HOXA10 and HOXA11 are homeobox genes that function as transcription factors essential to embryonic development. We have recently described a role for each of these two genes in regulating endometrial development in the adult during the course of a menstrual cycle. Both Hoxa10 and Hoxa11 are essential for implantation in the mouse and appear to play a similar role in women. To investigate the role of HOX genes in the endometrium of women with endometriosis, quantitative Northern blot analysis was performed on the endometrium of 40 normal cycling controls and 40 patients with documented endometriosis. Patients with endometriosis failed to show the expected mid-luteal rise in HOX gene expression as demonstrated in the controls. Aberrant HOX gene expression suggests that altered development of the endometrium at the molecular level may contribute to the aetiology of infertility in patients with endometriosis.

Diethylstilbestrol (DES) was widely used to treat pregnant women through 1971. The reproductive tracts of their female offspring exposed to DES in utero are characterized by anatomic abnormalities. Here we show that DES administered to mice in utero produces changes in the expression pattern of several Hox genes that are involved in patterning of the reproductive tract. DES produces posterior shifts in Hox gene expression and homeotic anterior transformations of the reproductive tract. In human uterine or cervical cell cultures, DES induces HOXA9 or HOXA10 gene expression, respectively, to levels approximately twofold that induced by estradiol. The DES-induced expression is not inhibited by cyclohexamide. Estrogens are novel morphogens that directly regulate the expression pattern of posterior Hox genes in a manner analogous to retinoic acid regulation of anterior Hox genes. Alterations in HOX gene expression are a molecular mechanism by which DES affects reproductive tract development. Changes in Hox gene expression are a potential marker for the effects of in utero drug use that may become apparent only at late stages of development.

Human chorionic gonadotropin (hCG), purified from the urine of 14 individuals with normal pregnancy, diabetic pregnancy, hydatidiform mole, or choriocarcinoma, plus two hCG standard preparations, was examined for concurrent peptide-sequence and asparagine (N)- and serine (O)-linked carbohydrate heterogeneity. Protein-sequence analysis was used to measure amino-terminal heterogeneity and the "nicking" of internal peptide bonds. The use of high-pH anion-exchange chromatography coupled with the increased sensitivity of pulsed amperometric detection (HPAE/PAD) revealed that distinct proportions of both hCG alpha- and beta-subunits from normal and aberrant pregnancy are hyperglycosylated, and that it is the extent of the specific subunit hyperglycosylation that significantly increases in malignant disease. Peptide-bond nicking was restricted to a single linkage (beta 47-48) in normal and diabetic pregnancy, but occurred at two sites in standard preparations, at three sites in hydatidiform mole, and at three sites in choriocarcinoma beta-subunit. In the carbohydrate moiety, alpha-subunit from normal pregnancy hCG contained nonfucosylated, mono- and biantennary N-linked structures (49.3 and 36.7%, means); fucosylated biantennary and triantennary oligosaccharides were also identified (7.3 and 6.9%). In choriocarcinoma alpha-subunit, the level of fucosylated biantennary increased, offset by a parallel decrease in the predominant biantennary structure of normal pregnancy (P < 0.0001). The beta-subunit from normal pregnancy hCG contained fucosylated and nonfucosylated biantennary N-linked structures; however, mono- and triantennary oligosaccharides were also identified (4.6 and 13.7%). For O-linked glycans, in beta-subunit from normal pregnancy, disaccharide-core structure predominated, whereas tetrasaccharide-core structure was also detected (15.6%). A trend was demonstrated in beta-subunit: the proportions of the nonpredominating N- and O-linked oligosaccharides increased stepwise from normal pregnancy to hydatidiform mole to choriocarcinoma. The increases were: for monoantennary oligosaccharide, 4.6 to 6.8 to 11.2%; for triantennary, 13.7 to 26.7 to 51.5% and, for O-linked tetrasaccharide-core structure, 15.6 to 23.0 to 74.8%. For hCG from individual diabetic pregnancy, the principal N-linked structure (34.7%) was consistent with a biantennary oligosaccharide previously reported only in carcinoma; and sialylation of both N- and O-linked antennae was significantly decreased compared to that of normal pregnancy. Taken collectively, the distinctive patterns of subunit-specific, predominant oligosaccharides appear to reflect the steric effect of local protein structure during glycosylation processes. The evidence of alternative or "hyperbranched" glycoforms on both alpha- and beta-subunits, seen at low levels in normal pregnancy and at increased or even predominant levels in malignant disease, suggests alternative substrate accessibility for Golgi processing enzymes, alpha 1,6 fucosyltransferase and N-acetylglucosaminyltrans...

Nicks, or missing peptide linkages, have been found in hCG beta-subunit between residues 44 and 45 and between residues 47 and 48. We examined the occurrence and biological and immunological activities of nicked hCG. As shown by sequence analysis, CR127 standard hCG is approximately 20% nicked, half at beta 44-45 and half at beta 47-48. Treatment with human leukocyte elastase increased the extent of nicking of CR127 standard hCG. The longer the incubation of CR127 standard with human leukocyte elastase (0, 2, and 21 h), the greater the extent of nicked hCG (20%, 46%, and 89%). As the extent of nicking increased, the receptor-binding ability diminished, as did the ability to stimulate progesterone production by rat corpus luteal cells in vitro (0.9, 0.74, and 0.29 microgram/microgram hCG, respectively). In a regression analysis, a linear relationship was indicated between the extent of nicking and receptor binding values (97% correlation) and between the extent of nicking and steroidogenic activity in vitro (99% correlation). From the intercepts of the regression lines, it was estimated that nicks reduced receptor binding by 11-fold and reduced the steroidogenic activity of hCG by 5-fold. We examined eight individual hCG preparations, three purified from pregnancy urine, three from urine from patients with hydatidiform mole, and two from urine from women with choriocarcinoma. In descending order, the eight individual hCG preparations were 100%, 100%, 85%, 76%, 42%, 41%, 0%, and 0% intact. Although no correlation was observed between the percent intact and the ability of the eight individual samples to displace 50% [125I]hCG in binding CG/LH receptor (r less than 0.5), a close correlation was noted between the percent intact and the steroidogenic activity in vitro (98% correlation). This separated the effects of nicking on receptor binding and steroidogenic activities and indicated that while multiple factors influence receptor binding, only nicking suppresses the steroidogenic activity of bound hCG. We examined the recognition of nicked hCG molecules by different hCG immunoassays. The Hybritech Tandem assay measured total hCG and did not distinguish nicked and intact hCG molecules (in a regression analysis, immunoactivity vs. percent intact hCG, r less than 0.5). In contrast, the immunometric assay using B109 hCG dimer-specific monoclonal antibody and anti-beta-peroxidase only detected the intact component of hCG (in a regression analysis, immunoreactivity vs. percent intact hCG, 98% correlation). We used these assays together to estimate the percentage of intact hCG and to deduce the extent of nicking.(ABSTRACT TRUNCATED AT 400 WORDS)

Human chorionic gonadotropin (hCG) exists in blood and urine as a variety of isoforms one of which contains peptide bond cleavages within its beta-subunit loop 2 and is referred to as nicked hCG (hCGn). This hCG isoform appears to be more prevalent in the urine of patients with certain malignancies and possibly in some disorders of pregnancy. Until now, only indirect immunoassays could be used to quantify hCGn. We report the development of two monoclonal antibodies (MAbs) to a form of hCGn isolated from a choriocarcinoma patient. This hCG isoform was not only 100% nicked, but also contained 100% tetrasaccharide-core O-linked carbohydrate moieties in its beta COOH-terminal region. Two-site immunometric assays have been developed using these new antibodies, B151 and B152. The former exhibits good specificity for hCGn independent of the source of the hCGn, the form excreted by choriocarcinoma patients or the form of hCGn from normal pregnancies. The latter antibody, B152, is sensitive to the carbohydrate moieties and possibly other differences in hCG isoforms, but is not for nicking of the beta-subunit. These two immunometric assays provide potential novel diagnostic tools for direct measurement of hCG isoforms which could not be accurately quantified earlier before development of the assays using these newly generated antibodies.