Unexplained Recurrent Abortion Research Articles

T-Helper 1-Type Immunity to Trophoblast in Women With Recurrent Spontaneous Abortion

To test the hypothesis that peripheral blood mononuclear cells in women with unexplained recurrent abortion (URA) produce T-helper 1 (TH1)-type cytokines in response to trophoblast antigens. Cohort study. Medical center. A total of 244 women with URA, 13 reproductively normal parous control women, and 10 men. Supernatants from trophoblast-activated peripheral blood mononuclear cells from all participants were tested for toxic effects on mouse embryos and by enzyme-linked immunosorbent assay (ELISA) for interferon gamma (IFN-gamma). Supernatants from 20 URA patients with embryotoxic activity and IFN-gamma, 13 reproductively normal parous women, and 10 men were further tested by ELISA for other TH1-type cytokines (interleukin-2 [IL-2], tumor necrosis factor-beta [TNF-beta]), TH2-type cytokines (IL-4, IL-10), and TNF-alpha. Embryotoxic activity was detected in supernatants from 160 of 244 URA patients and in none of the controls. Interferon gamma was detected in supernatants from 125 of 244 URA patients and was significantly associated with embryotoxicity (121 of 160 supernatants with embryotoxicity vs four of 84 supernatants without embryotoxicity [P < .001]). Of 20 supernatants from patients chosen for further study, all were positive for TNF-alpha, 17 for TNF-beta, two for IL-10, and one for IL-4. No cytokines were detected in supernatants from unstimulated or red blood cell membrane-activated cells of women with URA. In contrast, trophoblast-activated lymphocyte supernatants from reproductively normal women and men neither were embryotoxic nor contained TH1-type cytokines, but most contained the TH2-type cytokine IL-10. Three supernatants from reproductively normal women also contained IL-4. Whereas TH1-type immunity to trophoblast is associated with URA and may play a role in reproductive failure, TH2-type immunity may be a natural response to trophoblast contributing to successful pregnancy.

Preconceptional natural-killer-cell activity as a predictor of miscarriage

There is no immunological test for the prospective identification of alloimmune causes of miscarriage. We investigated whether activity of natural killer cells was predictive of subsequent abortion in women who had had unexplained recurrent abortions and had received no treatment. 24 women with high preconceptional NK activity defined as mean plus 1 SD of NK activity of 47 controls, had a significantly higher abortion rate in the next pregnancy than 44 women with normal levels of NK activity (71 vs 20%; relative risk 3·5; 95% CI 1·8–6·5).The preconceptional evaluation of NK activity in women with recurrent miscarriages may thus be predictive of the risk of pregnancy loss at the next conception.

Pregnancy: Seminal white blood cells and recurrent abortion*

The incidence of leukocytospermia (> 1 million white blood cells/ml semen) and concentrations of white blood cell populations in semen from men whose partners experience unexplained recurrent spontaneous abortion by conventional testing criteria are described and compared to those of men whose partners have normal reproductive histories. Comparable seminal granulocyte and total white blood cell concentrations between groups suggest that couples with unexplained recurrent abortion do not have an abnormally high incidence of subclinical genital tract infections for which leukocytospermia could serve as a marker. However, a significantly higher prevalence of elevated CD4+ and CD8+ T lymphocyte concentrations in semen from men whose partners have cellular immunity to sperm antigens (i.e. produce embryotoxic factors in response to sperm antigen stimulation) warrants further investigation.

Effects of Intravenous Immunoglobulins (IVIG) on Peripheral Blood B, NK, and T Cell Subpopulations in Women with Recurrent Spontaneous Abortions: Specific Effects on LFA-1 and CD56 Molecules

Polyspecific IgG given intravenously at high dose (IVIG) are increasingly used as an immunomodulating therapy in autoimmune diseases. However, very few studies have dealt with the action of IVIG on the expression of the leukocyte markers. During a clinical trial in which 13 young and healthy women received IVIG to prevent unexplained recurrent abortions we have evaluated by flow cytometry the action of IVIG on 17 clusters of leukocyte differentiation (CD). We found that the IVIG perfusions (0.5 g/kg) induced an increase in the number of polymorphonuclear and monocyte cells in the peripheral blood. This effect lasted 8 days. The IVIG treatment had no effect upon T cell populations stained with antibodies specific for CD2, CD3, CD4, CD8 and on CD4 +CD45RA +, CD4 +CD29 + ,CD8 +CD28 +, CD8 +CD28-subpopulations. A weak decrease in the B cell number was observed. The most striking phenomenon was the decrease in the number of CD56 + cells, whereas CD16 + and CD57 + cells were unaltered. By the double-staining technique we showed that CD56 +CD16 + cells became CD56 -CD16 + cells. Moreover, IVIG decrease the expression level of the LFA-1 molecule on monocytes and lymphocytes. The other adhesion molecules studied remained steady (CD11b, CD49d, CD49e, CD29, CD28, and CD62L). This study has shown that IVIG have no effect on 15 of 17 CD used but downmodulate two adhesion molecules playing a key role in the immune system.

Detection of Chlamydia trachomatis by the polymerase chain reaction in the cervices of women with acute salpingitis

OBJECTIVE: Our objective was to determine whether an increased prevalence of Chlamydia trachomatis could be detected by the polymerase chain reaction as opposed to culture in the cervices of women with acute salpingitis. STUDY DESIGN: Endocervical samples from 15 women with laparoscopy-verified acute salpingitis and 20 women seeking medical help for conditions other than pelvic pain were tested for Chlamydia trachomatis with the polymerase chain reaction. The oligonucleotide primer pairs used were specific for a 144 bp region of the major outer membrane protein that contained a single Eco RI endonuclease cleavage site. The detection of a 144 bp band that was cleaved by Eco RI to a 103 bp band denoted a Chlamydia trachomatis -positive sample. Cervical samples were cultured for Chlamydia trachomatis with the use of McCoy cells. The lymphocyte proliferative response to Chlamydia trachomatis elementary bodies was also determined. RESULTS: Nine of the 15 women (60%) with salpingitis had positive results when tested with the polymerase chain reaction for cervical Chlamydia trachomatis . Only two of these women (13%), both of whom had positive results when tested with the polymerase chain reaction, had cultures that were positive for Chlamydia trachomatis ( p Chlamydia . Those women plus two women with unexplained recurrent abortions had positive polymerase chain reaction test results for Chlamydia trachomatis . A lymphocyte proliferative response to Chlamydia trachomatis was detected in five of eight women with salpingitis, as well as three of the other four patients, all of whom had positive polymerase chain reaction test results; lymphocytes from the remaining women were unresponsive. Follow-up cervical samples were obtained 4 to 6 months after treatment from six of the patients with salpingitis who had positive polymerase chain reaction test results; at that time five had negative polymerase chain reaction test results for Chlamydia trachomatis . CONCLUSION: The polymerase chain reaction appeared to be more sensitive and more rapid than culture in detecting Chlamydia trachomatis in the cervices of women with acute salpingitis. This assay may be of value for the early diagnosis of chlamydial infections.

Anticardiolipin and Antinuclear Antibodies in Patients with Unexplained Recurrent Abortions

In a controlled study on a resident population in Kuwait, 103 patients with unexplained recurrent spontaneous abortions were investigated for the frequency of positive tests for anticardiolipin antiobodies (ACA) and antinuclear antibodies (ANA). The frequency of ACA was significantly (P<0.001) higher in patients (32.0%) than in controls (7.1%). Only four patients (3.8%) were positive for both IgG and IgM ACA. The frequency of positive tests for ANA was also significantly (P<0.001) higher in patients (13.6%) than in controls (1.2%). No difference was found between first and second trimester aborters in the frequency of positive tests for either ACA or ANA. Primary aborters did not differ from secondary aborters in the frequency of ACA. However, secondary aborters had significantly higher frequency of ANA. There was no concordance between ACA and ANAN positive tests. These results suggest that ACA may be associated with some cases of unexplained recurrent abortions and that tests for both IgG and IgM isotypes should be carried out in the investigations of these patients.

Antibodies to phospholipids and nuclear antigens in non-pregnant women with unexplained spontaneous recurrent abortions

In a collaborative study of 73 non-pregnant Kuwaiti women with unexplained spontaneous recurrent abortion (RSA), 30 control healthy non-pregnant multiparous Kuwaiti women and 20 North American women who received elective abortion(s), autoantibodies to 6 phospholipids and 9 nuclear antigens were measured. Women with recurrent spontaneous abortions demonstrated 3 times higher incidence of antibodies to phospholipids (30.1%) than controls (10% each) ( P = 0.029). The incidence of both IgM and IgA class antiphospholipid antibodies were significantly higher than those of controls. The incidence of antibodies to cardiolipin in women with recurrent spontaneous abortions (12.3%) was significantly higher than those of controls ( P = 0.035) and incidence of IgM but not IgG anticardiolipin antibody was significantly higher in women with RSAs than in controls ( P = 0.053). The incidences of anti-polyinosinic acid ( P = 0.035) and anti-histone I antibody ( P = 0.052) were significantly higher in women with recurrent spontaneous abortions than controls. There was no significant difference in the incidence of autoantibodies between primary and secondary aborters. However, women with a history of second trimester abortions showed a higher incidence of antiphospholipid antibodies than women with first trimester abortions only. Recurrent spontaneous abortion is associated with autoantibodies to phospholipid epitopes including IgA antiphospholipid antibodies.

Detection of Chlamydia trachomatis by the polymerase chain reaction in the cervices of women with acute salpingitis

Our objective was to determine whether an increased prevalence of Chlamydia trachomatis could be detected by the polymerase chain reaction as opposed to culture in the cervices of women with acute salpingitis. Endocervical samples from 15 women with laparoscopy-verified acute salpingitis and 20 women seeking medical help for conditions other than pelvic pain were tested for Chlamydia trachomatis with the polymerase chain reaction. The oligonucleotide primer pairs used were specific for a 144 bp region of the major outer membrane protein that contained a single EcoRI endonuclease cleavage site. The detection of a 144 bp band that was cleaved by EcoRI to a 103 bp band denoted a Chlamydia trachomatis-positive sample. Cervical samples were cultured for Chlamydia trachomatis with the use of McCoy cells. The lymphocyte proliferative response to Chlamydia trachomatis elementary bodies was also determined. Nine of the 15 women (60%) with salpingitis had positive results when tested with the polymerase chain reaction for cervical Chlamydia trachomatis. Only two of these women (13%), both of whom had positive results when tested with the polymerase chain reaction, had cultures that were positive for Chlamydia trachomatis (p < 0.02). Among the 20 other women, only two patients with cervicitis had positive cultures for Chlamydia. Those women plus two women with unexplained recurrent abortions had positive polymerase chain reaction test results for Chlamydia trachomatis. A lymphocyte proliferative response to Chlamydia trachomatis was detected in five of eight women with salpingitis, as well as three of the other four patients, all of whom had positive polymerase chain reaction test results; lymphocytes from the remaining women were unresponsive. Follow-up cervical samples were obtained 4 to 6 months after treatment from six of the patients with salpingitis who had positive polymerase chain reaction test results; at that time five had negative polymerase chain reaction test results for Chlamydia trachomatis. The polymerase chain reaction appeared to be more sensitive and more rapid than culture in detecting Chlamydia trachomatis in the cervices of women with acute salpingitis. This assay may be of value for the early diagnosis of chlamydial infections.

Influence of Immunotherapy on Antisperm Antibody Titer in Unexplained Recurrent Aborters

The mechanism of the beneficial effect of immunotherapy for human reproductive wastage remains to be elucidated. Because some women with unexplained recurrent spontaneous abortion are immunized with their partner's lymphocytes, it is important to determine whether such immunization results in elevation or enhancement of immunity to spermatozoa, because antigenic cross-reactivity between lymphocytes and spermatozoa has been reported. The present study was initiated to evaluate the changes in antisperm antibody titer and lymphocyte subsets after immunotherapy as compared to before immunotherapy. Antisperm antibody detection was performed by SpermCheck Assay, which is based on a modification of the immunobead test. Maternal lymphocyte subsets were analyzed in two-color flow-cytometric experiments. The percentage of antibody-positive sperm decreased significantly (P = 0.0008) after immunotherapy. The percentage of B(CD19+) cells (P = 0.0003), cytotoxic T(CD8+ and CD11b-) cells (P = 0.02) and the Th/Ts ratio (P = 0.005) decreased significantly, while suppressor T(CD8+ and CD11b+) cells increased significantly (P = 0.0002) after the immunotherapy. This suggests that cell-mediated immunosuppression was induced by immunotherapy. The data of the present study suggest that antisperm antibodies have potential for use as a marker for a deficiency in maternal genital tract immunosuppressor mechanisms and that immunotherapy could be an effective treatment for women with antisperm antibodies who have unexplained recurrent abortions.

Is an additional vaccination necessary for a successful second pregnancy in unexplained recurrent aborters who were successfully immunized with their husband's lymphocytes before the first pregnancy?

Is an additional immunotherapy necessary or not for patients who have obtained successful results after initial immunotherapy? The successive pregnancy outcome was analyzed in 22 patients out of 29 unexplained recurrent aborters who had undergone immunotherapy with their husband's lymphocytes according to our previously reported protocol (Takakuwa et al., Am J Reprod Immunol Microbiol. 10:1-9, 1986; Takakuwa et al., Am J Reprod Immunol. 23:37-41, 1990) and had obtained successful outcome between January 1983 and December 1989. In addition, the alteration of blocking antibodies (BAbs), which was evaluated by a one-way mixed lymphocyte culture reaction (MLR) blocking assay between the spouses, was analyzed in 26 patients out of 29. None of the 22 patients underwent further immunotherapy because a significantly high titer of MLR-BAbs had been detected before the new pregnancy. In 19 out of the 21 patients (90.5%), pregnancy was successful. Additional immunotherapy is not necessary for patients who have obtained successful results after the initial immunotherapy and are positive for MLR-BAbs after their first delivery.

The role of blocking factors and antipaternal lymphocytotoxic antibodies in the success of pregnancy in patients with recurrent spontaneous abortion

To elucidate the role of mixed lymphocyte reaction blocking factors (BF) and complement-dependent antipaternal lymphocytotoxic antibodies on the outcome of pregnancy in unexplained recurrent spontaneous aborters. A controlled study of immunotherapy in which the treated group was immunized with the husband's or a third party donor's lymphocytes and the control group received autologous lymphocytes. Tertiary care institution. Forty-three patients in the control group and 48 patients in the treated group. The before and after immunization levels of BF and antipaternal lymphocytotoxic antibodies were measured. The existence or changing pattern of BF and antipaternal lymphocytotoxic antibodies levels before and after immunization had no influence on the pregnancy outcome in either group of patients. Neither BF nor antipaternal lymphocytotoxic antibodies is essential for successful pregnancy. They probably reflect the immunological response of the mother to exposure to fetal antigens.

Analysis of HLA-DR types of unexplained recurrent spontaneous aborters in the Japanese population by oligonucleotide-DNA typing.

To examine whether unexplained recurrent spontaneous abortion (URSA), defined as 2 or more consecutive spontaneous abortions, is correlated with a particular DR type in the Japanese population, we determined the HLA-DR types of 82 primary aborters and 21 secondary aborters by DNA typing utilizing the polymerase chain reaction (PCR) and hybridization with sequence-specific oligonucleotides (SSOs). The DR gene frequencies of the patient group were compared with those of a normal group at three different levels of DR-definition (27, 13 and 11 DR types). At none of the three levels of comparison was any particular DR type with a frequency differing significantly between the patient and normal groups detected in Japanese URSA patients. Furthermore, we examined whether URSA was correlated with the degree of compatibility of HLA-DR antigen within patients and their husbands. Comparison of the DR compatibility between patients and normal couples was made in two different ways, i.e., comparison of the numbers of couples with mismatches and comparison of the average number of mismatches. For either of these two comparisons, we observed no difference in DR compatibility between patients and normal couples. Our results suggest that URSA is not correlated with any particular DR type and that the condition cannot be explained simply by DR compatibility between husband and wife.

Significant Compatibility Does Not Exist at the HLA‐DQB Gene Locus in Couples With Unexplained Recurrent Abortions

The polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) method was used for both examining compatibility at the HLA-DQB1 gene locus and determining HLA-DQ antigen polymorphism in spouses of unexplained recurrent abortions. Genomic DNA samples were prepared from peripheral mononuclear cells from patient and control couples. Two hundred and thirty base pair fragments of the second exon of the HLA-DQB genes were selectively amplified. Amplified DNAs were digested with the restriction endonucleases, Fok I, Hae III, Hha I, Rsa I and Sau3A I, and subjected to electrophoresis in a polyacrylamide gel. The RFLPs showed that habitual aborters and their husbands had neither significantly frequent alleles nor shared common alleles at the HLA-DQB locus when compared to the control group. Since significant HLA-DQB compatibility was not observed between the spouses and unexplained recurrent aborters, in order to determine whether or not HLA compatibility is responsible for the genesis of unexplained recurrent abortions, it is imperative to further examine the compatibility between other HLA gene loci.

Immunotherapy with paternal lymphocytes preceding in vitro fertilization-embryo transfer for patients with repeated failure of embryo transfer

Immunotherapy with paternal lymphocytes for unexplained recurrent aborters was applied, preceding IVF-ET, for infertile women with repeated failure of ET. In one patient showing close histocompatibility with the husband, the subsequent new IVF-ET was successful under positive MLR-blocking antibody induced by immunotherapy.

Influence of immunotherapy on the cellular immunity of unexplained recurrent aborters

Changes in lymphocyte subsets in whole blood were analyzed sequentially by flow cytometry with an automated leukocyte differential system in 15 patients with unexplained recurrent spontaneous abortions, each of whom underwent vaccination(s) with her husband's lymphocytes. Mitogen responses of peripheral blood lymphocytes (PBL) were also examined in these patients. The reactivity of PBL against mitogens revealed no significant change in each patient before and after vaccination(s) with her husband's lymphocytes. The CD4:8 ratio was observed to decrease significantly during 22 and 28 days after the first vaccination with a significant increase in the percentage of T suppressor-cytotoxic (CD8) cells. This change was also observed after the second vaccination. The percentages of other subsets did not change significantly after vaccination(s). In 11 patients out of 15, the pregnancy continued successfully and correlated with a predominance of Ts/c (CD8) over TH/1 (CD4) cells in the first trimester. These changes in lymphocyte subsets may indicate the induction of immune enhancing mechanisms and it is suggested that continuation of the predominance of Ts/c cells induced by immunotherapy might be important for the successful maintenance of pregnancy.

Result of Immunotherapy on Patients With Unexplained Recurrent Abortion: A Beneficial Treatment for Patients With Negative Blocking Antibodies

Thirty-nine unexplained recurrent aborters underwent vaccination using husband's lymphocytes according to the previously reported protocol. No mixed lymphocyte culture reaction-blocking antibodies (MLR-BAbs) were observed in these patients prior to vaccination. Of 35 newly pregnant patients after vaccination(s), pregnancy successfully continued in 28 (80.0%) and have already been terminated with a liveborn offspring. Pregnancy outcome was also analyzed in unexplained recurrent aborters who revealed positive MLR-BAbs without immunotherapy. In this group, out of eight pregnancies in seven patients, five (62.5%) continued beyond their critical period of 14 wks of gestation. Three infants born from these pregnancies, however, presented severe abnormalities. Furthermore, outcome of 14 pregnancies in 12 unexplained recurrent aborters with negative MLR-BAbs was analyzed since they had become pregnant without immunotherapy; pregnancy was successfully continued in only four cases (28.6%). Thus, vaccination using husband's lymphocytes on unexplained recurrent aborters with negative MLR-BAbs is suggested to be effective. In addition, it is suggested that immunotherapy for patients with positive MLR-BAbs should be carefully followed.

Paternal Mononuclear Cell Immunization Therapy for Repeated Miscarriage: Predictive Variables for Pregnancy Success

Pregnancy outcomes for 125 women with unexplained recurrent abortion conceiving after immunologic testing for possible paternal leukocyte immunization were analyzed. Pregnancy success was related to the number of previous miscarriages (relative risk 0.36 for each additional miscarriage after 3), a history of a late pregnancy loss (relative risk 0.18), any other relevant treated or untreatable diagnosis (relative risk 0.27), immunization with paternal mononuclear cells (relative risk 5.6), and time in weeks from test date to LMP of the next pregnancy (relative risk 0.93 for each additional week). The significant difference in pregnancy outcomes between women given a single immunization and nonimmunized women reflected a larger difference between those in each group conceiving within 12 weeks of initiating tests for inclusion in the treatment program. The latter observation suggests that any effect from a single immunization in prevention of recurrent miscarriage is of relatively short duration.

Autoimmune associated recurrent abortions.

A possible relationship between recurrent spontaneous abortions and autoimmune abnormalities was studied. Eight serological autoimmune or autoimmune-correlated parameters were investigated in 91 women with unexplained recurrent abortions (greater than or equal to 3 consecutive, spontaneous abortions) and 89 fertile control women. Five parameters were seen significantly more frequently in 19 women with at least one second trimester miscarriage which had been associated with severe intrauterine growth regardation (IUGR), than in controls. Seventeen of these 19 patients (89%) had at least one positive autoimmune parameter, compared to 15 of 72 patients (21%) with no second trimester abortions with IUGR (P less than 0.0001) and 14 (16%) of the controls (P less than 0.0001). No single autoantibody characterized patients who exhibited a significant accumulation of autoimmune parameters. These findings may suggest that women with recurrent abortions, in whom autoimmunity is thought to play a role, cannot be identified merely by one laboratory assay, such as that for cardiolipin antibodies, but must be defined by positivity of several criteria. Using our own test panel, preliminary clinical and serological criteria have been set up for the definition of an autoimmune-associated recurrent abortion condition. Twenty-three per cent of the patients in our material fulfilled these criteria, and seven out of nine of these women (78%) have to date been treated successfully with heparin/aspirin during pregnancy.

Cell-mediated immune responses to sperm antigens: effects on mouse sperm and embryos.

Sperm antigens were assessed for their ability to induce cell-mediated immune (CMI) responses. Purified fertilization antigen (FA-1), protamine, and the lithium diiodosalicylate (LIS)-solubilized sperm preparation activated presensitized lymphocytes to secrete soluble mediators that activated macrophages and significantly inhibited sperm motility and embryonic development. The FA-1, however, was the most potent antigen in inducing proliferative response as well as the release of soluble mediators. LIS-sperm preparation, which contained numerous antigens, showed the least activity. The unsensitized control spleen cells did not secrete any factor(s) when activated with the antigen. In conclusion, these results indicate that sperm antigens can specifically induce (CMI) factors that have detrimental effects on sperm motility and preimplantation embryos. These findings may have potential clinical implications for humans, especially in immunologic and unexplained infertility, recurrent abortions, and development of antisperm contraceptive vaccines.

Autoantibodies to soluble cellular antigens in unexplained recurrent abortion and infertility

In 36 women with unexplained primary recurrent abortion, 13 with secondary unexpained recurrent abortion, 25 with primary unexplained infertility, 7 with secondary unexplained infertility and two groups of control women, autoantibodies to soluble cellular antigens were measured by Western blotting to a disaggregated HeLa cell antigen preparation, by counter immunoelectrophoresis and by indirect immunofluorescence. Using Western blotting the women with primary infertility and those with secondary recurrent abortion had a significantly higher prevalence of autoantibodies ( P < 0.01 in each case). This was not shown using the other methods. It is possible that these antibodies could be causally related to the pathology of the conditions studied.