Endometrial Cell Populations Research Articles

P-498 Towards precision medicine: maternal genetic profiles as determinant of adequate decidualization and reproductive success; a prospective study in oocyte donation cycles.

Abstract Study question To determine the impact of genes related to decidualization and immune cell activation as predictors for reproductive success. Summary answer Women with unfavorable genetic profiles for Human Leukocyte Antigen (HLA)-F and Killer Immunoglobulin-like Receptor (KIR) genes showed decreased pregnancy rates. What is known already KIR genes encode receptors present on uterine Natural Killer (uNK) cells that are essential during placentation and early gestation. It has been established that women of KIR AA genotype show lower reproductive success than KIR BX, due to the lack of activation of these immune cells. Additionally, another gene, HLA-F, expressed in endometrial cells, also seems to be involved in the recruitment and supplementary activation of uNKs. Certain alleles of this gene appear to decrease its expression in the uterus and hinder the processes in which it is involved during the decidualization process, decreasing implantation rates and increasing time-to-pregnancy. Study design, size, duration We conducted a prospective cohort study with 75 patients, with no history of recurrent miscarriage or implantation failure, who attended our institution to undergo assisted reproduction treatments (ART) with donated oocytes, between January 2021 and December 2022. By using the egg donation model, we were able to investigate the endometrial cell population avoiding the oocyte factor bias Participants/materials, setting, methods All patients included were under 45 years of age (40.5 ± 2.9 years), had a normal BMI (21.6 ± 1.9 kg/m2) and had not been diagnosed with uterine pathologies or immunological conditions. All recipients were genotyped for the KIR and HLA-F genes and pregnancy, miscarriage (MR) and live birth rates (LBR) obtained in their egg donation cycles were recorded. Fisher's test was used to determine statistically significant differences (p < 0.05) between groups Main results and the role of chance Patients were divided into two groups according to their KIR genotype: 29 KIR AA (inhibitor) and 46 KIR BX (activator). In each group, allelic load was determined for the three HLA-F regions studied (RS1362126, RS2523405 and RS2523393). For all these regions, patients with favorable HLA-F alleles and KIR BX genotype had higher pregnancy rates than KIR AA patients with unfavorable HLA-F alleles (85.42 % vs 54.17% for RS1362126 region, OR = 4.956, p = 0.008; 83.33% vs 58.62% for RS2523405 region, OR = 3.529, p = 0.0295; 82.5% vs 57.69% for RS2523393 region, OR = 3.457, p = 0.0462). No significant differences were obtained for MR or LBR. This demonstrates the involvement of KIR and HLA-F in embryo implantation and that the coincidence of unfavorable alleles for both decreases reproductive success considerably, probably due to impaired decidualization and lack of uNK recruitment and activation. No significant differences were obtained for the rest of the genetic profiles, except when comparing pregnancy rate of KIR AA women with favorable HLA-F alleles to KIR AA women with unfavorable HLA-F alleles for the RS1362126 region (79.41% vs 54.17%, OR = 3.264, p = 0.04). The latter finding suggests that the lack of uNK cell activation, so evident in KIR AA patients, could be compensated by expressing favorable HLA-F alleles. Limitations, reasons for caution The low sample size of this study does not allow us to compare groups taking into account other genes, such as embryonic HLA-C, which also, and in combination with KIR genotypes, has been shown to play a role in proper implantation and placentation. Wider implications of the findings uNKs activation is required to promote embryo implantation. Combination of unfavorable genetic profiles involved in these immune mechanism leads to lower pregnancy rate. The study of these genetic profiles can help to predict reproductive success, thus identifying patients susceptible to receive immunological treatment before embryo transfer, possibly decreasing their time-to-pregnancy. Trial registration number Not applicable

Perspective: Can early embryonic losses be reduced in lactating dairy cows?

Perspective: Can early embryonic losses be reduced in lactating dairy cows?

O-046 Implantation and beyond, the endometrial perspective

Abstract text Early pregnancy failures are spread throughout the first few weeks, suggesting that impairment of normal biological processes can occur at sequential stages: epithelial attachment and breaching, early stromal and then decidual invasion, glandular and vascular invasion. Biopsy-based transcriptomics and proteomics have failed to demonstrate a reproducible or interpretable molecular signature for endometrial receptivity, but single cell RNAseq suggests a step change in the epithelial transcriptome in the mid secretory phase, consistent with the appearance of new cell phenotype(s). Previously unseen heterogeneity in both epithelial and stromal cell populations has become evident, but gene signatures have not yet achieved a level of resolution that allows insights clear enough to decode the biology of the receptive state. However, methodology for propagating and recombining endometrial cell populations into 3D engineered tissue models has advanced, so that new mechanistic questions can be asked. Initial acquisition of receptivity to implantation is followed by the development of a supportive environment for embryos that possess the capacity to progress, with maternal cell populations (epithelial, stromal, immune and vascular) acting cooperatively within a remodelling extracellular matrix (ECM). A balance must be achieved in the ECM between breakdown, with opening of hydrated spaces to allow expansion of the embryonic sac while maintaining a substrate for stable physical attachment to allow invasion by extravillous trophoblast. The extent and nature of cellular trafficking to and from the uterus is important before and during early pregnancy. There is evidence that regulation of a resident senescent cell subpopulation by uterine NK cells occurs in concert with ECM remodelling to achieve a functionally supportive environment in the early first trimester. Access of bone marrow-derived cells to vessel walls is regulated by placental-endothelial signalling to initiate remodelling for the increased blood flow to the conceptus that is required in later pregnancy. Thus ‘receptivity’ and ‘supportiveness’ require normal cell proportions and functional phenotypes in multiple endometrial cell populations and their physical environment in order to allow a well-calibrated sequential developmental response in the conceptus.

Adenomyosis pathogenesis: insights from next-generation sequencing.

BACKGROUNDAdenomyosis, characterized by the presence of islands of endometrial tissue surrounded by hypertrophic smooth muscle cells within the myometrium, is one of the most challenging uterine disorders in terms of diagnosis and management. Adenomyosis presents with pelvic pain, excessive uterine bleeding, anemia and infertility. The relative contributions of abnormal endometrial tissue and myometrial smooth muscle cells to the development and growth of adenomyosis are not well understood. Moreover, there is continuing debate on the origins of adenomyosis; two competing theories describe the invagination of basal endometrium into the myometrium or the metaplastic differentiation of remnant endometrial stem/progenitor cells within the myometrium.OBJECTIVE AND RATIONALEA recent series of next-generation sequencing (NGS) studies have provided the best scientific evidence thus far regarding the cellular origins of adenomyosis and the contributions of new signaling pathways to its pathogenesis, survival, and growth. These seminal studies on endometrium, adenomyosis and endometriosis demonstrate or support the following key points. (i) Mutations of KRAS map to both intracavitary endometrial tissue and proximally located adenomyotic samples, supporting the invagination theory of pathogenesis. Driver mutations found in smooth muscle cells of uterine fibroids are absent in adenomyosis. (ii) KRAS and other less frequent mutations are limited to endometrial-type epithelial cells. They are also observed in endometriosis, indicating that the disease process in adenomyosis is similar to that in endometriosis and distinct from that of uterine fibroids. (iii) Activating mutations of KRAS stimulate specific pathways to increase cell survival and proliferation and are associated with progesterone resistance in adenomyosis. Together, these findings suggest that distinct cell populations in eutopic endometrial tissue play key roles in the etiology of adenomyosis. Dependence on ovarian steroids and ovulatory cycles for disease severity is a unique feature of adenomyosis. In this context, common patterns of aberrant gene expression have been reported both in adenomyosis and endometriosis. These include pathways that favor increased estrogen biosynthesis, decreased estradiol metabolism, a unique estrogen receptor beta (ESR2)-driven inflammatory process, and progesterone resistance due to decreased progesterone receptor expression. Since adenomyosis exhibits a uniquely estrogen-driven inflammatory process and progesterone resistance, we discuss the interactions between these molecular characteristics and signaling pathways induced by the newly discovered KRAS mutations.SEARCH METHODSWe conducted a comprehensive search using PubMed for human and animal studies published until 2020 in the following areas: adenomyosis, endometriosis, endometrium, NGS, whole-exome sequencing, whole-genome sequencing, RNA sequencing, targeted deep sequencing, epigenetics, driver mutation, KRAS, progesterone resistance, estrogen action and steroid production.OUTCOMESTargeted deep sequencing analyses of epithelial cells in adenomyosis and adjacent basalis endometrial glands demonstrated recurring KRAS mutations in both cell types. This finding suggests that adenomyosis originates from basalis endometrium. Epithelial cells of the endometrium, adjacent adenomyosis and co-occurring endometriosis also share identical KRAS mutations. These findings suggest both adenomyosis and endometriosis are oligoclonal tissues that arise from endometrial cell populations carrying a specific driver mutation that most commonly affects the KRAS gene.WIDER IMPLICATIONSAdenomyosis usually follows an event such as pregnancy that has disrupted the integrity of the endometrial–myometrial junction followed by repetitious menstrual episodes that increase the likelihood of the entrapment of the basalis endometrium within the myometrium. Glandular epithelial cells carrying KRAS mutations and located within the deep crypts of basalis endometrium may become entrapped and invade myometrial tissue to give rise to adenomyosis. Evidence suggests that KRAS mutations may be responsible, in part, for previously observed phenomena such as prolonged cell survival and progesterone resistance in adenomyosis.

Studies of uterine secretions and products of primary cultures of endometrial cells in pigs

Abstract The uterus plays a central role in the reproductive biology of mammals. Adaptation of the uterus from an oviparous to a viviparous nature required changes that involved production of a uterine environment that could support the development of the embryo and fetus. Production of a suitable environment includes the synthesis and secretion of products by the uterine endometrium. However, the uterine endometrium is not a single homogeneous unit, but rather consists of several cell populations. Recent accomplishments in cell culture techniques provide a means for examining the contributions and secretory control of different endometrial cell populations. Furthermore, it is possible to recombine specific cell types to study their interaction. It is clear that the luminal epithelium, glandular epithelium and endometrial stroma produce different secretory products. Some secretions (for example uteroferrin) are secreted by only one cell type; others (for example prostaglandins, PGs) are secreted by all types of cell. There is much to be learned about the functions and regulations of endometrial secretions and there are important aspects of the role of the endometrium in pregnancy that present concepts do not address. For example, there is no explanation for the required synchrony between the embryo and uterus before day 10 and the implications of control of the uterine environment by progesterone from day 4 to day 10 are not understood. Almost all of the uterine secretory proteins are produced after day 10. In this review, we consider the protein and prostaglandin products from the different cell populations of the pig endometrium and propose a model to explain the integration of multiple sources of PGs and multiple regulators of PG secretion. Our purpose is to facilitate a more complete understanding of the individual uterine cell populations and a better understanding of how these cell types interact to function as a complete unit.

ENDOMETRIAL STROMAL CELLS: ISOLATION, EXPANSION, MORPHOLOGICAL AND FUNCTIONAL PROPERTIES

We aimed to study biological properties of human endometrial stromal cells in vitro. The endometrium samples (n = 5) were obtained by biopsy at the first phase of the menstrual cycle from women with endometrial hypoplasia. In all cases, a voluntary written informed consent was obtained from the patients. Endometrial fragments were dissociated by enzymatic treatment. The cells were cultured in DMEM/F12supplemented with 10% FBS, 2mМ L-glutamine and 1ng/ml FGF-2in a multi-gas incubator at 5% CO2 and 5% O2. At P3the cells were subjected to immunophenotyping, multilineage differentiation, karyotype stability and colony forming efficiency. The cell secretome was assessed by BioRad Multiplex immunoassay kit. Primary population of endometrial cells was heterogeneous and contained cells with fibroblast-like and epithelial-like morphology, but at P3 the majority of cell population had fibroblast-like morphology. The cells possessed typical for MSCs phenotype CD90+CD105+CD73+CD34-CD45-HLA-DR-. The cells also expressed CD140a, CD140b, CD146, and CD166antigents; and were negative for CD106, CD184, CD271, and CD325. Cell doubling time was 29.6 ± 1.3h. The cells were capable of directed osteogenic, adipogenic and chondrogenic differentiation. The cells showed 35.7% colony forming efficiency and a tendency to 3D spheroid formation. The GTG-banding assay confirmed the stability of eMSC karyotype during long-term culturing (up to P8). After 48h incubation period in serum-free medium eMSC secreted anti-inflammatory IL-1ra, as well as IL-6, IL-8and IFNγ, angiogenic factors VEGF, GM-CSF and FGF-2, chemokines IP-10and MCP-1. Thus, cultured endometrial stromal cells meet minimal ISCT criteria for MSC. Proliferative potential, karyotype stability, multilineage plasticity and secretome profile make eMSC an attractive object for the regenerative medicine use.

Mapping of estradiol binding sites through receptor micro-autoradiography in the endometrial stroma of early pregnant mice.

Estradiol triggers key biological responses in the endometrium, which rely on the presence and levels of its cognate receptors on target cells. Employing the receptor micro-autoradiography (RMAR) technique, we aimed to provide a temporal and spatial map of the functional binding sites for estradiol in the mouse endometrial stroma during early pregnancy. Uterine samples from days 1.5 to 7.5 of pregnancy were collected 1h after tritiated- (3H-) estradiol administration and prepared for RMAR analysis. Autoradiographic incorporation of 3H-thymidine (after 1-h pulse) was evaluated over the same gestational interval. Combined RMAR with either histochemistry with Dolichus biflorus (DBA) lectin or immunohistochemistry for detection of the desmin further characterized 3H-estradiol binding pattern in uterine Natural Killer (uNK) and decidual cells, respectively. 3H-estradiol binding levels oscillated in the pregnant endometrial stroma between the mesometrial and antimesometrial regions as well as the superficial and deep domains. Although most of the endometrial stromal cells retained the hormone, a sub-population of them, as well as endothelial and uNK cells, were unable to do so. Rises in the levels of 3H-estradiol binding preceded endometrial stromal cell proliferation. 3H-estradiol binding and 3H-thymidine incorporation progressively decreased along the development of the antimesometrial decidua. Endothelial proliferation occurred regardless of 3H-estradiol binding, whereas pericytes proliferation was associated with high levels of hormone binding. Endometrial cell populations autonomously control their levels of 3H-estradiol binding and retention, a process associated with their proliferative competence. Collectively, our results illustrate the intricate regulatory dynamic of nuclear estrogen receptors in the pregnant mouse endometrium.

Physiologic, health, and production responses of dairy cows supplemented with an immunomodulatory feed ingredient during the transition period

This study compared physiological, health, and productive parameters in dairy cows supplemented or not with Omnigen-AF (OMN; Phibro Animal Health, Teaneck, NJ) during the transition period. Thirty-eight nonlactating, multiparous, pregnant Holstein × Gir cows were ranked by body weight (BW) and body condition score (BCS), and assigned to receive (n=19) or not (CON; n=19) OMN at 56 g/cow daily (as-fed basis) beginning 35 d before expected date of calving. Before calving, cows were maintained in single drylot pen with ad libitum access to corn silage, and received (as-fed basis) 3kg/cow daily of a concentrate. After calving, cows were moved to an adjacent drylot pen, milked twice daily, offered (as-fed basis) 35kg/cow daily of corn silage, and individually received a concentrate formulated to meet their nutritional requirements after both milkings. Cows received OMN individually as top-dressing in the morning concentrate feeding. Before calving, cow BW and BCS were recorded weekly and blood samples were collected every 5 d beginning on d −35 relative to expected calving date. After calving and until 46 d in milk, BW and BCS were recorded weekly, individual milk production was recorded, and milk samples were collected daily for total solids and somatic cell count analyses. Blood was sampled daily from 0 to 7 d in milk, every other day from 9 to 21 d in milk, and every 5 d from 26 to 46 d in milk. On 30 and 46 d in milk, cows were evaluated for endometritis via cytobrush technique, based on % of polymorphonuclear (PMN) cells in 100 total cell count (PMN + endometrial cells). On 48.7±1.6 d in milk, 9 cows/treatment received a lipopolysaccharide (LPS) injection (0.25μg/kg of BW), and blood was sampled hourly from −2 to 8 h, at 12-h intervals from 12 to 72 h, and at 24-h intervals form 96 to 120 h relative to LPS administration. No treatment differences were detected on BW, BCS, serum concentrations of cortisol, fatty acids, insulin, glucose, haptoglobin, cortisol, and insulin-like growth factor-I. Cows receiving OMN had greater milk yield (30.3 vs. 27.1kg/d) and percentage of PMN cells in endometrial cell population (12.2 vs. 3.9%) compared with CON cows. After LPS administration, cows receiving OMN had greater mean serum haptoglobin (212 vs. 94 µg/mL), as well as greater serum concentration of tumor necrosis factor α at 1, 2, and 3 h relative to LPS injection compared with CON cows. In conclusion, OMN supplementation during the transition period enhanced innate immunity parameters and increased milk production in dairy cows.

Mesenchymal Stem/Progenitors and Other Endometrial Cell Types From Women With Polycystic Ovary Syndrome (PCOS) Display Inflammatory and Oncogenic Potential

Endometrium in polycystic ovary syndrome (PCOS) presents altered gene expression indicating progesterone resistance and predisposing to reduced endometrial receptivity and endometrial cancer. We hypothesized that an altered endocrine/metabolic environment in PCOS may result in an endometrial "disease phenotype" affecting the gene expression of different endometrial cell populations, including stem cells and their differentiated progeny. This was a prospective study conducted at an academic medical center. Proliferative-phase endometrium was obtained from 6 overweight/obese PCOS (National Institutes of Health criteria) and 6 overweight/obese controls. Microarray analysis was performed on fluorescence-activated cell sorting-isolated endometrial epithelial cells (eEPs), endothelial cells, stromal fibroblasts (eSFs), and mesenchymal stem cells (eMSCs). Gene expression data were validated using microfluidic quantitative RT-PCR and immunohistochemistry. The comparison between eEP(PCOS) and eEP(Ctrl) showed dysregulation of inflammatory genes and genes with oncogenic potential (CCL2, IL-6, ORM1, TNAIFP6, SFRP4, SPARC). eSF(PCOS) and eSF(Ctrl) showed up-regulation of inflammatory genes (C4A/B, CCL2, ICAM1, TNFAIP3). Similarly, in eMSC(PCOS) vs eMSC(Ctrl), the most up-regulated genes were related to inflammation and cancer (IL-8, ICAM1, SPRR3, LCN2). Immunohistochemistry scoring showed increased expression of CCL2 in eEP(PCOS) and eSF(PCOS) compared with eEP(Ctrl) and eSF(Ctrl) and IL-6 in eEP(PCOS) compared with eEP(Ctrl). Isolated endometrial cell populations in women with PCOS showed altered gene expression revealing inflammation and prooncogenic changes, independent of body mass index, especially in eEP(PCOS) and eMSC(PCOS), compared with controls. The study reveals an endometrial disease phenotype in women with PCOS with potential negative effects on endometrial function and long-term health.

Caprine endometrial stromal cells modulate the effects of steroid hormones on cytokine secretion by endometrial epithelial cells in vitro

The main purpose of this study was to examine the effects of 17β-estradiol (E2) and progesterone (P4) on cytokine secretion by caprine endometrial epithelial cells (EEC) in vitro. Epithelial cells grown alone or in co-culture with stromal cells (ESC) were treated with E2 or P4, or both. Homogeneity of the endometrial cell populations was ascertained immunocytochemically. The quantities of cytokines secreted in this system were assessed by ELISA and their protein expression by Western blot. The exposure of EEC to P4 alone or in combination with E2 significantly increased the amount of TGF-β1, TNF-α and IL-18 secretion, whereas E2 had no effect on the synthesis of these cytokines. When epithelial cells were co-cultured with ESC, the secretion of TGF-β1, TNF-α and IL-18 by EEC significantly increased compared to that by EEC alone. However, the treatment with both steroids decreased the secretion of TNF-α, IL-18 and TGF-β1 by EEC in the presence of ESC. In contrast to TGF-β1, TNF-α and IL-18, the secretion of leukemia inhibitory factor (LIF) by EEC was not affected by E2 and/or P4 either directly or indirectly. The present results indicate that the interactions between caprine endometrial stromal and epithelial cells can modulate the secretion of TGF-β1, TNF-α and IL-18 by EEC exposed to E2 and/or P4in vitro.

Endometrial biology during trophoblast invasion

Attainment of successful implantation depends upon the synchronized changes in the endometrium before and after the arrival of blastocyst into the uterine cavity. The cues obtained from the receptive endometrium helps in proliferation and differentiation of the trophoblast cells. During the course of invasive differentiation, the trophoblast cells undergo several morphological, biochemical and molecular changes to gain the invasive capabilities. In turn, close apposition of the developing embryo brings out functional and morphological changes into the hormone primed receptive endometrium. Global gene expression profiling of the endometrium in response to the developing embryo or in response to the pregnancy hormone, human chorionic gonadotropin, in primate and human models, suggest that the endometrial-embryo cross-talk mainly influences three biological processes. Biological processes getting influenced by the blastocyst "signals" are associated with immunomodulation, biosensing and invasion. Pro- and anti-invasive paracrine factors expressed by different endometrial cell populations regulate the trophoblast invasion through activation of diverse signaling pathways. Identification of the gene signatures involved in embryo-endometrial dialogue would enhance our understanding about the pathologies like miscarriages and endometriosis.

Spatiotemporal Coupling of Focal Extracellular Matrix Degradation and Reconstruction in the Menstrual Human Endometrium

Coupling of focal degradation and renewal of the functional layer of menstrual endometrium is a key event of the female reproductive biology. The precise mechanisms by which the various endometrial cell populations control extracellular matrix (ECM) degradation in the functionalis while preserving the basalis and the respective contribution of basalis and functionalis in endometrium regeneration are still unclear. We therefore compared the transcriptome of stromal and glandular cells isolated by laser capture microdissection from the basalis as well as degraded and preserved areas of the functionalis in menstrual endometria. Data were validated by in situ hybridization. Expression profile of selected genes was further analyzed throughout the menstrual cycle, and their response to ovarian steroids withdrawal was studied in a mouse xenograft model. Immunohistochemistry confirmed the results at the protein level. Algorithms for sample clustering segregated biological samples according to cell type and tissue depth, indicating distinct gene expression profiles. Pairwise comparisons identified the greatest numbers of differentially expressed genes in the lysed functionalis when compared with the basalis. Strikingly, in addition to genes products associated with tissue degradation (matrix metalloproteinase and plasmin systems) and apoptosis, superficial lysed stroma was enriched in gene products associated with ECM biosynthesis (collagens and their processing enzymes). These results support the hypothesis that fragments of the functionalis participate in endometrial regeneration during late menstruation. Moreover, menstrual reflux of lysed fragments overexpressing ECM components and adhesion molecules could easily facilitate implantation of endometriotic lesions.

Endometrial stem cells

The human endometrium is a dynamic tissue, which undergoes cycles of growth and regression with each menstrual cycle. Endometrial regeneration also follows parturition and extensive resection and occurs in postmenopausal women taking estrogen replacement therapy. It is likely that adult stem/progenitor cells are responsible for this remarkable regenerative capacity. This review discusses the first published evidence for the existence of endometrial stem/progenitor cells in human and mouse endometrium. Functional approaches have been used to identify candidate endometrial epithelial and stromal stem/progenitor cells, due to lack of known specific endometrial stem cell markers. Rare clonogenic cells and side population cells have been identified in human endometrial cell populations. In mouse endometrium, rare label-retaining cells have also been identified. The ability of transplanted human endometrial cells to grow endometrial tissue in animal hosts also suggests the presence of stem/progenitor cells. These initial studies providing the first functional evidence for epithelial and stromal stem/progenitor cells in human and mouse endometrium lay the groundwork for further studies to characterize their stem cell properties. They also provide the impetus to discover specific markers that will enable their prospective isolation and allow their location in normal and pathological endometrium to be determined.

Endometriosis--a stem cell disease?

Endometriosis is an estrogen-dependent and chronic disease with an unknown etiology and pathogenesis. It is however likely and well accepted that retrograde menstruation of endometrial cells into the pelvic cavity is the origin of this disease in many cases. Here we discuss a model in which retrogradely menstruated endometrial cells have different inherent developmental properties because they represent in fact a mixture of different developmental cell stages. These stages can be distinguished in part by the expression of marker proteins such as cytokeratin (intermediate filament protein of epithelial cells) or E-cadherin (intercellular adhesion protein of epithelial cells and metastasis suppressor molecule). Cytokeratin-positive E-cadherin negative cells, for example, would be less differentiated epithelial cells than cytokeratin-positive E-cadherin positive cells. In analogy to findings in other cell systems we assume that the cells which are undifferentiated or not fully differentiated still have the potential to give rise to differentiated daughter cells and, on the other hand, could be maintained as a pool of rather undifferentiated cells and capable of self renewal. This feature would be similar to stem cells (SC) and cells with plasticity. Interestingly we find epithelial cells of different developmental stages in deep infiltrating (e. g. of colon) or peritoneal endometriotic lesions. Therefore we conclude that less differentiated cells in retrogradely menstruated endometrial cell populations possibly representing SC features or plasticity might be the cellular source of primary endometriotic lesions and those present in lesions may contribute to the persistence of the disease by detaching and forming secondary lesions.

Stromal-epithelial interactions modulate estrogen responsiveness in normal human endometrium.

The coculture of endometrial epithelial cells (EEC) with stromal cells (ESC) allows achievement of an improved in vitro system for studying interactions between cells via soluble signals. The purpose of this study was to investigate whether 17beta-estradiol and insulin can induce proliferation of EEC through ESC-secreted factors. No evidence of estrogen-induced EEC proliferation has been reported so far in the conventional culture methods. To this end, we used an in vitro bicameral coculture model where human EEC were grown on extracellular matrix-coated inserts applied in dishes containing ESC. Proliferation was assessed by tritiated thymidine incorporation. Homogeneity of endometrial cell populations was ascertained immunocytochemically. 17beta-estradiol did not induce any proliferative effect on EEC cultured alone. Endometrial epithelial cell proliferation was significantly enhanced in EEC/ESC cocultures; moreover, it was further increased by 17beta-estradiol addition. Insulin increased proliferation in EEC cultured alone, but again the effect was more pronounced in EEC/ESC cocultures. Coincubation of 17beta-estradiol and an antibody against insulin-like growth factor I (IGF I) led to neutralization of ESC-mediated EEC proliferation. This work provides evidence that the effect of 17beta-estradiol on human EEC proliferation may be mediated at least in part through ESC-secreted IGF I. We also showed that insulin effect is also partially due to ESC activation.

High-density ovine endometrial cells exhibit natural killer activity during early pregnancy

Natural killer (NK)-like activity was assessed for peripheral blood lymphocytes (PBL) and unfractionated and fractionated endometrial cells recovered from ewes during the estrus cycle (Days 12 to 14) and early pregnancy (Days 16 to 18). The PBL and endometrial cells (each designated as effector cells) were cocultured with chromium-51 ( 51Cr) labeled NK-sensitive K-562 target cells in effector: target cell ratios ranging from 25:1 to 200:1, respectively. Lytic activity (i.e., release of 51Cr into the medium) was assessed at 22 h of culture. A high-density ( >-1.088 g/mL) population of endometrial cells from the pregnant ewes exhibited NK-like activity, whereas endometrial cells from the cyclic ewes failed to exhibit activity. Lytic activity of these cells was greater (P < 0.05) for pregnant than for cyclic ewes (12.0 and 2.1%, respectively) at the effector:target cell ratio of 100:1, respectively. For both groups of ewes, PBL exhibited NK-like activity. These data indicate that the ovine endometrium contains NK-like cells with lytic activity between Days 16 and 18 of pregnancy.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) targets myeloid leukocytes in the uterus during the post-mating inflammatory response in mice

Factors in seminal plasma elicit a surge of GM-CSF expression in uterine epithelial cells after mating in mice. This study investigates the nature of the endometrial cell populations targeted by epithelial GM-CSF. In quantitative RT-PCR studies, expression of the α-subunit of the GM-CSF receptor (GM-CSF-R) parallelled GM-CSF expression, being maximal during the 48 h period after mating and declining thereafter. Expression of mRNA encoding β-common chain (AIC2B) also increased after mating and remained high until the time of embryo implantation on day 4 of pregnancy. Cells expressing GM-CSF receptors were identified in sections of uterus on the day after mating using 125I-GM-CSF, and were located predominantly in the endometrial stroma subjacent to the luminal epithelium, co-localising with abundant populations of myeloid leukocytes. Cells expressing GM-CSF receptor were identified as macrophages, granulocytes and putative dendritic cells by flow cytometric analysis using lineage and receptor subunit specific antibodies. Recombinant GM-CSF injected into the uterine lumen of ovariectomised mice was found to elicit a dose-dependant accumulation of macrophages and granulocytes in the endometrium, in a pattern of distribution comparable to that seen in uteri after natural mating. Together, these data indicate a role for epithelial cell-derived GM-CSF in mediating the recruitment and potentially in modifying the behaviour of uterine leukocytes during the post-mating inflammatory response in mice.

Leucocyte populations and cytokine regulation in human uteroplacental tissues.

Human endometrial tissue and the decidualized endometrium in pregnancy contain relatively large numbers of leucocytes, the proportions of which vary during both the menstrual cycle and early pregnancy. CD3+ T-cells, CD14+ macrophages and a population of phenotypically unusual CD3-CD16-CD56++ large granular lymphocytes (LGL) are present, whereas B-cells are virtually absent. Relative T-cell numbers decrease in first-trimester decidua; T-cells are therefore unlikely to have an important role in the immunological maintenance of pregnancy but could be more important in implantation, when their relative numbers are greater. Extensive numbers of class II MHC-positive tissue macrophages in both the endometrium and placenta will provide an immediate antigen non-specific host defence to infection at this important site. Nevertheless, most attention has focused on a role for the predominant LGL endometrial cell population in the implantation and maintenance of pregnancy because, at the time of implantation, these LGLs comprise 70-80% of all leucocytes in the endometrium. It is now well recognized that there is substantial and complex cytokine activity within human uteroplacental tissues; both leucocytic and non-leucocytic cells have been shown to be capable of producing a significant array of cytokines. However, to avoid excessive pathological sequelae, such cytokine activity must be locally regulated. This has been highlighted by recent reports indicating that abnormal Th1-type cytokine responses could be a reason for immunological reproductive failure in women. Key cytokines controlling differentiation into a Thl (interleukin 12) or Th2 (interleukin 4) type pattern both exist in unusual molecular forms at the human maternal-fetal tissue interface and hence might be fundamental regulatory elements controlling cytokine action locally by an antagonistic action.

Effect of human chorionic gonadotropin on cytokine production from human endometrial cells in vitro.

To examine whether human chorionic gonadotropin (hCG) is involved in the regulation of interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, and leukemia inhibitory factor (LIF) secretion from cultured human endometrial cells. A mixed population of endometrial cells from six in vitro fertilization/embryo transfer patients was cultured and incubated with various doses of hCG (0, 1, 10, 50, 100, and 500 IU/ml) for 24 hr. IL-6, TNF-alpha, and LIF levels in the culture medium were measured with enzyme-linked immunosorbent assay. IL-6 and TNF-alpha levels were stimulated by hCG in a dose-dependent manner. Stimulation of IL-6 and TNF-alpha levels by 500 IU/ml of hCG increased their production by 3.7- and 2.8-fold, respectively (P < 0.05). Stimulation of IL-6 by 100 IU/ml of hCG was also significant (P < 0.05). However, there was no significant effect of hCG on LIF secretion by endometrial cells (P = 0.31). hCG is involved in the regulation of endometrial cytokine production from human endometrial cells in vitro. This finding supports the recently emerging notion that hCG could have important local roles within the uterus besides its well-known luteotrophic role on the corpus luteum for maintenance of pregnancy.

Apoptosis of human ovary and uterine endometrium during the menstrual cycle.

To investigate apoptotic changes in the ovary or uterine endometrium, we studied the cleavage of DNA in these tissues obtained from regularly cycling women by in situ analysis of DNA integrity and quantitative end labeling of DNA gel fractionation. In situ analysis of several sized atretic follicles of ovaries revealed that granulosa cells showed positive staining to some extent, however, these methods do not discriminate between cells undergoing apoptosis and those undergoing necrosis. Total DNA extracted from human corpora lutea (CL) of the early luteal phase contained predominantly high molecular weight DNA, whereas CL of the midluteal phase exhibited the appearance of DNA cleavage into low molecular weight ladders characteristic of apoptosis. Although apoptotic DNA cleavage of human CL increased from the midluteal phase to the late luteal phase, CL of early pregnancy did not exhibit apoptotic DNA fragmentation. Both large and small luteal cells were the primary cell type exhibiting DNA cleavage in human CL of the midluteal and late luteal phases and in regressive CL. Biochemical analysis of human endometrium revealed that the ladder pattern cleavage of DNA was identified at three different phases of the menstrual cycle, namely the early proliferative, late secretory, and menstrual phases. Cells undergoing apoptosis were scattered in the functional layer of the early proliferative endometrium, but not in the late proliferative phase to midsecretory phase: At the beginning of the late secretory phase, apoptosis reappeared in the stromal cells and spread gradually to almost all components of the functional layer. By contrast, cells in the basal layer showed no evidence of apoptosis throughout the menstrual cycle. The present findings suggest that: (1) human luteal regression may be mediated by apoptosis; (2) CL of early pregnancy may be rescued from luteolysis through inhibition of the occurrence of apoptotic luteal cell death, and (3) apoptosis occurs in specific populations of endometrial cells during the human endometrial cycle. In conclusion, apoptosis might play an important role in the regulation of the menstrual cycle in women.