Infections and Unexplained Infertility

NCT03581617 | Completed

• 1 other identifier: PRUAlGR-2013-00000084
• Study Type: observational
• Target: 143
• Locations: 0 countries
• Sites: N/A

Brief Summary

1. 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. 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. 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. 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

Keywords

Infertility; NK cells; HLA-G

Outcome Measures

Primary Outcomes (2)

• Prognostic value of NK cells in female idiopathic infertility

  Percentage of (e)NK CD56brightCD16-

  20 months

• Herpesvirus detection

  Presence of HHVs infection in endometrial cells

  16 months

Secondary Outcomes (3)

• Levels of sHLA-G and sHLA-E cellule pNK e eNK

  16 months

• HLA-G and HLA-E genetic polymorphisms

  10 months

• Cytokines levels in endometrial flushing samples

  10 months

Study Arms (2)

Infertile women

Inclusion criteria for the study group will be as follows: 21-38 years old, primary infertility (no live birth), regular menstrual cycle (24-35 days), body mass index (BMI) ≤ 25, FSH \<10 mUI/mL, E2 \< 50 pg/ml on day 2-3 of the menstrual cycle. They will be recruited at admission for tubal patency assessment.

Diagnostic Test: NK cell analysis; Diagnostic Test: Herpesvirus detection; Diagnostic Test: sHLA-G analysis; Diagnostic Test: HLA-G 14bp INS/DEL typing; Diagnostic Test: sHLA-E analysis

Fertile women

Inclusion criteria for control group will be as follows: 21-35 years old, almost one live birth, regular menstrual cycle (24-35 days), BMI ≤ 25, FSH \<10 mUI/mL, E2 \< 50 pg/mL on day 2-3 of the menstrual cycle. Women with endometritis, endometriosis, tubal factor, ovulatory dysfunction, anatomical uterine pathologies will be excluded.

Diagnostic Test: NK cell analysis; Diagnostic Test: Herpesvirus detection; Diagnostic Test: sHLA-G analysis; Diagnostic Test: HLA-G 14bp INS/DEL typing; Diagnostic Test: sHLA-E analysis

Interventions

NK cell analysis DIAGNOSTIC_TEST

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

Fertile women; Infertile women

Herpesvirus detection DIAGNOSTIC_TEST

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

Fertile women; Infertile women

sHLA-G analysis DIAGNOSTIC_TEST

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)

Fertile women; Infertile women

HLA-G 14bp INS/DEL typing DIAGNOSTIC_TEST

Genomic DNA will be genotyped by RealTime PCR

Fertile women; Infertile women

sHLA-E analysis DIAGNOSTIC_TEST

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)

Fertile women; Infertile women

Eligibility Criteria

• Age: 21 Years - 38 Years
• Gender Eligibility Details: Infertile women
• Healthy Volunteers: Yes
• Age Groups: Adult (18-64)
• Sampling Method: Non-Probability Sample

Study Population

This will be a prospective non-interventional clinical study, based on biological specimens sampled during current standard clinical practice. Based on our preliminary results, we will enroll 100 infertile and 30 control women to achieve a 0.8 power with 5% alpha error in detecting differences between infertile and fertile women.

You may qualify if:

• years old,
• primary infertility (no live birth),
• regular menstrual cycle (24-35 days),
• body mass index (BMI) ≤ 25, FSH \<10 mUI/mL,
• E2 \< 50 pg/ml on day 2-3 of the menstrual cycle. Recruitment at admission for tubal patency assessment.
• years old,
• almost one live birth,
• regular menstrual cycle (24-35 days),
• BMI ≤ 25, FSH \<10 mUI/mL,
• E2 \< 50 pg/mL on day 2-3 of the menstrual cycle

You may not qualify if:

• endometritis,
• endometriosis,
• tubal factor,
• ovulatory dysfunction,
• anatomical uterine pathologies.

Sponsors & Collaborators

• University Hospital of Ferrara: lead

Related Publications (1)

• Rizzo R, Lo Monte G, Bortolotti D, Graziano A, Gentili V, Di Luca D, Marci R. Impact of soluble HLA-G levels and endometrial NK cells in uterine flushing samples from primary and secondary unexplained infertile women. Int J Mol Sci. 2015 Mar 10;16(3):5510-6. doi: 10.3390/ijms16035510.

  PMID: 25764161 BACKGROUND

MeSH Terms

Conditions

Infertility, Female; Infertility

Condition Hierarchy (Ancestors)

Genital Diseases, Female; Female Urogenital Diseases; Female Urogenital Diseases and Pregnancy Complications; Urogenital Diseases; Genital Diseases

Study Officials

• Giuseppe Lo Monte, M.D.

  Università di Ferarra

  PRINCIPAL INVESTIGATOR

Study Design

• Study Type: observational
• Observational Model: COHORT
• Time Perspective: PROSPECTIVE
• Sponsor Type: OTHER
• Responsible Party: PRINCIPAL INVESTIGATOR
• PI Title: Doctor

Study Record Dates

• First Submitted: June 4, 2018
• First Posted: July 10, 2018
• Study Start: November 1, 2014
• Primary Completion: November 1, 2015
• Study Completion: November 1, 2016
• Last Updated: July 10, 2018

Record last verified: 2018-06

Data Sharing

• IPD Sharing: Will not share