Infections and Unexplained Infertility

Summary

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.

Conditions

• Infertility, Female

Interventions

DIAGNOSTIC_TEST

NK cell analysis

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

DIAGNOSTIC_TEST

Herpesvirus detection

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

DIAGNOSTIC_TEST

sHLA-G analysis

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)

DIAGNOSTIC_TEST

HLA-G 14bp INS/DEL typing

Genomic DNA will be genotyped by RealTime PCR

DIAGNOSTIC_TEST

sHLA-E analysis

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)

Sponsors & Collaborators

• University Hospital of Ferrara

  lead OTHER

Principal Investigators

• Giuseppe Lo Monte, M.D. · Università di Ferarra

Eligibility

Min Age

21 Years

Max Age

38 Years

Sex

FEMALE

Healthy Volunteers

Yes

Timeline & Regulatory

Start

2014-11-01

Primary Completion

2015-11-01

Completion

2016-11-01