Infertility, Female Clinical Trial
Official title:
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.
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.
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