Human Reproduction, Vol. 14, No. 12, 2974-2979,
December 1999
© 1999 European Society of Human Reproduction and Embryology
The prognostic value of anti-paternal antibodies and leukocyte immunizations on the proportion of live births in couples with consecutive recurrent miscarriages
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
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Anti-paternal antibodies directed towards paternal leukocytes have been used to predict the prognosis for the subsequent pregnancy in women with consecutive recurrent miscarriages (CRM) and also to determine if the patient has become immune after paternal leukocyte immunization. The predictive value is controversial, as these antibodies are not essential for pregnancy to develop, and only occur in a minority of parous women. This study tried to determine the predictive value of these antibodies when assessed separately for women with five or more abortions and compared to women with three or four abortions. The patients were assessed separately so that the higher live birth rate in the latter group would not obscure meaningful results in the former group with a poor prognosis. Antibody production, whether spontaneous, or induced by immunization, raised the live birth rate in primary and tertiary aborters with three, four, five or more abortions. Anti-paternal antibodies increased the proportion of live births from 18.5 to 53.7% (P
0.01) and from 44.4 to 67.5% (P 0.001) in primary aborters with 
5 CRM and 3–4 CRM respectively. Both immunization with paternal leukocytes per se and the ability to express anti-paternal antibodies were associated with an increased proportion of live births in the next pregnancy. Multivariate analysis showed that that the odds ratio for a live birth was approximately four times greater in women who were immunized and produced anti-paternal antibodies than in control patients. The lack of anti-paternal antibodies at initial testing could serve as a marker for the benefit of immunization with paternal leukocytes; the subsequent presence as a prognostic marker for the subsequent pregnancy.
Key words: anti-paternal antibodies/consecutive recurrent miscarriages/immunization
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Recurrent miscarriage is usually defined as the loss of three or more consecutive pregnancies prior to 20 or even 28 weeks of pregnancy (Salat Baroux, 1988
; Crosignani and Rubin, 1991). However, within this definition is a large and heterogeneous group of patients with many different causes of miscarriage. If these patients are taken as a whole, they have a relatively good prognosis, with subsequent live birth rates quoted to be 60–70% (ETEP, 1995; Clifford et al., 1997). With such odds it is difficult to show that any treatment improves the live birth rate, or that any investigation or laboratory test provides a reliable diagnosis of cause. The difficulties in assessing treatment have been widely discussed (Clark and Daya, 1991; Carp, 1996), as have the difficulties in reaching an accurate diagnosis (Hatasaka, 1991). At present, no reliable laboratory test is available to diagnose immunologically mediated miscarriages. Therefore immunomodulation has been used as treatment for all women with three or more unexplained pregnancy losses, and controversy has raged whether immunomodulation, by paternal leukocytes or intravenous immunoglobulin improves the live birth rate.
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.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Patients
Six hundred and fourteen patients who presented to our service with consecutive recurrent miscarriages (CRM) between 1987 and 1998 were assessed. Of these, 167 couples had 5–11 CRM and 447 couples had three or four CRM. The clinical features of each patient and her miscarriages were recorded, paying particular attention to whether the previous miscarriages were primary, secondary or tertiary. Patients were classified as primary aborters if they had no previous live births, secondary if there was a live birth followed by miscarriages. We also classified a third group of patients whom we have termed tertiary aborters (Carp et al., 1994). They had miscarriages followed by a live birth and at least three subsequent miscarriages. Patients having elective terminations of pregnancy prior to or between the spontaneous miscarriages were not included in the trial.
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 3–4 CRM group elected to be in the control group (Table I).
|
Immunization procedure
This procedure has been fully described elsewhere (Carp et al., 1990
). Briefly, paternal leukocyte immunizations were prepared from 100 ml of paternal blood. Approximately 80–85x106 mononuclear cells were harvested and isolated by Ficoll–Hypaque density centrifugation. The leukocytes were resuspended in 4 ml of 0.9% Hartman's solution. Aliquots of 0.5 ml were injected into the forearm at each of four intradermal and two subcutaneous sites. Immunization was repeated after 3–4 weeks. Two weeks after the second immunization the patient's serum was tested for anti-paternal antibodies by cross matching. If positive, the patient was advised to become pregnant. Immunization was boosted when pregnancy was diagnosed. If negative, booster immunizations were performed. Seventy-three women were immunized in the 5 CRM group and 201 women were immunized in the 3–4 CRM group.
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 2–3 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.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Six hundred and fourteen patients on our register had their results assessed: 167 couples had 5–11 CRM and 447 couples had three or four CRM. Four hundred and thirteen women were immunized with paternal lymphocytes, 104 of the
5 CRM group and 309 of the 3–4 CRM group. Two hundred and one patients comprised the control group, 63 of the 5 CRM and 138 of the 3–4 CRM groups respectively (Table I
). The age distribution of patients and time taken to conceive were similar in all the subgroups (Table I). The importance of anti-paternal antibody status per se is depicted in Table II. The presence of anti-paternal antibodies raised the live birth rate from 45.5 to 70.2% (P 0.001), in patients with three or four abortions, and from 29.8 to 50.9% after five or more abortions (P 0.013). This difference was apparent whether anti-paternal antibodies were formed by pregnancy or by immunization. The highest live birth rate was observed among women who had produced anti-paternal antibodies as a result of immunization (70.2 and 55.9% for the 3–4 CRM and 5 CRM groups respectively, Table III). The proportion of live births in women with anti-paternal antibodies at initial testing was 69.6 and 31.6% for the 3–4 CRM and 5 CRM groups respectively (Table III). Both these figures were significantly higher than in anti-paternal antibody negative patients (40.9 and 27.2% for the 3–4 CRM and 5 CRM groups respectively).
|
|
Eighty-one patients were primary, 51 were secondary and 35 were tertiary aborters in the
5 CRM group, and 284, 147 and 16 were primary, secondary and tertiary aborters in the 3–4 CRM group respectively. The influence of anti-paternal antibodies was apparent in primary, secondary and tertiary miscarriage groups (Table IV). Seroconversion from anti-paternal antibody negative to anti-paternal antibody positive was concomitant with an increased proportion of live births from 18.5 to 53.7% (P 0.004) in the 5 primary miscarriage group, and from 44.4 to 67.5% (P 0.001) in the 3–4 primary miscarriage group. This influence was not apparent in the secondary aborters in the 5 CRM group. However, the proportion of live births in the secondary 3–4 CRM group was associated with an increase from 50.7 to 77.8% (P = 0.001) in the women who seroconverted from anti-paternal antibody negative to positive. The same trend was observed in the tertiary CRM group. However, the figures did not reach statistical significance, possibly due to the small number of patients in this group.
|
Table V
summarizes the net effect of the predictive variables for a live birth using logistic regression. Multivariate analysis showed that if there were five or more abortions, the odds ratio for a live birth decreased by 0.47 of that of a patient with three or four abortions. All patients, whether immunized or expressing anti-paternal antibodies, had a greater chance of a live birth than control patients who were anti-paternal antibody negative and not immunized [OR = 2.07 (CI 1.11–3.83), for immunized patients remaining anti-paternal antibody negative, 3.99 (CI 2.61–6.10) for immunized patients who seroconverted; and 2.30 (CI 1.15–4.39) for patients with anti-paternal antibodies at initial testing].
|
The secondary aborters had a higher odds ratio (1.44) for a live birth than the primary aborters, and the tertiary aborters had an odds ratio of 1.09 for a live birth compared to the primary aborters. The odds ratio was 0.97 less for each additional month taken to conceive.
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.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
This work investigated the presence of anti-paternal antibodies and its bearing on the outcome of subsequent pregnancies in women with recurrent miscarriages. Expression of anti-paternal antibodies was positively associated with a higher live birth rate, whether induced by immunization or previous pregnancies, demonstrating the importance of the production of antibodies on the outcome of pregnancy in immunized patients. Another independent variable influencing the prognosis for a live birth was the number of abortions. This analysis confirms our previous finding (Carp, 1997
) and others (Daya and Gunby, 1994) that patients with five or more miscarriages have a poorer prognosis than patients with three or four miscarriages. Both groups (Daya and Gunby, 1994; Carp, 1997) have shown that the subsequent live birth rate decreases as the number of previous miscarriages increases. Additionally, these patients are less likely to have their results influenced by confounding factors (Christiansen, 1994). Therefore, the results of patients with five or more miscarriages were analysed separately from those of patients with three or four miscarriages. The difference in prognosis between the patients with three to four and five or more miscarriages demonstrates the importance of analysing these patients separately and not as a single homogeneous group. However, placing the boundary between the `good' and `poor' prognosis groups between four and five miscarriages is somewhat arbitrary. We chose this cut-off because there were enough patients with five or more miscarriages to allow statistical analysis. However, even patients with five or more abortions do not constitute a homogeneous group. The results were not corrected for possible fetal karyotypic abnormalities. These abnormalities were reported to cause up to 60% of recurrent miscarriages reported (Stern et al., 1996) and 35% of recurrent miscarriages in our preliminary (unpublished) series. Additionally, the results could not be corrected for the other numerous conditions that have been associated with pregnancy loss, including those described previously (Wheeler, 1991; ETEP, 1995), inappropriate luteinizing hormone, thrombophilias such as activated protein C resistance, homocysteinaemia, methyl tetrahydrofolate reductase (MTHFR) deficiency, and heavy metal poisoning (Gerhard et al., 1998). Hence, the results of the present study could be affected by karyotypic abnormalities, or some of the factors listed above. 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.
|
Notes |
|---|
4 To whom correspondence should be addressed
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Aoki, K., Kajiura, S., Matsumoto, Y. et al. (1995) Preconceptional natural-killer-cell activity as a predictor of miscarriage. Lancet, 345, 1340–1342.
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, 987–999.
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, 143–146.
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, 193–197.
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, 261–274.
Carp, H.J.A. (1996) Pitfalls in interpreting therapy for early pregnancy. Early Pregnancy, 2, 96–101.
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, 16–21.
Carp, H.J.A., Toder, V. and Mashiach, S. (1993) Immunotherapy of habitual miscarriage. Am. J. Reprod. Immunol., 28, 281–284.
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, 360–362.
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, 250–255.
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, 793–796.
Christiansen. O.B. (1996) A fresh look at the causes and treatments of recurrent miscarriage, especially its immunological aspects. Hum. Reprod. Update, 2, 271–293
Clifford, K, Rai, R. and Regan, L. (1997) Future pregnancy outcome in unexplained recurrent first trimester miscarriage. Hum. Reprod., 12, 387–389.
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 9–11, 1990. Hum. Reprod., 6, 609–610.
Daya, S. and Gunby, J. (1994) The effectiveness of allogeneic leukocyte immunization in unexplained primary recurrent spontaneous miscarriage. Am. J. Reprod. Immunol., 32, 294–302.
ETEP (1995) Euro-Team Early Pregnancy (ETEP) protocol for recurrent miscarriage. Hum. Reprod., 10, 1516–1520.
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, 301–309.
Hatasaka, H.H. (1991) Recurrent miscarriage: epidemiological factors, definitions and incidence. Clin. Obst. Gynecol., 37, 625–634.
Hill, J.A., Polgar, K., Anderson, D.J. (1995) T-helper 1-type immunity to trophoblast in women with recurrent spontaneous abortion. JAMA, 273, 1933–1936.
Huang, Y.T., Hsieh, Y.S. Phan, M.C. et al. (1997) Evaluation of the HLA antibodies in placental blood. Clin. Lab. Haematol., 19, 273–276.
Komlos, L., Zamir, R., Joshuah, H. et al. (1977) HLA antigens in couples with repeated miscarriages. Clin. Immunol. Immunopathol., 7, 330–335.
Mittal, K.K., Mickey, J.F., Singall, D.P. et al. (1968) Serotyping for homotransplantation. XVIII. Refinement of microdroplet lymphocyte cytotoxicity test. Transplantation, 6, 913–927.
Mowbray, J.F., Gibbings, C.R., Sidgwick, A.S. et al. (1983) Effects of transfusions in women with recurrent spontaneous abortion. Transplant. Proc., 15, 896–899.
Oksenberg, J.R., Persitz, E. and Amar, A. (1984) Maternal-paternal histocompatibility: lack of association with habitual miscarriages. Fertil. Steril., 42, 389–395.
Raghupathy, R. (1997) ThI-type immunity is incompatible with successful pregnancy. Immunol. Today, 18, 478–482.
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. 23–37.
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, 1555–1568.
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, 250–253.
Taylor, C. and Page Faulk, W. (1981) Prevention of recurrent miscarriage with leukocyte transfusions. Lancet, ii, 68–69.
Wheeler, J.M. (1991) Epidemiologic aspects of recurrent pregnancy loss. Infertil. Reprod. Med. Clin. N. Am., 2, 1–17.
Submitted on March 11, 1999;
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||