Molecular Human Reproduction, Vol. 7, No. 2, 175-183,
February 2001
© 2001 European Society of Human Reproduction and Embryology
Uterine physiology |
The NF-
B pathway in human endometrium and first trimester decidua
1 MRC Human Reproductive Sciences Unit and 2 Obstetrics and Gynaecology, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh, EH3 9ET, UK
Abstract
Nuclear factor kappa B (NF-
B) regulates proinflammatory genes and may be involved in inflammation associated with reproductive events e.g. menstruation, implantation. Activation of NF-B involves several protein kinases and subsequent degradation of an endogenous inhibitor, IB
. This study details expression of NF-B pathway intermediates in human endometrium and first trimester decidua. Messenger RNA was detected for IB, and IB kinase 
(IKK, a scaffolding protein) and the protein kinases, IKK, IKKß, NF-B inducing kinase (NIK), mitogen-activated protein kinase Erk kinase kinase 1 (MEKK1) and TANK-binding kinase 1 (TBK1) using real-time quantitative polymerase chain reaction (PCR). IB and TBK1 mRNA were increased in the perimenstrual phase of the menstrual cycle. This suggests that there is activation of NF-B due to premenstrual progesterone withdrawal, since NF-B activity increases IB gene expression. Differential expression of NF-B pathway intermediates occurred when progesterone concentrations increased in early pregnancy; IKK and NIK mRNA levels increased in decidua whilst IKKß and MEKK1 mRNA levels declined. Expression profiles of IKK and NIK proteins were determined immunohistochemically. Both were detected in glandular epithelium and endothelium of endometrium. In decidua, both were present in epithelium and decidualized stromal cells. The results of this study suggest that NF-B is activated during menstruation. During early pregnancy, NF-B may also be activated (via IKK–NIK) and may regulate the expression of molecules vital for implantation and successful pregnancy. However, pro-inflammatory signalling to NF-B (via IKKß–MEKK1) may be down-regulated in early pregnancy, contributing to the immunosuppressive mechanisms which prevail at this time.
endometrium/menstruation/NFB/pregnancy/progesterone
Introduction
Several functions of the human endometrium are associated with inflammatory-like responses, e.g. implantation and menstruation (Finn, 1986
). These events involve the increased expression of proinflammatory molecules, the infiltration of leukocytes and tissue remodelling (Kelly, 1994). Inappropriate activation of inflammatory pathways is also likely to be associated with pathophysiological complaints, e.g. menstrual dysfunction.
Increased expression of inflammatory mediators, e.g. interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), tumour necrosis factor (TNF) and prostaglandins have been associated with menstruation (Tabibzadeh et al., 1995b; Baird et al., 1996; Jones et al., 1997; Critchley et al., 1999). Similarly, interleukin-6 (IL-6), leukaemia inhibitory factor (LIF) and the interleukin-1 (IL-1) system are thought to have a role in implantation (Simón et al., 1994; Stewart, 1994; Tabibzadeh et al., 1995a). Although the sex steroids are likely to be involved in the regulation of inflammatory mediator expression in endometrium, the molecular mechanisms involved are unclear.
Nuclear factor B (NF-B) is a transcription factor involved in inflammatory and immune responses (Baldwin, 1996). There are several members of the NFB subunit family, including p50, p65, c-Rel and Rel B. These proteins form homo- and heterodimers with the classic NFB heterodimer containing p50 and p65. NFB is held inactive in the cytoplasm by its endogenous inhibitor, IB, until stimulated by an extracellular signal, e.g. lipopolysaccharide (LPS) and IL-1. The sequential activation of a series of protein kinases is thought to lead to the phosphorylation of IB which is then degraded via a ubiquitin–proteasome pathway (Figure 1). Recent studies suggest that IB is likely to be phosphorylated by an IB kinase (IKK) complex (DiDonato et al., 1997; Mercurio et al., 1997). This complex is composed of the protein kinases, IKK (DiDonato et al., 1997) and IKKß (Woronicz et al., 1997; Zandi et al., 1997), and the scaffolding protein, IKK (Rothwarf et al., 1998). Additionally, an inducible IKK has been identified in mouse macrophages (Shimada et al., 1999) and a similar protein, TANK binding kinase 1 (TBK1), has been found in human cells (Pomerantz and Baltimore, 1999). IKK itself is thought to be phosphorylated by a mitogen-activated protein kinase kinase kinase (MAP3K). In particular, NF-B inducing kinase (NIK) and MAPK Erk kinase kinase 1(MEKK1) have been shown to be capable of activating IKK (Malinin et al., 1997).
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Steroid hormones have been found to interact with the NF-
B pathway. Glucocorticoids are thought to antagonize the actions of the pathway and it is likely that progesterone has similar actions (McKay and Cidlowski, 1999). Progesterone has been shown to increase the expression of IB (Wissink et al., 1998) and the progesterone receptor can interact directly with NF-B possibly resulting in mutual antagonism (Kalkhoven et al., 1996).
There is little data regarding the role of the NF-B pathway in endometrium, although a recent study suggested that it may be involved in the up-regulation of LIF and IL-6 during the implantation window (Laird et al., 2000). The present study investigates the NF-B pathway in human endometrium and first trimester decidua.
Materials and methods
Tissue collection
Endometrial biopsies (n = 32) were collected from women undergoing gynaecological procedures for benign conditions. All women reported regular menstrual cycles (25–35 days) and had not received hormonal treatment in the 3 months preceding biopsy. Biopsies were dated from the patient's last menstrual period (LMP). Additionally, serum was separated from venous blood samples collected at the time of biopsy and oestradiol and progesterone concentrations were measured by radioimmunoassay. Histological dating according to the previously described criteria (Noyes et al., 1950) and circulating sex steroid concentrations were consistent with the date of LMP. For analysis of polymerase chain reaction (PCR), the biopsies were considered as three groups: (i) perimenstrual; (ii) proliferative; and (iii) secretory phases. The term perimenstrual describes biopsies from the premenstrual and menstrual phases of the cycle. Although the histological appearance of premenstrual and menstrual biopsies differs, the concentrations of circulating oestradiol and progesterone at these times are very similar. The decline in progesterone concentrations premenstrually initiates menstruation and so, clinically, the most relevant time to investigate the mechanisms involved in menstruation is immediately prior to and during menstruation. Sex steroid concentrations relating to the endometrial biopsies used in real-time PCR experiments are shown in Figure 2.
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) and progesterone (
) concentrations relating to perimenstrual (peri), proliferative (prol) and secretory (sec) phase endometrial biopsies included in the polymerase chain reaction (PCR) studies. The term perimenstrual describes endometrial biopsies from the premenstrual and menstrual phases of the cycle.First trimester decidua (n = 6) was collected by curettage of the uterine wall away from the site of implantation prior to suction termination of pregnancy. Trophoblastic villi (n = 5) was also collected at this time. Decidual parietalis (without trophoblast) was confirmed by examination of haematoxylin and eosin stained sections and immunohistochemical staining for cytokeratin.
All tissue samples were collected in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma, Poole, Dorset, UK). Additionally, endometrial and decidual biopsies were fixed in 10% neutral-buffered formalin (NBF) overnight at 4°C, stored in 70% ethanol and then embedded in wax.
Written informed consent was obtained from all patients prior to biopsy collection and ethical approval was received from Lothian Research Ethics Committee.
Cell culture
T47D cells were cultured in RPMI 1640 medium (Sigma) supplemented with 10% fetal calf serum (Mycoplex; PAA Laboratories, Teddington, UK), penicillin (50 µg/ml; Sigma), streptomycin (50 µg/ml; Sigma) and gentamycin (5 µg/ml; Sigma). Fetal calf serum was charcoal stripped of steroid hormones prior to addition to culture medium. Cells were cultured in 25cm3 cell culture flasks (Corning Costar, High Wycombe, UK) at a concentration of 2x105 cells/ml. After passaging, cells were incubated for 24h to allow adherence to flasks. Cells were then treated with or without progesterone (10–6 mol/l) and incubated for 0, 2, 4, 8 and 24 h. After incubation cells were trypsinized, centrifuged at ~300 g for 3 min to form a pellet and resuspended in Tri reagent. RNA was extracted and RT–PCR was performed as detailed below.
RT–PCR
Tissue samples were immersed in Ultraspec (Ultraspec RNA Isolation System, Biogenesis Ltd, Poole, UK) or Tri reagent (Sigma) at the time of collection and samples were homogenized. RNA was extracted from tissue homogenates and T47D cells as detailed in the manufacturers' protocols.
Real-time quantitative PCR was used to determine amounts of the following NF-B signalling pathway intermediates: IB, IKK, IKKß, IKK, NIK, MEKK-1 and TBK1. This PCR method monitors progress of the PCR via detection of a fluorescent signal released, by the action of Taq polymerase, from a specific probe which contains both fluorescent dye and quencher. The amount of specific amplicon present is then related to ribosomal 18S (which remains constant relative to the amount of cDNA present) and subsequently, to an internal control. Throughout these PCR studies, a proliferative endometrial sample was used as the internal control.
Details of the reverse transcription and quantitative PCR have been described previously (King et al., 2000). Briefly, RNA samples were reverse transcribed using random primers. PCR reaction mixtures were made containing forward and reverse primers (both 300 nmol/l) and the specific probe (200 nmol/l) for the pathway intermediate along with primers and probe for ribosomal 18S. All samples were measured in triplicate and no template controls were included in all runs. The PCR reactions were run on ABI Prism 7700 using standard conditions. ß-actin signal was measured in all RNA samples (without reverse transcription) to detect genomic DNA contamination. All samples with a ß-actin measurement which fell below an arbitrary level of 27 (i.e. more than 3 SD from the mean of the other tissue samples) were excluded from the analysis.
Primers and probes were designed using the PRIMER express program (PE Biosystem; Table I). The concentrations of primers and probes used in the PCR reactions were optimized and linearity of response was validated using serial dilution of a standard pool of cDNA. Within assay variations of the PCR measurement of specific amplicons in cDNA were calculated from six replicates.
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Immunohistochemistry
NIK
Tissue sections were dewaxed in histoclear (National Diagnostics, Atlanta, GA, USA) and rehydrated in descending grades of alcohol. After washing in phosphate-buffered saline (PBS) sections were subjected to a microwave antigen retrieval step. Sections were heated in 0.01 mol/l sodium citrate for 10 min and then incubated for 20 min in the oven. Non-specific endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide (BDH Laboratory Supplies, Poole, UK) in distilled water for 10 min at room temperature. All tissue sections were subjected to a non-immune block with diluted normal goat serum (Vectastain 4002; Vector Laboratories, Peterborough, UK) for 20 min in a humidified chamber at room temperature. Tissue sections were then incubated overnight at 4°C with 50 µl of rabbit anti-NIK antibody (1:200 in goat serum; H-248, Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA). In negative control sections the primary antibody was substituted with an equimolar concentration of rabbit immunoglobulin (Ig; Vector Laboratories). Sections were then incubated with biotinylated goat-anti rabbit IgG (Vector Laboratories) followed by an avidin–biotin peroxidase (ABC) detection system (both for 60 min at room temperature; Dako Ltd, Cambridge, UK). The peroxidase substrate diaminobenzidine (DAB; Dako Ltd) was used to identify positive staining. Sections were then counterstained with Harris's haematoxylin (Pioneer Research Chemicals Ltd, Colchester, UK), dehydrated in ascending grades of alcohol and mounted from xylene in Pertex (Cellpath plc, Hemel Hempsted, UK).
IKK
Immunolocalization of IKK was performed using an identical protocol to that of NIK with the exception that the primary antibody used was rabbit anti-IKK (1:800 in goat serum; H-744, Santa Cruz Biotechnology) and that incubations with the secondary antibody and ABC were for 40 min only.
Scoring of immunohistochemistry
Intensity and location of NIK and IKK immunoreactivity was determined by a semi-quantitative scoring system. 0 = absence of immunoreactivity; 1 = faint immunoreactivity; 2 = strong immunoreactivity and 3 = very strong immunoreactivity. Each section was scored by two observers who had been blinded to the stage of menstrual cycle.
Statistical analysis
Significant difference was determined by analysis of variance (ANOVA; Statview 3.0) and individual differences were assigned using Fisher's protected least squares differences (PLSD) test.
Results
Differential expression of NF-B pathway intermediates
Messenger RNA was detected in endometrium and first trimester decidua for the following NF-B pathway intermediates: IB, IKK, IKKß, IKK, NIK, MEKK-1 and TBK1.
IB mRNA levels were found to be significantly greater in perimenstrual endometrium compared with endometrium from all other stages of the cycle (P < 0.05; Figure 3). A trend towards increased expression of IB was observed in secretory endometrium compared with proliferative endometrium with a further increase in first trimester decidua (proliferative/decidua P < 0.05; Figure 3).
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IKK
and NIK mRNA expression was increased in first trimester decidua relative to endometrium (P < 0.03; Figures 4a,b). These increases were significant when decidua was compared with all stages of the menstrual cycle with the exception of IKK in endometrial biopsies from the proliferative phase.
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MEKK1 mRNA expression was decreased in decidua compared with endometrium from all stages of the cycle (P < 0.05; Figure 4d
) while IKKß mRNA expression was decreased in decidua relative to perimenstrual endometrium (P < 0.02; Figure 4c). IKK mRNA was expressed throughout the menstrual cycle and in first trimester decidua. Expression was higher in the secretory phase than in first trimester decidua (P < 0.05; Figure 4e). Expression of TBK1 mRNA was increased in the perimenstrual phase relative to the secretory phase (P < 0.03; Figure 4f).
Immunolocalization of NIK and IKK
NIK and IKK showed similar expression profiles in endometrium throughout the menstrual cycle and in first trimester decidua. Both proteins were localized mainly to the endometrial glandular epithelium and endothelium. Luminal epithelial immunoreactivity was also apparent in some biopsies. Stromal immunoreactivity was observed only in the stroma immediately adjacent to the luminal epithelium. In decidua, epithelial staining was apparent. Both proteins were also detected in the decidualized stromal cells although NIK immunoreactivity was faint. Only IKK was detected in the decidual endothelium (Figure 5).
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No variations in the intensity or localization of NIK or IKK
immunoreactivity were observed during the menstrual cycle. Reduced IKK stromal staining was found in late secretory phase biopsies compared with proliferative phase and decidual biopsies, but this was likely to be due to sloughing of the stroma nearest the lumen.
IKK immunoreactivity showed a trend towards increased intensity in all compartments in the functionalis layer of endometrium compared with the basalis region. This gradient was observed at all stages of the menstrual cycle. However, this only reached significance in the stromal compartment in the proliferative phase.
Progesterone regulation of NFB pathway intermediates in T47D cells
T47D cells constitutively express the progesterone receptor at levels several powers greater than endometrial epithelial cell lines. Hence, they constitute a good cell model for investigation of the effects of progestrone on NFB pathway intermediates. Figure 6 details the mRNA expression of NFB pathway intermediates (IB, IKK, IKKß, IKK, NIK, MEKK-1 and TBK1) in T47D cells treated with and without progesterone. Expression of mRNA for each intermediate was measured at 0, 2, 4, 8 and 24 h. At all time-points, progesterone treatment increased IB mRNA expression relative to control values. Progesterone had no effect on IKK, IKKß, IKK, NIK, MEKK-1 and TBK1 mRNA expression.
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Discussion
This study details the expression of molecules involved in the activation of the proinflammatory transcription factor, NF-B, in human endometrium and first trimester decidua (see Figure 7). Messenger RNA for the intermediary molecules IB, IKK, IKKß, IKK, NIK, MEKK1 and TBK1 was measured and immunoreactivity for IKK and NIK proteins was detected. The NF-B signal transduction pathway is involved in the up-regulation of inflammatory genes, including cyclo-oxygenase-2 (COX-2) and chemokines, and as such, may have a role in the control of the inflammatory events associated with implantation and menstruation.
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The key molecule involved in the activation of NF-
B is the endogenous inhibitor, IB. The degradation of IB results in activation of NF-B and expression of IB has been found to be increased by activation of NF-B (resulting in a negative feedback loop) (Sun et al., 1993) and by the steroids, progesterone and dexamethasone (Auphan et al., 1995; Scheinman et al., 1995; Wissink et al., 1998). In addition to IB, there are several other members of the IB family including IBß and IB. Although these inhibitors are also likely to be involved in the control of NFB in endometrium, IB was of particular interest due to its reported up-regulation by progesterone, as confirmed in this study. This study also showed that there is a significant increase in the amount of IB mRNA in endometrium from the perimenstrual phase of the menstrual cycle compared with the proliferative and secretory phases. However, progesterone is unlikely to be directly responsible for this increase since at this time progesterone concentrations have declined due to the demise of the corpus luteum. Instead, it is possible that the NF-B pathway is activated in the perimenstrual phase resulting in stimulation of IB gene expression. Progesterone has been reported to increase IB and inhibit NF-B activation and so, progesterone withdrawal premenstrually may result in stimulation of the pathway. The increased expression of proinflammatory molecules, e.g. COX-2, IL-8 and MCP-1 (Jones et al., 1997) has been reported in perimenstrual endometrium and upon progesterone withdrawal in an in-vivo model (Critchley et al., 1999). NF-B has been reported to regulate expression of these mediators in other systems (Mauviel et al., 1992; Adcock et al., 1997; Martin et al., 1997). Therefore, activation of the NF-B pathway as a result of progesterone withdrawal premenstrually may provide a mechanism for the resulting up-regulation of these molecules in endometrium. It should be noted that mediators known to activate NFB are present in endometrium during menstruation (e.g. IL-1, TNF) and these are likely to contribute to the stimulation of NFB at this time. Further clarification of the role of the NFB pathway in endometrium was obtained by studying the kinases that could affect IB concentration.
The signal leading to the degradation of IB is phosphorylation followed by ubiquitination. The IKK complex responsible for the phosphorylation consists of the protein kinases, IKK and IKKß, and the scaffolding protein, IKK. IKK and IKKß have been suggested to have different functions with IKK being involved in morphogenesis (Hu et al., 1999; Takeda et al., 1999) while IKKß is thought to be primarily responsible for proinflammatory signalling to NF-B (Delhase et al., 1999; Li et al., 1999). It is interesting to speculate that in human endometrium and first trimester decidua the kinases may have different roles. At the end of the menstrual cycle, in the event of pregnancy, progesterone levels continue rising and decidualization is extended. Alternatively, in the absence of pregnancy, progesterone concentrations decrease rapidly and menstruation ensues. Our results show that the outcome of the menstrual cycle has an effect on IKK and IKKß mRNA expression. IKK mRNA is expressed in endometrium throughout the menstrual cycle with increased expression in decidua. In contrast, the amount of mRNA for IKKß was decreased in decidua relative to expression in perimenstrual endometrium. This suggests that the mRNA expression of the two kinases is differentially regulated and implies that different functions are likely. The increased levels of IKK mRNA may be related to the changes in differentiation taking place in decidua at this time. Alternatively, IKK may be involved, via NF-B and COX-2, in the expression of mediators, e.g. prostaglandin E2, which are vital for implantation and successful pregnancy (Psychoyos et al., 1995). The reduced amount of IKKß mRNA in decidua may suggest a mechanism to decrease proinflammatory signalling to NF-B at a time when local immunosuppression is necessary. Messenger RNA for the scaffolding protein, IKK, is expressed at a higher level in endometrium from the secretory phase than in first trimester decidua. IKK is reported to interact primarily with IKKß and it has been suggested that its function is to mediate the interaction between IKKß containing IKK complexes and upstream components of the activation pathway (Rothwarf et al., 1998; Mercurio et al., 1999). Thus, it is interesting that both IKKß and IKK mRNA expression is reduced in decidua.
The IKK complex is phosphorylated by the MAPKKKs, NIK and MEKK1. It has been reported that, although both IKK and IKKß are phosphorylated during activation of the pathway, NIK preferentially phosphorylates IKK while MEKK1 preferentially phosphorylates IKKß (Ling et al., 1998; Nakano et al., 1998). Our data show that the amount of mRNA for NIK is increased in decidua relative to endometrium while MEKK1 is decreased in decidua. This mirrors the patterns observed for IKK and IKKß mRNA expression and suggests that NIK and MEKK1 are similarly differentially expressed.
Immunohistochemical localization of IKK and NIK provides supportive evidence of the PCR results. Both proteins are present in the decidualized stromal cells of first trimester decidua but absent from the endometrial stroma. This may explain the increase in mRNA expression of both kinases in decidua. Both proteins are localized predominantly to the glandular epithelium of endometrium. The endometrial epithelium expresses several mediators which may be regulated by the NF-B pathway, e.g IL-1, TNF and prostaglandins (Tabibzadeh and Sun, 1992; Tabibzadeh et al., 1995b; Baird et al., 1996). A previous study reported that Rel A, a component of the NF-B heterodimer, is present in the glandular epithelium and provided evidence that the activation of NF-B in these cells may be responsible for the increase in the expression of IL-6 and LIF during the implantation window (Laird et al., 2000). Our data show that two of the kinases involved in the activation of the NF-B pathway are also expressed in the glandular epithelium. In addition, IKK and NIK are expressed in the endothelium of endometrium. Activation of NF-B in endothelial cells has been reported to modulate expression of molecules involved in angiogenesis (Yoshida et al., 1997). The presence of the pathway in endometrial endothelial cells is interesting as angiogenesis is involved in both implantation and menstruation (Rogers et al., 1992; Smith, 1998).
TBK1 is a kinase with 48% homology to an inducible IKK (Pomerantz and Baltimore, 1999). The mRNA levels of this protein were significantly increased in endometrium from the perimenstrual phase of the cycle when compared with the secretory phase. This implies that the progesterone withdrawal which occurs prior to menstruation may upregulate this kinase and suggests that TBK1 is the kinase most likely to have a role in the phosphorylation of IB at menstruation. After phosphorylation, IB is ubiquitinated prior to degradation by the proteasome. It has been reported that, in late secretory endometrium, ubiquitin immunoreactivity increases in the glandular epithelial cells (Bebington et al., 1999). However, this is unlikely to be related to increased ubiquitination of IB (and, hence, NFB activation) as the ubiquitin was detected in the nuclei, rather than the cytoplasm, of the cells.
As discussed above premenstrual progesterone withdrawal results in increased expression of IB. However, the effects of progesterone production on IB expression in endometrium remain unclear. There is a trend towards increased IB mRNA expression in the secretory phase of the cycle with a further increase apparent in first trimester decidua. This is consistent with the increasing concentrations of progesterone present in the second half of the cycle and in early pregnancy and suggests that progesterone may increase IB expression in the endometrium. This would result in decreased activity of NF-B, thus contributing to the local immunosuppressive activity of progesterone in the endometrium during the phase of the cycle receptive to implantation as well as in early pregnancy. Alternatively, increased IB mRNA expression in first trimester decidua could be an indirect result of NF-B activation via IKK and NIK. In favour of the former hypothesis, progesterone was found to increase IB mRNA expression in T47D cells. This is consistent with a previous study (Wissink et al., 1998) and would support a role for progesterone in increasing IB expression in decidua. The other pathway intermediates discussed were unchanged in the presence of progesterone. This suggests that the effects of progesterone on the pathway intermediates in decidua may be indirect. Alternatively, regulation of the pathway in T47D cells may differ to that in endometrium. IB stimulation by progesterone is likely to be chronic and it is possible that effects of progesterone on other intermediates may be found to occur at earlier timepoints.
In summary, this study provides evidence supporting a role for the NF-B pathway in the inflammatory events associated with menstruation and in the regulation of mediators crucial to successful pregnancy. We also suggest that the proinflammatory arm of the NF-B activation pathway is likely to be suppressed in first trimester decidua, in the absence of infection, thus contributing to the immunosuppressive mechanisms which prevail during pregnancy.
Acknowledgments
We are grateful to Dr A.R.W.Williams for his expert histological assessment of the endometrial biopsies. We also thank Mr I.Swanston for performing progesterone and oestradiol radioimmunoassays.
Notes
3 To whom correspondence should be addressed. E-mail: A.E.King-1{at}sms.ed.ac.uk 
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Submitted on September 27, 2000; accepted on November 23, 2000.
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