Publications by authors named "Steven L Young"

91 Publications

Vitamin D and uterine leiomyomata: Is it time to let the sunshine in?

Fertil Steril 2021 Feb 4;115(2):340-341. Epub 2020 Dec 4.

University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

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http://dx.doi.org/10.1016/j.fertnstert.2020.09.151DOI Listing
February 2021

Endometrial epithelial ARID1A is critical for uterine gland function in early pregnancy establishment.

FASEB J 2021 Feb 22;35(2):e21209. Epub 2020 Nov 22.

Department of Obstetrics, Gynecology & Reproductive Biology, Michigan State University, Grand Rapids, MI, USA.

Though endometriosis and infertility are clearly associated, the pathophysiological mechanism remains unclear. Previous work has linked endometrial ARID1A loss to endometriosis-related endometrial non-receptivity. Here, we show in mice that ARID1A binds and regulates transcription of the Foxa2 gene required for endometrial gland function. Uterine-specific deletion of Arid1a compromises gland development and diminishes Foxa2 and Lif expression. Deletion of Arid1a with Ltf-iCre in the adult mouse endometrial epithelium preserves the gland development while still compromising the gland function. Mice lacking endometrial epithelial Arid1a are severely sub-fertile due to defects in implantation, decidualization, and endometrial receptivity from disruption of the LIF-STAT3-EGR1 pathway. FOXA2 is also reduced in the endometrium of women with endometriosis in correlation with diminished ARID1A, and both ARID1A and FOXA2 are reduced in nonhuman primates induced with endometriosis. Our findings describe a role for ARID1A in the endometrial epithelium supporting early pregnancy establishment through the maintenance of gland function.
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http://dx.doi.org/10.1096/fj.202002178RDOI Listing
February 2021

WNK1 regulates uterine homeostasis and its ability to support pregnancy.

JCI Insight 2020 Nov 19;5(22). Epub 2020 Nov 19.

Reproductive and Developmental Biology Laboratory and.

WNK1 (with no lysine [K] kinase 1) is an atypical kinase protein ubiquitously expressed in humans and mice. A mutation in its encoding gene causes hypertension in humans, which is associated with abnormal ion homeostasis. WNK1 is critical for in vitro decidualization in human endometrial stromal cells, thereby demonstrating its importance in female reproduction. Using a mouse model, WNK1 was ablated in the female reproductive tract to define its in vivo role in uterine biology. Loss of WNK1 altered uterine morphology, causing endometrial epithelial hyperplasia, adenomyotic features, and a delay in embryo implantation, ultimately resulting in compromised fertility. Combining transcriptomic, proteomic, and interactomic analyses revealed a potentially novel regulatory pathway whereby WNK1 represses AKT phosphorylation through protein phosphatase 2A (PP2A) in endometrial cells from both humans and mice. We show that WNK1 interacted with PPP2R1A, the alpha isoform of the PP2A scaffold subunit. This maintained the levels of PP2A subunits and stabilized its activity, which then dephosphorylated AKT. Therefore, loss of WNK1 reduced PP2A activity, causing AKT hypersignaling. Using FOXO1 as a readout of AKT activity, we demonstrate that there was escalated FOXO1 phosphorylation and nuclear exclusion, leading to a disruption in the expression of genes that are crucial for embryo implantation.
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http://dx.doi.org/10.1172/jci.insight.141832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710275PMC
November 2020

Cystic fibrosis carrier screening using next generation sequencing: A cautionary tale.

Fertil Steril 2020 Sep 7;114(3):495-496. Epub 2020 Aug 7.

Department of Obstetrics and Gynecology, University of North Carolina School of Medicine, Chapel Hill, North Carolina; UNC Fertility, Raleigh, North Carolina.

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http://dx.doi.org/10.1016/j.fertnstert.2020.06.005DOI Listing
September 2020

Bioinformatic jujutsu to defeat an endometrial transcriptomic foe.

Authors:
Steven L Young

Fertil Steril 2020 06 6;113(6):1163-1164. Epub 2020 May 6.

Department of Obstetrics and Gynecology, University of North Carolina School of Medicine, Chapel Hill, North Carolina.

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http://dx.doi.org/10.1016/j.fertnstert.2020.02.102DOI Listing
June 2020

Diagnostic and therapeutic options in recurrent implantation failure.

F1000Res 2020 25;9. Epub 2020 Mar 25.

Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Recurrent implantation failure (RIF) is an uncommon, imprecisely defined clinical disorder characterized by failure to achieve pregnancy after repeated embryo transfers. The diverse etiologies and incomplete understanding of RIF provide significant diagnostic and therapeutic challenges to patients and providers. Careful clinical evaluation prior to assisted reproduction can uncover many treatable causes, including thyroid dysfunction, submucosal myomas, and tobacco use. The more-subtle causes often require a more-targeted assessment. Undetected, small polyps or small areas of intrauterine synechiae are relatively common and easily treated contributors to RIF. Molecular and cellular abnormalities pose a greater therapeutic challenge. Putative causes of RIF, including progesterone resistance, shifted window of receptivity, decreased integrin expression, and immunologic disturbances, should be considered in the evaluation of a patient with otherwise unexplained RIF. It may also be true that a more complex and standardized definition of RIF would be helpful in these cases. In this paper, we review the diagnostic and therapeutic approaches to RIF, with emphasis on disorders of endometrial receptivity.
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http://dx.doi.org/10.12688/f1000research.22403.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7101014PMC
July 2020

Large, Non-Cavity Distorting Intramural Leiomyomas Decrease Leukemia Inhibitory Factor in the Secretory Phase Endometrium.

Reprod Sci 2020 02 1;27(2):569-574. Epub 2020 Jan 1.

Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, USA.

Despite mounting evidence that large intramural leiomyomas decrease fecundity during in vitro fertilization cycles, few studies have demonstrated a mechanism for this impact. We hypothesize that large intramural leiomyomas (IM) decrease the expression of endometrial implantation factors during the window of implantation. We prospectively recruited sub-fertile patients with IM 3 cm or greater in size planning myomectomy and performed endometrial biopsies the day of planned myomectomy (n = 9). Preoperative screening demonstrated no intercavitary lesions. Control endometrial samples were obtained from young, normally menstruating women free of uterine leiomyomas (n = 8). Endometrial samples were obtained in the mid-secretory phase (average cycle day for control patients and intramural leiomyoma patients were 24.5 and 21.3, respectively). Expression of implantation markers HOXA10, leukemia inhibitory factor (LIF), ER-α, and PR was compared using quantitative immunohistochemistry. Standard descriptive statistics were used to compare H-scores between the cohorts. Patients with intramural leiomyomas were found to have decreased LIF compared to controls (p value < 0.001). Expressions of HOXA10 and PR were no different between cohorts; however, ER-α showed a trend toward increased expression in the fibroid cohort (p value 0.07). LIF is downregulated in the endometrium of patients with large IM. This study is among the first to show decreased LIF expression in patients with uterine leiomyomas. We hypothesize that this difference from previously published work is due to sampling the endometrium at the height of LIF expression. Further work is needed to show if LIF downregulation is corrected with leiomyoma resection.
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http://dx.doi.org/10.1007/s43032-019-00056-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539805PMC
February 2020

Differential Expression of KRAS and SIRT1 in Ovarian Cancers with and Without Endometriosis.

Reprod Sci 2020 01 1;27(1):145-151. Epub 2020 Jan 1.

Department of Obstetrics and Gynecology, University Medical Group, Greenville Hospital System, Greenville, SC, 29605, USA.

Accumulating research shows that ovarian cancer progression can be influenced by both gene mutations and endometriosis. However, the exact mechanism at hand is poorly understood. In the current study, we explored the expression of KRAS and SIRT1, two genes previously identified as altered in endometriosis and ovarian cancer. Human endometrial samples were obtained from regularly cycling women with endometriosis, ovarian cancer, and endometriosis-associated ovarian cancer between 18 and 50 of age undergoing hysterectomy, and immunohistochemical analyses were performed. The cytoplasmic expression of KRAS was low in eutopic endometrium from women without endometriosis or ovarian cancer; however, it was elevated in those who have been diagnosed with endometriosis, as well as ovarian cancer with or without the presence of endometriosis. Nuclear and cytoplasmic SIRT1 expression was also low within endometrium without either disease. However, nuclear SIRT1 expression was increased in those with endometriosis and ovarian cancer associated with endometriosis. Nuclear but not the cytoplasmic expression of SIRT1 correlated with KRAS expression in ovarian cancers associated with endometriosis. These results suggest roles of KRAS and SIRT1 in endometriosis and endometriosis-associated ovarian cancer. Cytoplasmic KRAS expression proves to be a key biomarker in both diseases, while nuclear SIRT1 may be a new biomarker specific to those with endometriosis and those with both endometriosis and ovarian cancer. Further research of these genes could aid in determining the pathogenesis of both diseases and help in clarifying the development of endometriosis-associated ovarian cancer.
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http://dx.doi.org/10.1007/s43032-019-00017-4DOI Listing
January 2020

Neutrophil recruitment and function in endometriosis patients and a syngeneic murine model.

FASEB J 2020 01 2;34(1):1558-1575. Epub 2019 Dec 2.

Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON, Canada.

Endometriosis is a chronic inflammatory, gynecological disease characterized by the presence of endometrial-like tissue lesions outside of the uterus. Neutrophils are elevated in the systemic circulation and peritoneal fluid of endometriosis patients; however, whether and how neutrophils contribute to endometriosis pathophysiology remain poorly understood. With emerging roles for neutrophils in chronic and sterile inflammatory conditions, we sought to provide in-depth characterization of neutrophil involvement in endometriosis. We demonstrate that neutrophils reside within patient endometriotic lesions and that patient lesions possess a microenvironment that may influence neutrophil recruitment and function. We also provide the first evidence that systemic circulating neutrophils from endometriosis patients display distinct transcriptomic differences compared neutrophils from healthy control subjects. Time course characterization of our syngeneic, immunocompetent mouse model of endometriosis revealed that neutrophils are rapidly recruited to the peritoneal environment early after endometriotic lesion establishment and remain present in murine lesions long term. In vivo neutrophil depletion altered the systemic and peritoneal immune microenvironment of mice with endometriosis as demonstrated by changes in pro-inflammatory and angiogenic mediators. Taken together, these findings highlight a novel role for neutrophils in early events such as angiogenesis and modulation of the local inflammatory environment associated with endometriosis pathogenesis.
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http://dx.doi.org/10.1096/fj.201902272RDOI Listing
January 2020

Human Endometrial Transcriptome and Progesterone Receptor Cistrome Reveal Important Pathways and Epithelial Regulators.

J Clin Endocrinol Metab 2020 04;105(4)

Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, Durham, North Carolina.

Context: Poor uterine receptivity is one major factor leading to pregnancy loss and infertility. Understanding the molecular events governing successful implantation is hence critical in combating infertility.

Objective: To define Progesterone Receptor (PGR)-regulated molecular mechanisms and epithelial roles in receptivity.

Design: RNA-sequencing and PGR-ChIP-seq were conducted in parallel to identify PGR-regulated pathways during the Window of implantation (WOI) in endometrium of fertile women.

Setting: Endometrial biopsies from the proliferative and mid-secretory phases were analyzed.

Patients Or Other Participants: Participants were fertile, reproductive aged (18-37 years) women with normal cycle length, and without any history of dysmenorrhea, infertility, or irregular cycles. In total, 42 endometrial biopsies obtained from 42 women were analyzed in this study.

Interventions: There were no interventions during this study.

Main Outcome Measures: Here we measured the alterations in gene expression and PGR occupancy in the genome during the WOI, based on the hypothesis that PGR binds uterine chromatin cycle dependently to regulate genes involved in uterine cell differentiation and function.

Results: 653 genes were identified with regulated PGR binding and differential expression during the WOI. These were involved in regulating inflammatory response, xenobiotic metabolism, epithelial mesenchymal transition, cell death, interleukin/Signal Transducer And Activator Of Transcription (STAT) signaling, estrogen response, and Mammalian target of rapamycin complex 1 (MTORC1) response. Transcriptome of the epithelium identified 3052 differentially expressed genes, of which 658 were uniquely regulated. Transcription factors Interferon Regulatory Factor 8 (IRF8) and Myocyte Enhancer Factor 2C (MEF2C) were found to be regulated in the epithelium during the WOI at the protein level, suggesting potentially important functions that are previously unrecognized.

Conclusion: PGR binds the genomic regions of genes regulating critical processes in uterine receptivity and function.
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http://dx.doi.org/10.1210/clinem/dgz117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105253PMC
April 2020

Extracellular vesicles from endometriosis patients are characterized by a unique miRNA-lncRNA signature.

JCI Insight 2019 09 19;4(18). Epub 2019 Sep 19.

Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada.

With multifactorial etiologies, combined with disease heterogeneity and a lack of suitable diagnostic markers and therapy, endometriosis remains a major reproductive health challenge. Extracellular vesicles (EVs) have emerged as major contributors of disease progression in several conditions, including a variety of cancers; however, their role in endometriosis pathophysiology has remained elusive. Using next-generation sequencing of EVs obtained from endometriosis patient tissues and plasma samples compared with controls, we have documented that patient EVs carry unique signatures of miRNAs and long noncoding RNAs (lncRNAs) reflecting their contribution to disease pathophysiology. Mass spectrophotometry-based proteomic analysis of EVs from patient plasma and peritoneal fluid further revealed enrichment of specific pathways, as well as altered immune and metabolic processes. Functional studies in endometriotic epithelial and endothelial cell lines using EVs from patient plasma and controls clearly indicate autocrine uptake and paracrine cell proliferative roles, suggestive of their involvement in endometriosis. Multiplex cytokine analysis of cell supernatants in response to patient and control plasma-derived EVs indicate robust signatures of important inflammatory and angiogenic cytokines known to be involved in disease progression. Collectively, these findings suggest that endometriosis-associated EVs carry unique cargo and contribute to disease pathophysiology by influencing inflammation, angiogenesis, and proliferation within the endometriotic lesion microenvironment.
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http://dx.doi.org/10.1172/jci.insight.128846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6795291PMC
September 2019

Should we stop offering endometrial scratching prior to in vitro fertilization?

Fertil Steril 2019 06;111(6):1094-1101

Department of Obstetrics and Gynaecology, University of Auckland, Auckland, New Zealand; Fertility Plus, Auckland District Health Board, Auckland, New Zealand.

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http://dx.doi.org/10.1016/j.fertnstert.2019.04.017DOI Listing
June 2019

Msx Homeobox Genes Act Downstream of BMP2 to Regulate Endometrial Decidualization in Mice and in Humans.

Endocrinology 2019 07;160(7):1631-1644

Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, Illinois.

Endometrial stromal cells differentiate to form decidual cells in a process known as decidualization, which is critical for embryo implantation and successful establishment of pregnancy. We previously reported that bone morphogenetic protein 2 (BMP2) mediates uterine stromal cell differentiation in mice and in humans. To identify the downstream target(s) of BMP2 signaling during decidualization, we performed gene-expression profiling of mouse uterine stromal cells, treated or not treated with recombinant BMP2. Our studies revealed that expression of Msx2, a member of the mammalian Msx homeobox gene family, was markedly upregulated in response to exogenous BMP2. Interestingly, conditional ablation of Msx2 in the uterus failed to prevent a decidual phenotype, presumably because of functional compensation of Msx2 by Msx1, a closely related member of the Msx family. Indeed, in Msx2-null uteri, the level of Msx1 expression in the stromal cells was markedly elevated. When conditional, tissue-specific ablation of both Msx1 and Msx2 was accomplished in the mouse uterus, a dramatically impaired decidual response was observed. In the absence of both Msx1 and Msx2, uterine stromal cells were able to proliferate, but they failed to undergo terminal differentiation. In parallel experiments, addition of BMP2 to human endometrial stromal cell cultures led to a robust enhancement of MSX1 and MSX2 expression and stimulated the differentiation process. Attenuation of MSX1 and MSX2 expression by small interfering RNAs greatly reduced human stromal differentiation in vitro, indicating a conservation of their roles as key mediators of BMP2-induced decidualization in mice and women.
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http://dx.doi.org/10.1210/en.2019-00131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6591014PMC
July 2019

Integrative analysis of the forkhead box A2 (FOXA2) cistrome for the human endometrium.

FASEB J 2019 07 5;33(7):8543-8554. Epub 2019 Apr 5.

Division of Animal Sciences, University of Missouri, Columbia, Missouri, USA.

The pioneer forkhead box (FOX)A2 transcription factor is specifically expressed in the glands of the uterus, which are central to endometrial function and fertility. In mice, FOXA2 is a critical regulator of uterine gland development in the neonate and gland function in the adult. An integrative approach was used here to define the FOXA2 cistrome in the human endometrium. Genome-wide mapping of FOXA2 binding intervals by chromatin immunoprecipitation sequencing was performed using proliferative (P)- and midsecretory (MS)-phase endometrium and integrated with the transcriptome determined by RNA sequencing. Distinctive FOXA2 binding intervals, enriched for different transcription factor binding site motifs, were detected in the P and MS endometrium. Pathway analysis revealed different biologic processes regulated by genes with FOXA2 binding intervals in the P and MS endometrium. Thus, FOXA2 is postulated to regulate gene expression in concert with other transcription factors and impact uterine gland development and function in a cycle phase-dependent manner. Analyses also identified potential FOXA2-regulated genes that influence uterine receptivity, blastocyst implantation, and stromal cell decidualization, which are key events in pregnancy establishment.-Kelleher, A. M., Behura, S. K., Burns, G. W., Young, S. L., DeMayo, F. J., Spencer, T. E. Integrative analysis of the forkhead box A2 (FOXA2) cistrome for the human endometrium.
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http://dx.doi.org/10.1096/fj.201900013RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6593875PMC
July 2019

What exactly is endometrial receptivity?

Fertil Steril 2019 04;111(4):611-617

Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

Endometrial receptivity is a complex process that provides the embryo with the opportunity to attach, invade, and develop, culminating in a new individual and continuation of the species. The window of implantation extends 3-6 days within the secretory phase in most normal women. In certain inflammatory or anatomic conditions, this window is narrowed or shifted to preclude normal implantation, leading to infertility or pregnancy loss. Of the factors that prevent normal implantation and pregnancy, embryo and endometrial quality share responsibility. In this review, we highlight the advances in the study of implantation from the perspective of the endometrium, normally a barrier to implantation. New advances will allow the early identification of defects in endometrial receptivity and provide new avenues for treatment that promote successful establishment of pregnancy.
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http://dx.doi.org/10.1016/j.fertnstert.2019.02.009DOI Listing
April 2019

Loss of HDAC3 results in nonreceptive endometrium and female infertility.

Sci Transl Med 2019 01;11(474)

Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, Grand Rapids, MI 49534, USA.

Endometriosis is a disease in which tissue that normally grows inside the uterus grows outside the uterus and causes chronic pelvic pain and infertility. However, the exact mechanisms of the pathogenesis of endometriosis-associated infertility are unknown. Epigenetic dysregulation has recently been implicated in infertility. Here, we report a reduction of histone deacetylase 3 (HDAC3) protein amounts in eutopic endometrium of infertile women with endometriosis compared to a control group. To investigate the effect of HDAC3 loss in the uterus, we generated mice with conditional ablation of in progesterone receptor (PGR)-positive cells ( ; ). Loss of in the uterus of mice results in infertility due to implantation failure and decidualization defect. Expression microarray and ChIP-seq analyses identified and as direct targets of HDAC3 in both mice and humans. Reduction of abrogated decidualization in a primary culture of human endometrial stromal cells (hESCs) similar to that observed in infertile patients with endometriosis. Whereas attenuation of resulted in p300 recruitment to and genes in the uterus of mice as well as hESCs, inhibition of p300 permitted hESCs to undergo decidualization. Collectively, we found attenuation of HDAC3 and overexpression of collagen type I in the eutopic endometrium of infertile patients with endometriosis. HDAC3 loss caused a defect of decidualization through the aberrant transcriptional activation of and genes in mice and and genes in humans. Our results suggest that HDAC3 is critical for endometrial receptivity and decidualization.
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http://dx.doi.org/10.1126/scitranslmed.aaf7533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650287PMC
January 2019

FOXO1 regulates uterine epithelial integrity and progesterone receptor expression critical for embryo implantation.

PLoS Genet 2018 11 19;14(11):e1007787. Epub 2018 Nov 19.

Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States of America.

Successful embryo implantation requires a receptive endometrium. Poor uterine receptivity can account for implantation failure in women who experience recurrent pregnancy loss or multiple rounds of unsuccessful in vitro fertilization cycles. Here, we demonstrate that the transcription factor Forkhead Box O1 (FOXO1) is a critical regulator of endometrial receptivity in vivo. Uterine ablation of Foxo1 using the progesterone receptor Cre (PgrCre) mouse model resulted in infertility due to altered epithelial cell polarity and apoptosis, preventing the embryo from penetrating the luminal epithelium. Analysis of the uterine transcriptome after Foxo1 ablation identified alterations in gene expression for transcripts involved in the activation of cell invasion, molecular transport, apoptosis, β-catenin (CTNNB1) signaling pathway, and an increase in PGR signaling. The increase of PGR signaling was due to PGR expression being retained in the uterine epithelium during the window of receptivity. Constitutive expression of epithelial PGR during this receptive period inhibited expression of FOXO1 in the nucleus of the uterine epithelium. The reciprocal expression of PGR and FOXO1 was conserved in human endometrial samples during the proliferative and secretory phase. This demonstrates that expression of FOXO1 and the loss of PGR during the window of receptivity are interrelated and critical for embryo implantation.
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http://dx.doi.org/10.1371/journal.pgen.1007787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277115PMC
November 2018

SOX17 regulates uterine epithelial-stromal cross-talk acting via a distal enhancer upstream of Ihh.

Nat Commun 2018 10 24;9(1):4421. Epub 2018 Oct 24.

Reproductive and Development Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA.

Mammalian pregnancy depends on the ability of the uterus to support embryo implantation. Previous studies reveal the Sox17 gene as a downstream target of the Pgr-Gata2-dependent transcription network that directs genomic actions in the uterine endometrium receptive for embryo implantation. Here, we report that ablating Sox17 in the uterine epithelium impairs leukemia inhibitory factor (LIF) and Indian hedgehog homolog (IHH) signaling, leading to failure of embryo implantation. In vivo deletion of the SOX17-binding region 19 kb upstream of the Ihh locus by CRISPR-Cas technology reduces Ihh expression specifically in the uterus and alters proper endometrial epithelial-stromal interactions, thereby impairing pregnancy. This SOX17-binding interval is also bound by GATA2, FOXA2, and PGR. This cluster of transcription factor binding is common in 737 uterine genes and may represent a key regulatory element essential for uterine epithelial gene expression.
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http://dx.doi.org/10.1038/s41467-018-06652-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200785PMC
October 2018

Elevated levels of adrenomedullin in eutopic endometrium and plasma from women with endometriosis.

Fertil Steril 2018 06 2;109(6):1072-1078. Epub 2018 Jun 2.

Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, North Carolina. Electronic address:

Objective: To test adrenomedullin (Adm, ADM) as a downstream target of signal transducer and activator of transcription 3 (STAT3) in endometrial cells and to test midregional proadrenomedullin (MR-proADM) as a biomarker of endometriosis.

Design: Cross-sectional analysis of Adm expression in eutopic endometrium and of MR-proADM in plasma from women with and without endometriosis; and prospective study of MR-proADM levels in women with endometriosis undergoing surgical resection of ectopic lesions.

Setting: Academic medical centers.

Patient(s): Fifteen patients with endometriosis and 11 healthy control subjects who donated eutopic endometrial biopsies; and 28 patients with endometriosis and 19 healthy control subjects who donated plasma for MR-proADM analysis.

Intervention(s): None.

Main Outcome Measure(s): Adm mRNA levels according to quantitative real-time polymerase chain reaction after activation of STAT3 by interleukin-6 (IL-6) in Ishikawa cells; immunohistochemistry for ADM in eutopic endometrial biopsies from women with endometriosis compared with healthy donors; and MR-proADM levels measured by commercial immunoassay in plasma from healthy women and women with endometriosis who subsequently underwent surgical resection of ectopic lesions.

Result(s): Activation of STAT3 by IL-6 up-regulated Adm mRNA expression in Ishikawa cells. ADM protein levels were elevated in the eutopic endometrium of women with endometriosis. MR-proADM concentrations were higher in women with endometriosis but were not correlated with disease stage, corrected by surgery, or predictive of fertility outcome.

Conclusion(s): MR-proADM may be able to serve as a biomarker of endometriosis, but it is unknown whether elevated MR-proADM levels are secondary to the estrogenic and inflammatory properties of endometriosis or an inciting pathogenic factor.
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http://dx.doi.org/10.1016/j.fertnstert.2018.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6015786PMC
June 2018

Androgens and endometrium: new lessons from the corpus luteum via the adrenal cortex?

Authors:
Steven L Young

Fertil Steril 2018 04;109(4):623-624

Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

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http://dx.doi.org/10.1016/j.fertnstert.2018.01.027DOI Listing
April 2018

Effects of histone methyltransferase inhibition in endometriosis.

Biol Reprod 2018 08;99(2):293-307

Department of Basic Sciences-Microbiology Division, Ponce Health Sciences University, Ponce, Puerto Rico, USA.

Although the histone methyltransferase EZH2 and its product H3K27me3 are well studied in cancer, little is known about their role and potential as therapeutic targets in endometriosis. We have previously reported that endometriotic lesions are characterized by global enrichment of H3K27me3. Therefore, we aimed to (1) characterize the expression levels of EZH2 in endometriotic tissues; (2) assess H3K27me3 enrichment in candidate genes promoter regions; and (3) determine if pharmacological inhibition of EZH2 impacts migration, proliferation, and invasion of endometriotic cells. Immunohistochemistry of an endometriosis-focused tissue microarray was used to assess the EZH2 protein levels in tissues. Chromatin immunoprecipitation-qPCR was conducted to assess enrichment of H3K27me3 in candidate gene promoter regions in tissues. Immunofluorescence was performed to assess the effect of an EZH2-specific pharmacological inhibitor on H3K27me3 global enrichment in cell lines. To measure effects of the inhibitor in migration, proliferation, and invasion in vitro we used Scratch, BrdU, and Matrigel assays, respectively. Endometriotic lesions had significantly higher EZH2α nuclear immunostaining levels compared to eutopic endometrium from patients (glands, stroma) and controls (glands). H3K27me3 was enriched within promoter regions of candidate genes in some but not all of the endometriotic lesions. Inhibition of EZH2 reduced H3K27me3 levels in the endometriotic cells specifically, and also reduced migration, proliferation but not invasion of endometriotic epithelial cells (12Z). These findings support future preclinical studies to determine in vivo efficacy of EZH2 inhibitors as promising nonhormonal treatments for endometriosis, still an incurable gynecological disease.
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http://dx.doi.org/10.1093/biolre/ioy030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6373837PMC
August 2018

Interleukin-33 modulates inflammation in endometriosis.

Sci Rep 2017 12 20;7(1):17903. Epub 2017 Dec 20.

Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, K7L 3N6, Canada.

Endometriosis is a debilitating condition that is categorized by the abnormal growth of endometrial tissue outside the uterus. Although the pathogenesis of this disease remains unknown, it is well established that endometriosis patients exhibit immune dysfunction. Interleukin (IL)-33 is a danger signal that is a critical regulator of chronic inflammation. Although plasma and peritoneal fluid levels of IL-33 have been associated with deep infiltrating endometriosis, its contribution to the disease pathophysiology is unknown. We investigated the role of IL-33 in the pathology of endometriosis using patient samples, cell lines and a syngeneic mouse model. We found that endometriotic lesions produce significantly higher levels of IL-33 compared to the endometrium of healthy, fertile controls. In vitro stimulation of endometrial epithelial, endothelial and endometriotic epithelial cells with IL-33 led to the production of pro-inflammatory and angiogenic cytokines. In a syngeneic mouse model of endometriosis, IL-33 injections caused systemic inflammation, which manifested as an increase in plasma pro-inflammatory cytokines compared to control mice. Furthermore, endometriotic lesions from IL-33 treated mice were highly vascularized and exhibited increased proliferation. Collectively, we provide convincing evidence that IL-33 perpetuates inflammation, angiogenesis and lesion proliferation, which are critical events in the lesion survival and progression of endometriosis.
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http://dx.doi.org/10.1038/s41598-017-18224-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5738435PMC
December 2017

RBPJ mediates uterine repair in the mouse and is reduced in women with recurrent pregnancy loss.

FASEB J 2018 05 8;32(5):2452-2466. Epub 2018 Jan 8.

Department of Obstetrics, Gynecology, and Reproductive Biology, Michigan State University, Grand Rapids, Michigan, USA.

Unexplained recurrent pregnancy loss (uRPL) is associated with repeated embryo loss and endometrial repair with elevated endometrial expression of inflammatory cytokines, including IFN-γ. Notch signaling through its transcription factor recombination signal binding protein Jκ (RBPJ) regulates mechanisms including the immune response and repair after tissue injury. Initially, null mutation of RBPJ in the mouse uterus ( PgrRbpj; Rbpj c-KO) results in subfertility, but we have found that these mice become infertile after pregnancy as a result of dysfunctional postpartum uterine repair, including delayed endometrial epithelial and myometrial regeneration. RNA sequencing of postpartum uterine repair sites revealed global up-regulation of inflammatory pathways, including IFN signaling. Consistent with elevated IFN-γ, macrophages were recruited and polarized toward an M1-cytotoxic phenotype, which is associated with preventing repair and promoting further tissue injury. Through embryo transfer experiments, we show that dysfunctional postpartum repair directly impairs future embryo implantation in Rbpj c-KO mice. Last, we clinically correlated our findings from the Rbpj c-KO mouse in women diagnosed with uRPL. Reduced RBPJ in women with uRPL was associated with increased levels of IFN-γ. The data, taken together, indicate that RBPJ regulates inflammation during endometrial repair, which is essential for future pregnancy potential, and its dysregulation may serve as an unidentified contributor to uRPL in women.-Strug, M. R., Su, R.-W., Kim, T. H., Mauriello, A., Ticconi, C., Lessey, B. A., Young, S. L., Lim, J. M., Jeong, J.-W., Fazleabas, A. T. RBPJ mediates uterine repair in the mouse and is reduced in women with recurrent pregnancy loss.
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http://dx.doi.org/10.1096/fj.201701032RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6207278PMC
May 2018

The endometria of women with endometriosis exhibit dysfunctional expression of complement regulatory proteins during the mid secretory phase.

J Reprod Immunol 2018 02 27;125:1-7. Epub 2017 Oct 27.

Department of Obstetrics and Gynecology, University of South Carolina - Greenville, Greenville Health System, 890 West Faris Road, Suite 470, Greenville, SC 29605, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jri.2017.10.046DOI Listing
February 2018

Adrenomedullin improves fertility and promotes pinopodes and cell junctions in the peri-implantation endometrium.

Biol Reprod 2017 Sep;97(3):466-477

Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Implantation is a complex event demanding contributions from both embryo and endometrium. Despite advances in assisted reproduction, endometrial receptivity defects persist as a barrier to successful implantation in women with infertility. We previously demonstrated that maternal haploinsufficiency for the endocrine peptide adrenomedullin (AM) in mice confers a subfertility phenotype characterized by defective uterine receptivity and sparse epithelial pinopode coverage. The strong link between AM and implantation suggested the compelling hypothesis that administration of AM prior to implantation may improve fertility, protect against pregnancy complications, and ultimately lead to better maternal and fetal outcomes. Here, we demonstrate that intrauterine delivery of AM prior to blastocyst transfer improves the embryo implantation rate and spacing within the uterus. We then use genetic decrease-of-function and pharmacologic gain-of-function mouse models to identify potential mechanisms by which AM confers enhanced implantation success. In epithelium, we find that AM accelerates the kinetics of pinopode formation and water transport and that, in stroma, AM promotes connexin 43 expression, gap junction communication, and barrier integrity of the primary decidual zone. Ultimately, our findings advance our understanding of the contributions of AM to uterine receptivity and suggest potential broad use for AM as therapy to encourage healthy embryo implantation, for example, in combination with in vitro fertilization.
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http://dx.doi.org/10.1093/biolre/iox101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6248476PMC
September 2017

Evaluation of endometrial function: a Heraclean or Sisyphean task?

Authors:
Steven L Young

Fertil Steril 2017 10;108(4):604-605

Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

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http://dx.doi.org/10.1016/j.fertnstert.2017.07.1166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5683894PMC
October 2017

Early Endometriosis in Females Is Directed by Immune-Mediated Estrogen Receptor α and IL-6 Cross-Talk.

Endocrinology 2018 01;159(1):103-118

Receptor Biology Group, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina.

Endometriosis is a gynecological disease that negatively affects the health of 1 in 10 women. Although more information is known about late stage disease, the early initiation of endometriosis and lesion development is poorly understood. Herein, we use a uterine tissue transfer mouse model of endometriosis to examine early disease development and its dependence on estradiol (E2) and estrogen receptor (ER) α within 72 hours of disease initiation. Using wild-type and ERα knockout mice as hosts or donors, we find substantial infiltration of neutrophils and macrophages into the peritoneal cavity. Examining cell infiltration, lesion gene expression, and peritoneal fluid, we find that E2/ERα plays a minor role in early lesion development. Immune-mediated signaling predominates E2-mediated signaling, but 48 hours after the initiation of disease, a blunted interleukin (IL)-6-mediated response is found in developing lesions lacking ERα. Our data provide evidence that the early initiation of endometriosis is predominantly dependent on the immune system, whereas E2/ERα/IL-6-mediated cross-talk plays a partial role. These findings suggest there are two phases of endometriosis-an immune-dependent phase and a hormone-dependent phase, and that targeting the innate immune system could prevent lesion attachment in this susceptible population of women.
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http://dx.doi.org/10.1210/en.2017-00562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761597PMC
January 2018

Effect of randomized serum progesterone concentration on secretory endometrial histologic development and gene expression.

Hum Reprod 2017 09;32(9):1903-1914

Department of Obstetrics and Gynecology (CB#7570), 101 Manning Dr, University of North Carolina at Chapel Hill, NC, USA.

Study Question: What doses of secretory phase progesterone (P) in women are associated with altered endometrial structure and/or function?

Summary Answer: Consistently delayed histological maturation was seen at the lowest tested daily P dose (2.5 mg), whereas consistently altered functional response, as reflected by microarray analysis of gene expression was seen at both the 5 and 2.5 mg doses.

What Is Known Already: Progesterone is absolutely required for normal embryo implantation and pregnancy survival. Progesterone supplementation is beneficial in ART cycles.

Study Design, Size, Duration: In this case-control experimental trial, 46 healthy young female volunteers (age 19-34) underwent a single modeled endometrial cycle after GnRH down-regulation or monitored in natural cycles.

Participants/materials, Setting, Methods: In a university hospital, modeled cycles were obtained by GnRH agonist down-regulation, transdermal estradiol (E2) (0.2 mg/d), and daily injections of P in oil for 10 days: 2.5 mg (n = 6), 5 mg (n = 6), 10 mg (n = 12) or 40 mg (n = 12), after the 10th day of E2. Ten healthy, ovulatory women were used as controls. Endometrial biopsies were obtained on the 10th day of P exposure, or urinary LH surge (in controls). Analysis included histological dating, serum progesterone levels, microarray analysis of the whole genome, RT-PCR, western blot and comparison with the GEO database.

Main Results And The Role Of Chance: In endometrial biopsies, a morphological delay appears in the 2.5 mg/day of P group. Higher sub-physiological levels of P (≥5 mg/day) resulted in normal histology, but aberrant gene expression. P levels required for consistent histological delay were lower than those in all ovulatory women. Gene expression abnormalities occurred at higher sub-physiological P concentrations, without a change in histology, a functional-morphological disassociation. The expression of some endometrial receptivity-associated genes appeared multiphasic, with peak or nadir of mean or median expression levels between the lowest and highest doses, suggesting sustained supraphysiological doses seen in ART treatment cycles may not be optimal.

Large Scale Data: GEO DataSets ID: 200056980; GSE 56980.

Limitations, Reasons For Caution: These results were obtained in fertile women, who may respond differently from infertile subjects.

Wider Implications Of The Findings: The dose of P required for normal endometrial structure (5 mg/day) corresponds to a P concentration well below that seen in ovulatory women, suggesting that persistently delayed mid-secretory histology cannot be solely due to inadequate P concentrations in an ovulatory cycle. Endometrial gene expression is differentially regulated by different doses of progesterone. The apparent multiphasic response of some genes to P dose suggests the possibility that P concentration kinetics may play a role in normal endometrial preparation for receptivity. These findings strongly confirm that histologic development is not a reliable measure of endometrial P action.

Study Funding/competing Interest(s): Supported by The Eunice Kennedy Shriver National Institute for Child Health and Disease, National Institute of Health, USA (NICHD/NIH) (R01HD067721 and U54HD30476; SLY and BAL) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) 240239/2012-1 (RFS). All authors have no competing interests.
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http://dx.doi.org/10.1093/humrep/dex252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5850604PMC
September 2017

KRAS Activation and over-expression of SIRT1/BCL6 Contributes to the Pathogenesis of Endometriosis and Progesterone Resistance.

Sci Rep 2017 07 28;7(1):6765. Epub 2017 Jul 28.

Obstetrics and Gynecology, Greenville Health System, Greenville, SC, 29605, USA.

Endometriosis is an inflammatory condition that is associated with progesterone resistance and cell proliferation, resulting in pain, infertility and pregnancy loss. We previously demonstrated phosphorylation of STAT3 in eutopic endometrium of infertile women with this disorder leading to over-expression of the oncogene BCL6 and stabilization of hypoxia-induced factor 1 alpha (HIF-1α). Here we report coordinated activation of KRAS and over-expression of Sirtuin 1 (SIRT1), a histone deacetylase and gene silencer, in the eutopic endometrium from women with endometriosis throughout the menstrual cycle. The mice with conditional activation of KRAS in the PGR positive cells reveal an increase of SIRT1 expression in the endometrium compared to control mice. The expression of progesterone receptor target genes including the Indian Hedgehog pathway genes are significantly down-regulated in the mutant mice. SIRT1 co-localizes with BCL6 in the nuclei of affected individuals and both proteins bind to and suppress the promoter of GLI1, a critical mediator of progesterone action in the Indian Hedgehog pathway, by ChIP analysis. In eutopic endometrium, GLI1 expression is reduced in women with endometriosis. Together, these data suggest that KRAS, SIRT1 and BCL6 are coordinately over-expressed in eutopic endometrium of women with endometriosis and likely participate in the pathogenesis of endometriosis.
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http://dx.doi.org/10.1038/s41598-017-04577-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5533722PMC
July 2017

A balancing act: RNA binding protein HuR/TTP axis in endometriosis patients.

Sci Rep 2017 07 19;7(1):5883. Epub 2017 Jul 19.

Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, K7L 3N6, Canada.

Endometriosis, a major reproductive pathology affecting 8-10% of women is characterized by chronic inflammation and immune dysfunction. Human antigen R (HuR) and Tristetraprolin (TTP) are RNA binding proteins that competitively bind to cytokines involved in inflammation including: tumor necrosis factor alpha (TNF-α), granulocyte macrophage colony stimulating factor (GM-CSF), interleukin 6 (IL-6) among others, and stabilize and destabilize them, respectively. The aim of this study was to examine RNA binding protein (RNABP) HuR/TTP axis in endometriosis patients compared to menstrual stage matched healthy fertile controls in hopes of better understanding their contribution to the pathogenesis of endometriosis. Additionally, using a targeted in vitro siRNA approach, we examined whether knock-down of TTP can play a functional role on other RNABPs that competitively bind to inflammatory targets of TTP in both endometriotic and endometrial epithelial cell lines. Our results suggest that RNABPs TTP and HuR are dysregulated in endometriotic lesions compared to matched eutopic patient samples as well endometrium from healthy controls. Silencing of TTP in endometriotic and endometrial epithelial cells revealed differential response to inflammatory cytokines and other RNABPs. Our results suggest potential involvement of HuR/TTP RNA binding protein axis in regulation of inflammation in endometriosis.
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http://dx.doi.org/10.1038/s41598-017-06081-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5517625PMC
July 2017