Infections and Unexplained Infertility
Condition: Infertility, Female
MeSH terms: Infertility; Infertility, Female
Study type: Observational

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.
    

Detailed description: 
      Primary Objective The main challenge of this project is to define the potential role of NK
      cells as a prognostic marker for primary unexplained infertility. To achieve this objective
      we will perform a prospective study on a cohort of 100 unexplained infertile women.
      Peripheral blood and endometrial NK cells will be immune phenotyped and their activation
      status will be analyzed. Meanwhile, the presence of herpesvirus infection will be evaluated
      in peripheral blood and correlated with the results of NK analysis. These data will clarify
      the role of NK cells in infertile female conditions, evaluating the implication of
      herpesvirus infection as a cofactor for NK cell status. These data will provide a proof of
      principle of the use of NK cell analysis as a predictive marker for unexplained infertility.

      Secondary Objective The secondary objective is to evaluate the mechanisms at the basis of the
      NK cell status in infertility. Since HLA-G and HLA-E expression is modified by herpesvirus
      infection (9), as an immune escape mechanism, and these antigens are responsible of a correct
      embryo implantation (14), we will analyze the levels of these molecules in peripheral blood
      and endometrial environment. Meanwhile, HLA-G and HLA-E genetic polymorphisms will be
      analyzed. These results will be correlated with the presence of herpesvirus infection, KIR,
      LILRB and NKG2A receptor expression on pNK and eNK cells. These data will clarify the
      implication of HLA-G and HLA-E expression and genetic background in the control of NK cell
      activation and herpesvirus infection in infertile women.

      The achievement of these objectives will be obtained with 6 workpackages/aims (WP)

        1. Infertile women characterization (WP1) (1-20mth) We will recruit 100 unexplained
           infertile women and 30 control women. These women will be clinically evaluated,
           establishing a clinical database. From each woman, we will obtain peripheral blood
           samples, endometrium biopsies, and uterine flushing.

        2. NK cell immune-phenotype (WP2) (1-20mth) NK cells from peripheral blood and endometrium
           will be analyzed for subtype percentages (CD56, CD16, CD9, CD49a), and for the
           expression of KIR, LILRB-1 and -2 and CD94/NKG2A receptors, that are known to interact
           with HLA-G and HLA-E molecules producing inhibitory signals.

        3. Th1, Th2, Th17 and LIF (WP3). (1-20 mth) Th1, Th2, Th17 and LIF levels will be analyzed
           in plasma and uterine flushing samples, to evaluate activation status. The results will
           be correlated with NK cell count.

        4. Herpesvirus infection (WP4) (12-24mth) Herpesvirus DNA (HSV-1, HSV-2, EBV, CMV, HHV-6,
           HHV-7, VZV and HHV-8) presence will be analyzed in peripheral blood and uterine
           flushing.

        5. HLA-G (WP5) (12-24mth) HLA-G molecules are expressed as both membrane (HLA-G1) and
           soluble (HLA-G5, from mRNA alternative splicing; sHLA-G1, from membrane shedding)
           molecules. The expression of the membrane and soluble HLA-G will be evaluated in
           peripheral blood and endometrium. 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.

        6. HLA-E (WP6) (12-24mth) HLA-E molecules are expressed as both membrane and soluble
           molecules. The expression of the membrane and soluble HLA-E will be evaluated in
           peripheral blood and endometrium. Two non-synonymous alleles of HLA-E have been
           identified, HLA-ER (E*0101) and HLA-EG (E*0103) (16), where HLA-E expression of the EG
           protein was higher than ER. We will genotype the women for HLA-E polymorphisms, to
           evaluate the implication in infertility.
    

Intervention name(s): NK cell analysis; Herpesvirus detection; sHLA-G analysis; HLA-G 14bp INS/DEL typing; sHLA-E analysis
Intervention type(s): Diagnostic Test; Diagnostic Test; Diagnostic Test; Diagnostic Test; Diagnostic Test
Intervention description(s): 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; 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; 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); Genomic DNA will be genotyped by RealTime PCR; 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)
Observational model: Cohort
Time perspective: Prospective
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.
      
Keywords: Infertility; NK cells; HLA-G