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CompletedNCT04865367Ca2+Updated Jul 17, 2024

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

An observational study in Infertility, Infertility, Female and Aneuploidy, sponsored by ART Fertility Clinics LLC. Completed at 1 site in United Arab Emirates. Open to female participants aged 18 Years to 36 Years, including healthy volunteers. Per ClinicalTrials.gov, last updated 2024-07-17.

Sponsored by ART Fertility Clinics LLC · Observational

Study type
Observational
Model
Cohort
Time perspective
Prospective
Enrollment
81
Ages
18 Years to 36 Years
Sex
Female
01

Study summary

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

Read the 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.

02

Conditions studied

  • Infertility
  • Infertility, Female
  • Aneuploidy

Keywords

  • IVF
  • RNA
  • Ca2+
  • embryo
  • media
03

Who can participate

Ages eligible
18 Years to 36 Years
Sexes eligible
Female
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.

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

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)
04

Study design

Observational model
Cohort
Time perspective
Prospective
Enrollment
81 participants (actual)
Patient registry
No

Groups and cohorts

  • DEG n1: euploid medium

    culture media derived from euploid embryos

    Other: Exposure to culture media

  • DEG n2: aneuploid medium

    culture media derived from aneuploid embryos

    Other: Exposure to culture media

  • DEG n3: medium arrested embryos

    culture media derived from arrested embryos

    Other: Exposure to culture media

  • DEG n4: control culture medium

    pure culture media without contact to embryos

    Other: Exposure to culture media

Interventions

  • OtherExposure to culture media

    Exposure to culture media

05

What researchers measure

Primary outcomes

  1. 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

    Time frame: 1 day

  2. Change in peak and slope levels of intracellular calcium

    Change in peak and slope levels of intracellular calcium

    Time frame: 1 day

  3. Change in morphohology of cells after incubation with embryo media

    Change in morphohology of cells after incubation with embryo media

    Time frame: 1 day

Secondary outcomes

  1. Performance of ICSI

    Defined on day 0 as the number of injected oocytes/number of COCs assigned to the ICSI group

    Time frame: 1 day

  2. Embryo quality on day 3

    Defined by the number of blastomeres and their division pattern, fragmentation, presence of compaction, vacuoles, granulation and nuclei

    Time frame: 1 day

  3. 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)

    Time frame: 1 day

06

Study locations

1 site
  • ART Fertility Clinics LLC
    Abu Dhabi, 60202, United Arab Emirates
07

References and documents

Publications

  • 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. PubMed 16485018 ↗
  • 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. PubMed 21531996 ↗
  • 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. PubMed 23054067 ↗
  • 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. PubMed 20847090 ↗
  • 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. PubMed 12151421 ↗
  • 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. PubMed 17169205 ↗
  • 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. PubMed 23290997 ↗
  • 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. PubMed 22729284 ↗
  • 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. PubMed 18928407 ↗
  • 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. PubMed 11996897 ↗
  • 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. PubMed 27606670 ↗
  • 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. PubMed 27331863 ↗
  • 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. PubMed 17336650 ↗
  • 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. PubMed 24503642 ↗
  • 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. PubMed 28394884 ↗

Individual participant data

Plan to share: No

08

Registry details

Key details

Study ID
NCT04865367
Lead sponsor
ART Fertility Clinics LLC
Collaborators
University Women's Hospital Tübingen
Responsible party
Barbara Lawrenz (Scientific Director, ART Fertility Clinics LLC) — Principal investigator
First posted
Apr 29, 2021
Start date
Nov 4, 2021
Primary completion
Apr 16, 2023
Completion
May 15, 2023
Last update
Jul 17, 2024

Study contacts

BARBARA LAWRENZ, PhD
principal investigator · ART Fertility Clinics LLC

Oversight

Data monitoring committee
Yes
FDA-regulated drug
No
FDA-regulated device
No
View the source record on ClinicalTrials.gov ↗

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