Anti-paternal Cytotoxic Antibodies Research Articles

P-398 HLA expression profile - the picture of successful pregnancy

Abstract Study question Which the difference of HLA profile expression among normal and abnormal karyotype? Summary answer In missed abortion groups, a significant decrease in HLA-G expression is observed in the chorionic villus structures in comparison with normal karyotype group. What is known already The study of pregnant alloimmunity confirms the relationship of recurrent miscarriage with the individual characteristics of the HLA I and II class system (Human leukocyte antigen), which can lead to impaired production of the mixed lymphocyte reaction blocking antibodies (MLR-Bf), anti-paternalcyto-toxicantibodies (APCA) and anti-idiotypic antibodies, Ab2, which are necessary for successful implantation and pregnancy development. For example, HLA-G is mainly restricted to the fetal–maternal interface on the extravillous cytotrophoblast, to placenta, amnion when HLA II class system mostly determinates in dNK, T-cells, macrophages. Study design, size, duration It was a prospective complex cohort study from 2016-2020 years with cytogenetics and pathomorphological investigations. The purpose of this study is to investigate HLA-DR expression in gravid endometrium, HLA-G and KIR2DL4 expression in chorionic villous among 3 groups (study included 115 cases of abortion material): group 1 – 36 cases after missed abortion with normal karyotype, group 2–35 cases after missed abortion with polyploidy and group 3–44 cases after induced abortion group (normal pregnancy). Participants/materials, setting, methods Criteria of inclusion: abortive material from 3 groups of women with missed or after induced abortion; 6-12 weeks, singleton pregnancy, cytogenetic of chorionic villous was obligatorily - normal fetal karyotype and polyploidy of fetus. In this work, we used: cytogenetic method and morphological study (histological-H&E stain, immunohistochemical, morphometric method). Immunohistochemical examination included quantitative and qualitative assessment of the expression of Anti-HLA-DR (mouse monoclonal), Anti-HLA-G (mouse monoclonal) and AntiKIR2DL1+KIR2DL3 + KIR2DL4 + KIR2DS4 (rabbit polyclonal). Main results and the role of chance By IHC the expression of HLA-G in extravillous trophoblast cells but the expression of HLA-DR and KIR2DL4 were detected in stroma, glands, the endometrial epithelium, both in groups.The HLA-G expression area in both groups with missed abortion didn’t differ statistically. But the expression of HLA-G in 3 group was strictly higher (in group 1 with a normal karyotype 37.7, in group 2-36.4, in the control group 67.09, p < 0.001). When divided into subgroups depending on the karyotype, the following data were obtained: in the group with a non-developing pregnancy with a normal karyotype of male fetuses, there is a tendency to lower expression of HLA-G molecules compared to female fetuses (32.96 (10.22) and 41 .45(9.36), respectively). Expression of the KIR2DL4 receptor was verified in chorionic villi (in the cytotrophoblast and syncytiotrophoblast), as well as in the structures of the endometrium, both in the glands, stroma, and in the surface epithelium, regardless of the karyotype. The expression area of the KIR2DL4 receptor did not differ statistically between the three groups (29.6, 33.1 and 27.55, p = 0.75). The expression area of the HLA-DR was detected in each group in gravid endometrium, but it hadn’t statistically differences (44.1, 38.2 and 42.5, p = 0.82). Limitations, reasons for caution No reasons for cautions Wider implications of the findings In missed abortion groups,a significant decrease in HLA-G expression is observed in the chorionic villus structures in comparison with the control group.The mechanism of abortion in groups with missed abortion with both normal karyotype and pathological karyotype of the fetus is identical.HLA-G molecule has a leading role in successful implantation. Trial registration number АААА-А20-120041390025-9

Lymphocytes immunotherapy for preserving pregnancy: Mechanisms and Challenges.

Due to the expression of paternal antigens by the embryo, pregnancy is considered as a semi-allograft and so immunological dysregulation is considered as one of the important causes in repeated implantation failure (RIF) and recurrent pregnancy loss (RPL). It has been revealed that lymphocytes immunotherapy (LIT) could be an appropriate approach to prevent pregnancy loss in such patients. Various mechanisms have been suggested for effectiveness of LIT such as enhancing expression of anti-paternal cytotoxic antibodies (APCA), progesterone-induced blocking factor (PIBF), anti-idiotypic antibodies (Ab2), and mixed lymphocyte reaction blocking antibodies (MLR-Bf), as well as reduction in the T helper 1/T helper 2 ratio and deviation in the pattern of cytokines production. However, there are controversial results about the beneficial effect of LIT treatment in RIF and RPL patients. In the current study, we reviewed findings of LIT in RIF and RPL patients with a focus on possible mechanisms of alloimmunization in preserving pregnancy. Besides, we highlighted possible reasons for mixed results about the effectiveness of LIT and way of solving the problem. Also, we proposed potential laboratory and clinical criteria to recruit patients for LIT.

Correlation between serum zinc levels and successful immunotherapy in recurrent spontaneous abortion patients

BACKGROUND:Immunotherapy with paternal lymphocytes plays an important role in preventing recurrent spontaneous abortion (RSA) and is an effective treatment for it. This kind of treatment is performed as an immunotherapy method in several centers in the world. It attributes to the production of anti-paternal cytotoxic antibodies (APCAs) in women with RSA. Production of APCA after lymphocyte immunotherapy (LIT) in RSA patients gives them a better chance for successful pregnancy. Regarding the important effect of trace elements on the function of the immune system, we tried to investigate the correlation between serum zinc level and the success of LIT in RSA.MATERIALS AND METHODS:Serum zinc concentration was determined in two groups of RSA patients using atomic absorption spectrophotometer systems. Group (a) that responded to the paternal lymphocytes and their cross-match test was positive, and group (b) that had no response to the paternal lymphocytes immunizations and their cross-match test was negative.RESULTS:Serum zinc levels in group (a) patients were 74.98 ± 11.88 μg/dl, which was significantly higher than those in group (b) with the zinc concentration of 64.22 ± 9.22 μg/dl.CONCLUSIONS:Zinc deficiency may be one of the substantial causes of negative results for LIT in RSA patients. Therefore, compensation of zinc defect before LIT can be a promising approach to improve the immune response in patients.

REVIEW ARTICLE: Anti‐phospholipid Antibodies and Other Immunological Causes of Recurrent Foetal Loss – A Review of Literature of Various Therapeutic Protocols

An immune-based aetiology is one of the several accepted causes for recurrent foetal loss (RFL). However, most of the immunological theories have not fulfilled the criteria for causality. This is a review of the various immunological causes of RFL and the outcome of different treatment protocols. Both auto- and alloimmune maternal immunological abnormalities have been proposed to account for foetal loss. Among the autoimmune factors, anti-phospholipid antibodies (APAs) have been demonstrated to be the strongest risk factors for foetal loss, the prevalence of which is as high as 40% in women with RFL. Other autoimmune antibodies implicated in RFL are anti-nuclear antibodies (ANAs), anti-thyroid antibodies and anti-endothelial cell antibodies. The alloimmune factors implicated in pregnancy loss of unknown aetiology include abnormal natural killer (NK) cell activity, alteration in T helper 1 (Th1) and T helper 2 (Th2) ratios, presence of alloimmune antibodies like anti-paternal cytotoxic antibodies, anti-idiotypic antibodies, mixed lymphocyte reaction blocking antibodies and abnormal expression of HLA-G molecules. Management of patients with RFL is mainly based on immunomodulatory (prednisolone, intravenous immunoglobulins, plasma exchange, paternal lymphocyte therapy), anti-aggregation (aspirin) or anti-coagulation (unfractionated or low molecular weight heparin) agents. Low-molecular-weight heparin with low-dose aspirin has been found to be the most effective treatment for women with APAs and RFL. Differences in dosage, timing of treatment, inclusion criteria, outcome assessment parameters etc. are some of the factors which have resulted in discrepancies in various reports. Identification of the immunological mechanisms involved in pregnancy loss and the action of different therapeutic reagents is important so that effective therapies can be designed and investigated.

Limiting dilution analysis of the allo-MHC anti-paternal cytotoxic T cell response. II: Recurrent spontaneous abortion and the effect of immunotherapy.

Using limiting dilution analysis (LDA) we determined anti-paternal cytotoxic T lymphocyte precursor (CTLp) frequencies in the peripheral blood of 10 women with unexplained recurrent spontaneous abortion (RSA) before and after immunization with paternal lymphocytes. The women and their partners were HLA tissue-typed and none of the women had anti-paternal cytotoxic antibodies (APCA) before immunization. All other known causes of RSA were excluded. All 10 women were found to have high frequencies of specific anti-paternal cytotoxic T cells before immunization (range 1/1030 to 1/9574). Splitwell analysis showed that these cytotoxic cells were specific to paternal MHC antigens. These frequencies rose significantly following immunization (range 1/683 to 1/4652). The cytotoxic T lymphocyte frequencies against an HLA-mismatched third party varied from woman to woman, but were not affected by the immunization. The LDA data conformed to single-hit kinetics, indicating that only cytotoxic T cells were limiting in the assay. Our data are in sharp contrast to the previously held view that women with RSA may be hyporesponsive to paternal MHC antigens. Immunizing such women with paternal leucocytes further sensitizes them. These findings cannot be reconciled with a favourable outcome in the treatment of RSA with immunotherapy. We would argue that this treatment is at best of unproven value, and may even be harmful. That these women may sometimes have successful pregnancies following immunotherapy testifies to the effectiveness of the classical MHC antigen-deficient trophoblast as an immunological barrier between mother and fetus.

Lymphocyte immunotherapy and its probable mechanism in the maintenance of pregnancy in women with recurrent spontaneous abortion.

Most women with alloimmune cause of recurrent spontaneous abortion (RSA) includes increased sharing of human leukocyte antigens (HLA) that may prohibit the mother from making anti-paternal cyto-toxic antibodies (APCA), anti-idiotypic antibodies (Ab2) and mixed lymphocyte reaction blocking antibodies (MLR-Bf). Overactivity of T helper-1 (Th-1) cytokines and natural killer (NK) cells have been also reported to be the major alloimmune cause of recurrent spontaneous abortion (RSA). It was revealed from extensive updated analysis of this subject that paternal lymphocytes immunotherapy may play a significant role in the prevention of alloimmune cause of fetal loss in women with RSA. These alloimmune parameters are found to be suppressed in successful immunotherapy, which is comparable to normal pregnancy. Various studies represented that paternal lymphocyte immunotherapy was attributed to the high expression of APCA, Ab2, MLR-Bf and inhibition of Th-1 pattern of cytokines and NK cell activity in women with alloimmune cause of RSA. Present updated randomized clinical trials demonstrated that women with RSA of study group who have been treated with paternal lymphocyte immunotherapy had more successful outcomes (68%) as compared to women with RSA of control group who either received autologous lymphocytes/third party lymphocytes/normal saline or no therapy (54%), ( p<0.02). However, when the results of the randomized and nonrandomized studies were pooled together it was observed that 67% of women with RSA of study group who received paternal lymphocyte immunotherapy showed successful pregnancy outcome in comparison to 36% success in women with RSA of control group who either received autologous lymphocytes/third party lymphocytes/normal saline or no therapy ( p<0.05). These results advocate the role of paternal lymphocyte immunotherapy for the maintenance of pregnancy in women with RSA.

Immuno therapy In Recurrent Spontaneous Abortion: Randomized and Non-Randomized Trials

Objective: The present study was conducted to evaluate the efficacy of immunotherapy with husband's lymphocytes in women with recurrent spontaneous abortion (RSA). Methods: A total of 205 women with three or more consecutive abortions were screened for known causes of recurrent spontaneous abortion. Only 105 women were registered for immunotherapy after excluding the women who had one or the other known cause of abortion. 73 women were registered for immunotherapy against husband's lymphocytes. 32 women were registered under double blind randomized trial. These women were negative for antipaternal cytotoxic antibodies against their husband's cells. Success rate of immunotherapy was same in both the groups. Results: Immunotherapy with husband cells in 73 RSA women was carried out in non-randomized trial. Our results show that the success rate was significantly higher (86%) when compared to other groups of the present study i.e. RSA women who declined to enter the trial (33%), dropouts (50%) and antipaternal cytotoxicity negative RSA women (30%). Antipaternal cytotoxic antibodies (APCA) were taken as the measure of immuno-potentiation. Our results indicate that APCA can be taken as a good indicator for selecting patients for immunotherapy and also to measure whether the RSA women is adequately immunized or not. We have also seen the effect of husband's cells in double blind randomized trial group (32 RSA women). The success rate was 85%, which is comparable to non-randomized trial group. Conclusions: Our results indicate the importance of immunotherapy with husband's lymphocytes in RSA women and also show that APCA can be considered as one of the important immuno-potentiating factor.

Humoral immune response to an allogenic foetus in normal fertile women and recurrent aborters.

BackgroundSeveral humoral immune factors are responsible for a successful pregnancy. There are a few studies, which demonstrate the role of antipaternal cytotoxic antibodies (APCA) and mixed lymphocyte reaction blocking factor (MLR-Bf) in the maintenance of pregnancy. However, these factors have not been studied in women with history of recurrent spontaneous abortion (RSA). We designed this study to review the role of APCA and MLR-Bf in normal pregnancy as well as in women with a history of RSA.MethodsOne hundred and five women with unexplained recurrent spontaneous abortion were included in the present study. These women were screened for all other known causes of recurrent abortion. We also included 60 normal fertile women, fifteen from each trimester and fifteen women during the post partum period (up to six months). RSA and controls (normal pregnancy) were matched for age, caste, and socio economic background and also for parity. APCA and MLR-Bf were evaluated in all the groups. All women with RSA who conceived during the study period were on follow up.ResultsWe have analyzed the status of APCA and MLR-Bf in normal pregnancy (different gestational periods and during post partum), and in women with history of RSA. Our results show that APCA was significantly higher in controls as compared to RSA women. MLR-Bf was directed against the husbands' cells in normal pregnancy and was virtually absent in RSA women.ConclusionOur results indicate that there is a significantly low titer of APCA and MLR-Bf in women with recurrent spontaneous abortion. This highlights the role of these factors in the maintenance of successful pregnancy.

Retrospective case-control study of the outcome of pregnancy after paternal leukocytes transfusion in cases of recurrent pregnancy loss

Immunization therapy, for treatment of recurrent pregnancy loss (RPL), has been developed since the mid-1980's. The effect of this kind of therapy is still controversial The purpose of this retrospective, case-control study, was to evaluate the beneficial effect of immunotherapy for the treatment of patients with RPL and negative antipaternal cytotoxic antibodies. During a 3-year period (1992 to 1994), we were able to collect 25 such patients who conceived after having been immunized abroad. We compared the outcome of the pregnancies of those 25 patients with that of 41 unimmunized patients used as controls. There was no statistically significant difference between the two groups regarding maternal age, parity, number of previous abortions, gestational age, infant weight, and Apgar score at 5 minutes. The pregnancy success rate was higher in patients who were not immunized (80.5%) than in those who were immunized (56%). The difference bordered on, but did not reach, statistical significance (P=0.064). There was no significant difference whether immunization was performed before or during pregnancy or whether pregnancy occurred within 26 weeks of immunization or between 27 and 52 weeks. The only significant difference between the two groups was related to the drugs (Aspirin and progesterone) used in the antenatal period and this may explain the higher; although not significant, success rate in the control group. It is not yet clear whether immunotherapy has any substantial effect in preventing recurrent abortions. More controlled prospective studies are required to reach a final conclusion regarding this form of treatment.

Retrospective case-control study of the outcome of pregnancy after paternal leukocytes transfusion in cases of recurrent pregnancy loss

Abstract Immunization therapy, for treatment of recurrent pregnancy loss (RPL), has been developed since the mid-1980's. The effect of this kind of therapy is still controversial The purpose of this retrospective, case-control study, was to evaluate the beneficial effect of immunotherapy for the treatment of patients with RPL and negative antipaternal cytotoxic antibodies. During a 3-year period (1992 to 1994), we were able to collect 25 such patients who conceived after having been immunized abroad. We compared the outcome of the pregnancies of those 25 patients with that of 41 unimmunized patients used as controls. There was no statistically significant difference between the two groups regarding maternal age, parity, number of previous abortions, gestational age, infant weight, and Apgar score at 5 minutes. The pregnancy success rate was higher in patients who were not immunized (80.5%) than in those who were immunized (56%). The difference bordered on, but did not reach, statistical significance (P=0....

Outcome of pregnancy in women with recurrent spontaneous abortion following immunotherapy with allogeneic lymphocytes.

The efficacy of immunotherapy in the prevention of habitual abortion remains controversial. It has been suggested that the benefits are predominantly due to psychological factors. We have evaluated the success of pregnancy outcome following immunotherapy with allogeneic lymphocytes, in relation to the subsequent development of anti-paternal cytotoxic antibodies (APCA). It was observed that in women who developed an APCA titre of > or = 1:16, live births occurred in 16 out of 21 cases (76%), while only two out of seven (28%) women who failed to achieve an APCA titre of > or = 1:16 had successful pregnancies (P < 0.05). In eight women who had an APCA titre of 1:16 on initial screening, and were, therefore, excluded from the trial, successful pregnancy outcome was noted in 62.5% of cases. Although these results are based on a small sample and on an open, non-randomized trial, they show that the efficacy of immunotherapy is related to immune response to allogeneic lymphocytes, and is not simply a placebo effect. Measurement of APCA titre could serve as a marker for immunopotentiation.

Fc receptor blocking antibodies after active immunization for the treatment of recurrent spontaneous abortion

In a prospective study 140 couples who had at least three spontaneous abortions (RSA) were studied for the presence of Fc receptor blocking antibodies detected by the erythrocyte antibody rosette inhibition (EAI) assay, for anti-paternal cytotoxic antibodies (APCA), and for mixed lymphocyte culture inhibiting (MLCI) antibodies before and after active immunization with paternal lymphocytes. The comparative analysis revealed the EAI assay to possess a higher sensitivity than the APCA and/or MLCI tests in monitoring the specific immune response after active immunization. The success of pregnancy in EAI positive post-immunization patients was not influenced by the presence or absence of APCA or MLCI. In the light of a successful pregnancy outcome of 85.7% ( n = 37) in this study we conclude that the monitoring of Fc receptor blocking antibodies is useful in active immunization protocols for RSA patients.

Sharing of human leukocyte antigens in primary and secondary recurrent spontaneous abortions

The prevalence of recurrent fetal loss and its relationship to sharing of human leukocyte antigens within couples was examined in a large, ethnically homogeneous Chinese population in Taiwan: 91 couples with primary recurrent spontaneous abortion, 32 couples with secondary recurrent spontaneous abortion, and 51 normal fertile couples. There was an excess of human leukocyte antigen sharing in both types of recurrent aborters. The primary aborters shared human leukocyte A and DQ antigens and three or more of the human leukocyte A, B, DR, and DQ antigens. The secondary aborters did not have an excess of sharing at any one locus but they did share three or more of the human leukocyte A, B, DR, and DQ antigens. There was no excess of antipaternal cytotoxic antibodies in any group of aborters, and the primary aborters had a lower level of mixed lymphocyte reaction blocking factor than normal couples or secondary aborters. Previous studies showed that couples in whom a gestational trophoblastic tumor developed in the woman shared human leukocyte B and DQ antigens and three or more of the human leukocyte A, B, DR, and DQ antigens. These studies provide substantial support for the role of recessive genes linked to the major histocompatibility complex in the pathogenesis of recurrent spontaneous abortions and of gestational trophoblastic tumors.