Reduced homeobox protein MSX1 in human endometrial tissue is linked to infertility

Alan D. Bolnick, Jay M. Bolnick, Brian A. Kilburn, Tamika Stewart, Jonathan Oakes, Javier Rodriguez-Kovacs, Hamid Reza Kohan-Ghadr, Jing Dai, Michael P. Diamond, Yasushi Hirota, Sascha Drewlo, Sudhansu K. Dey, D. Randall Armant

Research output: Contribution to journalArticle

Abstract

STUDY QUESTION: Is protein expression of the muscle segment homeobox gene family member MSX1 altered in the human secretory endometrium by cell type, developmental stage or fertility?

SUMMARY ANSWER: MSX1 protein levels, normally elevated in the secretory phase endometrium, were significantly reduced in endometrial biopsies obtained from women of infertile couples.

WHAT IS KNOWN ALREADY: Molecular changes in the endometrium are important for fertility in both animals and humans. Msx1 is expressed in the preimplantation mouse uterus and regulates uterine receptivity for implantation. The MSX protein persists a short time, after its message has been down-regulated. Microarray analysis of the human endometrium reveals a similar pattern of MSX1 mRNA expression that peaks before the receptive period, with depressed expression at implantation. Targeted deletion of uterine Msx1 and Msx2 in mice prevents the loss of epithelial cell polarity during implantation and causes infertility.

STUDY DESIGN, SIZE DURATION: MSX1 mRNA and cell type-specific levels of MSX1 protein were quantified from two retrospective cohorts during the human endometrial cycle. MSX1 protein expression patterns were compared between fertile and infertile couples. Selected samples were dual-labeled by immunofluorescence microscopy to localize E-cadherin and β-catenin in epithelial cells.

PARTICIPANTS/MATERIALS, SETTING METHODS: MSX1 mRNA was quantified by PCR in endometrium from hysterectomies (n = 14) determined by endometrial dating to be in the late-proliferative (cycle days 10-13), early-secretory (cycle days 14-19) or mid-secretory (cycle days 20-24) phase. MSX1 protein was localized using high-throughput, semi-quantitative immunohistochemistry with sectioned endometrial biopsy tissues from fertile (n = 89) and infertile (n = 89) couples. Image analysis measured stain intensity specifically within the luminal epithelium, glands and stroma during the early-, mid- and late- (cycle days 25-28) secretory phases.

MAIN RESULTS AND THE ROLE OF CHANCE: MSX1 transcript increased 5-fold (P < 0.05) between the late-proliferative and early secretory phase and was then down-regulated (P < 0.05) prior to receptivity for implantation. In fertile patients, MSX1 protein displayed strong nuclear localization in the luminal epithelium and glands, while it was weakly expressed in nuclei of the stroma. MSX1 protein levels accumulated throughout the secretory phase in all endometrial cellular compartments. MSX1 protein decreased (P < 0.05) in the glands between mid- and late-secretory phases. However, infertile patients demonstrated a broad reduction (P < 0.001) of MSX1 accumulation in all cell types throughout the secretory phase that was most pronounced (∼3-fold) in stroma and glands. Infertility was associated with persistent co-localization of E-cadherin and β-catenin in epithelial cell junctions in the mid- and late-secretory phases.

LIMITATIONS, REASONS FOR CAUTION: Details of the infertility diagnoses and other patient demographic data were not available. Therefore, patients with uterine abnormalities (Mullerian) could not be distinguished from other sources of infertility. Antibody against human MSX2 is not available, limiting the study to MSX1. However, both RNAs in the human endometrium are similarly regulated. In mice, Msx1 and Msx2 are imperative for murine embryo implantation, with Msx2 compensating for genetic ablation of Msx1 through its up-regulation in a knockout model.

WIDER IMPLICATIONS OF THE FINDINGS: This investigation establishes that the MSX1 homeobox protein accumulation is associated with the secretory phase in endometrium of fertile couples, and is widely disrupted in infertile patients. It is the first study to examine MSX1 protein localization in the human endometrium, and supported by genetic findings in mice, suggests that genes regulated by MSX1 are linked to the loss of epithelial cell polarity required for uterine receptivity during implantation.

STUDY FUNDING/COMPETING INTERESTS: This research was supported by the NICHD National Cooperative Reproductive Medicine Network grant HD039005 (M.P.D.), NIH grants HD068524 (S.K.D.), HD071408 (D.R.A., M.P.D.), and HL128628 (S.D.), the Intramural Research Program of the NICHD, March of Dimes (S.K.D., S.D.) and JSPS KAKENHI grant 26112506 (Y.H.). There were no conflicts or competing interests.

Original languageEnglish (US)
Pages (from-to)2042-2050
Number of pages9
JournalHuman Reproduction
Volume31
Issue number9
DOIs
StatePublished - Sep 1 2016

Fingerprint

Homeodomain Proteins
Infertility
Endometrium
Proteins
Organized Financing
Epithelial Cells
National Institute of Child Health and Human Development (U.S.)
Catenins
Cell Polarity
Cadherins
Messenger RNA
Fertility
Epithelium
Reproductive Medicine
Biopsy
Intercellular Junctions
Muscle Proteins
Homeobox Genes
Microarray Analysis
Menstrual Cycle

Keywords

  • E-cadherin
  • Endometrium
  • Female infertility
  • Immunohistochemistry
  • Implantation
  • Menstrual cycle
  • Messenger RNA
  • Muscle segment homeobox 1
  • Secretory phase
  • Transcription factor

ASJC Scopus subject areas

  • Reproductive Medicine
  • Rehabilitation
  • Obstetrics and Gynecology

Cite this

Bolnick, A. D., Bolnick, J. M., Kilburn, B. A., Stewart, T., Oakes, J., Rodriguez-Kovacs, J., ... Armant, D. R. (2016). Reduced homeobox protein MSX1 in human endometrial tissue is linked to infertility. Human Reproduction, 31(9), 2042-2050. https://doi.org/10.1093/humrep/dew143

Reduced homeobox protein MSX1 in human endometrial tissue is linked to infertility. / Bolnick, Alan D.; Bolnick, Jay M.; Kilburn, Brian A.; Stewart, Tamika; Oakes, Jonathan; Rodriguez-Kovacs, Javier; Kohan-Ghadr, Hamid Reza; Dai, Jing; Diamond, Michael P.; Hirota, Yasushi; Drewlo, Sascha; Dey, Sudhansu K.; Armant, D. Randall.

In: Human Reproduction, Vol. 31, No. 9, 01.09.2016, p. 2042-2050.

Research output: Contribution to journalArticle

Bolnick, AD, Bolnick, JM, Kilburn, BA, Stewart, T, Oakes, J, Rodriguez-Kovacs, J, Kohan-Ghadr, HR, Dai, J, Diamond, MP, Hirota, Y, Drewlo, S, Dey, SK & Armant, DR 2016, 'Reduced homeobox protein MSX1 in human endometrial tissue is linked to infertility', Human Reproduction, vol. 31, no. 9, pp. 2042-2050. https://doi.org/10.1093/humrep/dew143
Bolnick AD, Bolnick JM, Kilburn BA, Stewart T, Oakes J, Rodriguez-Kovacs J et al. Reduced homeobox protein MSX1 in human endometrial tissue is linked to infertility. Human Reproduction. 2016 Sep 1;31(9):2042-2050. https://doi.org/10.1093/humrep/dew143
Bolnick, Alan D. ; Bolnick, Jay M. ; Kilburn, Brian A. ; Stewart, Tamika ; Oakes, Jonathan ; Rodriguez-Kovacs, Javier ; Kohan-Ghadr, Hamid Reza ; Dai, Jing ; Diamond, Michael P. ; Hirota, Yasushi ; Drewlo, Sascha ; Dey, Sudhansu K. ; Armant, D. Randall. / Reduced homeobox protein MSX1 in human endometrial tissue is linked to infertility. In: Human Reproduction. 2016 ; Vol. 31, No. 9. pp. 2042-2050.
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abstract = "STUDY QUESTION: Is protein expression of the muscle segment homeobox gene family member MSX1 altered in the human secretory endometrium by cell type, developmental stage or fertility?SUMMARY ANSWER: MSX1 protein levels, normally elevated in the secretory phase endometrium, were significantly reduced in endometrial biopsies obtained from women of infertile couples.WHAT IS KNOWN ALREADY: Molecular changes in the endometrium are important for fertility in both animals and humans. Msx1 is expressed in the preimplantation mouse uterus and regulates uterine receptivity for implantation. The MSX protein persists a short time, after its message has been down-regulated. Microarray analysis of the human endometrium reveals a similar pattern of MSX1 mRNA expression that peaks before the receptive period, with depressed expression at implantation. Targeted deletion of uterine Msx1 and Msx2 in mice prevents the loss of epithelial cell polarity during implantation and causes infertility.STUDY DESIGN, SIZE DURATION: MSX1 mRNA and cell type-specific levels of MSX1 protein were quantified from two retrospective cohorts during the human endometrial cycle. MSX1 protein expression patterns were compared between fertile and infertile couples. Selected samples were dual-labeled by immunofluorescence microscopy to localize E-cadherin and β-catenin in epithelial cells.PARTICIPANTS/MATERIALS, SETTING METHODS: MSX1 mRNA was quantified by PCR in endometrium from hysterectomies (n = 14) determined by endometrial dating to be in the late-proliferative (cycle days 10-13), early-secretory (cycle days 14-19) or mid-secretory (cycle days 20-24) phase. MSX1 protein was localized using high-throughput, semi-quantitative immunohistochemistry with sectioned endometrial biopsy tissues from fertile (n = 89) and infertile (n = 89) couples. Image analysis measured stain intensity specifically within the luminal epithelium, glands and stroma during the early-, mid- and late- (cycle days 25-28) secretory phases.MAIN RESULTS AND THE ROLE OF CHANCE: MSX1 transcript increased 5-fold (P < 0.05) between the late-proliferative and early secretory phase and was then down-regulated (P < 0.05) prior to receptivity for implantation. In fertile patients, MSX1 protein displayed strong nuclear localization in the luminal epithelium and glands, while it was weakly expressed in nuclei of the stroma. MSX1 protein levels accumulated throughout the secretory phase in all endometrial cellular compartments. MSX1 protein decreased (P < 0.05) in the glands between mid- and late-secretory phases. However, infertile patients demonstrated a broad reduction (P < 0.001) of MSX1 accumulation in all cell types throughout the secretory phase that was most pronounced (∼3-fold) in stroma and glands. Infertility was associated with persistent co-localization of E-cadherin and β-catenin in epithelial cell junctions in the mid- and late-secretory phases.LIMITATIONS, REASONS FOR CAUTION: Details of the infertility diagnoses and other patient demographic data were not available. Therefore, patients with uterine abnormalities (Mullerian) could not be distinguished from other sources of infertility. Antibody against human MSX2 is not available, limiting the study to MSX1. However, both RNAs in the human endometrium are similarly regulated. In mice, Msx1 and Msx2 are imperative for murine embryo implantation, with Msx2 compensating for genetic ablation of Msx1 through its up-regulation in a knockout model.WIDER IMPLICATIONS OF THE FINDINGS: This investigation establishes that the MSX1 homeobox protein accumulation is associated with the secretory phase in endometrium of fertile couples, and is widely disrupted in infertile patients. It is the first study to examine MSX1 protein localization in the human endometrium, and supported by genetic findings in mice, suggests that genes regulated by MSX1 are linked to the loss of epithelial cell polarity required for uterine receptivity during implantation.STUDY FUNDING/COMPETING INTERESTS: This research was supported by the NICHD National Cooperative Reproductive Medicine Network grant HD039005 (M.P.D.), NIH grants HD068524 (S.K.D.), HD071408 (D.R.A., M.P.D.), and HL128628 (S.D.), the Intramural Research Program of the NICHD, March of Dimes (S.K.D., S.D.) and JSPS KAKENHI grant 26112506 (Y.H.). There were no conflicts or competing interests.",
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note = "{\circledC} The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.",
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T1 - Reduced homeobox protein MSX1 in human endometrial tissue is linked to infertility

AU - Bolnick, Alan D.

AU - Bolnick, Jay M.

AU - Kilburn, Brian A.

AU - Stewart, Tamika

AU - Oakes, Jonathan

AU - Rodriguez-Kovacs, Javier

AU - Kohan-Ghadr, Hamid Reza

AU - Dai, Jing

AU - Diamond, Michael P.

AU - Hirota, Yasushi

AU - Drewlo, Sascha

AU - Dey, Sudhansu K.

AU - Armant, D. Randall

N1 - © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

PY - 2016/9/1

Y1 - 2016/9/1

N2 - STUDY QUESTION: Is protein expression of the muscle segment homeobox gene family member MSX1 altered in the human secretory endometrium by cell type, developmental stage or fertility?SUMMARY ANSWER: MSX1 protein levels, normally elevated in the secretory phase endometrium, were significantly reduced in endometrial biopsies obtained from women of infertile couples.WHAT IS KNOWN ALREADY: Molecular changes in the endometrium are important for fertility in both animals and humans. Msx1 is expressed in the preimplantation mouse uterus and regulates uterine receptivity for implantation. The MSX protein persists a short time, after its message has been down-regulated. Microarray analysis of the human endometrium reveals a similar pattern of MSX1 mRNA expression that peaks before the receptive period, with depressed expression at implantation. Targeted deletion of uterine Msx1 and Msx2 in mice prevents the loss of epithelial cell polarity during implantation and causes infertility.STUDY DESIGN, SIZE DURATION: MSX1 mRNA and cell type-specific levels of MSX1 protein were quantified from two retrospective cohorts during the human endometrial cycle. MSX1 protein expression patterns were compared between fertile and infertile couples. Selected samples were dual-labeled by immunofluorescence microscopy to localize E-cadherin and β-catenin in epithelial cells.PARTICIPANTS/MATERIALS, SETTING METHODS: MSX1 mRNA was quantified by PCR in endometrium from hysterectomies (n = 14) determined by endometrial dating to be in the late-proliferative (cycle days 10-13), early-secretory (cycle days 14-19) or mid-secretory (cycle days 20-24) phase. MSX1 protein was localized using high-throughput, semi-quantitative immunohistochemistry with sectioned endometrial biopsy tissues from fertile (n = 89) and infertile (n = 89) couples. Image analysis measured stain intensity specifically within the luminal epithelium, glands and stroma during the early-, mid- and late- (cycle days 25-28) secretory phases.MAIN RESULTS AND THE ROLE OF CHANCE: MSX1 transcript increased 5-fold (P < 0.05) between the late-proliferative and early secretory phase and was then down-regulated (P < 0.05) prior to receptivity for implantation. In fertile patients, MSX1 protein displayed strong nuclear localization in the luminal epithelium and glands, while it was weakly expressed in nuclei of the stroma. MSX1 protein levels accumulated throughout the secretory phase in all endometrial cellular compartments. MSX1 protein decreased (P < 0.05) in the glands between mid- and late-secretory phases. However, infertile patients demonstrated a broad reduction (P < 0.001) of MSX1 accumulation in all cell types throughout the secretory phase that was most pronounced (∼3-fold) in stroma and glands. Infertility was associated with persistent co-localization of E-cadherin and β-catenin in epithelial cell junctions in the mid- and late-secretory phases.LIMITATIONS, REASONS FOR CAUTION: Details of the infertility diagnoses and other patient demographic data were not available. Therefore, patients with uterine abnormalities (Mullerian) could not be distinguished from other sources of infertility. Antibody against human MSX2 is not available, limiting the study to MSX1. However, both RNAs in the human endometrium are similarly regulated. In mice, Msx1 and Msx2 are imperative for murine embryo implantation, with Msx2 compensating for genetic ablation of Msx1 through its up-regulation in a knockout model.WIDER IMPLICATIONS OF THE FINDINGS: This investigation establishes that the MSX1 homeobox protein accumulation is associated with the secretory phase in endometrium of fertile couples, and is widely disrupted in infertile patients. It is the first study to examine MSX1 protein localization in the human endometrium, and supported by genetic findings in mice, suggests that genes regulated by MSX1 are linked to the loss of epithelial cell polarity required for uterine receptivity during implantation.STUDY FUNDING/COMPETING INTERESTS: This research was supported by the NICHD National Cooperative Reproductive Medicine Network grant HD039005 (M.P.D.), NIH grants HD068524 (S.K.D.), HD071408 (D.R.A., M.P.D.), and HL128628 (S.D.), the Intramural Research Program of the NICHD, March of Dimes (S.K.D., S.D.) and JSPS KAKENHI grant 26112506 (Y.H.). There were no conflicts or competing interests.

AB - STUDY QUESTION: Is protein expression of the muscle segment homeobox gene family member MSX1 altered in the human secretory endometrium by cell type, developmental stage or fertility?SUMMARY ANSWER: MSX1 protein levels, normally elevated in the secretory phase endometrium, were significantly reduced in endometrial biopsies obtained from women of infertile couples.WHAT IS KNOWN ALREADY: Molecular changes in the endometrium are important for fertility in both animals and humans. Msx1 is expressed in the preimplantation mouse uterus and regulates uterine receptivity for implantation. The MSX protein persists a short time, after its message has been down-regulated. Microarray analysis of the human endometrium reveals a similar pattern of MSX1 mRNA expression that peaks before the receptive period, with depressed expression at implantation. Targeted deletion of uterine Msx1 and Msx2 in mice prevents the loss of epithelial cell polarity during implantation and causes infertility.STUDY DESIGN, SIZE DURATION: MSX1 mRNA and cell type-specific levels of MSX1 protein were quantified from two retrospective cohorts during the human endometrial cycle. MSX1 protein expression patterns were compared between fertile and infertile couples. Selected samples were dual-labeled by immunofluorescence microscopy to localize E-cadherin and β-catenin in epithelial cells.PARTICIPANTS/MATERIALS, SETTING METHODS: MSX1 mRNA was quantified by PCR in endometrium from hysterectomies (n = 14) determined by endometrial dating to be in the late-proliferative (cycle days 10-13), early-secretory (cycle days 14-19) or mid-secretory (cycle days 20-24) phase. MSX1 protein was localized using high-throughput, semi-quantitative immunohistochemistry with sectioned endometrial biopsy tissues from fertile (n = 89) and infertile (n = 89) couples. Image analysis measured stain intensity specifically within the luminal epithelium, glands and stroma during the early-, mid- and late- (cycle days 25-28) secretory phases.MAIN RESULTS AND THE ROLE OF CHANCE: MSX1 transcript increased 5-fold (P < 0.05) between the late-proliferative and early secretory phase and was then down-regulated (P < 0.05) prior to receptivity for implantation. In fertile patients, MSX1 protein displayed strong nuclear localization in the luminal epithelium and glands, while it was weakly expressed in nuclei of the stroma. MSX1 protein levels accumulated throughout the secretory phase in all endometrial cellular compartments. MSX1 protein decreased (P < 0.05) in the glands between mid- and late-secretory phases. However, infertile patients demonstrated a broad reduction (P < 0.001) of MSX1 accumulation in all cell types throughout the secretory phase that was most pronounced (∼3-fold) in stroma and glands. Infertility was associated with persistent co-localization of E-cadherin and β-catenin in epithelial cell junctions in the mid- and late-secretory phases.LIMITATIONS, REASONS FOR CAUTION: Details of the infertility diagnoses and other patient demographic data were not available. Therefore, patients with uterine abnormalities (Mullerian) could not be distinguished from other sources of infertility. Antibody against human MSX2 is not available, limiting the study to MSX1. However, both RNAs in the human endometrium are similarly regulated. In mice, Msx1 and Msx2 are imperative for murine embryo implantation, with Msx2 compensating for genetic ablation of Msx1 through its up-regulation in a knockout model.WIDER IMPLICATIONS OF THE FINDINGS: This investigation establishes that the MSX1 homeobox protein accumulation is associated with the secretory phase in endometrium of fertile couples, and is widely disrupted in infertile patients. It is the first study to examine MSX1 protein localization in the human endometrium, and supported by genetic findings in mice, suggests that genes regulated by MSX1 are linked to the loss of epithelial cell polarity required for uterine receptivity during implantation.STUDY FUNDING/COMPETING INTERESTS: This research was supported by the NICHD National Cooperative Reproductive Medicine Network grant HD039005 (M.P.D.), NIH grants HD068524 (S.K.D.), HD071408 (D.R.A., M.P.D.), and HL128628 (S.D.), the Intramural Research Program of the NICHD, March of Dimes (S.K.D., S.D.) and JSPS KAKENHI grant 26112506 (Y.H.). There were no conflicts or competing interests.

KW - E-cadherin

KW - Endometrium

KW - Female infertility

KW - Immunohistochemistry

KW - Implantation

KW - Menstrual cycle

KW - Messenger RNA

KW - Muscle segment homeobox 1

KW - Secretory phase

KW - Transcription factor

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