Global endometrial DNA methylation analysis reveals insights into mQTL regulation and associated endometriosis disease risk and endometrial function.

Endometriosis is a leading cause of pain and infertility affecting millions of women globally. Herein, we characterize variation in DNA methylation (DNAm) and its association with menstrual cycle phase, endometriosis, and genetic variants through analysis of genotype data and methylation in endometrial samples from 984 deeply-phenotyped participants. We estimate that 15.4% of the variation in endometriosis is captured by DNAm and identify significant differences in DNAm profiles associated with stage III/IV endometriosis, endometriosis sub-phenotypes and menstrual cycle phase, including opening of the window for embryo implantation. Menstrual cycle phase was a major source of DNAm variation suggesting cellular and hormonally-driven changes across the cycle can regulate genes and pathways responsible for endometrial physiology and function. DNAm quantitative trait locus (mQTL) analysis identified 118,185 independent cis-mQTLs including 51 associated with risk of endometriosis, highlighting candidate genes contributing to disease risk. Our work provides functional evidence for epigenetic targets contributing to endometriosis risk and pathogenesis. Data generated serve as a valuable resource for understanding tissue-specific effects of methylation on endometrial biology in health and disease.

Plasticity and therapeutic potential of cAMP and cGMP-specific phosphodiesterases in Toxoplasma gondii.

Toxoplasma gondii is a common zoonotic protozoan pathogen adapted to intracellular parasitism in many host cells of diverse organisms. Our previous work has identified 18 cyclic nucleotide phosphodiesterase (PDE) proteins encoded by the parasite genome, of which 11 are expressed during the lytic cycle of its acutely-infectious tachyzoite stage in human cells. Here, we show that ten of these enzymes are promiscuous dual-specific phosphodiesterases, hydrolyzing cAMP and cGMP. TgPDE1 and TgPDE9, with a Km of 18 µM and 31 µM, respectively, are primed to hydrolyze cGMP, whereas TgPDE2 is highly specific to cAMP (Km, 14 µM). Immuno-electron microscopy revealed various subcellular distributions of TgPDE1, 2, and 9, including in the inner membrane complex, apical pole, plasma membrane, cytosol, dense granule, and rhoptry, indicating spatial control of signaling within tachyzoites. Notably, despite shared apical location and dual-catalysis, TgPDE8 and TgPDE9 are fully dispensable for the lytic cycle and show no functional redundancy. In contrast, TgPDE1 and TgPDE2 are individually required for optimal growth, and their collective loss is lethal to the parasite. In vitro phenotyping of these mutants revealed the roles of TgPDE1 and TgPDE2 in proliferation, gliding motility, invasion and egress of tachyzoites. Moreover, our enzyme inhibition assays in conjunction with chemogenetic phenotyping underpin TgPDE1 as a target of commonly-used PDE inhibitors, BIPPO and zaprinast. Finally, we identified a retinue of TgPDE1 and TgPDE2-interacting kinases and phosphatases, possibly regulating the enzymatic activity. In conclusion, our datasets on the catalytic function, physiological relevance, subcellular localization and drug inhibition of key phosphodiesterases highlight the previously-unanticipated plasticity and therapeutic potential of cyclic nucleotide signaling in T. gondii.

Transcriptomic analysis supports collective endometrial cell migration in the pathogenesis of adenomyosis.

RESEARCH QUESTION: Adenomyosis is a common uterine disorder of uncertain causes. Can transcriptomic analyses of the endometrium and myometrium reveal potential mechanisms underlying adenomyosis pathogenesis? DESIGN: Transcriptomic profiles of eutopic endometrium and myometrium from women with and without diffuse adenomyosis and with symptomatic FIGO type 2-5 fibroids in the proliferative phase of the menstrual cycle were assessed using RNA sequencing and bioinformatic analysis. Differentially expressed genes (DEG) and potential pathways were validated by quantitative reverse transcription polymerase chain reaction, immunoblotting and Masson staining, using additional clinical samples. RESULTS: Top biological processes in the endometrium of women with versus without adenomyosis, enriched from DEG, comprised inflammation, extracellular matrix (ECM) organization, collagen degradation and hyaluronan synthesis, which are key in cell migration and cell movement. Top biological processes enriched from DEG in the myometrium of women with versus without adenomyosis revealed ECM organization dysfunction, abnormal sensory pain perception and gamma aminobutyric acid (GABA) synaptic transmission. Dysregulation of prolactin signalling was also enriched in eutopic endometrium and in the myometrium of women with adenomyosis. CONCLUSIONS: Overall, our results support the invasive endometrium theory in the pathogenesis of adenomyosis, in which inflammation induces ECM remodelling resulting in a track for subsequent endometrial collective cell migration and onset of adenomyosis. Moreover, abnormal myometrial GABA synaptic transmission may contribute to dysmenorrhoea in women with adenomyosis and is a possible target for novel therapeutic development. Prolactin signalling abnormalities may serve as another opportunity for therapeutic intervention.

Deep immunophenotyping reveals endometriosis is marked by dysregulation of the mononuclear phagocytic system in endometrium and peripheral blood.

BACKGROUND: Endometriosis is a chronic, estrogen-dependent disorder where inflammation contributes to disease-associated symptoms of pelvic pain and infertility. Immune dysfunction includes insufficient immune lesion clearance, a pro-inflammatory endometrial environment, and systemic inflammation. Comprehensive understanding of endometriosis immune pathophysiology in different hormonal milieu and disease severity has been hampered by limited direct characterization of immune populations in endometrium, blood, and lesions. Simultaneous deep phenotyping at single-cell resolution of complex tissues has transformed our understanding of the immune system and its role in many diseases. Herein, we report mass cytometry and high dimensional analyses to study immune cell phenotypes, abundance, activation states, and functions in endometrium and blood of women with and without endometriosis in different cycle phases and disease stages. METHODS: A case-control study was designed. Endometrial biopsies and blood (n = 60 total) were obtained from women with (n = 20, n = 17, respectively) and without (n = 14, n = 9) endometriosis in the proliferative and secretory cycle phases of the menstrual cycle. Two mass cytometry panels were designed: one broad panel and one specific for mononuclear phagocytic cells (MPC), and all samples were multiplexed to characterize both endometrium and blood immune composition at unprecedented resolution. We combined supervised and unsupervised analyses to finely define the immune cell subsets with an emphasis on MPC. Then, association between cell types, protein expression, disease status, and cycle phase were performed. RESULTS: The broad panel highlighted a significant modification of MPC in endometriosis; thus, they were studied in detail with an MPC-focused panel. Endometrial CD91+ macrophages overexpressed SIRPα (phagocytosis inhibitor) and CD64 (associated with inflammation) in endometriosis, and they were more abundant in mild versus severe disease. In blood, classical and intermediate monocytes were less abundant in endometriosis, whereas plasmacytoid dendritic cells and non-classical monocytes were more abundant. Non-classical monocytes were higher in severe versus mild disease. CONCLUSIONS: A greater inflammatory phenotype and decreased phagocytic capacity of endometrial macrophages in endometriosis are consistent with defective clearance of endometrial cells shed during menses and in tissue homeostasis, with implications in endometriosis pathogenesis and pathophysiology. Different proportions of monocytes and plasmacytoid dendritic cells in blood from endometriosis suggest systemically aberrant functionality of the myeloid system opening new venues for the study of biomarkers and therapies for endometriosis.

Whole-Tissue Deconvolution and scRNAseq Analysis Identify Altered Endometrial Cellular Compositions and Functionality Associated With Endometriosis.

The uterine lining (endometrium) exhibits a pro-inflammatory phenotype in women with endometriosis, resulting in pain, infertility, and poor pregnancy outcomes. The full complement of cell types contributing to this phenotype has yet to be identified, as most studies have focused on bulk tissue or select cell populations. Herein, through integrating whole-tissue deconvolution and single-cell RNAseq, we comprehensively characterized immune and nonimmune cell types in the endometrium of women with or without disease and their dynamic changes across the menstrual cycle. We designed metrics to evaluate specificity of deconvolution signatures that resulted in single-cell identification of 13 novel signatures for immune cell subtypes in healthy endometrium. Guided by statistical metrics, we identified contributions of endometrial epithelial, endothelial, plasmacytoid dendritic cells, classical dendritic cells, monocytes, macrophages, and granulocytes to the endometrial pro-inflammatory phenotype, underscoring roles for nonimmune as well as immune cells to the dysfunctionality of this tissue.

The protozoan parasite Toxoplasma gondii encodes a gamut of phosphodiesterases during its lytic cycle in human cells.

Cyclic nucleotide signaling is pivotal to the asexual reproduction of Toxoplasma gondii, however little do we know about the phosphodiesterase enzymes in this widespread obligate intracellular parasite. Here, we identified 18 phosphodiesterases (TgPDE1-18) in the parasite genome, most of which form apicomplexan-specific clades and lack archetypal regulatory motifs often found in mammalian PDEs. Genomic epitope-tagging in the tachyzoite stage showed the expression of 11 phosphodiesterases with diverse subcellular distributions. Notably, TgPDE8 and TgPDE9 are located in the apical plasma membrane to regulate cAMP and cGMP signaling, as suggested by their dual-substrate catalysis and structure modeling. TgPDE9 expression can be ablated with no apparent loss of growth fitness in tachyzoites. Likewise, the redundancy in protein expression, subcellular localization and predicted substrate specificity of several other PDEs indicate significant plasticity and spatial control of cyclic nucleotide signaling during the lytic cycle. Our findings shall enable a rational dissection of signaling in tachyzoites by combinatorial mutagenesis. Moreover, the phylogenetic divergence of selected Toxoplasma PDEs from human counterparts can be exploited to develop parasite-specific inhibitors and therapeutics.

Steroid hormones regulate genome-wide epigenetic programming and gene transcription in human endometrial cells with marked aberrancies in endometriosis.

Programmed cellular responses to cycling ovarian-derived steroid hormones are central to normal endometrial function. Abnormalities therein, as in the estrogen-dependent, progesterone-"resistant" disorder, endometriosis, predispose to infertility and poor pregnancy outcomes. The endometrial stromal fibroblast (eSF) is a master regulator of pregnancy success. However, the complex hormone-epigenome-transcriptome interplay in eSF by each individual steroid hormone, estradiol (E2) and/or progesterone (P4), under physiologic and pathophysiologic conditions, is poorly understood and was investigated herein. Genome-wide analysis in normal, early and late stage eutopic eSF revealed: i) In contrast to P4, E2 extensively affected the eSF DNA methylome and transcriptome. Importantly, E2 resulted in a more open versus closed chromatin, confirmed by histone modification analysis. Combined E2 with P4 affected a totally different landscape than E2 or P4 alone. ii) P4 responses were aberrant in early and late stage endometriosis, and mapping differentially methylated CpG sites with progesterone receptor targets from the literature revealed different but not decreased P4-targets, leading to question the P4-"resistant" phenotype in endometriosis. Interestingly, an aberrant E2-response was noted in eSF from endometriosis women; iii) Steroid hormones affected specific genomic contexts and locations, significantly enriching enhancers and intergenic regions and minimally involving proximal promoters and CpG islands, regardless of hormone type and eSF disease state. iv) In eSF from women with endometriosis, aberrant hormone-induced methylation signatures were mainly due to existing DNA methylation marks prior to hormone treatments and involved known endometriosis genes and pathways. v) Distinct DNA methylation and transcriptomic signatures revealed early and late stage endometriosis comprise unique disease subtypes. Taken together, the data herein, for the first time, provide significant insight into the hormone-epigenome-transcriptome interplay of each steroid hormone in normal eSF, and aberrant E2 response, distinct disease subtypes, and pre-existing epigenetic aberrancies in the setting of endometriosis, provide mechanistic insights into how endometriosis affects endometrial function/dysfunction.

Progestins Related to Progesterone and Testosterone Elicit Divergent Human Endometrial Transcriptomes and Biofunctions.

Progestins are widely used for the treatment of gynecologic disorders and alone, or combined with an estrogen, are used as contraceptives. While their potencies, efficacies and side effects vary due to differences in structures, doses and routes of administration, little is known about their effects on the endometrial transcriptome in the presence or absence of estrogen. Herein, we assessed the transcriptome and pathways induced by progesterone (P4) and the three most commonly used synthetic progestins, medroxyprogesterone acetate (MPA), levonorgestrel (LNG), and norethindrone acetate (NETA), on human endometrial stromal fibroblasts (eSF), key players in endometrial physiology and reproductive success. While there were similar transcriptional responses, each progestin induced unique genes and biofunctions, consistent with their structural similarities to progesterone (P4 and MPA) or testosterone (LNG and NETA), involving cellular proliferation, migration and invasion. Addition of estradiol (E2) to each progestin influenced the number of differentially expressed genes and biofunctions in P4 and MPA, while LNG and NETA signatures were more independent of E2. Together, these data suggest different mechanisms of action for different progestins, with progestin-specific altered signatures when combined with E2. Further investigation is warranted for a personalized approach in different gynecologic disorders, for contraception, and minimizing side effects associated with their use.

Macrophages display proinflammatory phenotypes in the eutopic endometrium of women with endometriosis with relevance to an infectious etiology of the disease.

OBJECTIVE: To phenotype transcriptomically M1 macrophages (Mϕ1) and M2 macrophages (Mϕ2) in the endometrium of women with endometriosis. DESIGN: Prospective experimental study. SETTING: University research laboratory. PATIENT(S): Six women with endometriosis and five controls without disease, in the secretory phase of the menstrual cycle. INTERVENTION(S): Mϕ1, Mϕ2, uterine natural killer, and T regulatory cells were isolated from human endometrium using a uniquely designed cell-specific fluorescence activating cell sorting panel. Transcriptome profiles were assessed by RNA high sequencing, bioinformatics, and biological pathway analyses. MAIN OUTCOMES MEASURE(S): Differential gene expression between Mϕ1 and Mϕ2 in women with and without endometriosis and in Mϕ1 versus Mϕ2 in each group was determined and involved different biologic and signaling pathways. RESULT(S): Flow cytometry analysis showed no significant differences in total numbers of leukocytes between control and endometriosis groups, although Mϕ1 were higher in the endometriosis group versus controls. Statistical transcriptomic analysis was performed only in Mϕ1 and Mϕ2 populations due to larger sample sizes. Bioinformatic analyses revealed that in women with endometriosis, endometrial Mϕ1 are more proinflammatory than controls and that Mϕ2 paradoxically have a proinflammatory phenotype. CONCLUSION(S): As Mϕ are phenotypically plastic and their polarization state depends on their microenvironment, the altered endometrial environment in women with endometriosis may promote endometrial Mϕ2 polarization and an Mϕ1 proinflammatory phenotype. Moreover, aberrant phenotypes of Mϕ may contribute to abnormal gene expression of the eutopic endometrium and a proinflammatory environment in women with endometriosis relevant to the pathophysiology of the disease and compromised reproductive outcomes.

Aberrant Endometrial DNA Methylome and Associated Gene Expression in Women with Endometriosis.

Endometriosis is an estrogen-dependent, progesterone-resistant disorder largely derived from retrograde transplantation of menstrual tissue/cells into the pelvis, eliciting an inflammatory response, pelvic pain, and infertility. Eutopic endometrium (within the uterus), giving rise to pelvic disease, displays cycle-dependent transcriptomic, proteomic, and signaling abnormalities, and although its DNA methylation profiles dynamically change across the cycle in healthy women, studies in endometriosis are limited. Herein, we investigated the DNA methylome and associated gene expression in three phases of the cycle in eutopic endometrium of women with severe endometriosis versus controls, matched for ethnicity, medications, smoking, and no recent contraceptive steroid use. Genome-wide DNA methylation and gene expression were coassessed in each sample. Cycle phase was determined by histology, serum hormone levels, and unsupervised principal component and hierarchical cluster analyses of microarray data. Altered endometrial DNA methylation in endometriosis was most prominent in the midsecretory phase (peak progesterone), with disruption of the normal pattern of cycle-dependent DNA methylation changes, including a bias toward methylation of CpG islands, suggesting wide-range abnormalities of the chromatin remodeling machinery in endometriosis. DNA methylation changes were associated with altered gene expression relevant to endometrial function/dysfunction, including cell proliferation, inflammation/immune response, angiogenesis, and steroid hormone response. The data provide insight into epigenetic reprogramming and steroid hormone actions in endometrium contributing to the pathogenesis and pathophysiology of endometriosis.

Global Transcriptome Abnormalities of the Eutopic Endometrium From Women With Adenomyosis.

OBJECTIVE: Adenomyosis is a clinical disorder defined by the presence of endometrial glands and stroma within the myometrium, the pathogenesis of which is poorly understood. We postulate that dysregulation of genes and pathways in eutopic endometrium may predispose to ectopic implantation. No study, to our knowledge, has examined the global transcriptome of isolated eutopic endometrium from women with clinically significant adenomyosis. DESIGN: Laboratory-based study with full institutional review board approval and consents. MATERIAL AND METHODS: Endometrial sampling was performed on hysterectomy specimens (proliferative phase) from symptomatic women with pathologically confirmed diffuse adenomyosis (n = 3). Controls (n = 5) were normo-ovulatory patients without adenomyosis. All patients were free from leiomyoma, endometriosis, and hormonal exposures. Isolated purified total RNA was subjected to microarray analysis using the Gene 1.0 ST Affymetrix platform. Data were analyzed with GeneSpring and Ingenuity Pathway analysis. Validation of several genes was undertaken by quantitative real-time reverse transcriptase polymerase chain reaction. RESULTS: Comparison of transcriptomes of proliferative endometrium from women with and without adenomyosis revealed 140 upregulated and 884 downregulated genes in samples from women with adenomyosis compared to controls. Highly differentially expressed genes include those involved in regulation of apoptosis, steroid hormone responsiveness, and proteins involved in extracellular matrix remodeling as well as microRNAs of unknown significance. Affected canonical pathways included eukaryotic initiation factor 2 signaling, oxidative phosphorylation, mitochondrial dysfunction, estrogen receptor signaling, and mammalian target of rapamycin signaling. CONCLUSION: The eutopic endometrium in patients with adenomyosis has fundamental abnormalities that may predispose to invasion and survival beyond the myometrial interface.

Cryopreservation and recovery of human endometrial epithelial cells with high viability, purity, and functional fidelity.

OBJECTIVE: To develop a protocol for cryopreservation and recovery of human endometrial epithelial cells (eECs) retaining molecular and functional characteristics of endometrial epithelium in vivo. DESIGN: In vitro study using human endometrial cells. SETTING: University research laboratory. PATIENT(S): Endometrial biopsies were obtained from premenopausal women undergoing benign gynecologic procedures. INTERVENTION(S): Primary eECs were cryopreserved in 1% fetal bovine serum/10% dimethylsulfoxide in Defined Keratinocyte Serum-Free Medium (KSFM). Recovered cells were observed for endometrial stromal fibroblast (eSF) contamination and subsequently evaluated for morphology, gene expression, and functional characteristics of freshly cultured eECs and in vivo endometrial epithelium. MAIN OUTCOME MEASURE(S): Analysis of eEC morphology and the absence of eSF contamination; evaluation of epithelial-specific gene and protein expression; assessment of epithelial polarity. RESULT(S): Endometrial epithelial cells recovered after cryopreservation (n = 5) displayed epithelial morphology and expressed E-cadherin (CDH1), occludin (OCLN), claudin1 (CLDN1), and keratin18 (KRT18). Compared with eSF, recovered eECs displayed increased (P<.05) expression of epithelial-specific genes AREG, CDH1, DEFB4A, MMP7, and WNT7A, while exhibiting low-to-undetectable (P<.05) stromal-specific genes COL6A3, HOXA11, MMP2, PDGFRB, and WNT5A. Recovered eECs secreted levels of cytokines and growth factors similarly to freshly cultured eECs. Recovered eECs could form a polarized monolayer with high transepithelial electrical resistance (TER) and impermeability to small molecules, and expressed apical/basolateral localization of CDH1 and apical localization of OCLN. CONCLUSION(S): We have developed a protocol for cryopreservation of eECs in which recovered cells after thawing demonstrate morphologic, transcriptomic, and functional characteristics of human endometrial epithelium in vivo.

Diversity of the vaginal microbiome correlates with preterm birth.

Reproductive tract infection is a major initiator of preterm birth (PTB). The objective of this prospective cohort study of 88 participants was to determine whether PTB correlates with the vaginal microbiome during pregnancy. Total DNA was purified from posterior vaginal fornix swabs during gestation. The 16S ribosomal RNA gene was amplified using polymerase chain reaction primers, followed by chain-termination sequencing. Bacteria were identified by comparing contig consensus sequences with the Ribosomal Database Project. Dichotomous responses were summarized via proportions and continuous variables via means ± standard deviation. Mean Shannon Diversity index differed by Welch t test (P = .00016) between caucasians with PTB and term gestation. Species diversity was greatest among African Americans (P = .0045). Change in microbiome/Lactobacillus content and presence of putative novel/noxious bacteria did not correlate with PTB. We conclude that uncultured vaginal bacteria play an important role in PTB and race/ethnicity and sampling location are important determinants of the vaginal microbiome.

Coculturing human endometrial epithelial cells and stromal fibroblasts alters cell-specific gene expression and cytokine production.

OBJECTIVE: To determine the effects of coculturing endometrial epithelial cells (eEC) with paired endometrial stromal fibroblasts (eSF) on cell-specific gene expression and cytokine secretion patterns. DESIGN: In vitro study. SETTING: University research laboratory. PATIENT(S): Endometrial biopsies were obtained from premenopausal women. INTERVENTION(S): Polarized eEC and subject-paired eSF were cultured for 12.5 hours alone (monoculture) or combined in a two-chamber coculture system without cell-cell contact. Cells and conditioned media were analyzed for global gene expression and cytokine secretion, respectively. Purified, endometrial tissue-derived eEC and eSF isolated by fluorescent activated cell sorting (FACS) were used as noncultured controls. MAIN OUTCOME MEASURE(S): Cell-specific global gene expression profiling and analysis of secreted cytokines in eEC/eSF cocultures and respective monocultures. RESULT(S): Transepithelial resistance, diffusible tracer exclusion, expression of tight junction proteins, and apical/basolateral vectorial secretion confirmed eEC structural and functional polarization. Distinct transcriptomes of eEC and eSF were consistent with their respective lineages and their endometrial origin. Coculture of eEC with eSF resulted in altered cell-specific gene expression and cytokine secretion. CONCLUSION(S): This coculture model provides evidence that interactions between endometrial functionally polarized epithelium and stromal fibroblasts affect cell-specific gene expression and cytokine secretion underscoring their relevance when modeling endometrium in vitro.

Initial predictors associated with outcome in injured multiple traumatic limb amputations: a Kandahar-based combat hospital experience.

INTRODUCTION: Improvised explosive devices (IEDs) are the defining mechanism of injury during Operation Enduring Freedom. This is a retrospective analysis of initial management for IED blast injuries presenting with bilateral, traumatic, lower-extremity (LE) amputations with and without pelvic and perineal involvement. METHODS: A database of trauma admissions presenting to a North Atlantic Treaty Organization (NATO) Role 3 combat hospital in southern Afghanistan over a 7-month period was created to evaluate the care of this particular injury pattern. Patients were included if they were received from point of injury with at least bilateral traumatic LE amputations and had vital signs with initial resuscitation efforts. RESULTS: Thirty-two presented with double LE amputations (36%) and nine with triple amputations (10%). After excluding 10 patients who failed to meet the inclusion criteria, 22 patients were analysed. The mean age was 29 years, and the average ISS and admission haemoglobin were 22 and 11.3mgl(-1), respectively. Patients received an average of 54 units of blood products and underwent 1.6 operations with a mean operative time of 142.5min. The pattern of injury was associated with an increase in the total blood products required for resuscitation (pelvis n=12, p=0.028, gastrointestinal tract (GI) n=14, p=0.02, perineal n=15, p=0.036). There was no relationship between ISS or admission haemoglobin and the need for massive transfusion. Low Glasgow Coma Scale (GCS) was associated with increased 30-day mortality. Hollow viscus injury and operative hemipelvectomy were also associated with mortality. CONCLUSIONS: Early 30-day follow-up demonstrated that IED injuries with bilateral LE amputations with and without pelvic and perineal involvement are survivable injuries. Standard measures of injury and predictors of survival bore little relationship to observed outcomes and may need to be re-evaluated. Long-term follow-up is needed to assess the extent of functional recovery and overall morbidity and mortality.

The dynamics of the vaginal microbiome during infertility therapy with in vitro fertilization-embryo transfer.

PURPOSE: To determine the vaginal microbiome in women undergoing IVF-ET and investigate correlations with clinical outcomes. METHODS: Thirty patients had blood drawn for estradiol (E(2)) and progesterone (P(4)) at four time points during the IVF-ET cycle and at 4-6 weeks of gestation, if pregnant. Vaginal swabs were obtained in different hormonal milieu, and the vaginal microbiome determined by deep sequencing of the 16S ribosomal RNA gene. RESULTS: The vaginal microbiome underwent a transition during therapy in some but not all patients. Novel bacteria were found in 33% of women tested during the treatment cycle, but not at 6-8 weeks of gestation. Diversity of species varied across different hormonal milieu, and on the day of embryo transfer correlated with outcome (live birth/no live birth). The species diversity index distinguished women who had a live birth from those who did not. CONCLUSIONS: This metagenomics approach has enabled discovery of novel, previously unidentified bacterial species in the human vagina in different hormonal milieu and supports a shift in the vaginal microbiome during IVF-ET therapy using standard protocols. Furthermore, the data suggest that the vaginal microbiome on the day of embryo transfer affects pregnancy outcome.

Biobanking human endometrial tissue and blood specimens: standard operating procedure and importance to reproductive biology research and diagnostic development.

OBJECTIVE: To develop a standard operating procedure (SOP) for collection, transport, storage of human endometrial tissue and blood samples, subject and specimen annotation, and establishing sample priorities. DESIGN: The SOP synthesizes sound scientific procedures, the literature on ischemia research, sample collection and gene expression profiling, good laboratory practices, and the authors' experience of workflow and sample quality. SETTING: The National Institutes of Health, University of California, San Francisco, Human Endometrial Tissue and DNA Bank. PATIENT(S): Women undergoing endometrial biopsy or hysterectomy for nonmalignant indications. INTERVENTION(S): Collecting, processing, storing, distributing endometrial tissue and blood samples under approved institutional review board protocols and written informed consent from participating subjects. MAIN OUTCOME MEASURE(S): Standard operating procedure. RESULT(S): The SOP addresses rigorous and consistent subject annotation, specimen processing and characterization, strict regulatory compliance, and a reference for researchers to track collection and storage times that may influence their research. CONCLUSION(S): The comprehensive and systematic approach to the procurement of human blood and endometrial tissue in this SOP ensures the high quality, reliability, and scientific usefulness of biospecimens made available to investigators by the National Institutes of Health, University of California, San Francisco, Human Endometrial Tissue and DNA Bank. The detail and perspective in this SOP also provides a blueprint for implementation of similar collection programs at other institutions.

Gene expression analysis of endometrium reveals progesterone resistance and candidate susceptibility genes in women with endometriosis.

The identification of molecular differences in the endometrium of women with endometriosis is an important step toward understanding the pathogenesis of this condition and toward developing novel strategies for the treatment of associated infertility and pain. In this study, we conducted global gene expression analysis of endometrium from women with and without moderate/severe stage endometriosis and compared the gene expression signatures across various phases of the menstrual cycle. The transcriptome analysis revealed molecular dysregulation of the proliferative-to-secretory transition in endometrium of women with endometriosis. Paralleled gene expression analysis of endometrial specimens obtained during the early secretory phase demonstrated a signature of enhanced cellular survival and persistent expression of genes involved in DNA synthesis and cellular mitosis in the setting of endometriosis. Comparative gene expression analysis of progesterone-regulated genes in secretory phase endometrium confirmed the observation of attenuated progesterone response. Additionally, interesting candidate susceptibility genes were identified that may be associated with this disorder, including FOXO1A, MIG6, and CYP26A1. Collectively these findings provide a framework for further investigations on causality and mechanisms underlying attenuated progesterone response in endometrium of women with endometriosis.

Cytotrophoblast induction of arterial apoptosis and lymphangiogenesis in an in vivo model of human placentation.

We studied the vascular effects of invasive human cytotrophoblasts in vivo by transplanting placental villi to the fifth mammary fat pads or beneath the kidney capsules of Scid mice. Over 3 weeks, robust cytotrophoblast invasion was observed in both locations. The architecture of the mammary fat pad allowed for detailed analysis of the cells' interactions with resident murine blood vessels, which revealed specific induction of apoptosis in the endothelial cells and smooth muscle walls of the arterioles. This finding, and confirmation of the results in an in vitro coculture model, suggests that a parallel process is important for enabling cytotrophoblast endovascular invasion during human pregnancy. Cytotrophoblast invasion of the kidney parenchyma was accompanied by a robust lymphangiogenic response, while in vitro, the cells stimulated lymphatic endothelial cell migration via the actions of VEGF family members, FGF, and TNF-alpha. Immunolocalization analyses revealed that human pregnancy is associated with lymphangiogenesis in the decidua since lymphatic vessels were not a prominent feature of the nonpregnant endometrium. Thus, the placenta triggers the development of a decidual lymphatic circulation, which we theorize plays an important role in maintaining fluid balance during pregnancy, with possible implications for maternal-fetal immune cell trafficking.

The immune environment in human endometrium during the window of implantation.

PROBLEM: Changes in the immune environment in the endometrium are believed to be important for successful implantation and maintenance of pregnancy. We have previously investigated global gene profiling in human endometrium during the window of implantation by oligonucleotide microarray technology, and analysis of these data underscore the regulation of a group of immune-related genes. The present study was therefore conducted to examine the pattern of expression and regulation of these genes including decay accelerating factor (DAF), indoleamine 2,3 dioxygenase (IDO), interleukin-15 (IL-15), IL-15 receptor alpha subunit (IL-15Ralpha), interferon regulatory factor-1 (IRF-1), lymphotactin (Lpn), natural killer-associated transcript 2 (NKAT2) and NKG5 in secretory and proliferative human endometrium. METHOD OF STUDY: Endometrial biopsies were obtained from normally cycling women in the late proliferative and mid-secretory phase of the menstrual cycle. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analysis were used to determine the expression and regulation of these genes in secretory and proliferative human endometrium. Cellular localization of NKG5, Lpn and IDO by in situ hybridization in secretory-phase endometrium was also examined. RESULTS: Semi-quantitative RT-PCR and Northern blot results demonstrate that there is a coordinated upregulation of this group of genes during the window of implantation. CONCLUSIONS: We demonstrate the upregulation of immune-related genes IL-15Ralpha, Lpn and NKG5 in secretory versus proliferative human endometrium. We also demonstrate a similar upregulation in secretory endometrium of other immune-related genes, viz, DAF, IDO, IL-15, IRF-1 and NKAT2. The functions of these genes include stimulation of proliferation of uterine natural killer (uNK) cells, inhibition of cytolytic activity of uNK cells, inhibition of cell growth of T cells and other pathogens and inhibition of the classical complement pathway. Upregulation of these immune-related genes in the window of implantation suggests their role during the process of implantation and in immune tolerance of the implanting conceptus.