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

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

Detailed Description
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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.

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

Conditions
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Infertility, Infertility, Female, Aneuploidy

Intervention / Treatment
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* Other: Exposure to culture media


Participation Criteria
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Eligibility Criteria
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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)

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 36 Years

Sexes Eligible for Study
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Female

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| DEG n1: euploid medium<br>culture media derived from euploid embryos | Other: Exposure to culture media<br>* Exposure to culture media<br>|
| DEG n2: aneuploid medium<br>culture media derived from aneuploid embryos | Other: Exposure to culture media<br>* Exposure to culture media<br>|
| DEG n3: medium arrested embryos<br>culture media derived from arrested embryos | Other: Exposure to culture media<br>* Exposure to culture media<br>|
| DEG n4: control culture medium<br>pure culture media without contact to embryos | Other: Exposure to culture media<br>* Exposure to culture media<br>|

What is the study measuring?
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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 | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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RNA, Ca2+, embryo, media, IVF