptin or leptin receptor antibody resulted in no signal in the glands or syncytiotrophoblast cells.
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Discussion
For the first time, to the best of our knowledge, the results presented here suggest the human endometrium as a novel endocrine target for leptin (Alfer et al., 1999b). Leptin itself is not expressed in this tissue, as shown by negative RT–PCR. We believe that the immunohistologically-detected leptin is of adipose tissue origin. The absence of any staining signal after pre-incubation of leptin receptor and leptin antibody with their blocking peptides (antigens for immunization) shows that they really identify their antigens. The functional long isoform of leptin receptor is permanently expressed in the human endometrium as shown by RT–PCR, contrary to the rat uterus, where only the short isoforms were detected (Chien et al., 1997). We could obtain only a small tissue biopsy from subfertile patients because their endometium was <3.5mm in thickness. So we decided to identify the receptor protein, because its mRNA is permanently expressed, as we have shown for fertile patients. The weak and atypical staining for the leptin receptor in some biopsies of subfertile patients may reflect its inadequate expression, as evidence for the endometrial defect. The very weak staining for leptin in the first biopsy of a subfertile patient, with negative staining for the long isoform leptin receptor, may possibly be a leptin bound to a short isoform of the leptin receptor or only background. Normally the leptin staining pattern corresponds to that of the leptin receptor.
The long isoform of the leptin receptor is able to activate the signal transducer and activator of transcription (STAT) pathway, whereas the short isoforms do not (Baumann et al., 1996; Bjorbaek et al., 1997). Our observation identifies the human endometrium as another leptin importing and binding tissue. The relevance of leptin in fertility has been shown convincingly in female and male mice (Chehab et al., 1996; Mounzih et al., 1997). In leptin deficient ob–/ob– female mice, which are grossly obese and infertile, food-restriction until the normal weight was restored, did not correct the phenotype of infertility. However, fertility could be restored by administration of recombinant leptin (Chehab et al., 1996).
It may be that the endometrium needs leptin in order to develop into a receptive tissue ready for implantation of a blastocyst at the mid-luteal phase of the cycle. Importantly, however, leptin is described as being involved in angiogenesis and blood vessel growth. The stimulation of endothelial cells by leptin leads to a marked enhancement of angiogenesis (Bouloumié et al., 1998). An angiogenic effect in the cornea of normal rats has been demonstrated, whereas leptin receptor-deficient fa–/fa– Zucker fatty rats lacked any corneal angiogenesis response (Sierra-Honigmann et al., 1998). As a positive control, vascular endothelial growth factor (VEGF) leads to a strong microvessel growth response in these fa–/fa– rats.
From our point of view, there is evidence that angiogenesis is one of the differentiation processes which are mediated by the leptin receptor and its ligand in the endometrial tissue. Further investigations are in progress to confirm this hypothesis. It is possible that the leptin receptor supports essentially molecular pathways leading to the differentiation of the endometrium. Interestingly, VEGF expression in the human endometrium demonstrates a spatial and temporal staining pattern (Classen-Linke et al., 2000) that is reminiscent of leptin receptor, with strongest signals in follicular and mid luteal phase.
In conclusion, we have shown for the first time that the long isoform of the leptin receptor is expressed in the human endometrium throughout the menstrual cycle and that leptin is not expressed in this tissue. Additionally, we demonstrated that a certain group of normally ovulating subfertile patients lack the leptin receptor in investigated cycles, which may make them unable to develop a `receptive' endometrium to accomplish embryo implantation.
Acknowledgments
The authors acknowledge the technical assistance of Sabine Eisner and Diana Seelis-Schmidt. This work was kindly supported by the START program of the School of Medicine, RWTH University of Aachen; further support was received by Schering AG Berlin, and last but not least by the Deutsche Forschungsgemeinschaft, Bonn (Grant Cl 88/3–2).
Notes
4 To whom correspondence should be addressed at: Department of Anatomy and Reproductive Biology, Wendlingweg 2, RWTH University of Aachen, 52057 Aachen, Germany. E-mail: jalfer{at}post.klinikum.rwth-aachen.de 
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Submitted on December 22, 1999; accepted on April 3, 2000.
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