1 Division of Transplantation Immunology, Tissue Typing Laboratory, 2 Department of Clinical Epidemiology and 3 Department of Obstetrics and Gynecology, Sheba Medical Center, Tel Hashomer 52621 and Department of Embryology, Tel Aviv University, Israel
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Abstract |
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Key words: anti-paternal antibodies/consecutive recurrent miscarriages/immunization
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Introduction |
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However, as three miscarriages can occur by chance, we considered that it is much more valid to assess a more homogeneous group of patients with a poor prognosis. Therefore we have assessed treatment in patients with five or more miscarriages. Numerous reports (Carp et al., 1993, 1996
, 1997
; Daya and Gunby, 1994
) have shown that the beneficial effect of immunopotentiation is greater in patients with a higher number of miscarriages. However, even within this group of patients, there is no laboratory test to diagnose which patients have immunologically mediated miscarriages or are suitable for immunization.
The initial reports on paternal leukocyte immunization used the sharing of HLA antigens between spouses as a criterion for immunization (Beer et al., 1981; Taylor and Page Faulk, 1981
). This excess sharing was considered to be increased in recurrently aborting couples (Komlos et al., 1977
), and responsible for the hyporesponsiveness to paternal antigens leading to miscarriage. This hyporesponsiveness was considered to be shown by a lower incidence of anti-paternal antibody in recurrently aborting couples (Mowbray et al., 1983
). Subsequent reports have not confirmed excess HLA antigen sharing between spouses in recurrently aborting couples (Caudle et al., 1983
; Oksenberg et al., 1984
) and controversy remains concerning the lack of anti-paternal antibodies. The lack of anti-paternal antibodies has even been reported to be physiological (Regan, 1987, 1988
). We considered that the confusion about these results might be due to confounding factors in patients with three or four miscarriages. Therefore reanalysis of the results of cross matching for anti-paternal antibodies was performed separately for patients with five or more miscarriages. The data were compared to the results for patients with three or four miscarriages, and analysed for primary and secondary and tertiary aborters separately. Multivariate analysis was also performed to assess anti-paternal antibody production and immunization, to determine the influence of each factor independently from the other confounding factors.
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Materials and methods |
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Patients were only included after other presumptive aetiological factors were found to be normal, i.e. (i) karyotype of both parents; (ii) glucose tolerance test; (iii) toxoplasmosis serology; (iv) hysterosalpingogram, thereby excluding anatomical abnormalities, intrauterine adhesions and cervical incompetence; (v) thyroid function; (vi) serum prolactin; (vii) normal luteal phase of at least 12 days and plasma progesterone >24.8 nmol/ml; (viii) anti-nuclear factor was tested by using rat liver as substrate and fluoresceinated rabbit anti-human IgG, or by immunofluorescence using a `Hep 2000' kit (Immunoconcepts, MI, USA); (ix) anticardiolipin antibody by enzyme-linked immunosorbent assay testing [cut-off value <13 IgG phospholipid units (GPLu)/ml and <7.6 IgM phospholipid units (MPLu)/ml], and lupus anticoagulant, according to the kaolin clotting time (KCT).
All women with three or more unexplained pregnancy losses were offered immunization, if none of the above causes of miscarriage was present. The same criteria were used whether patients were immunized or assigned to the control group. All patients were informed of the possible risks and benefits of immunization, and all signed an informed consent form.
Patients and spouses undergoing immunization were screened for syphilis, hepatitis B antigen, hepatitis C, cytomegalovirus (CMV) antibodies and human immunodeficiency virus (HIV) antibodies. Spouses with hepatitis B antigen, hepatitis C or CMV IgM antibodies were not used as immunizers.
Control group
The control group for the immunization study comprised of couples who refused the immunization procedure. Some were advised by their own physician not to accept experimental treatment. Forty-nine women in the 5 CRM group and 96 patients in the 34 CRM group elected to be in the control group (Table I
).
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Anti-paternal antibodies
The presence of anti-paternal antibodies was determined by cross matching between maternal undiluted fresh serum with paternal peripheral lymphocytes (Mittal et al., 1968). An immune response to paternal antigens was recorded when seroconversion occurred from crossmatch negative to positive. A positive crossmatch was recorded when maternal serum killed freshly drawn paternal peripheral lymphocytes at a proportion >40% of the control negative serum. Rabbit complement was used as the complement source. Crossmatching was tested prior to first immunization and 23 weeks after each immunization.
Statistical analysis
The results were analysed to determine the effect of anti-paternal antibody production on the outcome of the subsequent pregnancy for the group as a whole and separately for primary, secondary, or tertiary aborter status, with three, four, five or more miscarriages. The odds ratio for a live birth was calculated with 95% confidence intervals. Analysis was performed by Pearson's test and Fisher's exact test when the numbers were small. Multivariate analysis was performed using stepwise logistic regression for the following variables: number of abortions (five or more compared to three or four), anti-paternal antibody status correlated to immunization, primary, secondary or tertiary aborter status, time taken to conceive, and maternal age at the subsequent pregnancy. Non-significant variables were excluded from the final model.
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Results |
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Stepwise logistic regression showed the age at pregnancy to be a non-significant predictor of a subsequent live birth, it was therefore excluded from the final analysis.
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Discussion |
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In this series, patients who were anti-paternal antibody positive had a higher live birth rate. Although the presence of anti-paternal antibodies at initial testing was not a significant factor in the multivariate analysis, when analysed as an isolated factor, it raised the live birth rate from 45.5 to 70.2% in women with three or four abortions. This indicates that anti-paternal antibody positive patients with three or four abortions might not require immunization. If these patients are included in the control group of a trial of immunization, as many trials have done, the control group will have a high live birth rate. This is probably why, if all patients with recurrent abortion are treated as a homogeneous whole, there is a subsequent 60% live birth rate.
Patients who had no previous anti-paternal antibodies and who seroconverted to an anti-paternal antibody positive status had the highest live birth rate, with an odds ratio almost four times higher than control patients who were anti-paternal antibody negative and not immunized. However, women who were immunized had a higher live birth rate than non-immunized women even if anti-paternal antibody was not expressed (OR = 2.07 for a live birth even if there was no seroconversion). This could have a number of explanations: (i) a 40% cell kill in the cross match was chosen as a cut-off point between anti-paternal antibody positive and negative. Some of the `anti-paternal antibody negative' patients may have had a lower level of antibodies which may have been sufficient to improve the live birth rate; (ii) immunization per se may have altered other parameters, for which there are no available markers at present, and are not reflected by anti-paternal antibody production; (iii) self-selection for immunization might have introduced bias in that immunized patients might have been more psychologically motivated or influenced by other unknown factors.
The fact that immunized women who did not seroconvert had an odds ratio of 2.07 for a live birth and an odds ratio of 3.99 after seroconversion might indicate that immunization of a patient who is anti-paternal antibody negative is insufficient for achieving live birth in some patients with recurrent miscarriages. It is necessary to ensure that seroconversion has occurred. If not, booster immunizations may be required. The predictive value of anti-paternal antibodies was most marked in primary and tertiary aborters, while in the secondary aborters the predictive value was unclear.
It must be stressed that anti-paternal antibodies are not an essential prerequisite for pregnancy to develop. These antibodies are only present in 16.4% of sera obtained from placental blood clots (mainly maternal blood collected at the time of delivery) (Huang et al., 1997), and usually only become apparent near term in normal pregnancy (Regan, 1988
). In the work-up and treatment of recurrent miscarriages, anti-paternal antibodies might be just one indicator of an immune response. Other tests of the immune response have been used such as natural killer cells (Aoki et al., 1995
), TH1 and TH2 cytokines (Hill et al., 1995
; Raghupathy, 1997
), anti-idiotype antibodies to HLA (Behar et al., 1991
) and suppression by T-cells (Behar et al., 1993
). Further clinical trials are necessary to determine whether these tests are superior to testing for anti-paternal antibody status.
Maternal age did not have a significant influence on the outcome of the subsequent pregnancy. The time to conceive was significant statistically, but the time to conceive is a reflection on infertility and the efficacy of fertility treatment. Therefore the effect of `time to conceive' might not be significant biologically.
It must be borne in mind that this study was not placebo controlled or blinded, but patients self-selected; therefore, although immunization improved the live birth rate, the results must be interpreted with caution. A double blind controlled study that controls for anti-paternal antibody status and stratifies for the number of abortions appears to be necessary. The results should then be corrected for other confounders such as karyotypic anomalies in the fetus.
In conclusion, although there is still no test that is pathognomic for immunologically mediated abortion, anti-paternal antibodies seem to have a prognostic value in the assessment of miscarriages of unknown aetiology. Additionally, anti-paternal antibodies might indicate the need for immunization. However clinical skill and judgement are necessary in interpreting their significance in different groups of patients.
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Notes |
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References |
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Beer, A.E., Quebbman, J.F., Ayres, J.W.T et al. (1981) Major histocompatibility complex antigens. Maternal and paternal immune responses and chronic habitual miscarriages. Am. J. Obstet. Gynecol., 141, 987999.[ISI][Medline]
Behar, E., Carp, H.J.A. and Gazit, E. (1991) Anti-idiotypic antibodies to anti HLA class-I antibodies in habitual abortion. Am. J. Reprod. Immunol., 26, 143146.[ISI][Medline]
Behar, E., Carp, H.J.A., Livneh, A. et al. (1993) Differential suppression activity induced by paternal leukocyte immunization in habitual abortion. Gynecol. Obstet. Invest., 36, 193197.[ISI][Medline]
Carp, H.J.A. (1994) Abstracts of contributors' individual data submitted to the worldwide prospective observation study on immunotherapy for treatment of recurrent spontaneous abortion. Am. J. Reprod. Immunol., 32, 261274.[ISI]
Carp, H.J.A. (1996) Pitfalls in interpreting therapy for early pregnancy. Early Pregnancy, 2, 96101.[Medline]
Carp, H.J.A. (1997) Investigation and treatment for recurrent pregnancy loss. In Rainsbury, P. and Vinniker, D. (eds), A Practical Guide to Reproductive Medicine. Parthenon, Carnforth, Lancs, UK.
Carp, H.J.A., Toder, V., Gazit, E. et al. (1990) Immunization by paternal leucocytes for prevention of primary habitual abortion: results of a matched controlled trial. J. Gynaecol. Obstet. Invest., 29, 1621.
Carp, H.J.A., Toder, V. and Mashiach, S. (1993) Immunotherapy of habitual miscarriage. Am. J. Reprod. Immunol., 28, 281284.[ISI]
Carp, H.J.A., Ahiron, R., Mashiach, S. et al. (1996) Intravenous immunoglobulin in women with five or more miscarriages. Am. J. Reprod. Immunol., 35, 360362.[ISI][Medline]
Carp, H.J.A., Torchinsky, A., Portuguese, S. et al. and The Recurrent Miscarriage Immunotherapy Trialists Group (1997) Paternal leukocyte immunization after five or more miscarriages. Hum. Reprod., 12, 250255.[Abstract]
Caudle, M.R., Rote, N.S., Scott, J.R et al. (1983) Histocompatibility in couples with recurrent spontaneous miscarriage and normal fertility. Fertil. Steril., 39, 793796.[ISI][Medline]
Christiansen. O.B. (1996) A fresh look at the causes and treatments of recurrent miscarriage, especially its immunological aspects. Hum. Reprod. Update, 2, 271293[Abstract]
Clifford, K, Rai, R. and Regan, L. (1997) Future pregnancy outcome in unexplained recurrent first trimester miscarriage. Hum. Reprod., 12, 387389.[Abstract]
Crosignani P.C. and Rubin, B.L. (1991) Recurrent spontaneous miscarriage. The recommendations of the ESHRE workshop on recurrent spontaneous miscarriage held in Anacapri on September 911, 1990. Hum. Reprod., 6, 609610.[ISI][Medline]
Daya, S. and Gunby, J. (1994) The effectiveness of allogeneic leukocyte immunization in unexplained primary recurrent spontaneous miscarriage. Am. J. Reprod. Immunol., 32, 294302.[ISI][Medline]
ETEP (1995) Euro-Team Early Pregnancy (ETEP) protocol for recurrent miscarriage. Hum. Reprod., 10, 15161520.[Abstract]
Gerhard, I., Waibel, S., Daniel, V. and Runnebaum, B. (1998) Impact of heavy metals on hormonal and immunological factors in women with repeated miscarriages. Hum. Reprod. Update, 3, 301309.[ISI]
Hatasaka, H.H. (1991) Recurrent miscarriage: epidemiological factors, definitions and incidence. Clin. Obst. Gynecol., 37, 625634.
Hill, J.A., Polgar, K., Anderson, D.J. (1995) T-helper 1-type immunity to trophoblast in women with recurrent spontaneous abortion. JAMA, 273, 19331936.[Abstract]
Huang, Y.T., Hsieh, Y.S. Phan, M.C. et al. (1997) Evaluation of the HLA antibodies in placental blood. Clin. Lab. Haematol., 19, 273276.[ISI][Medline]
Komlos, L., Zamir, R., Joshuah, H. et al. (1977) HLA antigens in couples with repeated miscarriages. Clin. Immunol. Immunopathol., 7, 330335.[ISI][Medline]
Mittal, K.K., Mickey, J.F., Singall, D.P. et al. (1968) Serotyping for homotransplantation. XVIII. Refinement of microdroplet lymphocyte cytotoxicity test. Transplantation, 6, 913927.[ISI][Medline]
Mowbray, J.F., Gibbings, C.R., Sidgwick, A.S. et al. (1983) Effects of transfusions in women with recurrent spontaneous abortion. Transplant. Proc., 15, 896899.[ISI]
Oksenberg, J.R., Persitz, E. and Amar, A. (1984) Maternal-paternal histocompatibility: lack of association with habitual miscarriages. Fertil. Steril., 42, 389395.[ISI][Medline]
Raghupathy, R. (1997) ThI-type immunity is incompatible with successful pregnancy. Immunol. Today, 18, 478482.[ISI][Medline]
Regan, L. (1988) A prospective study of habitual miscarriage. In Beard, R.W. and Sharp, F. (eds), Early Pregnancy Loss: Mechanisms and Treatment. RCOG, London, pp. 2337.
Regan, L. and Braude, P.R. (1987) Is anti-paternal cytotoxic antibody a valid marker in the management of recurrent abortion? (letter). Lancet, II, 1280.
Salat-Baroux, J. (1988) Recurrent spontaneous miscarriages. Reprod. Nutr. Dev., 28, 15551568.[ISI][Medline]
Stern, J.J., Dorfman, A.D., Gutierez-Najar, M.D. et al. (1996) Frequency of abnormal karyotype among abortuses from women with and without a history of recurrent spontaneous abortion. Fertil. Steril., 65, 250253.[ISI][Medline]
Taylor, C. and Page Faulk, W. (1981) Prevention of recurrent miscarriage with leukocyte transfusions. Lancet, ii, 6869.
Wheeler, J.M. (1991) Epidemiologic aspects of recurrent pregnancy loss. Infertil. Reprod. Med. Clin. N. Am., 2, 117.
Submitted on March 11, 1999;