How Secreted-embryo-derived Trypsin Initiates, Maintains and Terminates Ca2+ Signals in Uterine Epithelial Cells (Ca2+)

To Investigate How Secreted-embryo-derived Trypsin Initiates, Maintains and Terminates Ca2+ (Intracellular Calcium) Signals in Uterine Epithelial Cells

To develop a deeper understanding of endometrial-embryo crosstalk through basic research, uncover therapeutic targets and to improve reproductive outcome.

Study Overview

• Status: Active, not recruiting
• Conditions: Infertility; Aneuploidy; Infertility, Female
• Intervention / Treatment: Other: Exposure to culture media

Detailed Description

Pregnancy is a complex and highly coordinated physiological process that involves implantation of a hatched blastocyst into a decidualizing endometrium. The main purpose of implantation is to ensure that the blastocyst firmly anchors into the decidual stroma, which allows further development by enabling placentation. Although a multitude of cellular events and molecular pathways involved in embryo-uterine crosstalk have been identified in mouse models, a comprehensive understanding of human embryo-uterine interaction is still missing. Our work indicates that endometrial epithelial Ca2+ signalling in response to serine proteases released by human embryos plays an important role in maternal recognition and selection of the conceptus at implantation. Previous studies have demonstrated that trophoblast spheroids can elevate [Ca2+]i in human uterine epithelial cell line (Ishikawa) by activating Ca2+ entry via mechano-sensitive Ca2+ permeable channels leading to the induction of epithelial adhesiveness. However, the mechanism(s) mediating the protease-induced [Ca2+]i transients in human uterine epithelium have not been studied to date. Investigators hypothesise that Na+ entry into the intravillous space via trypsin-activated ENaC will depolarise the cellular membrane and increase [Na+]v sufficiently high to reverse the sodium/calcium exchanger providing means for Ca2+ entry into the intravillous space. Ca2+ diffusion from the microvilli into the bulk cytoplasm will increase [Ca2+]i and, in parallel with SOCE, act as a source for re-filling of the ER. Increased [Ca2+]i will also activate the BK channels leading to repolarisation and termination of Ca2+ entry via the NCX.

By using spent medium from embryos, which will undergo pre-implantation genetic testing, it will become possible to determine, whether the above mentioned mechanisms are influenced by the ploidy status of the embryo.

• Study Type: Observational
• Enrollment (Actual): 81

Contacts and Locations

Study Locations

• United Arab Emirates
  • Abu Dhabi, United Arab Emirates, 60202
    • ART Fertility Clinics LLC

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years to 36 years (Adult)
• Accepts Healthy Volunteers: Yes

• Sampling Method: Non-Probability Sample

Study Population

Arab ethnicity, with primary / secondary infertility, who undergo an ICSI treatment with preimplantation genetic testing for aneuploidies.

Description

Inclusion Criteria:

• Couples with primary / secondary infertility who are planned to undergo ICSI treatment with PGT-A
• Age of each partner above 18 years

Exclusion Criteria:

• Couples with consanguinity (couple who is 1st or 2nd degree cousins)
• Couples in whom the female partner has a history of:
• Chemotherapy or radiation which impacts the ovarian reserve
• Surgery at the ovaries / adnex region
• Endometriosis
• Couples in whom the male partner has a history of:
• Chemotherapy / Radiation which impacts the semen result
• Surgery at the testicles
• Vasectomy
• Surgery for reversal of vasectomy
• Semen obtained by fine needle aspiration (FNA) or Testicular sperm extraction (TESE)

Study Plan

How is the study designed?

Number of groups / cohorts

4

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
DEG n1: euploid medium; culture media derived from euploid embryos	Other: Exposure to culture media; Exposure to culture media
DEG n2: aneuploid medium; culture media derived from aneuploid embryos	Other: Exposure to culture media; Exposure to culture media
DEG n3: medium arrested embryos; culture media derived from arrested embryos	Other: Exposure to culture media; Exposure to culture media
DEG n4: control culture medium; pure culture media without contact to embryos	Other: Exposure to culture media; Exposure to culture media

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Change in markers of protein	Change in markers of protein (PAR2, (p) and SGK1, NFkB, ORAI1-3 and STIM1-2 and COX2) using Western blotting	1 day
Change in peak and slope levels of intracellular calcium	Change in peak and slope levels of intracellular calcium	1 day
Change in morphohology of cells after incubation with embryo media	Change in morphohology of cells after incubation with embryo media	1 day

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Performance of ICSI	Defined on day 0 as the number of injected oocytes/number of COCs assigned to the ICSI group	1 day
Embryo quality on day 3	Defined by the number of blastomeres and their division pattern, fragmentation, presence of compaction, vacuoles, granulation and nuclei	1 day
Embryo quality on day 5 (Gardner and Schoolcraft,1999)	Gardner and Schoolcraft,1999) defined by: The expansion stage of the blastocyst; Quality of the ICM and TE; Day on which the biopsy is performed (day 5,6 or 7); Pregnancy outcomes (miscarriages/ectopic pregnancy/neonatal outcome)	1 day

Collaborators and Investigators

• Sponsor: ART Fertility Clinics LLC
• Collaborators: University Women's Hospital Tübingen
• Investigators: Principal Investigator: Barbara Lawrenz, PhD, ART Fertility Clinics LLC

Publications and helpful links

General Publications

• Heijnen EM, Eijkemans MJ, De Klerk C, Polinder S, Beckers NG, Klinkert ER, Broekmans FJ, Passchier J, Te Velde ER, Macklon NS, Fauser BC. A mild treatment strategy for in-vitro fertilisation: a randomised non-inferiority trial. Lancet. 2007 Mar 3;369(9563):743-749. doi: 10.1016/S0140-6736(07)60360-2.
• Wang H, Dey SK. Roadmap to embryo implantation: clues from mouse models. Nat Rev Genet. 2006 Mar;7(3):185-99. doi: 10.1038/nrg1808.
• Kaneko Y, Day ML, Murphy CR. Integrin beta3 in rat blastocysts and epithelial cells is essential for implantation in vitro: studies with Ishikawa cells and small interfering RNA transfection. Hum Reprod. 2011 Jul;26(7):1665-74. doi: 10.1093/humrep/der128. Epub 2011 Apr 30.
• Mertzanidou A, Wilton L, Cheng J, Spits C, Vanneste E, Moreau Y, Vermeesch JR, Sermon K. Microarray analysis reveals abnormal chromosomal complements in over 70% of 14 normally developing human embryos. Hum Reprod. 2013 Jan;28(1):256-64. doi: 10.1093/humrep/des362. Epub 2012 Oct 9.
• Teklenburg G, Salker M, Heijnen C, Macklon NS, Brosens JJ. The molecular basis of recurrent pregnancy loss: impaired natural embryo selection. Mol Hum Reprod. 2010 Dec;16(12):886-95. doi: 10.1093/molehr/gaq079. Epub 2010 Sep 16.
• Quenby S, Vince G, Farquharson R, Aplin J. Recurrent miscarriage: a defect in nature's quality control? Hum Reprod. 2002 Aug;17(8):1959-63. doi: 10.1093/humrep/17.8.1959.
• Aplin JD. Embryo implantation: the molecular mechanism remains elusive. Reprod Biomed Online. 2006 Dec;13(6):833-9. doi: 10.1016/s1472-6483(10)61032-2.
• Zhang S, Lin H, Kong S, Wang S, Wang H, Wang H, Armant DR. Physiological and molecular determinants of embryo implantation. Mol Aspects Med. 2013 Oct;34(5):939-80. doi: 10.1016/j.mam.2012.12.011. Epub 2013 Jan 2.
• Ruan YC, Guo JH, Liu X, Zhang R, Tsang LL, Dong JD, Chen H, Yu MK, Jiang X, Zhang XH, Fok KL, Chung YW, Huang H, Zhou WL, Chan HC. Activation of the epithelial Na+ channel triggers prostaglandin E(2) release and production required for embryo implantation. Nat Med. 2012 Jul;18(7):1112-7. doi: 10.1038/nm.2771.
• Rossier BC, Stutts MJ. Activation of the epithelial sodium channel (ENaC) by serine proteases. Annu Rev Physiol. 2009;71:361-79. doi: 10.1146/annurev.physiol.010908.163108.
• Marunaka Y, Niisato N. Effects of Ca(2+) channel blockers on amiloride-sensitive Na(+) permeable channels and Na(+) transport in fetal rat alveolar type II epithelium. Biochem Pharmacol. 2002 Apr 15;63(8):1547-52. doi: 10.1016/s0006-2952(02)00880-8.
• Salker MS, Hosseinzadeh Z, Alowayed N, Zeng N, Umbach AT, Webster Z, Singh Y, Brosens JJ, Lang F. LEFTYA Activates the Epithelial Na+ Channel (ENaC) in Endometrial Cells via Serum and Glucocorticoid Inducible Kinase SGK1. Cell Physiol Biochem. 2016;39(4):1295-306. doi: 10.1159/000447834. Epub 2016 Sep 8.
• Singh Y, Shi X, Zhang S, Umbach AT, Chen H, Salker MS, Lang F. Prolyl hydroxylase 3 (PHD3) expression augments the development of regulatory T cells. Mol Immunol. 2016 Aug;76:7-12. doi: 10.1016/j.molimm.2016.06.003. Epub 2016 Jun 19.
• Brosens JJ, Salker MS, Teklenburg G, Nautiyal J, Salter S, Lucas ES, Steel JH, Christian M, Chan YW, Boomsma CM, Moore JD, Hartshorne GM, Sucurovic S, Mulac-Jericevic B, Heijnen CJ, Quenby S, Koerkamp MJ, Holstege FC, Shmygol A, Macklon NS. Uterine selection of human embryos at implantation. Sci Rep. 2014 Feb 6;4:3894. doi: 10.1038/srep03894.
• Turco MY, Gardner L, Hughes J, Cindrova-Davies T, Gomez MJ, Farrell L, Hollinshead M, Marsh SGE, Brosens JJ, Critchley HO, Simons BD, Hemberger M, Koo BK, Moffett A, Burton GJ. Long-term, hormone-responsive organoid cultures of human endometrium in a chemically defined medium. Nat Cell Biol. 2017 May;19(5):568-577. doi: 10.1038/ncb3516. Epub 2017 Apr 10.

Study record dates

Study Major Dates

• Study Start (Actual): November 4, 2021
• Primary Completion (Actual): November 16, 2022
• Study Completion (Estimated): January 15, 2024

Study Registration Dates

• First Submitted: April 26, 2021
• First Submitted That Met QC Criteria: April 26, 2021
• First Posted (Actual): April 29, 2021

Study Record Updates

• Last Update Posted (Actual): July 25, 2023
• Last Update Submitted That Met QC Criteria: July 24, 2023
• Last Verified: July 1, 2023

More Information

Terms related to this study

• Keywords: IVF; media; RNA; embryo; Ca2+
• Additional Relevant MeSH Terms: Pathologic Processes; Chromosome Aberrations; Female Urogenital Diseases; Female Urogenital Diseases and Pregnancy Complications; Urogenital Diseases; Genital Diseases; Genital Diseases, Female; Infertility; Aneuploidy; Infertility, Female
• Other Study ID Numbers: 2012-ABU-014-BL

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No