32 Abstract / Journal of Reproductive Immunology 101–102 (2014) 18–39

on days 12.5 and 14.5 of gestation. Adult offspring wereused as donors for BM transplantation or underwent theasthma protocol without transplantation. Congenic CD45.2C56Bl/6J mice of the same age were lethally irradiated andserved as recipients. After hematopoietic reconstitution,experimental asthma was induced in chimeric animalsby sensitizing the animals with ovalbumin (OVA). Subse-quently, eosinophilia was quantified in broncho-alveolarlavage (BAL) using flow cytometry, airway inflammation inlungs was histomorphologically assessed in hematoxylinand eosin (H&E)-stained lung sections and total IgE wasmeasured in serum.

Results: Prenatally stressed animals without BMtransplantation revealed increased eosinophilia in BAL(52.2 ± 11.6%), more cell infiltration in the lung (46% with asevere inflammation score), and higher IgE levels in serum(3.3 ± 1.6 ng/ml) compared with controls (37.7 ± 10.9%eosinophils; 30% with severe inflammation score, and1.9 ± 1.1 ng/ml IgEs). Recipients of BM from prenatallystressed mice showed less severe asthma symptoms(41.4 ± 21% eosinophils; 0% with a severe inflammationscore and 1.6 ± 0.6 ng/ml IgEs) compared with recipientsof BM from controls (52.9 ± 18.8% eosinophils; 50% with asevere inflammation score and 3.9 ± 2.9 ng/ml IgEs).

Conclusion: Increased airway inflammation is observedmore profoundly in mice in which the respiratory systemwas exposed to prenatal stress. Thus, stress-induced mod-ification of the developing lung rather than the immunesystem may render an individual more prone to developingan asthmatic phenotype in later life.

http://dx.doi.org/10.1016/j.jri.2013.12.103

O29

B cell development undergoes profound changes duringpregnancy

Damián O. Muzzio ∗, Federico Jensen

Research Laboratory, Department of Obstetrics and Gynecol-ogy, Greifswald University, Germany

Introduction: Mammalian pregnancy represents achallenge to the rules of immunology. The immune sys-tem must tolerate the growing semi-allogeneic fetus at thesame time as both mother and fetus have to be protectedagainst the threat of pathogens. B cells actively participatein immune responses, mainly through the production ofantibodies. Their role in pregnancy is, however, an issue tobe elucidated.

Objective: To analyze the impact of gravity on B celldevelopment and function in mice.

Methods: C57Bl/6 females were allogeneically matedwith BALB/c males. B cell populations were analyzed inbone marrow (BM), blood, spleen, para-aortic lymph node(PLN) and the peritoneal cavity (PerC) by flow cytometry atearly pregnancy (day 7), mid-pregnancy (day 14), and latepregnancy (day 18). Non-pregnant C57Bl/6 females wereused as controls. Levels of IgM, IgA, IgE, and IgG in serumwere analyzed by ELISA. Localization of the marginal zoneand follicular B cells in the spleen was further analyzed byimmunofluorescence.

Results: Pregnancy induced a significant reduction inB lymphopoiesis depicted by a lower number of pre/pro Bcells as well as immature B cells in BM of pregnant micecompared with non-pregnant controls. However, matureB cells and plasma cells were accumulated in this tissueduring pregnancy, indicating B cell activation at the periph-ery. In keeping with this, higher numbers of marginal zoneB cells and antibody-producing plasma cells were foundin the spleen. Higher titers of IgM, IgA, IgE and IgG foundin serum of pregnant mice compared with non-pregnantcontrol mice clearly confirmed B cell activation duringpregnancy. Finally, significantly higher numbers of matureB cells were found in PLN and PerC of pregnant mice thanin non-pregnant females.

Conclusion: We clearly demonstrated in this work thatB cell development and function are highly modified dur-ing pregnancy. This piece of information was missing inour understanding of how the maternal immune systemis adapted to the presence of the semi-allogeneic fetus,thereby opening new avenues to be explored.

http://dx.doi.org/10.1016/j.jri.2013.12.104

O30

Alterations in peripheral blood lymphocytes before andafter successful pregnancy of patients with a history ofunexplained recurrent miscarriage

Pei-Yan Liang 1,2,3,∗, Guan-Gui Li 1,2,3, Liang-Hui Diao 1,2,3,Cui-Cui Liu 1,2,3, Xian Chen 1,2,3, Chun-Yu Huang 1,2,3,Zong Zeng 1,2,3

1 Fertility Center, Shenzhen Zhongshan Urology Hospital,Shenzhen 518048, China2 Shenzhen Key Laboratory of Reproductive Immunology forPeri-implantation, Shenzhen 518045, China3 Shenzhen Zhongshan Institute for Reproductive Medicineand Genetics, Shenzhen 518045, China

Introduction: The immunological aspects play a signif-icant role in unexplained recurrent miscarriage. The aim ofthis study was to investigate the immunological changesin peripheral blood lymphocytes before and after success-ful pregnancy in patients with a history of unexplainedrecurrent miscarriage.

Materials and methods: Forty-three patients with ahistory of unexplained recurrent miscarriage were treatedby immunotherapy after signing the informed consent.The treatments include paternal lymphocyte immunother-apy, 5%IVIg and/or TNF inhibitor before pregnancy. Afterimmunotherapy, 35 patients successfully delivered ahealthy child at full term, while 8 patients suffered anothermiscarriage (5 patients had fetal chromosomal abnormal-ities). The immunological parameters of 35 patients whodelivered successfully were detected by flow cytometryat three time points: the mid-luteal phase before theimmunotherapy and pregnancy, at week 12 and at week24 of the pregnancy. The tests include: the percentages andabsolute counts of T, B and NK cells as well as the CD4 andCD8 subpopulations of T cells in peripheral blood; NK cyto-toxicity; and the intracellular cytokine of CD4+T helper cell(IL-10, IFN-� and TNF-�).

Abstract / Journal of Reproductive Immunology 101–102 (2014) 18–39 33

Results: Compared with the value of the mid-lutealphase before pregnancy, the ratio of TNF-�/IL-10, NK cyto-toxicity, and the absolute count of CD3+ T cells and NK cellswere significantly decreased at both week 12 and week 24of the pregnancy. The TNF-� cytokine and absolute count ofCD4+ T cells were significantly decreased at week 12 of thepregnancy; the IFN-� cytokine, the ratio of IFN-�/IL-10, thepercentage of NK cells and the absolute count of B cells weresignificantly decreased whereas the percentages of CD3 + Tcells, CD4+ T cells, CD69+ T cells, and CD69+ NK cells weresignificantly increased at week 24 of the pregnancy.

Conclusions: The decrease in TNF-�/IL-10 ratio, NKcytotoxicity, NK cell and T cell counts probably contributeto the immune tolerance to the fetus and successful preg-nancy in patients with a history of unexplained recurrentmiscarriage. Furthermore, these parameters could be thepotential markers for immunotherapy for unexplainedrecurrent miscarriage.

http://dx.doi.org/10.1016/j.jri.2013.12.105

O31

Immunological methods to investigate alloimmunehabitual abortion

David Fekete 1,∗, Eszter Ujhelyi 1, Gyozo Petrányi 1, ÁdámGalamb 2, András Széll 3, Attila Pajor 2, István Vályi-Nagy 1

1 United St. István and St. László Hospital and OutpatientClinic, Budapest, Hungary2 Semmelweis University, 2nd Department of Obstetrics andGynaecology, Budapest, Hungary3 G1 Laboratory, Budapest, Hungary

Recurrent spontaneous abortion (RSA) is repeated preg-nancy loss at 6–12th week of gestation, despite normalimplantation and normal embryonic development. In sev-eral cases where no anatomical, genetic, hematological,endocrine or infectious cause can be identified we speakof RSA of unknown cause. These cases can occur againsta background of autoimmune/alloimmune conditions ordisorders of immune regulation. Clinical observations sug-gest that immunotherapies used to treat RSA are effectiveonly in those cases, where the immunological cause ofRSA is accurately assessed. For this reason a national RSAcommittee was founded in 2013 at the 2nd Departmentof Obstetrics and Gynaecology. In cooperation with thiscommittee we introduced a complex protocol for the inves-tigation of immunological RSA. The protocol consists of thefollowing:

Flowcytometry cross-match: Detection of IgGmolecules in the patient serum that react with the Tor B lymphocytes of the partner showing HLA sensitizationor immune regulation.

Mixed leukocyte culture (MLC) and blocking antibodyanalysis: With this method we can determine the cellularreactivity of the patient against the partner. We can alsomeasure whether the patients’ serum contains any factorthat modifies this reactivity.

Determination of Th1–Th2 ratio: The determination ofTh1 vs. Th2 cell ratio is based on measuring their char-

acteristic cytokines. Th1 dominance points to elevatedcellular immune response while Th2 dominance representshumoral immune response and suppressive immunoregu-lation.

Analysis of natural killer (NK) cells: If elevated NK cellnumbers are detected in the blood and/or their reactiv-ity is elevated indicates a generally upregulated immunereactivity of the patient.

In the present work we summarize our experience withthe 104 RSA patients investigated so far in our laboratory.We analyze the efficiency of the different measurementsand emphasize the importance of the integrated evalua-tion of the results. Most informative results were concludedfrom the MLC measurement, where 32% of the patientsshowed hyper-reactivity against the partner and 77% ofpatients’ serum contained factors that enhanced the reac-tivity. In contrast, only 21 patients had components in theirserum that blocked the reaction against the partner cells.

http://dx.doi.org/10.1016/j.jri.2013.12.106

O32

Macrophages are crucial for embryo implantation

Kenneth D. Beaman ∗, Mukesh K. Jaiswal

Department of Microbiology and Immunology, RosalindFranklin University of Medicine and Science, North Chicago,IL, USA

Among immune cells, the macrophages have the mostpotential as regulators of tissue homeostasis under variousinflammation-associated conditions. During pregnancyproper, immune response is crucial to support trophoblastproliferation and differentiation for successful placen-tal development. Our data show that semen initiates areceptive immune/inflammatory reaction in a maternalenvironment that will be vital for successful implantation.The sperm’s expression of vacuolar ATPase, particularlythe a2 isoform, plays an important role in commencingprogressive changes in a composition of immunocom-petent cells at the endometrial mucosa, such as at thelevels and in the phenotypes of endometrial macrophages.Once initiated, these changes result in establishment ofthe critical balance of inflammation at the maternal–fetalinterface. This balance also allows the angiogenic portionof the inflammatory reaction to prevail, while limiting thedestructive aspects of the immune response.

An alteration of vacuolar ATPase expression in the malemouse via siRNA knock down or by monoclonal antibodyadministration results in changes related to the functionalcharacteristics of testicular macrophages, namely modi-fied cytokine production. These experiments mimic ourearlier findings using the classic CBA/DBA model in arole of cytokine profile (systemic or testicular?) in malemouse determinative between success and failure in thisrecurrent miscarriage model and not an assumed adap-tive immune difference. Perhaps the most interesting factis that the alteration of cytokine production by testi-cular macrophages manifests in poor pregnancy outcomedespite viable blastocysts being implanted.