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Problem: To compare inflammatory- and immune-associated peritoneal cytokines of adolescents and adults with and without endometriosis., Methods of Study: In a nested case-control study in multiple university-affiliated scientific centers, ten adolescents and thirteen adults with visually and histologically confirmed endometriosis (cases), thirteen adolescents with visually suspected endometriosis but indeterminate (seven patients) or negative (six patients) histology, and fifteen adults undergoing surgery for non-malignant gynecologic disease without endometriosis (controls) underwent laparoscopic aspiration of peritoneal fluid (PF), from which PF and conditioned medium (CM) cytokine levels were assayed., Results: Compared to adults with endometriosis, MCP-3, IL-12p40, MIP-1β, and IL-15 were significantly higher among adolescents with endometriosis, while TNF-β and CTACK were lower among adolescents. These differences were similar comparing adolescents with endometriosis to adult controls except for MIP-1β, which was not statistically different. MIP-1β was, however, the only cytokine observed to differ between adult cases and controls. There were no significant differences in CM cytokines among the three groups. Results were similar when analyses were restricted to samples collected (a) during menstrual cycle days 1-10, (b) from patients unexposed to exogenous hormones, or (c) from all adolescents despite presence or absence of histologic endometriosis., Conclusion: Biologically relevant and statistically significant differences in six PF cytokines were observed and suggest a more pro-invasion cytokine profile among adolescents with endometriosis. Adolescents with endometriosis have unique peritoneal cytokine profiles and molecular behavior when compared to adults with and without endometriosis., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Immune cell differentiation is critical for adequate tissue-specific immune responses to occur. Here, we studied differentiation of human uterine natural killer cells (uNK cells). These cells reside in a tissue undergoing constant regeneration and represent the major leukocyte population at the maternal-fetal interface. However, their physiological response during the menstrual cycle and in pregnancy remains elusive. By surface proteome and transcriptome analysis as well as using humanized mice, we identify a differentiation pathway of uNK cells in vitro and in vivo with sequential acquisition of killer cell immunoglobulin-like receptors and CD39. uNK cell differentiation occurred continuously in response to the endometrial regeneration and was driven by interleukin-15. Differentiated uNK cells displayed reduced proliferative capacity and immunomodulatory function including enhanced angiogenic capacity. By studying human uterus transplantation and monozygotic twins, we found that the uNK cell niche could be replenished from circulation and that it was under genetic control. Together, our study uncovers a continuous differentiation pathway of human NK cells in the uterus that is coupled to profound functional changes in response to local tissue regeneration and pregnancy., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Immune tolerance is crucial for the successful pregnancy, while immune effectors and their products are required to safeguard a fetus from the infectious pathogens. The key immune effectors, such as T, B, and natural killer (NK) cells, monocytes, macrophages, and dendritic cells take part in regulating the immune responses at the maternal-fetal interface. The immune effectors become involved in intraovarian reproductive processes as well, such as ovulation, production of corpus luteum (CL) and its degeneration and determine the quality and evolution of the oocyte during the folliculogenesis. In the cycling endometrium, NK cells are rapidly infiltrated into the endometrium after ovulation and participate in angiogenesis and spiral artery remodeling process. In this study, we reviewed the characteristics and action mechanisms of immune effectors and their products in the peripheral blood, ovary, and endometrium during the ovarian cycle, since a comprehensive understanding of immune responses during the ovarian cycle and the time of implantation can help us to predict the pregnancy outcome and take effective measures for the prevention of potential obstetrical complications., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Problem: To study the prevalence of HHV-6 in endometrial biopsies among women experiencing recurrent implantation failure (RIF) after IVF/ET compared with controls., Method of Study: Thirty women experiencing RIF after IVF/ET and 10 fertile women participated in the study. All women had endometrial biopsies taken in the luteal phase of their menstrual cycle for an endometrial immune profile (EIP) and HHV-6 mRNA as well as lymphocyte and granulocyte populations. The prevalence of HHV-6 in endometrial biopsies was determined, and biopsies for positive and negative expression of HHV-6 were compared with the results of their EIP and lymphocyte and granulocyte populations., Results: Thirty-seven percentage of women with a history of RIF and 0% of controls demonstrated the presence of HHV-6 in their endometrial biopsies. No associations were found when the results of the endometrial immune profile were compared with the presence or absence of HHV-6. Significant increase in neutrophil-specific CD16b mRNA was found in HHV-6-positive samples, and the levels of B cells-related CD19 mRNA were lower in biopsies from women with RIF in comparison with normal controls., Conclusion: HHV-6 infection is an important factor in RIF., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The control of complement activation within embryo-endometrium environment is critical for embryo survival. Cell evasion from complement attack requires interaction of complement regulatory proteins (CRPs) with cell adhesion αvβ3 integrin. We aim to compare the expression of CRPs in endometria of women with and without endometriosis and to examine the molecular interaction of decay accelerating factor (DAF) with αvβ3 integrin. Endometrial expression of Membrane cofactor protein (CD46), Decay accelerating factor (DAF), Membrane attack complex inhibitory factor (CD59) and β3 integrin subunit were determined through menstrual cycle by immunohistochemistry. DAF protein quantity was determined by Western blot and mRNA levels measured in epithelial cells isolated by laser capture microdissection (LCM). Using in vitro assay, we examined DAF and β3 integrin expression through paracrine regulation between endometrial compartments. To determine whether β3 integrin and DAF interacts in vivo, endometrial samples were subjected to immunoprecipitation and colocalization using dual immunofluorescence technique. DAF and β3 integrin expression were significantly low in samples from women with endometriosis during mid secretory phase. This observation was supported by decreased DAF protein quantity; faint DAF and β3 integrin interaction and reduced mRNA levels in cells dissected by LCM. Moreover epithelial DAF and β3 integrin expression through paracrine regulation by progesterone from stromal compartment was disrupted in endometriosis. Endometria from women with endometriosis exhibits aberrant expression of complement proteins. The abnormal DAF expression potentially compromises embryo survival, contributing to understand the implantation failure in women with endometriosis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Study Question: Is it possible to determine the receptivity status of an endometrium by combined quantitative reverse transcription PCR (RT-qPCR) expression analysis of genes involved in endometrial proliferation and immunity?, Summary Answer: The new ER Map®/ER Grade® test can predict endometrial receptivity status by RT-qPCR using a new panel of genes involved in endometrial proliferation and the maternal immune response associated to embryonic implantation., What Is Known Already: The human endometrium reaches a receptive status adequate for embryonic implantation around Days 19-21 of the menstrual cycle. During this period, known as the window of implantation (WOI), the endometrium shows a specific gene expression profile suitable for endometrial function evaluation. The number of molecular diagnostic tools currently available to characterize this process is very limited. In this study, a new system for human endometrial receptivity evaluation was optimized and presented for the first time., Study Design, Size, Duration: ER Map®/ER Grade® validation was achieved on 312 endometrial samples including fertile women and patients undergoing fertility treatment between July 2014 and March 2016. Expression analyses of 184 genes involved in endometrial receptivity and immune response were performed. Samples were additionally tested with an independent endometrial receptivity test., Participants/materials, Setting, Methods: A total of 96 fertile women and 120 assisted reproduction treatment (ART) patients participated in the study. Endometrial biopsy samples were obtained at LH + 2 and LH + 7 days in fertile subjects in a natural cycle and at the window of implantation (WOI) in patients in a hormone-replacement therapy (HRT) cycle. Total RNA was purified, quality-checked and reverse-transcribed. Gene expression was quantified by high-throughput RT-qPCR and statistically analyzed. Informative genes were selected and used to classify samples into four different groups of endometrial receptivity status., Main Results and the Role of Chance: Significantly different gene expression levels were found in 85 out of 184 selected genes when comparing LH + 2 and LH + 7 samples (paired t-test, P < 0.05). Gene ontology analyses revealed that cell division and proliferation, cell signaling and response, extracellular organization and communication, immunological activity, vascular proliferation, blood pressure regulation and embryo implantation are the most over-represented biological terms in this group of genes. Principal component analysis and discriminant functional analysis showed that 40 of the differentially expressed genes allowed accurate classification of samples according to endometrial status (proliferative, pre-receptive, receptive and post-receptive) in both fertile and infertile groups., Large Scale Data: N/A., Limitations, Reasons for Caution: To evaluate the efficacy of this new tool to improve ART outcomes, further investigations such as non-selection studies and randomized controlled trials will also be required., Wider Implications of the Findings: A new comprehensive system for human endometrial receptivity evaluation based on gene expression analysis has been developed. The identification of the optimal time for embryo transfer is essential to maximize the effectiveness of ART. This study is a new step in the field of personalized medicine in human reproduction which may help in the management of endometrial preparation for embryo transfer, increasing the chances of pregnancy for many couples., Study Funding/competing Interest(s): The authors have no potential conflict of interest to declare. No external funding was obtained for this study., (© The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com)

Previous transcriptome studies of the human endometrium have revealed hundreds of simultaneously up- and down-regulated genes that are involved in endometrial receptivity. However, the overlap between the studies is relatively small, and we are still searching for potential diagnostic biomarkers. Here we perform a meta-analysis of endometrial-receptivity associated genes on 164 endometrial samples (76 from 'pre-receptive' and 88 from mid-secretory, 'receptive' phase endometria) using a robust rank aggregation (RRA) method, followed by enrichment analysis, and regulatory microRNA prediction. We identify a meta-signature of endometrial receptivity involving 57 mRNA genes as putative receptivity markers, where 39 of these we confirm experimentally using RNA-sequencing method in two separate datasets. The meta-signature genes highlight the importance of immune responses, the complement cascade pathway and the involvement of exosomes in mid-secretory endometrial functions. Bioinformatic prediction identifies 348 microRNAs that could regulate 30 endometrial-receptivity associated genes, and we confirm experimentally the decreased expression of 19 microRNAs with 11 corresponding up-regulated meta-signature genes in our validation experiments. The 57 identified meta-signature genes and involved pathways, together with their regulatory microRNAs could serve as promising and sought-after biomarkers of endometrial receptivity, fertility and infertility.

Many women with endometriosis experience compromised fertility. This disease clearly exerts quantitative damage on the ovaries, and perhaps, also qualitative damage. However, it remains controversial whether endometrial receptivity is compromised. Here we review the evidence from basic transcriptomic signature data to clinical data from an oocyte donation model and find support for the concept that endometrial receptivity is not impaired in women with endometriosis when healthy embryos reach the endometrial cavity., (Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The endometrium maintains complex controls on proliferation and apoptosis as part of repetitive menstrual cycles that prepare the endometrium for the window of implantation and pregnancy. The reliance on inflammatory mechanisms for both implantation and menstruation creates the opportunity in the setting of endometriosis for establishment of chronic inflammation that is disruptive to endometrial receptivity, causing both infertility and abnormal bleeding. Clinically, there can be little doubt that the endometrium of women with endometriosis is less receptive to embryo implantation, and strong evidence exists to suggest that endometrial changes are associated with decreased cycle fecundity as a result of this disease. Here we provide unifying concepts regarding those changes and how they are coordinated to promote progesterone resistance and estrogen dominance through aberrant cell signaling pathways and reduced expression of key homeostatic proteins in eutopic endometrium of women with endometriosis., (Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Heavy menstrual bleeding (HMB) affects 30% of women of reproductive age and significantly interferes with quality of life. Altered endometrial vascular maturation has been reported in HMB and recurrent miscarriage, the latter associated with increased uterine natural killer (uNK) cell numbers. This study compared endometrial leukocyte populations in controls and women with HMB. Formalin-fixed paraffin-embedded endometrial biopsies from controls (without endometrial pathology) and HMB were immunostained for CD14 (macrophages), CD56 (uNK cells), CD83 (dendritic cells), FOXP3 (regulatory T cells/Tregs), CD3 and CD8 (T cells). Leukocyte numbers were analysed as a percentage of total stromal cells in five randomly selected fields of view in the stratum functionalis of each sample. In control women across the menstrual cycle, 2-8% of total stromal cells were CD3(+) cells, 2-4% were CD8(+) T cells and 6-8% were CD14(+) macrophages. Compared with controls, CD3(+) cells were reduced during the mid-secretory phase (4%, P<0.01) and increased in the late secretory phase (12%, P=0.01) in HMB. CD83(+) dendritic cells and FOXP3(+) Tregs were scarce throughout the menstrual cycle in both groups. In controls, 2% of stromal cells in proliferative endometrium were CD56(+) uNK cells, increasing to 17% during the late secretory phase. In HMB, CD56(+) uNK cells were increased in the proliferative (5%, P<0.01) and early secretory (4%, P<0.02) phases, but reduced (10%, P<0.01) in the late secretory phase. This study demonstrates dysregulation of uNK cells in HMB, the functional consequence of which may have an impact on endometrial vascular development and/or endometrial preparation for menstruation., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Most of the key physiological processes in the human reproductive tract involve a significant inflammatory component. These processes include follicle development, ovulation, implantation, pregnancy, labor, postpartum, remodeling and menstruation. In this context, the term 'inflammation' usually means an influx of leukocytes ('immune cells'), often of different types, into a reproductive tract tissue. These examples of inflammation are not overtly associated with any infective process. There may also be evidence that these invading leukocytes have altered their functions to take on specific and relevant local regulatory roles. Specific sequential changes in different leukocytes can be demonstrated within human endometrium during the different phases of the normal menstrual cycle. Leukocytes are fairly sparse in numbers through the proliferative phase, but increase substantially into and through the secretory phase, so much so that around 40% of all stromal cells in the premenstrual phase are leukocytes, mainly uterine natural killer cells, a large granulated lymphocyte. Other leukocytes which play key roles in menstruation appear to be macrophages, mast cells, dendritic cells, neutrophils, eosinophils and regulatory T cells. Premenstrual withdrawal of progesterone increases the endometrial expression of inflammatory mediators, including IL-8 and MCP-1, which are believed to drive endometrial leukocyte recruitment at this time. Macrophages and neutrophils are rich sources of defensins and whey acid protein motif proteins, which play important roles in ensuring microbial protection while the epithelial barrier is disrupted. Mast cells are increasingly activated as the menstrual phase approaches, and leukocyte proteases trigger a cascade of matrix metalloproteinases and degradation of extracellular matrix. Dendritic cells and other antigen-presenting cells (e.g. macrophages) almost certainly facilitate clearance of cellular debris from the uterine cavity, and reduce the amount of viable cellular material transiting the Fallopian tubes. All of these processes are influenced or controlled by regulatory T cells. Many of these leukocytes also have the potential to release regulatory molecules which stimulate endometrial repair mechanisms. Increasing recent evidence also implicates disturbances of immune cells and their cytokine mediators in contributing to symptoms of abnormal uterine bleeding and pelvic pain. These recent findings all point towards the importance of the 'inflammatory process' in both normal and abnormal endometrial bleeding.

Menstruation is a complex process dependent on premenstrual release of inflammatory mediators and proteolytic enzymes from endometrial cells. Endometrial leukocytes are traditionally considered to be the major source of the inflammatory factors. However, evidence is emerging to suggest a role for decidualized endometrial stromal cells in the premenstrual inflammatory cascade. We sought to determine if withdrawal of hormone support (estrogen and progesterone) from decidualized endometrial stromal cells, in a model mimicking the precise timing leading to menstruation, activated inflammatory signaling pathways and downstream release of inflammatory mediators. Human endometrial stromal cells decidualized gradually over 12 days of estradiol and progestin treatment as evidenced by an increase in prolactin secretion. Withdrawal of hormone support from decidualized stromal cells resulted in a decrease in cytoplasmic IkappaB and a progressive increase in nuclear accumulation of NF-kappaB, as demonstrated by Western immunoblot and immunocytochemical analyses. Concomitant with nuclear translocation of NF-kappaB, hormone withdrawal led to production of a host of inflammatory mediators by the decidualized stromal cells, including IFN-alpha, IL-6, CCL11, GM-CSF, CCL2, IL1-RA, CXCL10, CXCL8, IL-12, IL-15, VEGF, and CCL5. Elevation of inflammatory mediators was not observed, however, upon hormone withdrawal in cells treated with the NF-kappaB inhibitor BAY 11-7085. Decidualized stromal cells are likely highly sensitive sensors of changing hormone levels. This provides a mechanism by which decidualized stromal cells may recruit inflammatory leukocytes into the premenstrual endometrium and contribute to the intense inflammation underlying this unique physiological process.

Recent studies suggest that changes in certain uterine immune cell populations in endometrium of women with endometriosis are likely to precede changes at ectopic sites. This preliminary study is a first look into the function of uterine-draining lymph nodes (LNs) during the menstrual cycle and in the presence of endometriosis. Paraffin-embedded obturator LNs were obtained from women with (n = 7, mean age 44.3) and without (n = 9, mean age 38.4) endometriosis, who had undergone hysterectomy for cervical or ovarian cancer and in whom LN involvement was not detected. Immunohistochemical staining for endometrial stromal cells and a range of immune cell populations was performed. The CD10+ endometrial stromal cells were detected in uterine-draining LNs throughout the menstrual cycle with numbers peaking during menstruation. The inflammatory process of menstruation was also associated with increased numbers of CD3+, CD4+, Foxp3+, DC-Sign+, CD68+, CD20+, CD79+, and plasma cells. In endometriosis, CD10+ endometrial stromal cells were further increased in numbers, but CD3+, CD4+, DC-Lamp+, FoxP3+, and plasma cells were reduced. This study indicates that efficient immunological responses may be required to contain shed endometrial fragments within the draining uterine LNs thus preventing their further dissemination with establishment of ectopic lesions at distant sites.

The control of complement activation in the embryo-maternal environment has been demonstrated to be critical for embryo survival. Complement proteins are expressed in the human endometrium; however, the modulation of this expression by embryo signals has not been explored. To assess the expression of complement proteins in response to human chorionic gonadotropin (hCG), we designed an experimental study using in vivo and in vitro models. Twelve fertile women were treated with hCG or left untreated during the mid-luteal phase, and an endometrial biopsy was performed 24 hours later. The localizations of C3, membrane cofactor protein (MCP; CD46), decay-accelerating factor (DAF; CD55), and protectin (CD59) were assessed by immunohistochemistry, and the messenger RNA (mRNA) levels of these proteins were quantified by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in cells harvested from endometrial compartments using laser capture microdissection. Endometrial explants were cultured with or without hCG for 24 hours, and the C3 and DAF protein levels were measured by Western blotting. Elevated C3 mRNA levels in stromal cells and elevated DAF levels in epithelial luminal cells were detected after hCG treatment. In the endometrial explant model, the progesterone receptor antagonist RU486 inhibited the increases in the levels of C3 and DAF in response to hCG. The findings of this study indicate that hCG plays a role in embryo-endometrium communication and affects the expression of complement proteins in endometrial compartments during the implantation window.

The human endometrium is exposed to cyclical fluctuations of ovarian-derived sex steroids resulting in proliferation, differentiation (decidualization), and menstruation. An influx of leukocytes (up to 15% macrophages) occurs during the latter stages of the menstrual cycle, including menses. We believe the endometrial macrophage is likely to play an important role during the menstrual cycle, especially in the context of tissue degradation (menstruation), which requires regulated repair, regeneration, and phagocytic clearance of endometrial tissue debris to re-establish tissue integrity in preparation for fertility. The phenotype and regulation of the macrophage within the endometrium during the menstrual cycle and interactions with other cell types that constitute the endometrium are currently unknown and are important areas of study. Understanding the many roles of the endometrial macrophage is crucial to our body of knowledge concerning functionality of the endometrium as well as to our understanding of disorders of the menstrual cycle, which have major impacts on the health and well-being of women.

Problem: Throughout the reproductive cycle and into early pregnancy, the normal endometrium undergoes changes in a range of leukocytes, epithelia, stromal fibroblasts, and vascular structures caused by intersecting effects of hormone balance and embryo implantation. The direct investigation in humans of reproductive tract responses during normal and physiologically altered cycles is not practical or feasible. METHOD AND STUDY: The aim of this study was to define immunological and morphological changes through immunohistological and morphometric evaluation of the endometrium throughout the menstrual cycle and the decidua during early gestation in the rhesus monkey, a tractable experimental animal model., Results: A zone-dependent method for the immunohistological description of the rhesus uterine mucosa was established and showed that leukocyte infiltration, stromal cell decidualization, glandular and vascular responses were zone- and cell type-dependent, and changed throughout the cycle and early pregnancy. Morphological heterogeneity of uterine natural killer cells in the cycling endometrium and gestational decidua were consistent with the recent characterization of phenotypic subsets., Conclusions: These data establish a morphological platform upon which to further study the regulation of endometrial responses to the hormonal mileau of pregnancy, the control of local leukocyte populations, and the responses to threatened pregnancy, infection, and inflammation., (© 2012 John Wiley & Sons A/S.)

Problem: Chronic endometritis is an elusive entity that is often asymptomatic and undetectable by conventional endometrial biopsy and histological examination. Using immunohistochemistry for full-thickness endometrium, we sought for its clinicopathological features., Method of Study: Two hundred and thirty-four archival endometrial specimens obtained by hysterectomy were immunostained for the plasmacyte marker syndecan-1 to identify chronic endometritis. Endometrial morphology was dated by the standard criteria. The immunoreactive cells were enumerated in 10 non-overlapping endometrial stromal areas. The clinical parameters were obtained from the medical charts., Results: Chronic endometritis was identified in 11.1% of the samples examined. Its occurrence was similar between the proliferative phase and secretory phase. A total of 23.1% of the cases were asymptomatic. Stromal plasmacyte infiltration and morphological delay were more prominent in symptomatic chronic endometritis than in asymptomatic counterpart., Conclusions: Chronic endometritis is a common gynecological pathological condition and more often asymptomatic than ever expected. There was no menstrual cycle-dependent fluctuation in its occurrence., (© 2011 John Wiley & Sons A/S.)

Interleukin-23 (IL-23) is a novel cytokine involved in the regulation of organ-specific immune responses. We hypothesized that expression of IL-23 in the human endometrium is menstrual cycle and pregnancy dependent, and is involved in endometrial immune regulation. IL-23 expression and regulation was investigated in the human endometrium and placenta in vivo using immunohistochemistry and in vitro using Western blot and cell viability analyses. IL-23 immunoreactivity in endometrial glandular cells was highest in the late proliferative and early secretory phases, as compared to other cycle phases and first trimester tissues. Endometrial stromal cells (ESC) showed weak IL-23 immunoreactivity without significant changes in intensity and distribution throughout the menstrual cycle. First trimester decidual cells revealed significantly stronger IL-23 staining compared to ESC from non-pregnant endometrium. Both villous cytotrophoblasts and syncytiotrophoblasts also showed positive IL-23 immunoreactivity, with a higher staining in syncytiotrophoblasts. In the trophoblastic cell line HRT8, IL-23 expression increased in a time-dependent manner, but was undetectable in stromal cells under all treatment conditions. ESC treated with recombinant IL-23 showed significantly decreased IL-8 secretion and cell viability. These results suggest a possible regulatory role for IL-23 in the menstrual cycle and in early pregnancy, although the extent and function of this role are yet to be determined., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Problem: The number of uterus natural killer (NK) cells change through the menstrual cycle, but the origin of uterus NK cell was not unclear. Our aims are to study whether we can reproduce repetition of menstrual cycle and to reveal the origin of uterus NK cells., Method of Study: Endometrial samples were obtained from fertile women, and the tissues were transplanted into ovariectomized non-obese diabetic (NOD)/severe combined immunodeficiency (SCID)/γCnull (NOG) mice. Mice were treated with sex hormones which were in accord with human menstrual cycle., Results: The replants showed similar histological changes as in eutopic endometrium repeatedly. CD56-positive, CD16-negative NK cells increased significantly during the treatment with estradiol and progesterone combination., Conclusion: Histological assessment demonstrated that this model of NOG mice repeatedly exhibited regular menstrual cycles, and this model mimicked not 'ectopic endometrium', but 'eutopic endometrium' in humans. Change in number of NK cells suggested that NK cell might be derived from the endometrium., (© 2010 John Wiley & Sons A/S.)

Indoleamine 2,3-dioxygenase (IDO), an enzyme responsible for tryptophan catabolism, is thought to be required to prevent the rejection of the allogenic fetus by maternal T cells and to protect against intra- and extra-cellular pathogens. Consequently, we studied the expression of IDO in the endometrium of female Balb/c mice during the oestrous cycle. At each phase, the endometrium was peeled away and the relative expression of IDO mRNA was detected by semi-quantitative RT-PCR. The presence of IDO protein was confirmed in each phase by Western blotting and immunohistochemistry. Our results showed that IDO is expressed in the endometrium of cycling mice during all the phases of oestrous cycle. The expression of IDO was highest at the oestrus and lowest at the dioestrus. By means of Western blotting and immunohistochemistry, we obtained evidence that IDO protein is synthesised in the endometrium of cycling mice throughout the oestrous cycle. In accordance with RT-PCR results, IDO protein was predominant at the oestrus phase. IDO protein was mainly localised in the glandular and luminal epithelial cells. Our results support the concept of IDO providing a mechanism of innate immunity to protect from ascending infections of the female reproductive tract. In addition, considering the fact that mating only occurs during the oestrus phase, the high expression of IDO in this phase is likely to be a mechanism that induces immunological tolerance of the fetus.

Problem: The purpose of the study was to investigate the different expressions of toll-like receptors (TLRs) in the proliferative and the secretory phase of endometrial tissue., Method of Study: Eight infertile women were included in this study. The endometrial tissues of proliferative and secretory phase were obtained from each woman. The tissues were evaluated for the expression of mRNA for TLR1-10 by reverse transcribed polymerase chain reaction (RT-PCR). The proteins of TLR2, 3, 4, and 9 were evaluated by Western blot. The mRNA and protein level of proliferative and secretory endometrial tissue from the same woman was compared. The data were analysed with SPSS15.0., Results: TLR1-7, 9, and 10 mRNA were expressed throughout the menstrual cycle, but in the same woman, the expression of TLR2-6, 9, and 10 mRNA was higher during the secretory phase than that in the proliferative phase. The Western blot also showed that the protein expression of TLR2, 3, 4, and 9 was stronger in the secretory phase than that in the proliferative phase in the same woman., Conclusion: The expression of TLRs is cycle dependent in human endometrial tissue. The expressions of TLRs were higher in the secretory phase than that in the proliferative phase: this indicated that TLRs may be regulated by sex hormones throughout the menstrual cycle.

Objective: To investigate menstrual cycle changes in expression by eutopic endometrium of a nonclassic human leukocyte antigen, HLA-G, which binds to the killer immunoglobulin-like receptor (KIR) 2DL4 (CD158d) on natural killer (NK) cells. Such antigens have been linked to endometriosis., Design: Case-control study., Setting: University hospital., Patient(s): We examined 20 Japanese women undergoing hysterectomy for endometriosis and 17 undergoing hysterectomy for myoma., Intervention(s): Immunohistochemical HLA-G staining of eutopic endometrium and peritoneal fluid (PF) cells from women with and without endometriosis. Flow cytometric analysis of PF NK cells from women with and without endometriosis., Main Outcome Measure(s): HLA-G staining in eutopic endometrium was quantified by image analysis. The KIR2DL4-expressing NK cells in PF were investigated by flow cytometry., Result(s): The HLA-G was expressed by eutopic endometrium only in the menstrual phase and not in the late proliferative or secretory endometrium. Intensity of HLA-G staining did not differ significantly between women with and without endometriosis. The HLA-G- expressing cells were detected in PF during the menstrual period. These cells are morphologically and flow cytometrically different from mesothelial cells and NK cells., Conclusion(s): The HLA-G expression is observed in eutopic endometrium only during the menstrual phase, and no differences were observed between women with and without endometriosis. Epithelial cells bearing HLA-G may enter the peritoneal cavity during retrograde menstruation, allowing the antigen to react locally with KIR2DL4.

Background: Endometriosis is an inflammatory condition, characterized by the presence of endometrial-like tissue outside the uterus. The immune system provides a defence mechanism in response to foreign pathogens, and macrophages play important roles in this response. Activation of macrophages has been reported in peritoneal fluid and ectopic endometriotic lesions; however, controversy exists regarding the composition and function of macrophage populations in eutopic endometrium of women with and without endometriosis. This study aimed to quantify macrophages in eutopic endometrium of women with and without endometriosis, during the early, mid and late proliferative and menstrual phases of the cycle., Methods: Paraffin-embedded endometrial curettage blocks were selected from pathology archives. Seventy-six specimens from women with and without endometriosis were analysed using standard immunohistochemical techniques with CD68-PGM1 (phosphoglucomutase 1) clone antibody. Macrophages were counted according to their morphology over several fields of view., Results: A significant increase in macrophage cell numbers was shown in eutopic endometrium in women with endometriosis (mean +/- SD, 182.7 +/- 72.9/mm(2)) during all stages of the proliferative phase compared with normal controls (101.6 +/- 53.4/mm(2); P < 0.001). Significant increase in macrophage density occurred in the control group during the mid-menstrual phase, Days 3-4 (P < 0.01), which was not observed in women with endometriosis., Conclusions: This study further supports an association between immune changes in eutopic endometrium and presence of endometriosis. However, it remains uncertain if eutopic immune changes are primary or secondary occurrences.

Background: Dendritic cells (DCs) are specialized antigen presenting cells that are highly involved in the stimulation and modulation of the immune response within mucosal surfaces, including the female reproductive tract. DCs have been poorly characterized in the non-pregnant endometrium., Methods: Hysterectomy specimens were obtained from premenopausal women (n = 49) with histologically normal endometrium. Endometrial sections were stained immunohistochemically using antibodies for monoclonal mouse anti-human CD1a and CD83, two markers which are specific for populations of immature and mature DCs, respectively., Results: There was a significantly higher density of endometrial CD1a+ DCs than CD83+ DCs throughout the menstrual cycle (P < 0.001). The density of CD1a+ and CD83+ DCs did not vary between the fundus and isthmus of the uterus. There was a significant increase in the density of CD1a+ DCs, but not CD83+ DCs, in the basal layer of the endometrium through the phases of the menstrual cycle. The density of CD83+ was significantly greater in the basal layer compared with the functional layer during both the proliferative (P = 0.004) and secretory phases (P = 0.001), whereas for CD1a+ DCs, the greater density in the basal layer was only observed in the secretory phase (P < 0.001)., Conclusions: The highly coordinated cyclical changes in DC populations during the normal menstrual cycle reported in this study may be important for local regulatory mechanisms relevant to menstruation and implantation; alterations in this normal profile may contribute to the development of disturbances of function, fertility and even benign gynaecological disease.

The human endometrium is a unique tissue that undergoes dramatic monthly remodeling during the menstrual cycle in preparation for an implanting conceptus. This remodeling involves sequential proliferation and differentiation of endometrial stromal and epithelial cells, coupled with extensive angiogenesis and infiltration of a specific specialized immune cell subset. Increasing evidence points to an essential role for these maternal leukocytes in stimulating the endometrial angiogenesis, and we propose that they also play a key role in the decidual vascular transformation. Aberrant endometrial angiogenesis, decidualisation and vascular transformation is thought to underlie many pathologies of pregnancy, from infertility to the development of preeclampsia and Intra Uterine Growth Restriction. In this chapter we review the cellular processes associated with each stage of endometrial and decidual transformation, detailing the role of the immune cell populations and the angiogenic and chemotactic factors secreted by them.

Objective: The mechanism of action of intrauterine devices (IUDs) is not well understood. This investigation was intended to gain further insight into the role of leukaemia inhibitory factor (LIF) in intrauterine contraception. We evaluated the immunohistochemical distribution patterns of LIF in women wearing a copper T380A IUD., Methods: The immunohistochemical distribution patterns of LIF in women who had been using a copper T380A IUD for different periods of time, two months after removal of the IUD, and in normal fertile women were evaluated. Endometrial biopsies were obtained from four groups of patients according to the duration of T Cu380A IUD use (group I: <5 years, n = 15, and group II: > or = 5 years, n = 15), after IUD removal (group III, n = 15) and normal fertile women (controls, n = 15) during the window of implantation (WOI). Staining intensity of LIF was evaluated using semi-quantitative IRS-scores., Results: The lowest expression of LIF was observed in women using a copper T380A IUD, being statistically significant compared with the control group (p < 0.05). LIF immunostaining remained abnormal two months after IUD removal., Conclusions: Copper IUDs can inhibit expression of LIF and they may cause inhibition of the implantation stage, which is crucial for pregnancy.

Innate immunity in the endometrium has fundamental significance for reproduction. Although toll-like receptors (TLRs) play central roles in innate immune responses, their expression in the human endometrium remains to be fully elucidated. We have examined the gene expression of TLR2, TLR3, TLR4, and TLR9 in endometrial tissues by real-time quantitative PCR and in situ hybridization. The expression levels of the four genes in endometrial tissues varied in a similar pattern during the menstrual cycle; the levels were high in the perimenstrual period and low in the periovulatory period. Expression of the four genes was detected in both epithelial cells and stromal cells throughout the menstrual cycle. Expression levels were higher in epithelial cells for TLR3 and in stromal cells for TLR4, while they were comparable in epithelial cells and stromal cells for TLR2 and TLR9. These findings imply that differential spatio-temporal expression patterns of TLRs subserve proper innate immunity of the endometrium.

RCAS1 expression is related to the regulation of activated immune cells and to connective tissue remodeling within the endometrium. DFF45 seems to play an important role in the apoptotic process, most likely by acting through the regulation of DNA fragmentation. Its expression changes within the endometrium seem to be related to the resistance of endometrial cells to apoptosis. The aim of the present study was to evaluate RCAS1 and DFF45 endometrial expressions during ovulation and the implantation period. RCAS1 and DFF45 expression was assessed by the Western-blot method in endometrial tissue samples obtained from 20 patients. The tissue samples were classified according to the menstrual cycle phases in which they were collected, with a division into three phases: late proliferative, early secretory, and mid-secretory. The lowest level of RCAS1 and the highest level of DFF45 endometrial expression was found during the early secretory cycle phase. Statistically significantly higher RCAS1 and statistically significantly lower DFF45 endometrial expression was identified in the endometrium during the late proliferative as compared to the early secretory cycle phase. Moreover, statistically significantly higher RCAS1 and statistically significantly lower DFF45 expression was found in the endometrium during the mid-secretory as compared to the early secretory cycle phase. The preparation for implantation process in the endometrium is preceded by dynamic changes in endometrial ECM and results from the proper interaction between endometrial and immune cells. The course of this process is conditioned by the immunomodulating activity of endometrial cells and their resistance to immune-mediated apoptosis. These dynamic changes are closely related to RCAS1 and DFF45 expression alterations.

Quantity and peculiarity of lymphocytes in the endometrium in the proliferation phase of the normal menstrual cycle have been studied. Operative material from 14 women of reproductive age with the diagnosis of carcinoma colli uterine have been investigated. Our investigations included morphometric studies of the character, quantity and localization of the lymphocytes. General quantity of immunocompetent cells were represented by CD3+ T lymphocytes which exist in all layers of the endometrium, among them predominant was the CD8+T lymphocyte subpopulation. CD20+ B lymphocytes in fact were missing in the epithelium, but in the other layers of the endometrium they were found in insignificant quantity.

Unique CD16(-) natural killer (NK) cells appear in the human cycling endometrium and acutely increase in number after ovulation. Selective recruitment from peripheral blood (PB) CD16(-) NK cells is a potential mechanism for the postovulatory increase of these NK cells. The interaction between selectin L, an adhesion molecule playing a critical role in leukocyte extravasation, and its ligands may be involved in this phenomenon. We investigated the menstrual cycle-dependent fluctuation of selectin L expression on PB CD16(-) NK cells and selectin L ligand expression in the human endometrial endothelium. The expression of selectin L on PB CD16(-) NK cells was constantly high throughout the menstrual cycle compared with other PB CD16(+) NK cells and non-NK lymphocytes. Among eight selectin L ligands examined, podocalyxin-like, mucosal addressin cell adhesion molecule-1 (MADCAM1) and chondroitin sulfate proteoglycan 2 (CSPG2) were localized in the endometrial endothelium. Semiquantitative score of immunostaining intensity in the endometrial endothelium for MADCAM1 was highest in the late secretory phase, whereas that for CSPG2 peaked throughout the secretory phase. There was a strong positive correlation between the number of endometrial NK cells and the semiquantitative score for CSPG2. Three active isoforms of CSPG2 mRNA were detected in the human endometrium. These findings support the idea that the interaction between selectin L and selectin L ligands functions in the postovulatory selective recruitment of PB CD16(-) NK cells into the human endometrium.

Background: Clarifying the normal distribution of activation antigens will contribute to database construction studies of monoclonal-antibody-based therapies in endometrial disorders., Methods: In this study, endometrial tissue samples obtained during proliferative and secretory phases and decidual samples of early pregnancies were immunostained by the monoclonal antibodies anti-CD26, anti-CD30, anti-CD70, anti-CD71, and anti-CD98 using the indirect immunoperoxidase method., Results: CD26 is expressed on the glandular epithelium in the endometrium and decidua. Endothelial CD26 is expressed less in the decidua when compared to the endometrium. CD30 is strongly expressed by decidual cells. It is only weakly expressed on endometrial and decidual vessels. Glandular and endothelial CD70 expression is mainly seen in the proliferative phase of the menstrual cycle. Glandular CD71 expression is less in the decidua when compared to the endometrium. Its expression on stromal cells is more in the secretory phase of the menstrual cycle and in early pregnancy deciduae. It is expressed on endometrial vessels but not on decidual vessels. Glandular CD98 is expressed more in the decidua when compared to the endometrium. This antigen exists on endometrial lymphocytes. It is strongly expressed on the endothelium in the endometrium and decidua., Conclusion: It seems that CD26 and CD70 are not involved in the functions of endometrial and decidual stromal cells. CD30 and CD71 are thought to be involved in decidualization. Absence of activation antigens other than CD98 on lymphocytes indicated an antigenic profile for large granular lymphocytes that is different from regular lymphocytes.

Objective: Endometrium is a specialized organ in which phenomena controlling the level of cell proliferation and apoptosis are marked. The aim of our study was to determine the presence of proteins involved in apoptosis and proliferation: RCAS1, MT and the number of CD56-positive cells and their activity to elucidate their possible role in the development of adenocarcinoma and endometriosis., Materials and Methods: MT, RCAS1, CD56-positivity and CD69 expression were assessed in 55 tissue samples by Western blot and immunohistochemistry methods., Results: We found that endometrium during secretory menstrual cycle phase is characterized by significantly higher RCAS1 and higher MT expression than in proliferative phase. The number of CD56-positive cells and the CD69 antigen expression was significantly increased. Endometrial adenocarcinoma was characterized by significantly increased RCAS1 expression, while MT expression was comparable to the level found in the secretory phase. The number of CD56-positive cells was significantly decreased and their activity was comparable to the level found in the secretory phase. Endometriosis was accompanied by significantly lower RCAS1 and MT expressions, with lower number of CD-56 positive cells and lower expression of CD69 antigen in comparison to the secretory phase., Conclusions: The ability of endometrium to determine cytotoxic activity (RCAS1 expression changes) and high protection against DNA damage (MT expression) with concomitant changes in the number of immune cells and their activity, observed in normal endometrium during the menstrual cycle phases seems to be fundamental for pathological features of endometrial adenocarcinoma and endometriosis.

Toll-like receptor 3 (TLR3) responds to dsRNA, a product of most viral life cycles, and initiates production of proinflammatory and antiviral cytokines. The role of TLR3 in human mucosal immunity of the endometrium has not been examined. The effects of TLR3 ligation in endometrial epithelium could be significant as the endometrium is a significant site for viral entry and infection. Additionally, the cytokine milieu plays an essential role in normal functions of the endometrium such as uterine cycle progression, epithelial proliferation and shedding, and embryo implantation. In this study, we demonstrated cycle dependent expression of functional TLR3 in primary endometrial epithelial tissue and expression of intracellular TLR3 in human endometrial epithelial cell lines. We established that stimulation of TLR3-positive cell lines and primary human endometrial epithelial cells with dsRNA leads to TLR3-dependent expression of interleukin (IL)-6, IL-8, interferon (IFN)-inducible protein 10, RANTES, and IFN-beta. These results indicate that the cytokine profile of human endometrial epithelial cells can be modified through TLR3 stimulation. Our findings suggest that TLR3 is involved in the immune responses of endometrial epithelial cells after exposure to dsRNA and has the potential to alter the cytokine milieu and influence the outcome and consequences of infection.

A large number of CD16(-) natural killer (NK) cells appear in the human endometrium after ovulation. One possible explanation for this phenomenon is recruitment from peripheral blood (PB) CD16(-) NK cells. In this study, we examined whether IL-15 is involved in postovulatory recruitment from PB CD16(-) NK cells. The IL-15 receptor alpha chain was expressed on PB CD16(-) NK cells but not on CD16(+) NK cells. In an in vitro migration assay, recombinant human IL-15 enriched PB CD16(-) NK cells. Endometrial soluble protein fraction in the secretory phase, but not in the proliferative phase, also enriched these NK cells. Neutralization of IL-15 in the secretory phase endometrium with anti-IL-15 monoclonal antibody significantly lowered the enrichment of PB CD16(-) NK cells. In contrast, neutralization of other potential chemokines, including stromal cell-derived factor-1 or macrophage inflammatory protein-1alpha, had no significant effect. The IL-15 concentration in the endometrial soluble protein fraction was higher in the secretory phase than in the proliferative phase, with a peak in the midsecretory phase. These results support the idea that endogenous IL-15 in secretory phase endometrium plays a central role in postovulatory recruitment of PB CD16(-) NK cells into the human endometrium.

Problem: Macrophages are highly individualized in tissues and their activities are a reflection of systemic and local environmental signals. The expression of activation (CD69, CD71) and adhesion (CD54) molecules on the surface of CD14+ endometrial macrophages at various phases of the menstrual cycle was compared with the cell surface receptors of peritoneal fluid macrophages., Method of Study: Two-colour-flow cytometry was used to determine the peritoneal and endometrial macrophage phenotype., Results: Endometrium macrophages expressed a lower level of CD69+ and CD54+ macrophages than peritoneal macrophages. However, CD71 receptors displayed similar expression in both macrophage populations, endometrium and peritoneal, except during the proliferative phase., Conclusion: These findings demonstrate the differences between macrophages from endometrium and peritoneal fluid with regard to CD69, CD71 and CD54 expression. In addition, increased numbers of endometrial macrophages in the late secretory phase of the menstrual cycle suggest that they may play a role in menstruation.

Problem: Recent evidence of growth hormone (GH) receptor expression in rat endometrium and human myometrium have focused our attention on the role of the GH in endometrial development. We tested the expression of GH in the human endometrium throughout the menstrual cycle and during pregnancy., Method of Study: Immunohistochemical study was performed on endometrial specimens of fertile women in different periods of the menstrual cycle and in decidua of pregnant women., Results: Glandular cells of the human endometrium were positive for GH in the mid and late luteal phase. Furthermore, the glandular cells of decidua showed intense staining for GH, while the stromal cells were negative. No immunostaining was expressed in the proliferative or early luteal phase. The intensity levels of staining for GH in decidual specimens were significantly higher than in glandular cells of secretory endometrium specimens (P < 0.01)., Conclusions: The glandular cells of the human endometrium express GH from the late luteal phase throughout pregnancy in the decidual tissue. We suppose that GH plays an important role in blastocyst implantation.

Objective: To clarify the expression of 6Ckine, a potential chemoattractant for endometrial natural killer (NK) cells, in the human endometrium., Design: Experimental study., Setting: Department of obstetrics and gynecology at a medical university., Patient(s): Fifty-seven fertile women 25 to 52 years of age who had regular menstrual cycles and normal endometrium and were undergoing hysterectomy., Intervention(s): Endometrium was obtained from operative samples., Main Outcome Measure(s): Tissue was immunostained to determine the localization of 6Ckine in the endometrium throughout the menstrual cycle. The number of NK cells was counted in 10 nonoverlapping stromal areas. The concentration of 6Ckine in homogenized endometrium was determined by enzyme-linked immunosorbent assay., Result(s): Endometrial surface, glandular epithelial cells, and perivascular stromal cells were immunoreactive for 6Ckine throughout the menstrual cycle with some fluctuation. In addition, some T cells, NK cells, and macrophages in the stroma were immunoreactive for 6Ckine. The 6Ckine concentration was low in the proliferative phase but elevated in the secretory phase. It showed a moderate positive correlation with the number of endometrial NK cells., Conclusion(s): 6Ckine may be a potential chemoattractant for endometrial NK cells.

Remodelling of the human endometrium occurs during the normal menstrual cycle. This process involves the disintegration of the superficial or functionalis layer of the endometrium following the fall in progesterone resulting from the demise of the corpus luteum and the reconstruction of a new layer without scarring. The degradative properties of matrix metalloproteinases (MMP) and their presence in the endometrium during remodelling events suggests that they are effector molecules in this process. The features of menstruation parallel those of an inflammatory response and the abundance of leukocytes in the endometrium prior to the onset of menstruation indicates a role for these cells in the remodelling process. This review examines the relationship between leukocytes and the local production and activation of MMP within the endometrium.

Objective: Our purpose was to test the hypothesis that menstruation and intrapelvic injection of endometrium for the induction of endometriosis affect inflammatory parameters in peritoneal fluid from baboons., Study Design: In the first part of this study, 107 laparoscopies were performed in 62 female baboons with a normal pelvis during menstruation, the follicular phase, and the luteal phase. In the second part of this study, 21 baboons were studied during paired laparoscopies in the follicular phase and the luteal phase of the cycle. In the third part of this study, 11 baboons were studied by paired laparoscopies during menses and during the nonmenstrual phase of the cycle. In the fourth part of this study, paired laparoscopies were performed in 7 baboons before and after intrapelvic injection of endometrium. Peritoneal fluid was aspirated and measured in all laparoscopies and assessed for leukocyte concentration. In the third and fourth parts of the study, peritoneal fluid was analyzed for the concentrations of inflammatory cytokines and for the proportions of cells with immunohistochemical staining positive for these cytokines., Results: During menstruation, in comparison with nonmenstrual phases of the cycle, the leukocyte concentration of the peritoneal fluid was increased significantly, as were the proportions of peritoneal fluid cells with positive staining for tumor necrosis factor alpha, transforming growth factor beta(1), and intercellular adhesion molecule 1 and the peritoneal fluid concentrations of transforming growth factor beta(1) and interleukin 6. After intrapelvic injection of endometrium, the peritoneal fluid leukocyte concentration and the proportions of peritoneal fluid cells with positive staining for tumor necrosis factor alpha, transforming growth factor beta(1), CD3, and human leukocyte antigen (DR locus) significantly increased., Conclusion: Subclinical peritoneal inflammation occurs in baboons during menstruation and after intrapelvic injection of endometrium.

Interleukin-11 is a member of the IL-6 family of cytokines. Its presence in mouse decidua has been shown and experiments in genetically modified mice have suggested the importance of its receptor in stromal cell decidualization. In this study we used immunocytochemistry to determine expression of IL-11 in human endometrium. The effects of TNFalpha, IL-1alpha and TGFbeta on IL-11 production by epithelial and stromal cells was also investigated. Immunocytochemical staining in sections cut from 19 endometrial biopsies obtained throughout the menstrual cycle showed that IL-11 was expressed in both human endometrial epithelial and stromal cells, with epithelial staining being more intense than that seen in the stromal cells, at all times except the late secretory phase when the intensity was similar. Basal IL-11 production by cultured epithelial cells was greater than basal production by stromal cells. IL-1alpha, TNFalpha and TGFbeta (0.1-10 ng/ml) all caused a concentration-dependent increase in IL-11 production by both epithelial and stromal cells, but stimulated: basal values were greater for stromal than epithelial cells for all three cytokines. This work shows, for the first time, the presence of IL-11 within the human endometrium and that its production is controlled by other cytokines, which are postulated to play a role in implantation. Thus IL-11 may also play an important role in human endometrial function and embryo implantation.

Lymphoid aggregates (LA) develop during the proliferative phase of the menstrual cycle in the human uterine endometrium (EM). They contain mostly CD8+ T cells and B cells. As these LA are absent immediately following menses, they may arise by division of cells resident in the EM, or by division of a limited number of precursor cells that traffic into the EM during the early proliferative phase of the menstrual cycle. Alternatively, they may arise by the continuous trafficking of cells into the EM throughout the proliferative phase of the menstrual cycle. In this study we investigated the distribution and frequency of CD8+ T cells in the aggregates using expression of Vbeta2 or Vbeta8 as markers of clonality and Ki-67 as a marker of dividing cells. Confocal microscopic analysis of endometrial tissues showed the random distribution of CD8+ T cells within aggregates within the same sample and in aggregates from different samples. Furthermore, comparisons of the distribution of Vbeta2 and Vb8 with expected values predicted from Poisson distribution values were not significantly different, suggesting that CD8+ T cells do not arise by division from single precursors. A low level of T-cell division within LAs was confirmed by positive staining for Ki-67. Dividing T cells were randomly dispersed throughout the LA and the frequency of dividing cells did not vary greatly between aggregates within the same tissue. Nearest-neighbour analysis of dividing cells showed no statistically significant deviations from a random distribution. Taken together, these results suggest that LA develop during the menstrual cycle largely by the trafficking of cells to nucleation sites within the EM, rather than by division of a limited number of precursor cells.

Problem: To establish the relative proportions of endometrial lymphocyte subpopulations during the menstrual cycle., Method of Study: Lymphocytes were investigated by flow cytometry, during the early proliferative (EP), late proliferative (LP), early secretory (ES) and late secretory (LS) phases of the menstrual cycle., Results: Between the LP and LS phases, there was an increase in NK cells from 26.4% to 83.2% (P = 0.0017) of the CD45+ population. T cells decreased from 55.06% to 6.7% (P = 0.0017). Within the T-cell population, CD8+ cells decreased significantly from 63.0% in the LP phase to 54.2% in the LS phase (P = 0.04). In contrast, potential regulatory populations such as double negative (DN) T cells CD3+CD4-CD8- and natural T cells (NT) CD3+CD56+, increased significantly in the LS phase (P = 0.05; P = 0.03). gammadelta T cells, predominantly of the DN and NT phenotypes remained consistently low at all stages of the cycle (2.34%)., Conclusion: Endometrial lymphocyte fluctuations during the menstrual cycle may reflect hormonal regulation of maternal immunity, thereby promoting tolerance at the time of implantation.

Intraepithelial leukocytes (IEL) are recognized as an important component of most mucosal surfaces but have received scant attention in the human female reproductive tract. The aim of the present study was to characterize, quantify and compare IEL populations in normal endometrium (n = 30) and in eutopic and ectopic (endometriotic or adenomyotic lesions) endometrium from women with endometriosis (n = 30) or adenomyosis (n = 15) at different menstrual cycle phases in order to assess the role of IEL in these common but poorly understood disorders. IEL populations were examined in formalin-fixed, paraffin-embedded sections using a streptavidin-biotin-peroxidase complex technique and quantified in relation to epithelial cell numbers. IEL in control endometrium and eutopic endometrium in endometriosis and adenomyosis varied during the menstrual cycle, with CD45+, CD43+ and CD56+ cells increasing from the proliferative to the late secretory phase. IEL were elevated in surface compared with glandular epithelium in the proliferative and early secretory phases. Throughout the menstrual cycle there were no significant differences in IEL between eutopic and ectopic endometrium in adenomyosis. Endometriotic foci, however, contained elevated levels of CD45+, CD3+ and CD8+ cells and reduced numbers of CD56 + cells compared with the corresponding eutopic endometrium and these did not vary with menstrual cycle phase. In contrast, ectopic endometrium in adenomyosis showed some cyclical changes with CD56+ cells increasing significantly in the late secretory phase. It is possible these differences may play a role in the pathogenesis of endometriosis and the associated complications.

Flow cytometry studies of first-trimester normal human decidua have proposed that a proportion of endometrial granulated lymphocytes (eGL), the predominant leucocyte population in early human pregnancy, co-express CD69. The purpose of this study was to investigate CD69 expression by immunohistochemistry throughout the menstrual cycle and in first-trimester human decidua and to determine whether CD69 expression by eGL is an in vivo characteristic or whether the antigen is acquired in vitro after tissue disaggregation and cell purification. Single immunoenzymatic and double immunofluorescence labelling of tissue sections, supplemented with double immunoenzymatic staining of purified eGL and flow cytometry, indicated that eGL do not co-express CD69 in situ but that they acquire the molecule in vitro after cell purification and culture.

Problem: LIF is believed to be involved in human reproduction. Because little is known about the function of this cytokine in proliferative phase of cycle and because LIF is found to regulate the cellular growth we evaluated the possible relationship between endometrial LIF secretion and endometrial growth., Method: The present is prospective, blinded study with clinical and immunobiochemical correlation between endometrial LIF concentration and endometrial ultrasound pattern. Twenty-four patients who were candidates for IVF and oocyte donation are included in this study. At day 10 of their cycle the endometrial biopsy was performed, followed by vaginal sonographic measurements of endometrial thickness and pattern. Endometrial LIF concentration was measured by ELISA in supernatants taken from cultured explants., Results: Endometrial LIF production significantly negatively correlated with endometrial thicknesses (P < 0.05). There was a 4-fold elevation in LIF production when the endometrial thickness was below 5 mm. Strong correlation was found also between endometrial LIF production and sonographic endometrial pattern (P < 0.05). The most significant amount of LIF was found in nonfavorable endometria., Conclusion: In the proliferative phase of cycle there is a dynamic relationship between endometrial sonographic appearance and local LIF secretion. Specifically LIF production is negatively correlated with endometrial thickness and pattern. The low amount of cytokine is a normal uteral environment for endometrial development, whereas deregulation of cytokine production toward its overexpression may lead to a strong inhibitory effect on endometrial growth. This finding might have an important clinical implication.

Problem: Comparison and characterisation of different lymphocyte subsets in the endometrium of endometriosis patients and in healthy women on every day of the menstrual cycle with special emphasis on the CD4:CD8 ratio in the endometrium., Method: Immunohistochemical staining of 253 endometrial biopsies of infertile women with and without endometriosis with Anti-Leu4 (CD3), Anti-Leu3a (CD4), Anti-Leu2a (CD8), Anti-Leu7 and Anti-Human-B-cell (CD22) using the immune peroxidase reaction. Identification and counting of positive lymphocyte were performed on cryostat sections., Results: Endometrial lymphocyte subsets show equal quantity and distribution in endometriosis patients and in the control group. After a peak in the early proliferative phase the absolute number of T lymphocytes decreases while a predominance of T-suppressor/cytotoxic T lymphocytes (CD8) compared to T-helper/inducer lymphocytes (CD4) occurs towards the end of the menstrual cycle., Conclusion: Endometrium as the potential parent epithelia of endometriosis lesions seems not to be altered in its lymphatic cell content compared to healthy women. Furthermore, endometrium is clearly characterised as part of the mucosa associated lymphatic tissue (MALT). T lymphocytes show specific quantitative changes due to different phases of the menstrual cycle.

Endometrial lymphocytes were studied at all stages throughout the menstrual cycle and early pregnancy by flow cytometry to examine different lymphocyte subpopulations and the expression of the T- and NK-cell activation markers. After pregnancy, CD8+CD3+ lymphocytes were decreased in the decidua. In both endometrium and decidua, more T cells expressed CD69, CD71, HLA-DR, and CD38 antigens than in peripheral blood. After pregnancy, CD71+CD3+ lymphocytes were further increased. CD25+CD3+ lymphocytes decreased significantly in the endometrium and decidua of ectopic pregnancies, but not in the decidua of normal pregnancies. These findings indicate that T cells are regionally activated in the first trimester, and it may be the result of the stimulation by fetal antigens. NK cells were the most abundant cell type in the decidua, which expressed the phenotype CD16- CD56+, and CD57-CD56+. The proportion of activated decidual NK cells was increased in anembryonic pregnancies more than in normal pregnancies, although the total NK subpopulation was similar in both groups. This might result in increased NK cytotoxicity in anembryonic pregnancies. In conclusion, T cells are activated, but NK cytotoxicity is decreased in the decidua of early normal pregnancies. This might be important in the control of trophoblast growth and placental development.

The present study was undertaken to investigate the expression of beta1 integrins in human endometrium and decidua using flow cytometry, immunohistochemistry, and immunoprecipitation. Fluorescence-activated flow cytometry demonstrated the greater expression of the beta 1, alpha 1, alpha 2, and alpha 5 subunits of the beta1 integrin family in cultured stromal cells from the midsecretory phase, than in those of the early proliferative phase. The addition of estradiol (E2) and progesterone (P) to cultured stromal cells in the early proliferative phase increased the expression of beta1 integrins in vitro. The immunohistochemical distribution of beta1 integrins demonstrated predominantly glandular epithelial staining in the proliferative phase, and mesenchymal and glandular staining in the midsecretory phase. Flow cytometry also demonstrated the expression of the beta 1, alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6 subunits of beta 1 integrin family in cultured decidual cells, and the enriched-fraction of prolactin (PRL)-producing decidual cells isolated by Percoll gradients showed high levels of beta 1, integrins expression. Immunohistochemistry confirmed the beta 1 integrin cell surface phenotypes in cultured decidual cells observed by flow cytometry. Autoradiography of immunoprecipitate subjects to SDS-PAGE revealed three major polypeptides with molecular weights of 130 kDa (beta 1 subunit), 165 kDa (alpha 2 subunit), and 210 kDa (alpha 1 subunit) under reducing conditions. In summary, the present study demonstrated that endometrial stromal and decidual cells expressed beta1 integrin subunits at their surfaces. The expression exhibited a variability throughout the menstrual cycles, being predominantly detected in the secretory phase, and was maintained highly in the decidua. Thus, beta 1 integrins in human endometrium and decidua may be important in mediating the organization of extracellular matrix proteins derived from embryos during the early stage of implantation.

Problem: Integrins belong to a family of cell adhesion molecules that are present on virtually all cells. The temporal and spatial expression of these important proteins on the human endometrium suggests that certain integrins may participate in the cascade of molecular events leading to successful implantation., Methods: Using immunohistochemistry, we studied the expression of 12 different integrins in up to 600 samples of human endometrium throughout the menstrual cycle. Intensity and distribution of staining was determined using the semiquantitative HSCORE, with specific focus on the differences between glandular and luminal expression., Results: We noted that the glandular and luminal epithelium undergo independent alterations in integrin expression throughout the menstrual cycle. Specifically, glandular epithelium express certain integrins only during the window of implantation, while luminal epithelium down-regulate certain integrins during this time. The expression of one integrin (the alpha v beta 3 vitronectin receptor) on both luminal and glandular epithelium coincides with the time of embryo attachment; aberrant expression of this integrin is associated with infertility., Conclusion: It appears that the endometrium is a unique tissue with regard to the number of integrins that undergo temporal and spatial changes during the menstrual cycle. These data may offer new directions for the development of a novel contraceptive approach targeted to the endometrium as well as a better understanding of occult causes of infertility in women.

CD44 is a cell surface glycoprotein known to be a hyaluronate receptor, which functions in lymphocyte homing and cancer metastasis. We have previously shown that CD44 molecules are expressed in normal endometrial tissue. In this study we immunohistochemically examined expression of CD44 glycoproteins in normal human endometrial tissue to gain insight into roles of CD44 molecules. No expression of the CD44 standard form was observed with 11 endometria in the proliferative phase. On the contrary, 13 of 19 secretory phase endometria were stained intensely by anti-CD44 standard form anti-body; all CD44-positive cases were in the mid and late secretory phases. Among the 13 CD44-positive endometria, four also were positive for the CD44 variant with exon v6. CD44 molecules were present at all but the luminal cell surfaces. These results indicate that expression of CD44 depends on menstrual cycle, suggesting that CD44 molecules might be involved in implantation of fertilized ovum in endometrium.