SNPMiner Trials by Shray Alag


SNPMiner Trials: Clinical Trial Report


Report for Clinical Trial NCT03581617

Developed by Shray Alag, 2019.
SNP Clinical Trial Gene

Prognostic Markers in Women With Primary Unexplained Infertility

1. Background: In women, unexplained infertility has been associated with a range of cellular and molecular defects in the endometrium, adverse immune responses and immunological factors. Natural killer (NK) cells are included as they constitute the most abundant leukocyte population in the decidua. While decidua NK cells were extensively investigated, the study of endometrial eNK cells still lacks comprehensive researches. The reduction in eNK frequency has been associated with infertility status, in particular in the presence of a concomitant herpesvirus viremia. Since herpesviruses use as immune-escape HLA-G and HLA-E molecules, that are immune-inhibitory and important for a correct placentation, they could represent infertility co-factors. 2. Aims: Since lack of an accurate diagnosis in reproductive medicine leads to treatment failure, this proposal focuses on eNK cell characterization as a diagnostic factor for unexplained women infertility. We will evaluate also co-factors, taking into consideration herpesvirus infection and HLA-G and HLA-E expression. 3. Methods: Peripheral blood and endometrial NK cells will be immune-phenotyped and cell count and activation status (CD107a, IL-6, IL-10, IL-17) will be correlated with infertility condition. The implication of herpesvirus will be evaluated by DNA from peripheral blood and endometrial flushing samples analysis by HSV-1, HSV-2, EBV, CMV, HHV-6, HHV-7, VZV and HHV-8 specific primers an PCR technique. HLA-G and HLA-E expression will be analyzed in peripheral blood and endometrial environment by flow cytometry and ELISA tests and correlated by NK cell expression of their receptors (KIRs, LILRB1/2, NKG2A). 4. Expected results: On the basis of our preliminary results, we expect to identify NK cells as prognostic marker for primary unexplained infertility, with herpesvirus infection and HLA-G and HLA-E expression as co-factors. These data will be of importance in the management of infertile women.

NCT03581617 Infertility, Female
MeSH: Infertility Infertility, Female
HPO: Female infertility Infertility

5 Interventions

Name: NK cell analysis

Description: We selected to perform the study during the proliferative phase (day 9-11), where only resident eNK cells are present in the endometrium. The samples will be analyzed with the following methods, that are routinely used in the OUs of the proposal: - pNK cell analysis: PBMCs will be purified with Ficoll solution and NK cells will be analyzed by flow cytometry (FacsVantage, BD) with anti-CD56, CD16, CD9, CD49a, KIRs, LILRB1, LILRB2, CD94, CD107a eNK cell analysis: eNK cells will be obtained from endometrial biopsies during proliferative phase, determined by ultrasound scan and analyzed for NK cell subtypes and KIRs, LILRB1, LILRB2, CD94, CD107a expression by flow cytometry

Type: Diagnostic Test

Infertile women Fertile women

Name: Herpesvirus detection

Description: Herpesvirus detection: DNA will be analyzed by specific primers for HSV-1, HSV-2, EBV, CMV, HHV-6, HHV-7, VZV, and HHV-8, with PCR and nested PCR

Type: Diagnostic Test

Infertile women Fertile women

Name: sHLA-G analysis

Description: sHLA-G analysis: sHLA-G quantification in plasma and endometrial flushing will be performed by ELISA using anti-HLA-G (G233) and anti-beta2-microglobulin HRP-conjugated moAbs (Exbio)

Type: Diagnostic Test

Infertile women Fertile women

Name: HLA-G 14bp INS/DEL typing

Description: Genomic DNA will be genotyped by RealTime PCR

Type: Diagnostic Test

Infertile women Fertile women

Name: sHLA-E analysis

Description: sHLA-E analysis: sHLA-E quantification in plasma and endometrial flushing will be performed by ELISA using anti-HLA-E (3D12, eBioscience) and anti-beta2-microglobulin HRP-conjugated moAbs (Exbio)

Type: Diagnostic Test

Infertile women Fertile women


Primary Outcomes

Description: Percentage of (e)NK CD56brightCD16-

Measure: Prognostic value of NK cells in female idiopathic infertility

Time: 20 months

Description: Presence of HHVs infection in endometrial cells

Measure: Herpesvirus detection

Time: 16 months

Secondary Outcomes

Description: Levels of sHLA-G and sHLA-E in uterine flushing samples

Measure: Levels of sHLA-G and sHLA-E cellule pNK e eNK

Time: 16 months

Description: Frequency of HLA-G and HLA-E genetic polymorphisms

Measure: HLA-G and HLA-E genetic polymorphisms

Time: 10 months

Description: Levels of Th1 and Th2 cytokines

Measure: Cytokines levels in endometrial flushing samples

Time: 10 months

Time Perspective: Prospective

Cohort


There is one SNP

SNPs


1 rs66554220

HLA-G expression is controlled by a polymorphism of insertion/deletion (ins/del) of 14 base pairs (rs66554220), where the deletion of 14bp stabilizes the mRNA producing higher levels of HLA-G (15).

We will genotype the women for rs66554220 polymorphism, to evaluate the implication in HLA-G levels of expression in infertile condition.



HPO Nodes


HPO:
Female infertility
Genes 6
CYP19A1 PGR TLE6 HUWE1 FOXL2 TUBB8
Infertility
Genes 133
HJV MKRN3 SOX3 SNORD115-1 SOX9 AMH PATL2 PLCZ1 AMHR2 KLHL10 WDR66 MAGEL2 CFAP300 SRA1 ZMPSTE24 WWOX LHX4 GCM2 STUB1 DNAAF4 TUBB8 AXL DAZ4 CYP19A1 RBMY1A1 CFTR DUSP6 TSGA10 GATA4 WT1 MEIOB CCNO IPW PWRN1 CDH23 POU1F1 SRY DAZ1 NDN LRRC6 CFAP43 TSPY1 H6PD HUWE1 TEX14 NR3C1 USP9Y TEX11 CCDC141 GBA2 ZP3 QRICH2 CFAP298 CDC14A AR CCDC39 AK7 SNORD116-1 HESX1 FGF17 MAP3K1 ZFPM2 PGR MKRN3-AS1 DNAH1 FANCA DNAJB13 LHCGR ZP1 FSIP2 DNAAF5 DNAAF3 DNAI1 AURKC USP8 SLC26A8 PTPN11 OTX2 POLR3B CATSPER1 FOXL2 DMRT3 DHH LMNA BRAF CATSPER2 BRDT ZMYND10 RSPH1 ADGRG2 WDR11 FEZF1 GLI2 SPATA16 FSHB SPRY4 VAMP7 NR0B1 SUN5 DNAI2 NPAP1 TLE6 SOHLH1 RNF216 DDX3Y PIH1D3 WEE2 NR5A1 FLRT3 CTNS PWAR1 STRC HERC2 HSD17B3 DAZ3 DAZ2 DNAAF1 PLIN1 CFAP44 MAP2K1 IL17RD DNAH9 NANOS1 GNRH1 GNRHR SNRPN PRLR SEPTIN12 PMFBP1 CEP19 PROP1 SEMA3E CCDC40