is there really an association between cytotoxic natural killer cells and implantation failure in...

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Symposium Abstracts / Journal of Reproductive Immunology 90 (2011) 131–163 145 to investigate the level of colocalisation between HLA-G and HLA-E. Results: Surface expression of HLA-E, HLA-C and HLA- G has been determined and quantified on trophoblast cell lines JEG-3 and ACH-3P by flow cytometry which revealed that the numbers of HLA-C and HLA-G molecules are higher than HLA-E molecules. Total protein contents of HLA-E, HLA-C and HLA-G have been determined by immuno-blotting. The object based colocalisation analy- sis revealed that out of total HLA-E molecules expressed on trophoblast cell surface 18% of HLA-E molecules are colocalised with HLA-C. In the same study we found that 72% of HLA-E is colocalised with HLA-G. These results strongly support the evidence that HLA-E is dependent on its MHC class I counterparts for its surface expression. Furthermore, HLA-E complexes formed by class Ia signal sequences derived peptides have been shown to interact with CD94/NKG2A heterodimers on NK cells and inhibiting their killing activities. HLA-E complex with HLA-G signal peptide has been found to interact with CD94/NKG2C and activate NK cells. Hence, HLA-E works together with HLA-C and HLA-G to inhibit NK cytotoxicity and also activate NK cells to secrete cytokines thus regulating placentation and facilitating pregnancy progress. Conclusions: Trophoblast cell lines JEG-3 and ACH-3P show similar pattern of MHC class I expression. HLA-E molecules display physical interactions with HLA-C and HLA-G molecules on the cell-surface of both trophoblast cell lines. The interactions of HLA-E with other class members might be functionally important for example in signalling or the modulation of uterine NK cell lysis and NK-like LGL-mediated attack. doi:10.1016/j.jri.2011.06.027 S26 Role of DNA copy number variations in genetic predis- position to recurrent pregnancy loss L. Nagirnaja a,, L. Kasak a , P. Palta b,f , K. Rull a,c , O.B. Christiansen d , T. Esko e,f , M. Remm b,f , M. Metspalu e,f , M. Laan a a Human Molecular Genetics Research Group, Estonia b Department of Bioinformatics, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia c Department of Obstetrics and Gynaecology, Tartu University Clinics, Tartu, Estonia d Rigshospitalet, Copenhagen, Denmark e Estonian Genome Centre, University of Tartu, Tartu, Estonia f Estonian Biocentre, Tartu, Estonia Introduction: Recurrent miscarriage (RM) (3 mis- carriages before 22 gestational weeks or spontaneous abortion of fetus <500 g) is a heterogeneous condi- tion occurring in 1–2% of fertile couples. Despite a wide spectrum of known causes, approximately half of the cases are defined as idiopathic. The aims of the current study were to explore whether gene copy number variations (CNV) contribute to inheritable pre- disposition to RM, to identify novel loci involved in establishment of a successful pregnancy and conse- quently provide new insights into the mechanism of RM. Material and methods: Altogether 73 Estonian sam- ples (43 RM patients, 30 fertile controls, recruited at Tartu University Hospital, Estonia) were genotyped on Illumina Infinum platform. Twelve genomic loci with altered CNV frequencies in RM cases compared to con- trols were validated by TaqMan quantitative PCR. Two CNV regions for which the results supported Illumina array data were selected for replication in additional Estonian (n = 119 RM patients, n = 128 fertile controls) and Danish (n = 438 RM patients, n = 228 fertile con- trols) cohorts. Comparative control frequency data was obtained on 2500 genotyped individuals from Estonian Genome Project (EGP) representing average Estonian pop- ulation. Results: A 25.9 kb deletion on Chr 2 that has been implicated in RM previously shows no frequency differ- ences among Danish (3.1% controls, 2.7% RM patients) but is more prevalent in Estonian primary RM cases than in controls (8.7% and 4.8% respectively, p = 0.272 due to small sample size). Additionally, a 52.4 kb dupli- cation (up to 5 copies) on Chr 5 is four times more frequent among women with primary RM than controls and EGP samples (combined frequency 8.4% vs 2.0% and 1.5% respectively, p = 0.006). The duplicated region involves two genes associated with reproductive function for the first time. Conclusions: Two dosage sensitive genomic regions are found to possibly affect reproductive success: (i) deletion on Chr 2 indicates association with RM in a population- specific manner; (ii) duplication of novel fertility-related genes on Chr 5 increases the risk of primary RM in females in both, Estonian and Danish population. One of the genes duplicated on Chr 5 is linked to a pathway shown to be critical for placental growth, invasion and metabolism. Keywords: Recurrent miscarriage; Copy number varia- tion; Placental function doi:10.1016/j.jri.2011.06.028 S27 Is there really an association between cytotoxic natural killer cells and implantation failure in both blood and endometrium? I. Santillan , V. Verdu, J.M. Bajo GINEFIV, Madrid, Spain Introduction: Natural killer (NK) cells have been shown to be involved in embryo implantation. In humans but not mice or other species with post implantation decidualiza- tion, uterine natural killer (uNK) cells may contribute to blastocyst implantation and are of interest as therapeu- tic targets in female infertility. Uterine natural killer cells seem to have a special phenotype: CD56 bright and CD16 dim. In contrast, natural killer T (NKT) cells (CD56+CD3+) are known to be especially cytotoxic. However, the possi- ble association between NK cells and implantation failure

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Page 1: Is there really an association between cytotoxic natural killer cells and implantation failure in both blood and endometrium?

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Symposium Abstracts / Journal of Re

o investigate the level of colocalisation between HLA-Gnd HLA-E.

Results: Surface expression of HLA-E, HLA-C and HLA-has been determined and quantified on trophoblast

ell lines JEG-3 and ACH-3P by flow cytometry whichevealed that the numbers of HLA-C and HLA-G moleculesre higher than HLA-E molecules. Total protein contentsf HLA-E, HLA-C and HLA-G have been determined bymmuno-blotting. The object based colocalisation analy-is revealed that out of total HLA-E molecules expressedn trophoblast cell surface ∼18% of HLA-E molecules areolocalised with HLA-C. In the same study we found that72% of HLA-E is colocalised with HLA-G. These results

trongly support the evidence that HLA-E is dependentn its MHC class I counterparts for its surface expression.urthermore, HLA-E complexes formed by class Ia signalequences derived peptides have been shown to interactith CD94/NKG2A heterodimers on NK cells and inhibiting

heir killing activities. HLA-E complex with HLA-G signaleptide has been found to interact with CD94/NKG2C andctivate NK cells. Hence, HLA-E works together with HLA-Cnd HLA-G to inhibit NK cytotoxicity and also activate NKells to secrete cytokines thus regulating placentation andacilitating pregnancy progress.

Conclusions: Trophoblast cell lines JEG-3 and ACH-3Phow similar pattern of MHC class I expression. HLA-Eolecules display physical interactions with HLA-C andLA-G molecules on the cell-surface of both trophoblastell lines. The interactions of HLA-E with other classembers might be functionally important for example in

ignalling or the modulation of uterine NK cell lysis andK-like LGL-mediated attack.

oi:10.1016/j.jri.2011.06.027

26

ole of DNA copy number variations in genetic predis-osition to recurrent pregnancy loss

. Nagirnaja a,∗, L. Kasak a, P. Palta b,f, K. Rull a,c, O.B.hristiansen d, T. Esko e,f, M. Remm b,f, M. Metspalu e,f, M.aan a

Human Molecular Genetics Research Group, EstoniaDepartment of Bioinformatics, Institute of Molecular and Celliology, University of Tartu, Tartu, EstoniaDepartment of Obstetrics and Gynaecology, Tartu Universitylinics, Tartu, EstoniaRigshospitalet, Copenhagen, DenmarkEstonian Genome Centre, University of Tartu, Tartu, EstoniaEstonian Biocentre, Tartu, Estonia

Introduction: Recurrent miscarriage (RM) (≥3 mis-arriages before 22 gestational weeks or spontaneousbortion of fetus <500 g) is a heterogeneous condi-ion occurring in 1–2% of fertile couples. Despite aide spectrum of known causes, approximately half

f the cases are defined as idiopathic. The aims of

he current study were to explore whether gene copyumber variations (CNV) contribute to inheritable pre-isposition to RM, to identify novel loci involved in

ve Immunology 90 (2011) 131–163 145

establishment of a successful pregnancy and conse-quently provide new insights into the mechanism ofRM.

Material and methods: Altogether 73 Estonian sam-ples (43 RM patients, 30 fertile controls, recruited atTartu University Hospital, Estonia) were genotyped onIllumina Infinum platform. Twelve genomic loci withaltered CNV frequencies in RM cases compared to con-trols were validated by TaqMan quantitative PCR. TwoCNV regions for which the results supported Illuminaarray data were selected for replication in additionalEstonian (n = 119 RM patients, n = 128 fertile controls)and Danish (n = 438 RM patients, n = 228 fertile con-trols) cohorts. Comparative control frequency data wasobtained on 2500 genotyped individuals from EstonianGenome Project (EGP) representing average Estonian pop-ulation.

Results: A 25.9 kb deletion on Chr 2 that has beenimplicated in RM previously shows no frequency differ-ences among Danish (3.1% controls, 2.7% RM patients)but is more prevalent in Estonian primary RM casesthan in controls (8.7% and 4.8% respectively, p = 0.272due to small sample size). Additionally, a 52.4 kb dupli-cation (up to 5 copies) on Chr 5 is four times morefrequent among women with primary RM than controlsand EGP samples (combined frequency 8.4% vs 2.0% and1.5% respectively, p = 0.006). The duplicated region involvestwo genes associated with reproductive function for thefirst time.

Conclusions: Two dosage sensitive genomic regions arefound to possibly affect reproductive success: (i) deletionon Chr 2 indicates association with RM in a population-specific manner; (ii) duplication of novel fertility-relatedgenes on Chr 5 increases the risk of primary RM in femalesin both, Estonian and Danish population. One of the genesduplicated on Chr 5 is linked to a pathway shown to becritical for placental growth, invasion and metabolism.

Keywords: Recurrent miscarriage; Copy number varia-tion; Placental function

doi:10.1016/j.jri.2011.06.028

S27

Is there really an association between cytotoxic naturalkiller cells and implantation failure in both blood andendometrium?

I. Santillan ∗, V. Verdu, J.M. Bajo

GINEFIV, Madrid, Spain

Introduction: Natural killer (NK) cells have been shownto be involved in embryo implantation. In humans but notmice or other species with post implantation decidualiza-tion, uterine natural killer (uNK) cells may contribute toblastocyst implantation and are of interest as therapeu-tic targets in female infertility. Uterine natural killer cellsseem to have a special phenotype: CD56 bright and CD16

dim. In contrast, natural killer T (NKT) cells (CD56+CD3+)are known to be especially cytotoxic. However, the possi-ble association between NK cells and implantation failure
Page 2: Is there really an association between cytotoxic natural killer cells and implantation failure in both blood and endometrium?

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146 Symposium Abstracts / Journal of Re

is not yet clear. The relationship between blood and uter-ine NK cells is not completely understood. The object of thisstudy is to investigate the correlation between implanta-tion failure and the number and type of NK cells in bloodand in the endometrium.

Materials and methods: This is case-control prospec-tive study. So far, 17 women have been included. Caseswith implantation failure were defined as patients whodid not achieve pregnancy after at least three transfersof 6 or more good quality embryos. Embryo quality wasdefined according ASEBIR classification. Controls were fer-tile women under 30 years old with at least one vaginaldelivery. NK cell measurement was performed between the18th and the 22th day of a natural cycle. For this purpose, anendometrial biopsy was taken with a Cornier cannule andkept in formaldehyde 10%. Immunohistochemical stainingwas done for CD56, CD16, CD3, CD4, CD8 and CD25. Thesame day a blood test was performed to quantify NK cellsand NKT cells. All women gave written consent for the pro-cedure.

Results: The mean percentage of NK cells in blood inwomen with implantation failure was 12.5%. 36% of theseNK cells were CD3+CD56+ (NKT cells). In the endometrium,NK cells were on average 12% of the total number of cellsin the tissue. They were most commonly found aroundepithelial glands. 50% of the cases had more than 10%NK cells in the endometrium. A clear association betweenblood and endometrium NK cell levels was found in 80% ofwomen.

Conclusions: Although these are only preliminaryresults, there seems to be an association between a highnumber of NK cells in the blood and in the endometriumin women with implantation failure. Uterine NK cells aremainly found around epithelial glands. The phenotype ofthese cells could be especially cytotoxic. We conclude thatblood NK cell parameters may be predictive of endometrialNK cell parameters.

doi:10.1016/j.jri.2011.06.029

S28

Systemic inflammation perpetuates cellular influx viaup-regulation of ovarian VCAM-1 expression in a mousemodel of polycystic ovary syndrome (PCOS)

M.E. Solano a,∗, V.A. Sander b, H. Ho c, A.B. Motta d, P.C.Arck a

a Laboratory for Experimental Feto-Maternal Medicine,Department of Obstetrics and Fetal Medicine, University Med-ical Center Hamburg-Eppendorf, Hamburg, Germanyb IIB-INTECH, Buenos Aires, Argentinac Brain-Body-Institute, Department of Medicine, McMasterUniversity, Hamilton, Ontario, Canadad Laboratory of Ovarian Physiopathology. CEFYBO-UBA-CONICET, Buenos Aires, Argentina

Introduction: PCOS is an endocrine disease affecting a

wealth of women in their reproductive years. It is one ofthe main causes for anovulatory sterility and is associatedwith obesity, insulin resistance and chronic inflammation.

ve Immunology 90 (2011) 131–163

Rapidly emerging evidence suggests that the immune sys-tem aggravates the onset and/or clinical features of PCOSsymptoms at systemic and ovarian levels. Therefore, theaim of this study was to unveil alterations on immune,metabolic and endocrine PCOS-like symptoms in a mousemodel of dehydroepiandrosterone (DHEA)-induced PCOS.

Materials and methods: Twenty-three days old BALB/cfemale mice were injected daily with DHEA (60 mg/kg ofbody weight (BW) in neutral oil as vehicle) over a periodof 20 days. Control mice received vehicle alone. The stageof the estrous cycle, determined by observing the cell com-position of the vaginal smears, and the BW were assessedover the course of the injections. Then, mice were sacri-ficed and ovarian tissue and blood was harvested. Serumestradiol (E) levels were determined by ELISA and thefrequency and phenotype of peripheral blood cells wasevaluated by flow cytometry. Serial histological sectionsfrom one entire ovarian tissue specimen were stained byhematoxylin and eosin (H&E) for morphological analysis.Further, tissue sections from the second ovary were usedfor the immunohistochemical (IHC) detection of vascularcell adhesion molecule (VCAM)-1, intercellular adhesionmolecule (ICAM)-1, MHCII and CD4 positivity. Peritonealcells were stimulated for 24 h with PMA/ionomycin andcytokines determined in the supernatant by Cytomet-ric Bead Arrays. In subsequent experiments, naïve lymphnode-derived cells were incubated in vitro with and with-out DHEA (0.1, 1 and 10 �M) for 21 and 48 h, followed byflow cytometry analysis of cell frequency. Statistical anal-ysis of the data were performed using Mann–Whitney UTest, significance was set at p < 0.05.

Results: We observed a significant weight gain andinsulin resistance in DHEA-androgenized mice, whichstrengthens the validity of the PCOS mouse model. Thiswas further supported by the formation of follicular cystsin ovaries of DHEA-androgenized mice. As shown by IHC,the majority of ovarian cysts expressed VCAM-1 and ICAM-1 in the Granulosa Cell Layer (GCL), whereas VCAM-1 wasinduced also in the Theca Cell Layer (TCL) of all the folli-cles and cysts. These markers were rarely observed in theGCL of follicles from control ovaries. Further, an enhancedproduction of inflammatory cytokines as well as IL-10 byperitoneal cells could be observed in PCOS-mice, stronglysuggesting the association of chronic inflammation andPCOS. Also, the frequency and activation of CD4+ cells inblood was increased upon DHEA androgenization. The sub-sequent in vitro experiments were designed based on theseex vivo findings and confirmed that DHEA indeed not onlyinduced the activation of CD4+ cells, but also up-regulatedthe expression ligand for VCAM-1 and ICAM-1, VLA-4 andLFA-1. We could further confirm that CD4+ cells were alsopresent in the ovaries infiltrating the GCL of all the cysts,especially in areas of high VCAM-1 expression.

Conclusions: In the present study, we present novel evi-dence that the immune system is activated in the mousemodel for PCOS on a systemic and local level. We proposethat VCAM-1 is involved in aggravating PCOS symptoms,

such as altered follicular development and anovulatoryinfertility, by promoting leucocyte recruitment to theovaries and local inflammation. These findings offer novel