Fetal cells in maternal blood: recovery by charge flow separation.

Fetal blood cells can be recovered from the maternal circulation by charge flow separation (CFS), a method that obviates the risks associated with amniocentesis and chorionic villus sampling. By CFS, we processed blood samples from 13 women carrying male fetuses, 2 carrying fetuses with trisomy 21, and 1 who had delivered a stillborn infant with trisomy 18. On average more than 2000 fetal nucleated red blood cells were recovered per 20-ml sample of maternal blood. Recovery of fetal cells was confirmed by fluorescence in situ hybridization with probes for chromosomes Y, 18 and 21. After culturing of CFS-processed cells, amplification by the polymerase chain reaction revealed Y-chromosomal DNA in clones from four of six women bearing male fetuses, but not in clones from three women bearing female fetuses.

Identification of p53 mutations in endometrial adenocarcinoma by polymerase chain reaction-single-strand conformation polymorphism.

OBJECTIVE: We determined whether mutations in p53 exons 5-6-7-8, as detected in the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) test, might be correlated with stage or grade in endometrial adenocarcinoma. METHODS: We amplified sequences containing exons 5, 6, 7, or 8 in DNA from tumors and controls. Mutation within the amplified sequences was indicated by changes in electrophoretic mobility (band shifts) in the SSCP test. The results were analyzed statistically and compared with the results of other, similar studies. RESULTS: We identified 15 band shifts among 47 endometrial tumors studied (band shifts in two different exons in two cases) and none among 42 controls. Band shifts in exons 5 and 8 were associated uniformly with grade 2 or grade 3 histology. In other studies p53 mutations were correlated with advanced-stage malignancy. CONCLUSION: Further evaluation of particular p53 mutations and their relation to disease course in endometrial adenocarcinoma seems warranted. The PCR-SSCP test seems well-suited to this purpose.

SRVX, a sex reversing locus in Xp21.2-->p22.11.

Duplication within Xp21 causes female or intersexual development in human embryos with an XY chromosome complement. We have mapped the responsible gene, SRVX (sex reversal X), in XY-sex-reversed maternal half siblings who had inherited the duplication from their mother. The limited size of the duplication in our cases, relative to its extent in other similar cases, allows assignment of the SRVX locus to Xp21.2-->p22.11. We infer that SRVX is part of a pathway of sex-determining genes that includes SRY and SRA1, the latter recently assigned to chromosome 17q. If mutation of SRA1 or SRVX can reverse the sex of the XY fetus, this would explain why mutation within SRY is found only sporadically in women with XY gonadal dysgenesis.

Fetal cells in the maternal circulation: isolation by multiparameter flow cytometry and confirmation by polymerase chain reaction.

During pregnancy, nucleated fetal erythrocytes enter the maternal circulation and can be isolated efficiently from the maternal cells by multiparameter flow cytometry. Male DNA, implying presence of a male fetus, can be identified in flow-sorted maternal blood by polymerase chain reaction with oligonucleotide primers flanking single-copy Y-specific DNA sequences. Among flow-sorted samples, we correctly identified fetal sex in 17/18 (94%) pregnancies of 10-21 weeks gestation. Maternal blood thus provides a potential opportunity for prenatal diagnosis that could preclude the need for invasive procedures in current use.

Sex selection with monoclonal H-Y antibody.

H-Y antigen defined by antibody from male-sensitized female mice has been reported in male embryos of mouse, rat, cattle, goat, pig, and sheep. We now describe the use of monoclonal H-Y antibodies in identification of male and female during embryo transfer in cattle. Monoclonal H-Y antibodies were applied with fluorescein-isothiocyanate-conjugated goat antimouse immunoglobulin (Ig), and the embryos were scored for fluorescence under ultraviolet light. In alternative trials with 149 embryos, the method could be applied with 73% to 82% efficiency. Pregnancy rates for treated embryos were at least as high as those expected for untreated embryos. The calves that developed from the treated embryos are normal and growing.

On the expression of H-Y antigen in transsexuals.

Histocompatibility-Y (H-Y) antigen, the presumptive inducer of the mammalian testis, is present in the cells of normal males and not in the cells of normal females. Recent reports have implied that patients with transsexualism exhibit H-Y antigen phenotypes at variance with those of normal males and females and, thus, that H-Y serology might provide a tool for the diagnosis and study of the transsexual condition. We therefore evaluated blood and testicular cells from 21 male-to-female transsexuals using conventional and monoclonal H-Y antibodies. We found no evidence of abnormal H-Y phenotype. Five of the patients were interviewed postoperatively by two examiners and rated for the diagnosis of transsexualism. Three of the five were rated primary transsexual by one or both examiners, and two were rated secondary transsexual.

H-Y typing in the ELISA.

In a series of quantitative absorptions, biotin-conjugated monoclonal H-Y antibody was reacted with a plated source of H-Y antigen in the ELISA. H-Y was demonstrated in testis supernatant fluid of the mouse in this system, and in cells from males of the mouse, bovine and human and females of the chicken, thereby confirming its evolutionary persistence, and underscoring its presumptive role in the primary determination of vertebrate sex.

On the secretion of H-Y antigen.

It has been proposed that H-Y antigen secreted by cells of the Sertoli lineage is bound by receptors on these and other cells of the primordial gonad and thereby initiates formation of the testicular cords, and that H-Y is not an integral transmembrane component but a part of a ternary system with beta 2-microglobulin and products of the MHC. It follows that cultured Daudi cells, which lack beta 2-microglobulin and HLA, should secrete H-Y. This is consistent with evidence obtained with monoclonal H-Y antibody and an ELISA. By this method, free H-Y was demonstrable in the supernatant fluids of cultured Sertoli cells and Daudi cells. The assay provides a useful alternative to detection of H-Y in the complement-dependent cytotoxicity test.

H-Y antibodies recognize the H-Y transplantation antigen.

The question is raised whether the H-Y transplantation antigen, as defined by skin graft rejection, is the same as the "male-specific" antigen that is identified by H-Y antibodies. We have addressed that question by injecting female mice of the inbred C57BL/6 strain with serologically defined H-Y antigen, and measuring the response of those female mice to skin grafts from intrastrain male mice. The results of our study indicate that the "two" male antigens are crossreactive or identical.

H-Y antigen in the chicken.

To determine whether asymmetrical development of the chicken ovary could be related to differential expression or function of H-Y antigen, the putative ZW ovary inducer, we compared the ability of cells from the left and right gonad to absorb H-Y antibodies in 17-day chick embryos and in one-day-old hatchlings. In addition, we studied uptake of soluble H-Y antigen by gonadal cells in 17-day embryos, one-day-old hatchlings, and young adults. Indications are that H-Y is present in the left gonad of the female, and to a lesser extent in the right, and not in the testes of the male. Our preliminary results indicate moreover that the right ZW gonad may lack receptor sites for soluble H-Y at around the time of hatching. It may be inferred that expression of H-Y antigen is a prerequisite of ovarian development in birds, although it remains to be determined unambiguously whether H-Y is the primary inducer of the ZW gonad or an accessory molecule involved in some intermediate aspect of gonadal development.