Galina Kovalevskaya

Trophoblast origin of hCG isoforms: cytotrophoblasts are the primary source of choriocarcinoma-like hCG.

We have previously demonstrated that a hyperglycosylated isoform of chorionic gonadotropin (hCG) (B152 hCG) is detected in the blood and urine in early pregnancy and is subsequently rapidly replaced by the hCG isoform (B109 hCG) characteristic of later pregnancy. In the current study we have extended our work on the origin of these isoforms. We have used a combination of in situ and in vitro approaches. Localization studies in placental tissues showed that monoclonal antibody B109 stained very specifically syncytiotrophoblast (STBs) from first and second trimester tissues. At term, STBs exhibited no B109 staining at all. Immunostaining with B152 antibody, that recognize the hyperglycosylated isoform of hCG, revealed only punctate staining of STBs in most villi of first trimester tissue. Both antibodies B109 and B152 failed to stain cytotrophoblasts (CTBs). To assess the functional relevance of these observations we analyzed conditioned media from purified CTBs using two immunometric assays, one of which (B152-B207*) has primary specificity for the hyperglycosylated, choriocarcinoma-like hCG and the other (B109-B108*) having primary specificity for the later pregnancy hCG isoform. Regardless of gestational age, isolated CTBs secreted predominantly B152 hCG isoform in contrast to placental villi (predominantly STBs), which released primarily the B109 hCG isoform. Isolated CTBs, however, failed to immunostain with both B109 and B152 antibodies. To resolve this contradiction, we cultured CTBs in the presence of brefeldin A, a drug known to block secretion by inhibiting protein translocation from the endoplasmic reticulum to the Golgi vesicles. Brefeldin A treated CTBs stained strongly with B109 and did not stain or stained weakly with B152 antibody. We assume that treatment with brefeldin A impaired glycosylation of beta subunit and consequently inhibited the production of hyperglycosylated form of hCG recognized by B152. In summary, our in vitro experiments indicate that both isoforms of hCG are produced by villus CTBs and that the dominant isoform is the one recognized by antibody B152. STBs produce primarily the less glycosylated B109 hCG isoform. This data suggests that at the beginning of pregnancy villus CTBs are the major source of the B152 hCG isoform. This finding is supported by our clinical data that show that the dominant hCG isoform in the blood and urine of pregnant women in the first 6 weeks of pregnancy is recognized by B152 (). The inversion of the B152/B109 ratio observed after 6-7 weeks of pregnancy can be explained by the reduction of number of villus CTBs and/or by maturation of STBs.

Differential urinary gonadotrophin profiles in early pregnancy and early pregnancy loss

Early pregnancy loss (EPL), detected by patterns of human chorionic gonadotrophin (hCG) in urine, is the biomarker employed in investigations of the impact of personal, workplace or environmental reproductive toxins on human fertility. An issue central to these studies is what, in terms of urinary hCG expression, constitutes an EPL.

Metabolism of hCG and hLH to multiple urinary forms.

Human chorionic gonadotropin (hCG) is synthesized primarily in the placenta while human luteinizing hormone (hLH) is produced in the pituitary. Both hormones are highly homologous in structure and both appear to be altered to analogous molecular forms as the hormones are proteolytically processed, or metabolized, from tissue of origin, through the circulation, and finally to the urine. Placental hCG is excreted into urine as heterodimeric hormone, heterodimeric nicked hCG, free subunits (some nicked), and predominantly as the hCG beta core fragment. A pituitary form of heterodimeric hCG, which is partly sulfated as is pituitary hLH, was recently isolated and is likely the form of hCG observed in the urine of healthy postmenopausal women and nonpregnant premenopausal women as well. A pituitary form of the hLH beta core fragment, highly analogous in structure to that of urinary hCG beta core fragment, has been used to develop specific monoclonal antibody assays to measure urinary hLH beta core fragment which is excreted at significantly higher molar concentrations than is hLH in the urine of ovulating women 1 or 2 days after the LH surge. This fragment of LH appears in the urine of postmenopausal women as well. The development of the capability to distinguish the hCG beta core fragment from the hLH beta core fragment in urine may have useful applications in tumor marker assays, pregnancy tests, and menopause. While hCG urinary assays have been widely employed, urinary assays for hCG and hLH metabolites are much less used since the urinary molecular forms are only partly known. Our studies of hCG and hLH urinary metabolites are directed towards improvement of the utility of urinary measurements of molecules derived from these hormones. Since many of the molecular forms of these two hormones in urine differ from their forms in blood, it may be necessary to produce new immunoassays as well as novel urinary reference preparations to accurately measure these molecules within their urinary matrix.

Immunochemical Measurement of Early Pregnancy Isoforms of hCG: Potential Applications to Fertility Research, Prenatal Diagnosis, and Cancer

Human chorionic gonadotropin, the glycoprotein hormone of pregnancy, is found naturally in blood and urine in a variety of isoforms. These variants are related to both peptide bond cleavages (such as the nicked forms of hCG) and the beta core fragment urinary metabolite, as well as the larger variety of species resulting from carbohydrate heterogeneity. We have recently developed immunoassay systems that can measure nicked forms of hCG (antibody B151) as well as particular high carbohydrate variants (hyperglycosylated forms) of hCG (B152), which are associated with cancers producing hCG. Using the assay system for nicked hCG, we found that nicked hCG does not appear to be present as a significant hCG isoform during normal pregnancies if the urine specimens are well preserved. Applying the assay for hyperglycosylated hCG isoforms, we discovered that these forms are prevalent during very early pregnancy and decline rapidly to low concentration after the first 6 weeks of pregnancy. Persistence of these early pregnancy forms does not bode well for the pregnancy. Other investigators report that measurement of such hCG isoforms may aid in diagnosis of Down syndrome pregnancies. In summary, measurement of the hyperglycosylated hCG isoforms are useful for evaluation of healthy progress of normal pregnancy, as an additional detection marker for Down syndrome pregnancies, and as a potential new marker of trophoblastic malignancy. New reference preparations will soon be available for the calibration of assay systems for measurement of many of these hCG variants and metabolites.

Vasopressin deficiency and vasodilatory state in end-stage liver disease

OBJECTIVES Relative vasopressin deficiency, a contributor to vasodilatory septic shock, also may be a cause of the vasodilatory state in liver disease. This study assessed endogenous vasopressin levels in patients with liver disease and their hemodynamic response to exogenous vasopressin. DESIGN A prospective, observational study. SETTING A single-center, tertiary hospital. PARTICIPANTS Human subjects undergoing liver transplantation or major surgery. INTERVENTIONS Vasopressin levels were measured in 28 patients with liver disease undergoing liver transplantation and 7 control patients with normal liver function. Additionally, intravenous vasopressin was administered to 20 liver transplant recipients, and the hemodynamic response was observed. MEASUREMENTS AND MAIN RESULTS Patients with liver disease had significantly lower baseline vasopressin levels than controls (19.3 ± 27.1 pg/mL v 50.9 ± 36.7 pg/mL, p = 0.015). Patients with low vasopressin levels (≤20 pg/mL) were more likely to have lower baseline mean blood pressure (≤80 mmHg) than patients with high vasopressin levels (11/16 v 0/4, p = 0.013). Systemic vascular resistance increased by 33% 3 minutes after intravenous vasopressin. Thirteen of 16 patients with low vasopressin levels compared with 1 of 4 patients with high vasopressin levels responded to exogenous vasopressin, with an increase of mean blood pressure by more than 20% (p = 0.028). CONCLUSIONS Patients with liver disease have lower vasopressin levels than controls and respond with a brisk vasoconstrictor response to exogenous vasopressin. Therefore, relative endogenous vasopressin deficiency may contribute to vasodilatory shock in liver disease similar to what has been observed in septic shock.

HLH beta core fragment immunoreactivity in the urine of ovulating women: a sensitive and specific immunometric assay for its detection.

Recently, we isolated an hLH beta core fragment (hLHβcf) from human pituitaries. This molecule is homologous to the hCG beta core fragment (hCGβcf), which may be a marker of normal pregnancy, Down syndrome, and certain cancers. We now report antibodies to the hLHβcf, four of which have been applied in sensitive immunoradiometric assays for urinary measurements. One of the antibodies recognizes an epitope on the hLHβcf, which is not present on the hCGβcf, hLH, or hLHβ. This specific hLHβcf antibody acts cooperatively with other newly-developed antibodies reported here to produce an assay with a sensitivity of 1 fmol/ml of hLHβcf. The specificity of these new IRMA systems will make it possible to measure the hLHβcf in urine in the presence of hLH, hLH beta, or the hCGβcf. Although the hLHβcf used to develop specific antibodies was purified from pituitaries, the assays developed recognize this metabolite in urine. Measurements of heterodimeric hLH as compared to hLHβcf in the urine of cycling women indicated that the concentration of hLHβcf rose as high as 6–7 times the concentration of hLH starting a day after the midcycle surge. The new measuring systems allow the precise quantitation of this hLH metabolite in urine.