Contraception: Expression of progesterone receptor mRNA in the endometrium during the normal menstrual cycle and in Norplant(R) users

The expression of endometrial progesterone receptor mRNA duringthe human menstrual cycle and in Norplant users was studiedusing digoxigenin-labelled ribonucleic probes for in-situ hybridizationon 6 µm paraffin embedded endometrial sections. The stainingintensity was scored blind semi-quantitatlvely. Blood ovariansteroid concentrations were measured in Norplant users. Alldata were analysed by analysis of variance. Glandular progesteronereceptor mRNA concentrations were low during the menstrual-to-earlyproliferative stage but increased during the early-to-mid tolate-proliferative stage then declined non-significantly overthe secretory stage. No such variation was observed in stromalcells. Progesterone receptor mRNA concentrations were lowerin Norplant than controls during early-to-mid to late-proliferativestages (in glandular epithelium and stroma) and during secretorystage (in stroma only). Norplant subjects with amenorrhoea hadhigher concentrations of stromal progesterone receptor mRNAbut lower plasma oestrogen concentrations than subjects withbreakthrough bleeding. The pattern of variation in progesteronereceptor mRNA concentrations during the normal menstrual cycleresembles the published pattern for the receptor protein. Theresults demonstrate: (i) a differential sensitivity of glandularand stromal progesterone receptors to steroid regulation; (ii)in contrast to previous findings of an increase in immunoreactiveprogesterone receptor protein in Norplant endometrium, progesteronereceptor mRNA concentrations in these tissues were reduced;and (iii) there was significantly more progesterone receptormRNA in subjects with amenorrhoea than in those with breakthroughbleeding.     

Hormonal and embryonic regulation of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in the human endometrium and the human blastocyst

Chemokines are implicated in the implantation process. The aim of this study was to investigate mRNA expression and protein levels of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in human endometrium throughout the menstrual cycle, during HRT and in the human blastocyst. The regulation of chemokine receptors in the endometrial epithelium was also studied using an in-vitro model for the apposition phase of human implantation. We found up-regulation of endometrial CXCR1 mRNA (419-fold increase), CCR5 mRNA (612-fold increase) and CCR2B mRNA (657 fold-increase) during the luteal phase peaking in the pre-menstrual endometrium. CXCR4 mRNA levels presented a specific although modest (18-fold increase) up-regulation during the implantation window. These findings were corroborated at the protein level in natural and HRT cycles. Immunoreactive CCR5 and CCR2B receptors were detected in human blastocysts whereas CXCR4 and CXCR1 were not present. Chemokine receptors in cultured endometrial epithelial cells showed an up-regulation and polarization of CXCR1, CXCR4 and CCR5 receptors when a human blastocyst was present. The specific distribution and regulation of chemokine receptors in the endometrial epithelium and the human blastocyst suggest a possible implication of these receptors in the apposition and adhesion phases of human implantation.     

Assessment of the proliferative status of epithelial cell types in the endometrium of young and menopausal transition women

BACKGROUND: We determined protein and mRNA expressions of markers of normalhuman endometrial proliferation and hypothesized that dysregulationof the endometrial response to estradiol (E₂) and progesteronewould be observed in the older menopausal transition (MT) womencompared with mid-reproductive age (MRA) controls. METHODS: Endometrial biopsies were prospectively obtained from MRA andMT non-randomized healthy volunteers during proliferative (±exogenous E₂) and secretory (MRA only) menstrual cycle phases.mRNA and/or nuclear protein expressions of proliferative markers(MKI67, PCNA and MCM2), cell-cycle regulators (cyclins A1, E1and D1 and cyclin dependant kinase Inhibitor B; CCNA1, CCNE1,CCND1 and CDKN1B) and sex-steroid receptors [estrogen receptor(ER) and progesterone receptor (PR)] were assessed in endometriallumen, gland and stroma. RESULTS: MRA women had significantly higher proliferative than secretoryexpression of MKI67, PCNA, MCM2, CCNA1, CCNE1, ESR1 and PGRin lumen and gland (minimal stromal changes), whereas CDKN1Bprotein expression was higher during the secretory phase. E₂-treatmentof MT women led to relatively less MKI67 glandular protein expressioncompared with MRA women; no other age-related differences wereobserved. CONCLUSION: Although the MT does not appear to alter the proliferative cellphenotype of endometrial epithelium and stroma, the data suggestthat prior to the MT, age is associated with a decrease in someproliferative markers and steroid receptor expression statuswithin different endometrial cell types.     

Gene expression pattern and immunoreactive protein localization of LGR7 receptor in human endometrium throughout the menstrual cycle

Relaxin (RLX) is a pregnancy-associated polypeptide hormone. In non-pregnant women, the peak of circulating relaxin coincides with the window of endometrial receptivity and both in vivo and in vitro experiments showed that it plays a role in the decidualization process. Recently, two receptors, LGR7 and LGR8, have been identified as high affinity receptors for relaxin. Here we describe LGR7 mRNA and protein expression in human endometrium using semi-quantitative and quantitative fluorescent PCR (Q-PCR) and immunohistochemical analyses. Three different experimental designs were used. First, endometrial biopsies from five different phases of the menstrual cycle were analysed. Secondly, we assessed the early luteal phase in more detail. Finally we analysed the expression at LH+2 (2 days after the natural LH surge, pre-receptive endometrium) versus LH+7 (receptive endometrium) within the same menstrual cycle from the same patient to avoid inter-cycle or inter-person variations in gene expression. Our results indicate that there is no consistent regulation of LGR7 mRNA expression, neither during the menstrual cycle nor during the early-mid-luteal phase. In general, we observed a large degree of variation in LGR7 mRNA expression levels between patients. LGR7 immunoreactive protein was identified in all stages of the menstrual cycle. LGR7 protein was localized in both the epithelial and the stromal compartments, except for the mid-luteal phase when the expression was restricted to the endometrial epithelium. We conclude that no consistent regulation of LGR7 mRNA expression can be detected in human endometrium during the menstrual cycle.     

Progressive rise in the expression of interleukin-6 in human endometrium during menstrual cycle is initiated during the implantation window

In order to be prepared for implantation, human endometriumundergoes a predictable series of proliferative and secretorychanges. Cytokines play an important role in regulation of thesechanges. Therefore, in this study, we immunolocalized the cytokine,interleukin-6 (IL-6), its receptor and the signal transducergp130 in human endometrium throughout the menstrual cycle. Duringthe entire menstrual cycle, the IL-6 receptor and gp130 werefound primarily in the endometrial glands and to a lesser extentin the stroma. The immunoreactivity of these proteins did notchange in endometrial cells during the entire menstrual cyclewith an exception of reduced immunoreactivity of gp130 in endometrialglands during menstrual phase. Immunostaining showed that immunoreactiveIL-6 was weakly expressed in human endometrium during the proliferativephase. Strong immunoreactivity for IL-6 appeared in endometriumduring the putative 'implantation window'. Expression was byfar most pronounced both in the glandular and surface epithelialcells. The amount of immunoreactive IL-6 in the epithelium progressivelyincreased during the secretory/menstrual phases. During thelate secretory phase, only stromal cells in the upper functionalisexhibited immunoreactivity for IL-6. Western blot analysis corroboratedthe immunohistochemical data. Human endometrial IL-6 consistedof a protein with an apparent mobility of 26 kDa. The immunoreactiveband of IL-6 was weak in the proliferative phase. The expressionof this protein increased progressively during the secretory/menstrualphases. The findings show a cell-specific pattern of distributionfor immunoreactive IL-6 in human endometrium. The menstrualcycle-dependent expression of IL-6 suggests that this cytokinemay play a role in changes in endometrium that prepare thistissue for implantation and menstrual shedding.     

Variant progesterone receptor mRNAs are co-expressed with the wild-type progesterone receptor mRNA in human endometrium during all phases of the menstrual cycle

Progesterone receptor (PR) variant mRNAs in human endometrium could encode proteins with the potential to alter progesterone action in states of normal and abnormal endometrial development. We have assessed the expression levels of mRNA for the wild-type PR and splice variants of PR mRNA lacking exon 4 (del-4 PR), exon 6 (del-6 PR), exons 4 and 6 (del-4&6 PR), and part of exon 4 (del-p4 PR) or part of exon 6 (del-p6 PR) in the human endometrium throughout menstrual cycle development. Eighty-eight endometrial specimens (47 proliferative, 41 secretory) were collected from patients undergoing hysterectomy for benign gynaecologic causes. Measurements by RT-PCR indicated that mRNAs for wild-type PR, and splice variants del-4 PR, del-6 PR, del-4&6 PR, del-p6 PR, and a novel del-p4 PR were detected in all endometrial specimens throughout the menstrual cycle. Higher levels of wild-type PR and all PR variant mRNAs were found in the early and mid-proliferative endometrial phases than in secretory endometrium. The relative expression of mRNA for all PR variants compared to wild-type PR mRNA, however, did not change through all stages of endometrial development. We, therefore, found no evidence of differential co-expression of the PR variants compared with wild-type PR during normal menstrual development. Future studies will determine if the expression profile of PR variant mRNAs will be different in the endometrium of patients with infertility, recurrent pregnancy loss, or endometrial adenocarcinoma.     

Immunocytochemical study of progesterone receptors in the human endometrium during the menstrual cycle

Specific mouse monoclonal antibody (alpha PR6) against progesterone receptor was used with an avidin biotin complex technique to localize progesterone receptors in frozen sections of 26 normal cyclic human endometria. Progesterone receptor was detected in the nuclei of epithelial and stromal cells in both the functionalis and basalis layers. In the functionalis, the receptor content increased from the early to the late proliferative phase in both cell components. It remained high in the early secretory phase and decreased in the mid- and late secretory phases, comparatively more rapidly in the epithelium than in the stroma. In the latter, the predecidual cell nuclei were receptor-positive. The menstrual phase endometrium lacked receptors. The basalis was rich in progesterone receptors during the proliferative, early and midsecretory phases in both components and receptor-free during the late secretory and menstrual phases of the cycle. Myometrial smooth muscle cell nuclei contained progesterone receptors, whereas they were absent in endometrial and myometrial vessels. Overall, the epithelial progesterone receptor content seemed to correlate with the endometrial tissue levels of estradiol, possibly reflecting its estrogen sensitivity, whereas the stromal progesterone receptor content during the secretory phase at least, in part, may be constitutively synthetized.     

Endothelin synthesis and receptors in human endometrium throughout the normal menstrual cycle

This study was undertaken to investigate the presence of messengerRNA (mRNA) for prepro-endothelin-I (ET-1) and the known receptorsubtypes (ETA and ETB) in human endometrium at different stagesof the menstrual cycle obtained at hysterectomy. Northern blotanalysis revealed expression of ET-1 mRNA in human endometriumduring the normal menstrual cycle. The concentration of ET-1mRNA in endometrial tissue was greater during the menstrualand proliferative phases than during the ovulatory and secretoryphases. Immunoreactive ET-1 was secreted into the medium ofisolated endometrial stromal cells. Oestradiol and progesteronesignificantly attenuated ET-1 release in endometrial stromalcells cultured for 6 days. ETA and ETB mRNA were also presentin endometrial tissue of the normal cycle. The concentrationof ETA receptor mRNA was greater in the proliferative phasethan in the secretory phase, whereas expression of ETB mRNAincreased in menstrual phase. ET-1 significantly increased extracellularaccumulation of cyclic AMP (cAMP), intracellular generationof inositol phosphates and significantly enhanced DNA synthesisin cultured endometrial stromal cells from the proliferativephase. Our results showed that human endometrial cells synthesizedand released ET-1, and contained ETA and ETB receptors whichwere functionally coupled to phosphoinositide breakdown andto adenylate cyclase with the increase of cAMP by ET-1 stimulation.Our findings suggest that ET-1 may have a potential autocrineand/or paracrine function in human endometrial stromal cells.     

Cell cycle: Immunolocalization of bcl-2 protein in human endometrium in the menstrual cycle and simulated early pregnancy

Cell death by apoptosis is now regarded as an important featureof normal endometrial physiology. Recent reports have suggestedthat bcl-2, a proto-oncogene responsible for the suppressionof apoptosis, is expressed in endometrium and may be involvedin the regulation of menstruation. Using standard immunohistochemicalprocedures, the immunoreactivity of bcl-2 and progesterone receptorshas been investigated in normal human endometrium throughoutthe menstrual cycle (n = 25) as well as endometrium exposedto continued oestradiol and progesterone stimulation by ‘rescue’of corpus luteum (n = 4) with exogenous human chorionic gonadotrophin(HCG) administration (pseudopregnancy). Marked immunoreactivity,consistent with previous reports, was noted in the glandularepithelium during the proliferative phase of the cycle. Immunostainingpersisted in the glandular epithelium during the secretory phase,although the percentage and intensity of staining was markedlyreduced. Staining in the stromal compartment was only notedduring the late secretory phase of the cycle. Co-localizationwith an antibody against CD56 demonstrated that this immunoactivitylargely reflected the presence of lymphocytes in the stroma.Endometrium from subjects who underwent ‘luteal rescue’displayed limited immunostaining in either glands or stroma.The absence of significant bcl-2 expression in endocrinologicallymaintained endometrium makes it highly unlikely that bcl-2 isimportant in prolonging endometrial cell survival in the lutealphase of the menstrual cycle.     

Differential expression of angiopoietins 1 and 2 and their receptor Tie-2 in human endometrium

Angiogenesis, the growth of new capillaries from pre-existing blood vessels, is a physiological process involved in both normal menstrual cycling and implantation of the embryo. So far, very little is known about the expression of angiopoietins, growth factors involved in angiogenesis, in human endometrium. Both angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are ligands for the endothelial cell-specific receptor tyrosine kinase Tie-2. In this study we determined the mRNA expression of Ang-1, Ang-2 and Tie-2 by quantitative competitive RT/(QC)-PCR (including specifically designed competitor cDNA) in biopsied human endometrium throughout the menstrual cycle. We detected the mRNA for the angiopoietins in 30 out of 32 endometrial biopsies (94%), covering early proliferative (n = 4), mid proliferative (n = 12), late proliferative (n = 3), early secretory (n = 3), mid secretory (n = 5) and late secretory (n = 3) phases. Analysis of the target/competitor ratios (QC-PCR) revealed that Ang-1 mRNA expression was significantly up-regulated (P = 0.027) during the secretory phase of the menstrual cycle. In contrast, the expression levels of both Ang-2 mRNA and Tie-2 mRNA showed only minor variations at different cycle stages. These findings were confirmed by the relative expression ratio of Ang-1 versus Ang-2 in a multiplex PCR. The expression of Ang-1, Ang-2 and Tie-2 mRNA was detected in both isolated endometrial epithelial and stromal cell fractions. Immunohistochemical localization of the proteins revealed qualitative differences in both cell type and cycle stage expression. In conclusion, the enhanced Ang-1 expression during the secretory phase might serve to stabilize the newly developed blood vessels.     

Regulation of TNF-alpha mRNA expression in endometrial cells by TNF-alpha and by oestrogen withdrawal

During each menstrual cycle, the human endometrium undergoes a series of orchestrated and well controlled changes in anticipation of the arrival of the blastocyst. In the absence of implantation, the endometrium is shed. The underlying basis of the menstrual bleeding is not clear, however, it seems to be related to steroid hormone withdrawal. We showed that tumour necrosis factor-alpha (TNF-alpha) is released by human endometrium and that endometrial epithelial cells are a major source of TNF-alpha mRNA and protein. We show here that TNF-alpha mRNA shows a specific menstrual cycle-dependent expression. The expression of TNF-alpha is mostly minimal throughout the proliferative, early and mid-secretory phases. Expression of TNF-alpha mRNA, however, is increased in the human endometrium in the late secretory phase and during endometrial bleeding. Such a menstrual cycle-dependent expression suggests that specific signals regulate the expression of TNF-alpha mRNA in the human endometrium. In vitro, the expression of TNF-alpha mRNA in endometrial epithelial cells could be regulated by exogenous TNF-alpha. This induced expression was both time- and dose-dependent. In vitro, the TNF-alpha mRNA expression was not altered by oestrogen, progesterone, or both, in the endometrial epithelial cells under conditions that maintain the steroid hormone receptors. However, in vivo, oestrogen withdrawal led to an enhanced expression of TNF-alpha in endometrial epithelial cells. These findings suggest that the up-regulation of TNF-alpha in human endometrium in the late secretory phase may be related to the falling serum oestrogen concentration at the end of the menstrual cycle as well as the potentiating effect of released TNF-alpha on its own mRNA expression.     

Temporal and site-specific expression of transforming growth factor- beta4 in human endometrium

We recently identified a novel member of the transforming growth factor (TGF)-beta superfamily and showed that this gene, designated as endometrial bleeding associated factor (ebaf), or TGFbeta4, has a unique expression pattern in human endometrium. By Northern blot analysis, we showed that this gene was expressed in human endometrium during the late secretory and menstrual phases and was absent in proliferative, early and mid-secretory endometria. In this report, we show by in-situ hybridization that the mRNA of the TGF-beta4 is not expressed in the proliferative endometria. On the other hand, focal expression of the TGFbeta4 mRNA first appears in some endometrial glands in the mid-secretory phase. The TGFbeta4 mRNA is strongly expressed in the endometrial stroma during the late secretory and menstrual phases of the cycle. We raised a polyclonal rabbit antiserum against a peptide at the C terminal of the protein. Western blot analysis using affinity purified antiserum shows that the TGFbeta4 precursor detected in the endometrium as well as placenta is 41 kDa. Bands in the range of 45-51 kDa are also present in human endometrium, more predominantly during the late secretory phase. Immunohistochemical staining shows a low level of immunoreactivity for TGFbeta4 in the early, mid- and late proliferative and early and mid-secretory endometria. A strong immunoreactivity for TGFbeta4 is present in the stroma and to lesser extent in the endometrial glands in late secretory and menstrual endometria. The specificity of staining was shown by neutralizing the activity of the antibody with the synthetic peptide used for raising the antibody and by omitting the antibody. The findings show that TGFbeta4, both at the mRNA and protein levels, exhibits temporal and site specific expression in human endometrium.      

Cycle-dependent expression of macrophage migration inhibitory factor in the human endometrium

BACKGROUND: Macrophage migration inhibitory factor (MIF) isa multifunctional cytokine that was shown to promote angiogenesisand tissue remodelling. Our previous studies identified MIFas one of the principal bioactive molecules involved in endothelialcell proliferation released by ectopic endometrial cells. METHODSAND RESULTS: In the present study, we examined the expressionof MIF in the human endometrium and found an interesting distributionand temporal pattern of expression throughout the menstrualcycle. Immunoreactive MIF was predominant in the glands andsurface epithelium. Dual immunofluorescence analysis furtheridentified endothelial cells, macrophages and T-lymphocytesas cells markedly expressing MIF in the stroma. Quantitativeassessment of MIF protein showed a regulated cycle phase-dependentexpression pattern. MIF expression increased in the late proliferative/earlySecretory phase of the menstrual cycle was moderate during thereceptive phase or what is commonly called the implantationwindow before increasing again at the end of the cycle. Thispattern paralleled MIF mRNA expression determined by northernblot. CONCLUSION: The cycle phase-specific expression of MIFsuggests a tight regulation and perhaps different roles forthis factor in the reparative, reproductive and inflammatory-likeprocesses that occur in human endometrium during every menstrualcycle.     

Uterine glandular area during the menstrual cycle and the effects of different in-vitro fertilization related hormonal treatments

The human uterine glandular epithelium undergoes a sequenceof well characterized changes during the menstrual cycle thatpresumably play an important role in preparation for blastocystimplantation. The aim of this study was to measure objectivelyglandular volume over the entire menstrual cycle and comparethe results with eight different clinical superovulation orhormone replacement therapy (HRT) subject groups. Endometrialbiopsies were taken from control normal menstrual cycle subjects(n = 96), and eight other smaller groups of women who had receiveddifferent in-vitro fertilization (TVF) related treatments. Thetotal area of glandular epithelium was objectively measuredfrom routine histological slides using computerized image analysis.Control menstrual cycle results showed a significantly greatergland area in the early secretory stage of the cycle than atany time between the early proliferative through to the mid-lateproliferative stages (P < 0.05). FVF patients receiving clomiphenecitrate and human menopausal gonadotrophin had a significantlysmaller glandular area than those in the control groups at equivalentstages of the menstrual cycle. The use of progesterone supplementationremoved this significant difference. Patients on the ‘Flare"regime had the highest gland area, although this was not significantlydifferent from controls. Buserelin down-regulation gave a glandarea that was closest to the normal cycle controls. The threeHRT groups showed high variability in gland volume between patients.The results from this study demonstrate that superovulationcan cause significant alterations in endometrial gland volume,but that these do not necessarily preclude implantation.