Endocannabinoid Regulation in Human Endometrium Across the Menstrual Cycle

Humans produce endogenous cannabinoids (endocannabinoids), a group of molecules that activate the same receptors as tetrahydrocannabinol. Endocannabinoids play important roles in reproduction in multiple species, but data in human endometrium are limited. Because endocannabinoids such as anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) often act within tissues as paracrine factors, their effects can be modulated by changes in expression of locally produced synthetic and degradative/oxidative enzymes. The objective of this study was to localize and quantify expression of these key synthetic and degradative/oxidative enzymes for AEA and 2-AG in human endometrium throughout the menstrual cycle. Key synthetic enzymes include N-arachidonyl-phosphatidylethanolamine phospholipase-D (NAPE-PLD), diacylglycerol-lipase a (DAGL-α, and DAGL-β. Key degradative enzymes include fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL); cyclooxygenase 2 (COX2) is an oxidative enzyme. Endometrial samples were collected in 49 regularly cycling, normal women. Protein localization and expression were achieved by immunohistochemistry and messenger RNA (mRNA) expression by real-time reverse transcriptase polymerase chain reaction. No significant cycle-dependent mRNA expression was observed except that of COX2 (P = .002), which demonstrated maximum expression in the proliferative phase. During the secretory phase, NAPE-PLD protein had increased expression in luminal (P = .001), stromal (P = .007), and glandular (P = .04) epithelia, while FAAH had increased glandular (P = .009) and luminal (P = .01) expression. Increased expression in glandular epithelia was identified for MAGL (P = .03). The COX2 had increased luminal expression during the early secretory phase (P < .0001). In conclusion, maximal expression of degradatory/oxidative enzymes in the secretory phase may foster decreased endocannabinoid tone during implantation

Proteomic Analysis of the Luteal Endometrial Secretome

Endometrium attains a secretory architecture in preparation for embryo implantation, but the identity of most endometrial secretory products remains unknown. Our objective was to characterize the endometrial secretome and compare protein expression between prereceptive (luteinizing hormone [LH]+4) receptive (LH+9) and phase endometrium. Endometrial lavage was performed in 11 participants and analyzed by difference gel electrophoresis (DIGE). LH+4 and LH+9 specimens were labeled with cyanine fluorescent dyes Cy3 and Cy5 tags, respectively, and combined. Proteins were separated using 2-dimensional gel electrophoresis, isolated, trypsin-digested, and subjected to mass spectrometry. In all, 152 proteins were identified; 82 were differentially expressed. Most proteins with increased expression on LH+9 functioned in host defense, while proteins with decreased expression had many functions. A total of 14 proteins had changes suggesting altered posttranslational modification. This article describes the first application of proteomic analysis to endometrial secretions, allowing identification of novel endometrial proteins as well as those differentially secreted in prereceptive and receptive phases

G Protein-Coupled Estrogen Receptor (GPER) Expression in Normal and Abnormal Endometrium

Rapid estrogen effects are mediated by membrane receptors, and evidence suggests a role for both a membrane-associated form of estrogen receptor alpha (ESR1; ERα) and G-protein coupled receptor 30 (GPER; GPR30). Considering estrogen’s importance in endometrial physiology and endometriosis pathophysiology, we hypothesized that GPER could be involved in both cyclic changes in endometrial estrogen action and that aberrant expression might be seen in the eutopic endometrium of women with endometriosis. Using real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and immunohistochemical analysis of normal endometrium, endometrial samples demonstrated cycle-regulated expression of GPER, with maximal expression in the proliferative phase. Eutopic and ectopic endometrium from women with endometriosis overexpressed GPER as compared to eutopic endometrium of normal participants. Ishikawa cells, an adenocarcinoma cell line, expressed GPER, with increased expression upon treatment with estrogen or an ESR1 agonist, but not with a GPER-specific agonist. Decreased expression was seen in Ishikawa cells stably transfected with progesterone receptor A. Together, these data suggest that normal endometrial GPER expression is cyclic and regulated by nuclear estrogen and progesterone receptors, while expression is dysregulated in endometriosis

Endocannabinoid Regulation in Human Endometrium Across the Menstrual Cycle

Humans produce endogenous cannabinoids (endocannabinoids), a group of molecules that activate the same receptors as tetrahydrocannabinol. Endocannabinoids play important roles in reproduction in multiple species, but data in human endometrium are limited. Because endocannabinoids such as anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) often act within tissues as paracrine factors, their effects can be modulated by changes in expression of locally produced synthetic and degradative/oxidative enzymes. The objective of this study was to localize and quantify expression of these key synthetic and degradative/oxidative enzymes for AEA and 2-AG in human endometrium throughout the menstrual cycle. Key synthetic enzymes include N-arachidonyl-phosphatidylethanolamine phospholipase-D (NAPE-PLD), diacylglycerol-lipase a (DAGL-α, and DAGL-β. Key degradative enzymes include fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL); cyclooxygenase 2 (COX2) is an oxidative enzyme. Endometrial samples were collected in 49 regularly cycling, normal women. Protein localization and expression were achieved by immunohistochemistry and messenger RNA (mRNA) expression by real-time reverse transcriptase polymerase chain reaction. No significant cycle-dependent mRNA expression was observed except that of COX2 (P = .002), which demonstrated maximum expression in the proliferative phase. During the secretory phase, NAPE-PLD protein had increased expression in luminal (P = .001), stromal (P = .007), and glandular (P = .04) epithelia, while FAAH had increased glandular (P = .009) and luminal (P = .01) expression. Increased expression in glandular epithelia was identified for MAGL (P = .03). The COX2 had increased luminal expression during the early secretory phase (P < .0001). In conclusion, maximal expression of degradatory/oxidative enzymes in the secretory phase may foster decreased endocannabinoid tone during implantation

G Protein-Coupled Estrogen Receptor (GPER) Expression in Normal and Abnormal Endometrium

Rapid estrogen effects are mediated by membrane receptors, and evidence suggests a role for both a membrane-associated form of estrogen receptor alpha (ESR1; ERα) and G-protein coupled receptor 30 (GPER; GPR30). Considering estrogen’s importance in endometrial physiology and endometriosis pathophysiology, we hypothesized that GPER could be involved in both cyclic changes in endometrial estrogen action and that aberrant expression might be seen in the eutopic endometrium of women with endometriosis. Using real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and immunohistochemical analysis of normal endometrium, endometrial samples demonstrated cycle-regulated expression of GPER, with maximal expression in the proliferative phase. Eutopic and ectopic endometrium from women with endometriosis overexpressed GPER as compared to eutopic endometrium of normal participants. Ishikawa cells, an adenocarcinoma cell line, expressed GPER, with increased expression upon treatment with estrogen or an ESR1 agonist, but not with a GPER-specific agonist. Decreased expression was seen in Ishikawa cells stably transfected with progesterone receptor A. Together, these data suggest that normal endometrial GPER expression is cyclic and regulated by nuclear estrogen and progesterone receptors, while expression is dysregulated in endometriosis

The genetics of recurrent hydatidiform moles: new insights and lessons from a comprehensive analysis of 113 patients

Hydatidiform mole is an aberrant human pregnancy characterized by early embryonic arrest and excessive trophoblastic proliferation. Recurrent hydatidiform moles are defined by the occurrence of at least two hydatidiform moles in the same patient. Fifty to eighty percent of patients with recurrent hydatidiform moles have biallelic pathogenic variants in NLRP7 or KHDC3L. However, in the remaining patients, the genotypic types of the moles are unknown. We characterized 80 new hydatidiform mole tissues, 57 of which were from patients with no mutations in the known genes, and we reviewed the genotypes of a total of 123 molar tissues. We also reviewed mutation analysis in 113 patients with recurrent hydatidiform moles. While all hydatidiform moles from patients with biallelic NLRP7 or KHDC3L mutations are diploid biparental, we demonstrate that those from patients without mutations are highly heterogeneous and only a small minority of them are diploid biparental (8%). The other mechanisms that were found to recur in patients without mutations are diploid androgenetic monospermic (24%) and triploid dispermic (32%); the remaining hydatidiform moles were misdiagnosed as moles due to errors in the analyses and/or their unusual mechanisms. We compared three parameters of genetic susceptibility in patients with and without mutations and show that patients without mutations are mostly from non-familial cases, have fewer reproductive losses, and more live births. Our data demonstrate that patients with recurrent hydatidiform moles and no mutations in the known genes are, in general, different from those with mutations; they have a milder genetic susceptibility and/or a multifactorial etiology underlying their recurrent hydatidiform moles. Categorizing these patients according to the genotypic types of their recurrent hydatidiform moles may facilitate the identification of novel genes for this entity