Profiling the expression and function of ER46 in human endometrial tissues and uterine NK cells

AbstractStudy questionDoes the oestrogen receptor isoform, ER46, contribute to regulation of endometrial function?Summary answerER46 is expressed in endometrial tissues during the proliferative and secretory phases and is the predominant ERα isoform in first trimester decidua. ER46 is abundantly expressed in uterine NK (uNK) cells and localised to the cell membrane. Activation of ER46 regulates the function of human uNK cells by increasing cell motility.What is known alreadyOestrogens acting via their cognate receptors are essential regulators of endometrial function and play key roles in establishment of pregnancy. ER46 is a 46kDa truncated isoform of full length ERα (ER66, encoded by ESR1) that contains both ligand and DNA binding domains. Expression of ER46 in human endometrium has not been investigated previously. ER46 is located at the cell membrane of peripheral blood leukocytes and mediates rapid responses to oestrogens. UNK cells are a phenotypically distinct (CD56brightCD16-) population of tissue-resident immune cells that regulate vascular remodelling within the endometrium and decidua. We have shown that oestrogens stimulate rapid increases in uNK cell motility. Previous characterisation of uNK cells suggests they are ER66-negative but expression of ER46 has not been characterised. We hypothesise that uNK cells express ER46 and that rapid responses to oestrogens are mediated via this receptor.Study design, size, durationThis laboratory-based study used primary human endometrial (n=24) and decidual tissue biopsies (n=30) as well as uNK cells which were freshly isolated from first trimester human decidua (n=18).Participants/materials, setting, methodsPrimary human endometrial and first trimester decidual tissue biopsies were collected using methods approved by the local institutional ethics committee (LREC/05/51104/12 and LREC/10/51402/59). The expression of oestrogen receptors (ER66, ER46 and ERβ) was assessed by qPCR, western blot and immunohistochemistry. Uterine Natural Killer (uNK) cells were isolated from first trimester human decidua by magnetic bead sorting. Cell motility of uNK cells was measured by live cell imaging: cells were treated with oestradiol (E2)-BSA (10nM equivalent), the ERβ-selective agonist 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; 10nM) or vehicle control (DMSO).Main results and the role of chanceER46 was detected in proliferative and secretory phase tissues and was the predominant ERα isoform in first trimester decidua samples. Immunohistochemistry revealed ER46 was co-localised with ER66 in cell nuclei during the proliferative phase but detected in both the cytoplasm and cell membrane of stromal cells in the secretory phase and in decidua. Triple immunofluorescence staining of decidua tissues identified expression of ER46 in the cell membrane of CD56-positive uNK cells which were otherwise ER66-negative. Profiling of isolated uNK cells confirmed expression ER46 and localised ER46 protein to the cell membrane. Functional analysis of isolated uNK cells using live cell imaging demonstrated that activation of ER46 with E2-BSA significantly increased uNK cell motility.Limitations, reasons for cautionExpression patterns in endometrial tissue was only determined using samples from proliferative and secretory phases. Assessment of first trimester decidua samples was from a range of gestational ages which may have precluded insights into gestation specific changes in these tissues. Our results are based on in vitro responses of primary human cells and we cannot be certain that similar mechanisms occur in situ.Wider implications of the findingsE2 is an essential regulator of reproductive competence. This study provides the first evidence for expression of ER46 in human endometrium and decidua of early pregnancy. We describe a mechanism for regulating the function of human uNK cells via expression of ER46 and demonstrate that selective targeting with E2-BSA regulates uNK cell motility. These novel findings identify a role for ER46 in human endometrium and provide unique insight into the importance of membrane-initiated signalling in modulating the impact of E2 on uNK cell function in women.Study funding/competing interest(s)These studies were supported by MRC Programme Grants G1100356/1 and MR/N024524/1 to PTKS. HODC was supported by MRC grant G1002033.

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Profiling the expression and function of oestrogen receptor isoform ER46 in human endometrial tissues and uterine natural killer cells

Human Reproduction ◽

10.1093/humrep/dez306 ◽

2020 ◽

Vol 35 (3) ◽

pp. 641-651 ◽

Cited By ~ 2

Author(s):

Douglas A Gibson ◽

Arantza Esnal-Zufiaurre ◽

Cristina Bajo-Santos ◽

Frances Collins ◽

Hilary O D Critchley ◽

...

Keyword(s):

Cell Membrane ◽

Western Blot ◽

Cell Motility ◽

First Trimester ◽

Human Endometrium ◽

Decidual Tissue ◽

Rapid Responses ◽

Unk Cells ◽

Selective Targeting ◽

Unk Cell

Abstract STUDY QUESTION Does the oestrogen receptor isoform, ER46, contribute to regulation of endometrial function? SUMMARY ANSWER ER46 is expressed in endometrial tissues, is the predominant ER isoform in first trimester decidua and is localised to the cell membrane of uterine natural killer (uNK) cells where activation of ER46 increases cell motility. WHAT IS KNOWN ALREADY Oestrogens acting via their cognate receptors are essential regulators of endometrial function and play key roles in establishment of pregnancy. ER46 is a 46-kDa truncated isoform of full length ERα (ER66, encoded by ESR1) that contains both ligand- and DNA-binding domains. Expression of ER46 in the human endometrium has not been investigated previously. ER46 is located at the cell membrane of peripheral blood leukocytes and mediates rapid responses to oestrogens. uNK cells are a phenotypically distinct (CD56brightCD16−) population of tissue-resident immune cells that regulate vascular remodelling within the endometrium and decidua. We have shown that oestrogens stimulate rapid increases in uNK cell motility. Previous characterisation of uNK cells suggests they are ER66-negative, but expression of ER46 has not been characterised. We hypothesise that uNK cells express ER46 and that rapid responses to oestrogens are mediated via this receptor. STUDY DESIGN, SIZE, DURATION This laboratory-based study used primary human endometrial (n = 24) and decidual tissue biopsies (n = 30) as well as uNK cells which were freshly isolated from first trimester human decidua (n = 18). PARTICIPANTS/MATERIALS, SETTING, METHODS Primary human endometrial and first trimester decidual tissue biopsies were collected using methods approved by the local institutional ethics committee (LREC/05/51104/12 and LREC/10/51402/59). The expression of ERs (ER66, ER46 and ERβ) was assessed by quantitative PCR, western blot and immunohistochemistry. uNK cells were isolated from first-trimester human decidua by magnetic bead sorting. Cell motility of uNK cells was measured by live cell imaging: cells were treated with 17β-oestradiol conjugated to bovine serum albumin (E2-BSA, 10 nM equivalent), the ERβ-selective agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; 10 nM) or dimethylsulphoxide vehicle control. MAIN RESULTS AND THE ROLE OF CHANCE ER46 was detected in proliferative and secretory phase tissues by western blot and was the predominant ER isoform in first-trimester decidua samples. Immunohistochemistry revealed that ER46 was co-localised with ER66 in cell nuclei during the proliferative phase but detected in both the cytoplasm and cell membrane of stromal cells in the secretory phase and in decidua. Triple immunofluorescence staining of decidua tissues identified expression of ER46 in the cell membrane of CD56-positive uNK cells which were otherwise ER66-negative. Profiling of isolated uNK cells confirmed expression of ER46 by quantitative PCR and western blot and localised ER46 protein to the cell membrane by immunocytochemistry. Functional analysis of isolated uNK cells using live cell imaging demonstrated that activation of ER46 with E2-BSA significantly increased uNK cell motility. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Expression pattern in endometrial tissue was only determined using samples from proliferative and secretory phases. Assessment of first trimester decidua samples was from a range of gestational ages, which may have precluded insights into gestation-specific changes in these tissues. Our results are based on in vitro responses of primary human cells and we cannot be certain that similar mechanisms occur in situ. WIDER IMPLICATIONS OF THE FINDINGS E2 is an essential regulator of reproductive competence. This study provides the first evidence for expression of ER46 in the human endometrium and decidua of early pregnancy. We describe a mechanism for regulating the function of human uNK cells via expression of ER46 and demonstrate that selective targeting with E2-BSA regulates uNK cell motility. These novel findings identify a role for ER46 in the human endometrium and provide unique insight into the importance of membrane-initiated signalling in modulating the impact of E2 on uNK cell function in women. Given the importance of uNK cells to regulating vascular remodelling in early pregnancy and the potential for selective targeting of ER46, this may be an attractive future therapeutic target in the treatment of reproductive disorders. STUDY FUNDING/COMPETING INTEREST(S) These studies were supported by Medical Research Council (MRC) Programme Grants G1100356/1 and MR/N024524/1 to PTKS. H.O.D.C. was supported by MRC grant G1002033. The authors declare no competing interests related to the published work.

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First Trimester Histiotrophe Shows Altered Sialylation Compared with Secretory Phase Glycoconjugates in Human Endometrium

Placenta ◽

10.1016/j.placenta.2010.04.011 ◽

2010 ◽

Vol 31 (7) ◽

pp. 576-580 ◽

Cited By ~ 18

Author(s):

C.J.P. Jones ◽

J.D. Aplin ◽

G.J. Burton

Keyword(s):

First Trimester ◽

Human Endometrium ◽

Secretory Phase

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4307 Role of Pre-pregnancy Uterine Natural Killer Cells in Human Embryo Implantation

Journal of Clinical and Translational Science ◽

10.1017/cts.2020.316 ◽

2020 ◽

Vol 4 (s1) ◽

pp. 102-102

Author(s):

Jessica Kanter ◽

Sneha Mani ◽

Scott Gordon ◽

Monica Mainigi

Keyword(s):

Natural Killer ◽

Tunel Staining ◽

Spiral Artery ◽

Secretory Phase ◽

Potential Marker ◽

Unk Cells ◽

Artery Remodeling ◽

Unk Cell ◽

Uterine Natural Killer

OBJECTIVES/GOALS: Human placentation requires complex coordination between maternal and fetal cell types but remains incompletely understood. We hypothesize that uterine natural killer (uNK) cells, an immune cell type that increases in abundance during the implantation window, is essential for appropriate implantation and placentation. METHODS/STUDY POPULATION: We plan to examine stromal cell (SC) decidualization, spiral artery remodeling, and EVT invasion, processes vital for early pregnancy establishment, in the presence or absence of secretory phase uNK cells. Fetal extravillous trophoblasts (EVTs) will be isolated from first trimester pregnancy tissue; maternal SCs, endothelial cells (ECs) and uNK cells will be obtained from secretory phase uterine tissue. SCs will be placed in monoculture and coculture with uNK cells and prolactin will be measured to evaluate decidualization. To study EVT invasion, we will utilize our novel “implantation-on-a-chip” device to determine how addition of uNK cells affects EVT migration through a collagen-matrigel matrix. In this system, we will also examine spiral artery remodeling with or without uNK cells via TUNEL staining. RESULTS/ANTICIPATED RESULTS: We anticipate that uNK cell addition to SCs will lead to a significant increase in SC prolactin levels, suggesting a role of uNK cells in endometrial decidualization. In vitro, we expect the addition of uNK cells will increase EC apoptosis and promote EVT invasion. DISCUSSION/SIGNIFICANCE OF IMPACT: Although decidual NK cells are known to participate in placentation, the role of pre-pregnancy uNK cells is unknown. uNK cell involvement in processes important for the earliest stages of pregnancy would provide a potential marker for abnormal placentation and offer avenues for intervention to decrease placentation associated perinatal morbidity.

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Decidual NK cell-derived conditioned medium from miscarriages affects endometrial stromal cell decidualisation: endocannabinoid anandamide and tumour necrosis factor-α crosstalk

Human Reproduction ◽

10.1093/humrep/dez260 ◽

2020 ◽

Vol 35 (2) ◽

pp. 265-274 ◽

Cited By ~ 2

Author(s):

B M Fonseca ◽

S C Cunha ◽

D Gonçalves ◽

A Mendes ◽

J Braga ◽

...

Keyword(s):

Conditioned Medium ◽

Stromal Cell ◽

First Trimester ◽

Cytokine Profile ◽

Enzyme Linked Immunosorbent Assay ◽

Endometrial Stromal Cell ◽

Unk Cells ◽

Tnf Α ◽

The Impact ◽

Unk Cell

Abstract STUDY QUESTION What are the effects of endocannabinoid anandamide (AEA) in uterine natural killer (unK) cells from miscarriage decidua, regarding their cytokine profile and endometrial stromal cell (ESC) crosstalk? SUMMARY ANSWER uNK-conditioned media from miscarriage samples present high TNF-α levels which inhibit ESC decidualisation. WHAT IS KNOWN ALREADY AEA plasma levels are higher in women who have suffered a miscarriage. Moreover, AEA inhibits ESC proliferation and differentiation, although the levels and impact on the uNK cell cytokine profile at the feto-maternal interface remain elusive. STUDY DESIGN, SIZE, DURATION This laboratory-based study used human primary uNK cells which were isolated from first-trimester decidua (gestational age, 5–12 weeks) derived from 8 women with elective pregnancy termination and 18 women who suffered a miscarriage. PARTICIPANTS/MATERIALS, SETTING, METHODS The first-trimester placental tissues were assayed for AEA levels by UPLC-MS/MS and respective enzymatic profile by western blot. The uNK cells were isolated and maintained in culture. The expression of angiogenic markers in uNK cells was examined by quantitative PCR (qPCR). The uNK-conditioned medium was analysed for IFN-γ, TNF-α and IL-10 production by enzyme-linked immunosorbent assay, and the impact on ESC differentiation was assessed by measuring decidual markers Prl, Igfbp-1 and Fox01 mRNA expression using qPCR. MAIN RESULTS AND THE ROLE OF CHANCE AEA levels were higher in miscarriage decidua compared with decidua from elective terminations. The uNK cell-conditioned medium from the miscarriage samples exhibited high TNF-α levels and interfered with the decidualisation of ESCs. Exacerbated inflammation and elevated TNF-α levels at the feto-maternal interface may trigger AEA signalling pathways that, in turn, may impact decidualisation and the angiogenic ability of uNK cells. LARGE-SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Primary uNK cell responses are based on a simple in vitro model. Thus, in complex microenvironments, such as the feto-maternal interface, the mechanisms may not be exactly the same. Also, the inflammatory events of miscarriage that, in this study, have happened prior to processing of the samples may cause different responses to that observed. In addition, the magnitude of the inflammatory response, required to trigger the AEA pathways that impact decidualisation and the uNK angiogenic ability in vivo, is still unclear. WIDER IMPLICATIONS OF THE FINDINGS The endocannabinoid AEA is a modulator of reproductive competence. AEA not only may contribute to neuroendocrine homeostasis but also can take part in uterine changes occurring during early pregnancy. STUDY FUNDING/COMPETING INTEREST(S) The work was supported by UID/MULTI/04378/2019 with funding from Fundação para a Ciência e a Tecnologia (FCT)/MCTES through national funds and PORTUGAL 2020 Partnership Agreement, NORTE-01-0145-FEDER-000024. S.C. Cunha acknowledges FCT for the IF/01616/2015 contract. There are no conflicts of interest.

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P433 The uNK cell in human decidua of normal pregnancy and spontaneous abortion in the first trimester pregnancy and its angiogenic function regulated by progesterone and IL-15

International Journal of Gynecology & Obstetrics ◽

10.1016/s0020-7292(09)61925-1 ◽

2009 ◽

Vol 107 ◽

pp. S537-S537

Author(s):

W. Huang ◽

Y. Liu ◽

G. Li ◽

T. Hu ◽

H. Zhu

Keyword(s):

Spontaneous Abortion ◽

First Trimester ◽

Normal Pregnancy ◽

First Trimester Pregnancy ◽

Trimester Pregnancy ◽

Human Decidua ◽

Unk Cell

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Proliferation of Uterine Natural Killer Cells Is Induced by Human Chorionic Gonadotropin and Mediated via the Mannose Receptor

Endocrinology ◽

10.1210/en.2008-1309 ◽

2009 ◽

Vol 150 (6) ◽

pp. 2882-2888 ◽

Cited By ~ 92

Author(s):

Nicole Kane ◽

Rodney Kelly ◽

Philippa T. K. Saunders ◽

Hilary O. D. Critchley

Keyword(s):

Natural Killer ◽

Mannose Receptor ◽

Lh Receptor ◽

Uterine Natural Killer Cells ◽

Unk Cells ◽

The Impact ◽

Insight Into ◽

Unk Cell ◽

Uterine Natural Killer

The endometrial lining of the human uterus contains a population of phenotypically distinct (CD56bright, CD16dim), tissue-specific, natural killer [uterine natural killer (uNK)] cells that play a key role in the establishment of a successful pregnancy. An increase in the number of endometrial uNK cells occurs when the conceptus implants, and there is a further increase during the early stages of placentation. Here, we describe studies that have identified human chorionic gonadotrophin (hCG), a glycoprotein synthesized by the preimplantation conceptus, as a novel regulator of uNK cell proliferation. The impact of hCG on uNK cells was mediated via the mannose receptor (CD206) rather than by the classical hCG/LH receptor that was not expressed. The mannose receptor and hCG were colocalized on the surface of uNK cells, and proliferation did not occur if cells were incubated with deglycosylated hCG or intact hCG in the presence of excess d-Mannose. These novel observations provide new insight into the endocrine-immune dialogue that exists between the conceptus and immune cells within the receptive endometrium, and have implications for the role of uNK cell-trophoblast interactions and pregnancy outcome.

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