Predictive factors in recurrent spontaneous aborters--a multicenter study.

PROBLEM: Compare data from several centers relating to success rates in recurrent spontaneous miscarriage and assess the significance of indicators of subsequent pregnancy loss. METHOD: Data from 777 couples with unexplained recurrent spontaneous abortion from independent studies at seven centers were analyzed using logistic regression analysis. The following covariates were considered: age of patient, number of previous spontaneous abortions, length of previous abortions history, sub-fertility index (defined as the product of the number of spontaneous abortions and the abortion history), whether a patient was a primary or secondary aborter, and whether a patient had received leukocyte immunotherapy. RESULTS: There was a highly significant difference between the seven centers in success rates in the subsequent pregnancy and a highly significant association between success rate and each of the following covariates: the number of previous abortions, the length of the previous abortion history and the sub-fertility index. In particular, for each increase of 10 units in the value of the sub-fertility index, up to a value of 30, the odds in favor of a successful pregnancy decreased by a factor of 0.6, i.e., 40%. There was, however, little evidence of an association between the success rate in the subsequent pregnancy and age, parity, or immunization with cells from the husband. CONCLUSIONS: The sub-fertility index may be a useful measure of likelihood of success in a subsequent pregnancy.

Reference ranges for haematology parameters in pregnancy derived from patient populations.

Reference ranges for several haematology parameters in pregnancy were determined by the mathematical detection of Gaussian and Gamma distributions in partitioned but unselected patient data. For each trimester, red cell parameters were shown to be well described by Gaussian distributions. Platelet and white cell parameters were best described by Gamma distributions with the exception of eosinophil and basophil counts for which neither distribution was applicable. The reference ranges derived for each trimester are compared.

DNA methylation patterns of the gamma delta beta-globin genes in human fetal and adult erythroid tissues.

An investigation of the correlation between the gamma----beta-globin switch and DNA methylation was carried out. The restriction patterns obtained with methylation-sensitive and -insensitive enzymes indicated hypomethylation in the promoter region of the gamma-globin genes in fetal liver DNA but high methylation of the same region in all other samples (except in the presence of an elevated erythroblast count or leukemia). All samples appeared to be partially hypomethylated at the 5' end of the delta-globin gene and hypomethylated at the 3' region of the beta-globin gene. Although consistent with a role for DNA methylation in globin gene regulation, the results also suggest that other factors besides methylation may be required for regulation of the level of expression, and switching of the globin genes.

DNA polymorphisms of the human CD46 gene in fetal and adult tissues.

DNA polymorphisms of the CD46 gene were investigated using a full length cDNA probe and five different restriction enzymes in genomic DNA samples from human fetal tissues and adult peripheral blood leukocytes. A HindIII polymorphism was observed and was classed as Types I, II and III at a ratio of approximately 3.5:2:1, depending on the presence or absence of two polymorphic fragments of 4.5 and 2.0 kb. No variation of polymorphic distribution was evident between the fetal and adult samples. Whether variation in CD46 expression between maternal and fetal tissue is relevant to fetal viability in pregnancy remains to be established.

Predictors of pregnancy success in repeated miscarriage.

Factors that may have a bearing on subsequent pregnancy success or failure in patients with recurrent abortion were examined in 165 women with a history of three or more consecutive miscarriages in the first trimester. The overall success rate was 67.9%. Factors that were found to correlate significantly with success rate were length of abortion history, total number of abortions, interval from last miscarriage to present pregnancy, and whether there was any degree of subfertility. Logistic regression analysis showed that the abortion x years index and maternal age accounted for all the variation observed in our data. Where all other known causes of abortions are excluded, recurrent aborters can be subdivided into two populations--namely, those with a relatively good prognosis characterized by a short abortion history and absence of subfertility problems, compared to those with a poor prognosis namely those with a long abortion history or presence of subfertility problems. These data clearly demonstrate major differences in success rates in women depending on the number of abortions and the length of abortion history (abortion x year index), particularly in women over the age of 30 years.

Treatment of recurrent aborters by immunization with paternal cells--controlled trial.

A paired sequential trial was undertaken to establish whether paternal mononuclear cells improved the prognosis in couples with recurrent abortions. For this purpose, 10(7)-10(8) cells obtained from the blood of partners were injected intravenously, subcutaneously, and intra-dermally into women who had had three or more consecutive miscarriages with the same partner. Control women were given normal saline, injected in the same manner. The result of the sequential analysis showed that there was no significant beneficial effect of the cells compared to control. The overall success rate was 70% (32/46 couples). The success rate in patients given cells was 62% (13/21), while in those given saline it was 76% (19/25). While the overall success rate in this study compares with a number of other studies, we find an equally high success rate with non-immunized patients. We conclude that the value of immunization for the prevention of recurrent miscarriage has not been established.

Direct detection of beta thalassemic mutations: use of biotin-labelled allele specific probes.

Mutations at positions beta IVS1-6, beta IVS1-110, and beta 39 of the beta globin gene are responsible for the three most common thalassemic genes in the Mediterranean population. The polymerase chain reaction (PCR) was employed to amplify a 536 base pair segment surrounding this region. Nonradioactive labelling of an oligonucleotide probe, specific for the beta IVS1-110 mutation, was achieved by incorporation of biotin-16-dUTP into a standard 3'-end labelling procedure. This probe was subsequently hybridized with the PCR amplification product and permitted detection of the mutant gene in a homozygous beta thalassemic child by a simple colour detection method using a streptavidin-alkaline phosphatase conjugate and NBT/BCIP (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) substrate. A known cloned mutant gene was similarly detected. Results could be obtained within 48 hr. These findings suggest that such an approach could provide a rapid and specific means for detection of beta thalassemic mutations without the need for radioactive probes.

The lupus anticoagulant, anticardiolipin antibodies, and recurrent miscarriage.

The presence of antibodies to cardiolipin and the lupus anticoagulant were investigated in 189 couples with recurrent abortion. Abnormal coagulation results as measured by an elevation of the APTT or KCT were found in 6% and 7% of patients, respectively; however, the lupus anticoagulant was detected in only 3% of patients. Anticardiolipin antibody levels higher than normal were found in 15% of patients. There was no significant difference between parameters in primary as compared to secondary recurrent aborters or subfertile compared to fertile recurrent aborters. The success of the next pregnancy could not be predicted by any of the laboratory tests.

Point mutations at codon 12 of C-K-ras in human endometrial carcinomas.

Samples of human endometrial carcinomas of ranging degree of histological differentiation, and normal endometrial tissues were screened for the presence of single site DNA mutations at codon 12 of the C-K-ras gene using oligonucleotide hybridisation of DNA amplified by the polymerase chain reaction (PCR). Two instances of base substitution were detected; one resulting in a GLY-ALA amino acid replacement in a well differentiated (grade 1) tumour and the second causing a GLY-CYS replacement in a poorly differentiated (grade 3 tumour). The small number of samples tested does not permit conclusions to be drawn with respect to correlations between DNA point mutations and the clinical features, age of patient, tumour receptor status and degree of histological differentiation grading.

Identification of the major sources of error in estrogen receptor measurements for individual laboratories using both tissue and cytosol samples.

The major sources of error between laboratories performing estrogen receptor measurements in tissue samples were identified for 17 participating laboratories in a trial conducted by the Australasian Quality Assurance programme. Both tissue and cytosol samples were provided, and the In-House assays were compared with the ER-EIA kit (Abbott Laboratories, U.S.A.) as a reference assay. For both the In-House and Abbott assays, tissue samples resulted in a between laboratory CV of about 55% and a within laboratory CV of about 30%. In contrast to tissue samples, the between laboratory CV for cytosol samples was reduced to 41% for the In-House assays and to 33% for the Abbott assay, whereas the within laboratory CV was reduced to 10% for both types of assay. The different methods of tissue homogenization by themselves were not found to be sources of error, and protein extraction efficiency from tissue was strongly correlated with protein measurement (P less than 0.0005). The major sources of error due to protein measurement, cytosol preparation, In-House and Abbott assays were evaluated for individual laboratories. The results indicated absence of any major sources of error for four laboratories, while one, two and three or more sources were indicated for seven, three and three laboratories respectively. The conclusion that about half the participants need to improve their ER assays was confirmed by three independent reviews. Furthermore, the trial demonstrated that tissue samples are essential as quality assurance material for a realistic assessment of ER assays in biopsy specimens.

Sources of improvement in between laboratory variation in estrogen and progestin receptor measurement using tissue samples during the Australian Quality Assurance Programme.

Tissue and cytosol samples were compared as quality control material for assessment of between and within laboratory error in measurement of estrogen and progestin receptors (ER and PR) in a series of four trials for a total of 17 participating laboratories during the Australasian Quality Assurance Programme. For tissue samples, a substantial reduction in between laboratory CVs for both ER and PR from about 90 to 50% was achieved during the programme. In contrast, for cytosol samples a substantially lower between laboratory CV of about 30% was obtained. Tissue sample heterogeneity could be excluded as a major source of variation between laboratories. The likely source of the observed improvement in CV for tissue samples during the trials was due to a reduction of the initial under-estimation of receptor concentration in tissue samples by some of the participants. Although cytosol preparation from tissue samples was shown to be one major source of error, other sources of error such as the receptor assay itself and the associated protein measurements were identified. It is concluded that fragmented tissue samples are essential for a realistic assessment of between laboratory error in receptor measurements in biopsy material such as obtained from clinical breast cancer samples.

Histocompatibility antigens and habitual abortion.

Eighty-five couples with habitual abortion were studied: 43 were primary aborters (i.e., had no advanced pregnancy beyond 20 weeks gestation), and 42 were secondary aborters. The only significant finding in primary female aborters was a reduction in the incidence of B35. In contrast, in secondary female aborters there was a reduction in the incidence of DR3 and A1/B8/DR3 and an increase in the degree of homozygosity of HLA B. In the male partners of primary aborters, there was a reduced incidence of DR3 and A1/B8/DR3. In addition, in male partners of both primary and secondary aborters, there was increased HLA-B homozygosity and increased frequency of DR4-5 phenotype. Increased sharing of HLA antigens between partners was not found to be significantly different from the control population. These findings emphasize that primary and secondary female aborters form distinct populations, which could explain differences in immunological responses to various antigens, including fetal-related antigens. The closely related findings in female secondary aborters and male partners of primary aborters are intriguing but cannot be explained. Whether or not these genetic markers bear any relationship to reproductive success remains to be determined.

Immunogenetic studies in habitual abortion.

Recent evidence suggests that immunological factors could play a role in the aetiology of habitual abortion. In this study, 71 couples with habitual abortion (i.e. more than 3 successive abortions) were investigated. Sharing of HLA-A, B, DR between partners was not found to be significantly increased. Anti-HLA antibodies in women against their partners' lymphocytes were found in 18.3%. After immunization with 10(7)-10(8) lymphocytes obtained from husband or unrelated donor, there was a seroconversion rate of 61% in those without antibodies initially. This was not dependent on the number of cells injected, source of cells, degree of sharing of HLA antigens or whether the couple were primary aborters (i.e. having had no advanced pregnancy), or secondary aborters. Seven pregnancies have resulted following the immunization procedure so far, 5 occurring in cross-match positive patients and 2 in cross-match negative. Growth retarded infants resulted from the pregnancy of 1 primary aborter and 2 secondary aborters.

Racial variations in obstetric practice.

This study involves a retrospective analysis of 453 pregnant persons, with the aim of comparing certain disorders of pregnancy as well as infant and placental parameters in various racial groups within the same community. Significant variations were seen in the mean age of the patients, age at first pregnancy, frequency distribution of first pregnancy, infant weight as well as gravida: parity ratio. There was a 3-fold increase in incidence of preeclampsia in the Australian-born population compared to other racial groups. Mild anaemias (haemoglobin less than 11.5 g/dl) were found in up to 61% of the Australian-born population compared to 32% of the other racial groups; however, more significant degrees of anaemia were more commonly found in certain ethnic groups (e.g. Greek 16%, Italian 15%, Australian-born 6%). These studies emphasize that overall incidence studies in a polyglot population can have very limited meaning, and that greater attention must be paid to the actual racial variations within a population.

Changes in lymphocyte status and responsiveness in pregnancy detected by a fluorescent cell probe.

Lymphocyte reactivity assessed by a fluorescent lipophilic probe test of responsiveness to concanavalin A (con A) was shown to differ from normal in early pregnancy. The difference was most marked in multiparas. Abnormal reactivity was detected in the earliest pregnancy examined (5 weeks' gestation) and up to about the 20th week; after 20 weeks, reactivity was normal in all of the multiparas and most of the nulliparas studied. However, in pregnancy induced hypertension (PIH), a disorder of late pregnancy, the same responsiveness as in early pregnancy was found. When unstimulated lymphocytes were examined, abnormal reactivity associated with increased fluorescence was observed in early pregnancy and in PIH, compared with normal late pregnancy, reflecting alteration in lymphocyte membrane phospholipids. It is postulated that pregnancy is associated with sequential change in immunity, disturbance of which may result in immunologically-determined obstetric morbidity.

The immunoglobulin class of antispermatozoal antibodies in serum.

The aim of this study was to determine the immunoglobulin class of circulating antisperm antibody using a technique called the indirect immunobead test (IBT). In the IBT sperm bound antibodies are detected using polyacrylamide beads coated with rabbit antihuman immunoglobulin classes IgG, IgA, and IgM. Of the 20 infertile men with serum immobilizins, 100% were found to be positive for sperm-bound IgG, 50% positive for IgA, and 0% positive for IgM, using the IBT. Similarly, 20 infertile females with serum immobilizins showed 95% positivity for IgG, 60% for IgA, and 15% for IgM. Thus there was a good correspondence between the presence of serum immobilizins as determined by the sperm immobilization test (SIT) and the IBT. This study provides data that indicates that IgG and IgA are the two major immunoglobulin classes of sperm antibody in male and female immune sera as detected by a simple, sensitive immunological technique, the serum IBT.

Increased frequency of autoantibodies in men with sperm antibodies.

Autoantibodies to thyroid microsomes were more frequent in 102 infertile men with complement-dependent sperm-immobilizing activity (sperm immobilization test [SIT] ) in serum (11.8%) than in a control group of 277 men of comparable ages and semen quality without sperm antibodies (4.3%, P less than 0.05). Frequencies of organ-specific antibodies (antigastric parietal cell, antithyroglobulin, and antithyroid microsome) in 57 men with genital tract obstructions and positive SIT were similar to those for control subjects, and there were no significant differences in the frequencies of non-organ-specific autoantibodies (antinuclear antibody, rheumatoid factor, antimitochondrial, and anti-smooth muscle) in the three groups. Because in men without genital tract obstruction antithyroid microsomal autoantibodies were more common with sperm antibodies than without, the possibility of a genetic factor in the causation of sperm autoimmunity should be considered.