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

Sponsor

ART Fertility Clinics LLC (Other)

Overall Status

Recruiting

CT.gov ID

NCT04865367

Collaborator

University Women's Hospital Tübingen (Other)

Enrollment

Location

13.8

Anticipated Duration (Months)

2.2

Patients Per Site Per Month

Study Details

Study Description

Brief Summary

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

Condition or Disease	Intervention/Treatment	Phase
Infertility Infertility, Female Aneuploidy	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 Design

Study Type:

Observational

Anticipated Enrollment :

30 participants

Observational Model:

Cohort

Time Perspective:

Prospective

Official Title:

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

Actual Study Start Date :

Nov 4, 2021

Anticipated Primary Completion Date :

Oct 30, 2022

Anticipated Study Completion Date :

Dec 30, 2022

Arms and Interventions

Arm	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

Outcome Measures

Primary Outcome Measures

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

Secondary Outcome Measures

Performance of ICSI [1 day]
Defined on day 0 as the number of injected oocytes/number of COCs assigned to the ICSI group
Embryo quality on day 3 [1 day]
Defined by the number of blastomeres and their division pattern, fragmentation, presence of compaction, vacuoles, granulation and nuclei
Embryo quality on day 5 (Gardner and Schoolcraft,1999) [1 day]
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)

Eligibility Criteria

Criteria

Ages Eligible for Study:

18 Years to 36 Years

Sexes Eligible for Study:

Female

Accepts Healthy Volunteers:

Yes

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)