The secretion of human chorionic gonadotropin-like substance in women employing contraceptive measures.

To study the secretion pattern of a hCG-like substance (hCG') in normal women and those employing contraceptive measures, we assayed daily urine concentrates by RIAs using an anti-hCG beta-COOH-peptide serum and an anti-hCG serum to monitor hCG' and human LH (hLH), respectively. In eight cycles of four normal women, urinary hCG' was within the normal range (less than 100 ng/day), except that four samples in two women had marginal elevations of hCG' during the menstrual or premenstrual period, but not at the time of the midcycle hLH surge. Among seven cycles from three intrauterine device (IUD) users, six showed midcycle hLH surges, and in five, hCG' was prominent (0.17-3.6 micrograms/day) in the late luteal phase. In eight cycles of women ingesting steroid contraceptives on a conventional schedule, hCG' was found during the premenstrual and menstrual intervals. The pattern of hCG' secretion was episodic, and hCG' levels were in the range of 0.3-1.8 microgram/day. It was notable that hCG' was excreted at the highest levels during intervals when ingestion of steroids was withheld. These findings suggest that contraceptive steroid hormones may modulate pituitary hCG' secretion. From gel high pressure liquid chromatographic studies, the immunoactive hCG' detected in urinary extracts from normally cycling women and IUD users had a molecular size larger than hCG beta and hLH, but was slightly smaller than hCG (lot CR119). The hCG' identified in these eluents is unlikely to be a subunit of hCG. On the other hand, the immunoactive hCG' in the urinary extracts from oral contraceptive users was mainly the hCG beta-subunit-like substance. Both hCG alpha and hCG beta immunoactive substances were heterogeneous in size in contrast to those found in normally cycling women and IUD users. In summary, the hCG' excretion pattern was different in menstrual and postmenopausal women, and hCG' identified in these subjects was heterogeneous.

PIP: To study the secretion pattern of an hCG-like substance (hCG') in normal women and those employing contraception, the authors assayed daily urine concentrates by radioimmunoassays using an anti-hCGbeta-COOH-peptide serum and an anti-hCG serum to monitor hCG' and human luteinizing hormone (hLH), respectively. In 8 cycles of 4 normal women, urinary hCG' was within the normal range (100 ng/day), except that 4 samples in 2 women had marginal elevations of hCG' during the menstrual or premenstrual period, but not at the time of the midcycle hLH surge. Among 7 cycles from 3 IUD users, 6 showed midcycle hLH surges, and in 5, hCG' was prominent (0.17-3.6 mcg/day) in the late luteal phase. In 8 cycles of women ingesting steroid contraceptives on a conventional schedule, hCG' was found during the premenstrual and menstrual intervals. The pattern of hCG' secretion was episodic and hCG' levels were in the range of 0.3-1.8 mcg/day. It was notable that hCG' was excreted at the highest levels during intervals when steroid ingestion was withheld. These findings suggest that contraceptive steroids may modulate pituitary hCG' secretion. From gel high pressure liquid chromatographic studies, the immunoactive hCG' detected in urinary extracts from normally cycling women and IUD users had a molecular size larger than hCGbeta and hLH, but was slightly smaller than hCG (lot CR119). The hCG' identified in these eluents is unlikely to be a subunit of hCG. On the other hand, the immunoactive hCG' in the urinary extracts from OC users was mainly the hCG beta-subunit-like substance. Both hCGalpha and hCGbeta immunoactive substances were heterogeneous in size in contrast to those found in normally cycling women and IUD users. In summary, the hCG' excretion pattern was different in menstrual and postmenopausal women, and hCG' identified in these subjects was heterogeneous.

Immunohistochemical demonstration of placental protein 5 in trophoblastic tumours.

An immunoperoxidase technique was used for the detection of placental protein 5 (PP5) in tissue specimens from trophoblastic tumours. PP5 was detected in all of 15 cases of hydatidiform mole, in two out of six destructive moles and in one of seven choriocarcinomas. Though the biological importance of PP5 is not clear, its association with inactivation of plasmin suggests a role in the control of placental invasiveness.

Circulating placental proteins in pregnancies complicated by RH isoimmunization.

Nine pregnant women with Rh isoimmunization who delivered newborns with hydrops fetalis were studied. The placental proteins, pregnancy specific beta 1-glycoprotein (SP1), human placental lactogen, and placental protein 5 (PP5) were measured in maternal serum by radioimmunoassays. The results indicate that both the serum human placental lactogen and PP5 levels were significantly higher than those observed in normal pregnancy. The strikingly higher circulating PP5 levels found in all nine patients with Rh isoimmunization studied suggests that serum PP5 may be specifically elevated in pregnant patients with Rh isoimmunization and hydrops fetalis.

Placental protein 5 (PP5) in severe pre-eclampsia and eclampsia.

Circulating methods of placental protein 5 (PP5) were studied in patients with severe preeclampsia and eclampsia. Elevated PP5 levels were frequently observed in the eclampsia group. This elevation may be due to disturbance in endogenous coagulation mechanisms at the placental site.

Change in human chorionic gonadotropin in gestational trophoblastic disease observed during the course of chemotherapy.

This study investigates the physicochemical characteristics of human chorionic gonadotropin (hCG) in gestational trophoblastic disease (GTD), with special reference to the clinical course of chemotherapy and prognosis. In gel high performance liquid chromatography (HPLC), the hCG molecules from normal pregnancy and from the hydatidiform mole had the same molecular form as standard purified hCG, whereas hCG from choriocarcinoma was inconsistent in molecular form, containing molecules which are smaller, the same or larger than those of standard purified hCG. In two fatal choriocarcinoma patients, large hCG and large hCG alpha were found in the urine samples collected within one month prior to death. In a chromatofocusing study, the chromatofocusing pattern of hCG from GTD was acidic and similar to that of early pregnancy. The chromatofocusing patterns did not alter or were altered only slightly during the course of chemotherapy. In a Concanavalin A-Sepharose (Con A) chromatography study, the Con A binding shifted from low to high binding in patients with GTD who were responsive to chemotherapy. In summary, the molecular form, electric charge and Con A binding of hydatidiform mole hCG are similar to those of early pregnancy hCG and standard purified hCG, whereas the molecular form and Con A binding of choriocarcinoma are different from those of early pregnancy hCG and standard purified hCG. The presence of smaller or larger molecular forms of hCG may be an ominous sign, whereas the presence of high Con A binding may be a favorable sign. The chromatofocusing pattern seems to be unrelated to the clinical course of chemotherapy.

Rapid and simple immunoassays for measurement of human chorionic gonadotropin using monoclonal antibodies.

Two monoclonal antibodies, designated Gab-35 and Gab-144, were used in competitive radioimmunoassay (RIA) and solid-phase sandwich enzyme immunoassay. By SDS-polyacrylamide gel electrophoresis and protein blot enzyme immunobinding assay, Gab-35 and Gab-144 were shown to react specifically to the alpha-subunit and beta-subunit of human chorionic gonadotropin (hCG), respectively. The immunoglobulin class and subclass of these two monoclonal antibodies were found to be IgG1 by the Ouchterlony double gel immunodiffusion test. The beta-subunit specific monoclonal antibody of Gab-144 was used in competitive RIA for determination of serum hCG in the range of 5-500 mIU/ml. The assay results correlated well with the monoclonal antibody-based RIA system and commercial RIA kits using polyclonal antisera. Moreover, the present competitive RIA system showed a shorter incubation time (1 hour 20 minutes) and a shorter total assay time (1 hour 40 minutes), as compared to those of the commercial RIA kit. A solid-phase sandwich enzyme immunoassay was developed using a combination of the Gab-35 antibody-coated microtiter plate and the beta-subunit specific antibody of Gab-144 conjugated with horseradish peroxidase (HRP). The assay range was 20-200 mIU/ml, and the incubation time was only 30 minutes. In addition, the solid-phase sandwich enzyme immunoassay developed in the present study could also be used as a qualitative hCG assay with a cutoff point of 50 mIU/ml as an early pregnancy test at or just a few days before the missed period.

Placenta increta in the second trimester of pregnancy: report of a case.

A rare case of placenta increta in the second trimester of pregnancy is reported. The patient was at 15 weeks gestation when the pregnancy, which had been complicated by a maternal rubella infection, was terminated at a regional hospital. However, vaginal bleeding persisted after the operation in spite of medication to control bleeding. Curettage of the uterine cavity one month later failed to reveal any retained placental tissue or other pathology. Therefore, an exploratory laparotomy was performed, yet nothing particular was found in the peritoneal cavity. So, the patient was transferred to our department. Sonography revealed a lower uterine mass of 4.0 x 3.3 cm in size. A persistently low serum hCG titer was also found. Placenta accreta was highly suspected. Three doses of methotrexate were given to control bleeding, yet without results. Hysterectomy was finally performed. A histological study revealed placenta increta.

Experience of human chorionic gonadotropin assays in gestational trophoblastic tumors.

From May 1979 through December 1988, 146 patients with gestational trophoblastic tumors (71 hydatidiform mole, 3 partial mole, 15 choriocarcinoma and 57 persistent trophoblastic tumors) were studied. A total of 1178 daily urine samples were collected before and/or after treatment, and in the course of follow-up. H93 RIA (an HCG specific assay), H80 RIA (an assay detecting hCG and hLH) and a hCG alpha assay measured levels in the urine specimens. Three hCG declining patterns (pattern D, P and R) based on the H93 RIA assay were noted. Patients showing pattern D had the most favorable outcome (no mortality at all). However, pattern P and R had a 10% and 14.3% mortality rate, respectively. The ratios of H80/H93, hCG alpha/H93, hCG alpha/H80 in the urine specimens were similar in both pattern D and R (excluding samples from a patient who expired later). However, the ratios of H80/H93, hCG alpha/H93, hCG alpha/H80 of samples from the patient (CK) who expired later were significantly different from those of the pattern D and R. This was suggestive of a marked unbalanced secretion of hCG and its subunit in the urine specimens of patient CK. The molecular forms in pattern D were similar to the standard hCG. However, the molecular form in pattern R of 3 fatal choriocarcinomas showed a great variation, from smaller to larger than the standard hCG. The isoelectric points of hCG in pattern D and R were all acidic. In clinical practice, we can measure the ratios of H80/H93, hCG alpha/H93 and hCG alpha/H80, molecular forms, and isoelectric points of hCG.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of management of gestational trophoblastic tumor in National Taiwan University Hospital.

Advances in the management of gestational trophoblastic tumor have been made during the last three decades. Individualization of the therapy is one of the major advances. A number of risk factors has proved to predict accurately the prognosis of each patient. A few systems were currently in use, but difficult in putting them to practical use in the different geographical areas. At National Taiwan University Hospital from 1965 to 1979, 65 patients treated by chemotherapy were analyzed with respect of various prognostic factors. The score was assigned according to the mortality rate of each item to each prognostic factor, and thus established a scoring system which is suitable for the use in Taiwan. After establishment of our scoring system, 51 patients from 1980 to 1986 were treated according to the system and the appropriate therapeutic regimens. The outcome of these patients and toxicity of the different therapeutic regimens are presented.

Production of monoclonal antibodies to human chorionic gonadotropin.

Production of monoclonal antibodies against human chorionic gonadotropin (hCG) has been studied using hCG as an immunogen. Spleen cells of BALB/c mice immunized with hCG were fused with NS-1 mouse myeloma cells. This study reports the successful isolation of a hybrid clone secreting a monoclonal antibody specific for hCG. By using PEG 4,000 as a fusion agent, the fusion rates were between 42.0 and 50.2%. In total 842 hybridomas were produced. Among them, 403 hybridomas had hCG antibody production. After cloning twice by limiting dilution and alternately screening by enzyme immunoassay and by radioimmunoassay, there were 39 cell lines having specific antibody production. Among them, the No. 57-42-2 had the highest reactivity. By Ouchterlony test, the monoclonal antibody was shown to be IgG1. The affinity constant of the antibody to hCG was 0.6 x 10(9) 1/mole. In radioimmunoassay, the cross reactivity of the antibody to human luteinizing hormone (LH) and human follicle-stimulating hormone (FSH) was 1.5% and 0.7%, respectively. There was no cross reaction with human thyrotropin-stimulating hormone (TSH).

Cord blood hypertriglyceridemia in Chinese newborns: an index of fetal distress.

Cord serum triglyceride concentrations in 214 Chinese newborns were measured at birth with the Hitachi Model 716 autoanalyzer using the G-3-PDH UV end point method. The mean and the 95th percentile value in a normal population of 100 newborns were found to be 28 mg/100 ml and 48 mg/100 ml, respectively. Using 48 mg/100 (P95) as a cut-off point, maternal-fetal factors were evaluated in 35 hypertriglyceridemic and 35 normotriglyceridemic newborns. A significant association was demonstrated between high cord serum triglyceride levels and toxemia, overterm, meconium-stained amniotic fluid, deceleration of fetal heart rate, intrauterine growth retardation, low one-minute Apgar score (less than or equal to 7), and low umbilical arterial pH (less than 7.20). These findings pointed out that cord serum hypertriglyceridemia was an index of fetal distress and associated with many unfavorable intrauterine conditions. They also suggested that hypertriglyceridemia might be related to the activated sympathetic nervous system and the enhanced release of catecholamines.

Effect of estrogen (Premarin) replacement therapy on serum level of total estrogen and urinary excretion of calcium and hydroxyproline in postmenopausal Chinese women.

The study subjects included a total of 30 postmenopausal Chinese women, including 14 natural menopausal women, 13 castrated menopausal women and 3 post-irradiated menopausal women. Premarin 1.25 mg/day was given orally for 3 weeks and off one week, repeated for 6 cycles. Fasting morning urine and blood samples were collected before hormone treatment and at the end of 3 weeks, 11 weeks, and 23 weeks of Premarin therapy. Serum total estrogen level was measured by radioimmunoassay. Urinary calcium and creatinine were measured by atomic absorption and Jeffe's reaction, respectively. The concentration of urinary hydroxyproline was determined by Kivirikko's method. After Premarin therapy, the mean concentration of serum total estrogen increased 3 to 4 times from the pretreatment level of 71.7 pg/ml. On the other hand, the mean value of calcium/creatinine (Ca/Cr) molar ratio dropped down from 0.249 to 0.098. The mean value of hydroxyproline/creatinine (HOPr/Cr) molar ratio was reduced from 0.028 to 0.012. In view of the hormonal and biochemical changes after Premarin therapy, it is concluded that estrogen (Premarin) replacement should be effective in the treatment of enhanced bone loss or osteoporosis in postmenopausal women. The relationship of estrogen and calcitonin in the regulation of bone metabolism is also discussed.

Heterogeneity of human chorionic gonadotropin in various biological fluids as revealed by chromatofocusing.

This study demonstrates that chromatofocusing is powerful in analyzing multiple forms of hCG from biological fluids. For analyzing hCG from biological fluids, it is necessary to perform chromatofocusing, in the range of pH 6.2-3.0 and pH 9.0-6.0. By chromatofocusing, highly purified hCG (CR121) was found to be acidic, in the range of pI 4.22-3.8, and hCG beta was more acidic, in the range of pI 4.0-3.2. Moreover, hCG from the first trimester pregnancy, hydatidiform mole or choriocarcinoma was also mainly acidic. Therefore, chromatofocusing in the range of 6.2-3.0 was suitable for analyzing purified hCG, hCG beta, and urinary hCG from the first trimester pregnancy, hydatidiform mole and choriocarcinoma. On the other hand, because hCG in the third trimester pregnancy and the toxemia of pregnancy were mainly alkaline, the chromatofocusing system in the range of pH 9.0-6.0 was suitable for analyzing hCG from the third trimester pregnancy and the toxemia of pregnancy.

Purification and characterization of metal-binding proteins and the corresponding mRNA from human placentas.

Two metal-binding proteins, designated as PI and PII, were isolated and purified from normal term human placentas by gel filtration and ion-exchange chromatography. The molecular weights were determined to be 10,000 and 12,000 daltons, and isoelectric points (pI) were 4.8 and 5.9, respectively. The amino acid composition of these proteins was quite different from that of metallothionein. Total amount of acidic amino acid residues was in large excess over that of basic amino acid residues. Cadmium and zinc were the major metals bound to these proteins. The metal contents of cadmium and zinc in placental tissue were 39.34 and 22.23 ng/g placenta, respectively, as measured by flame atomic absorption spectrophotometry. The in vitro translated metal-binding proteins encoded by the corresponding mRNA were characterized by the purified rabbit antiserum against PI and PII. The demonstrated presence of these metal-binding proteins in human placenta suggests its possible role of detoxification activity and protective effect to the fetuses in utero.

Circulating placental proteins (hCG, SP1 and PP5) in trophoblastic disease.

Serum human chorionic gonadotrophin (hCG), pregnancy-specific beta 1-glycoprotein (SP 1) and placental proteins 5 (PP5) levels have been measured by radioimmunoassay in 20 patients (116 samples) with hydatidiform mole, one patient (nine samples) with invasive mole and 10 patients (103 samples) with choriocarcinoma. Measurement of both serum SP1 and hCG are useful in the monitoring of these diseases. The presence of PP5 in hydatidiform mole and its absence in choriocarcinoma support the hypothesis that PP5 is closely associated with the invasive activity of malignant trophoblast.

Studies of monoclonal antibodies against human chorionic gonadotropin. II. Applications of human chorionic gonadotropin monoclonal antibodies on immunoassays.

Monoclonal antibodies generated against human chorionic gonadotropin (hCG) were utilized in radio- and enzyme-immunoassays of this reproductive hormone in biological fluids. One of the monoclonal antibodies, beta-4D6, was shown to have extremely high affinity (Ka = 8 X 10(10) M-1)) and high specificity (less than or equal to 0.6% LH cross-reactivity) to beta-subunit of hCG and whole hCG. It was used in competitive radioimmunoassays (RIA) for the determination of low levels of serum hCG. An excellent correlation was obtained concerning the assay results between the monoclonal antibody-based system and others using conventional polyclonal anti-sera. In combination with another discrete hCG monoclonal antibody, solid-phase sandwich radiometric and enzyme immunoassays were established. These immunoassays could easily be performed, and they offer sensitivity and efficacy of hCG determination comparable to those of conventional ones.

Serum squamous cell carcinoma antigen in gynecologic malignancies with special reference to cervical cancer.

Squamous cell carcinoma (SCC) antigen was measured by double-antibody radioimmunoassay in the sera of 113 patients with gynecologic malignancies and 30 controls. The mean serum SCC antigen level was 9.24 ng/ml in those with cervical squamous cell carcinoma, 2.15 ng/ml in those with other gynecologic malignancies, and 1.25 ng/ml in controls. With a cutoff value of 2.23 ng/ml (2 SD above the mean of the control group), the rate of SCC antigen elevation was 54% in cervical cancer (78), 14% in vulvar or vaginal cancer (7), 22% in ovarian cancer (18), and 10% in endometrial cancer (10). In cervical squamous cell carcinoma, the rates of elevated SCC antigen level increased with disease advancement in stages 0, I, II, III, and IV, by 13, 50, 53, 78, and 100%, respectively. In early-stage cervical squamous cell carcinoma, SCC antigen was not sensitive enough for screening. However, if elevated, serum SCC antigen levels decreased rapidly after successful surgical treatment. One case with a serum SCC antigen level above 65 multiples of the cutoff value had widespread cancer and postoperative recurrence. In the advanced case, the sensitivity was much higher. In the recurrent case, the positive rate was 73%. Serum SCC antigen level is useful in predicting the prognosis and monitoring the course of cervical squamous cell carcinoma, especially in the detection of a recurrence.

Free alpha subunit of human chorionic gonadotropin in women with non-trophoblastic tumors.

Patterns of secretion of free alpha subunit in 242 women with non-trophoblastic tumors were studied. In 4 patients molecular forms of alpha subunit were investigated. In normal menstruating women, day to day fluctuations of alpha subunit (36 to 4871 ng/day) with a peak at midcycle were commonly observed. This makes measurement of the alpha subunit as a tumor marker difficult to interpret in this group of women. On the other hand, the secretion of alpha subunit in postmenopausal women was relatively stable (from 50 to 450 ng/day). Thus the incidence of elevated alpha subunit secretion (greater than 400 ng/day) in postmenopausal patients with non-trophoblastic tumors was as follows; cervical cancer (29.9%), ovarian cancer (50%), corpus cancer (37.5%), vulvar cancer (33.3%), lung cancer (53.3%), gastrointestinal tract cancer (80%). The elevation of the alpha subunit was unrelated to the histological type of cancer. Discordant secretion of hCG and its subunits were noted. The incidence of elevated hCG secretion (greater than 100 ng/day) for the same group of postmenopausal patients with non-trophoblastic tumors was 44.8% for cervical cancer, 100% for ovarian cancer, 62.5% for corpus cancer, 33.3% for vulvar cancer, 26.7% for lung cancer and 40% for gastrointestinal tract cancer. The incidence of isolated alpha subunit secretion in cervical cancer stages III and IV, lung cancer and gastrointestinal tract cancer (40-43%) was higher than that in other cancers (0-27%). For cervical cancer, the more advanced disease was associated with higher alpha subunit levels. The molecular forms of alpha subunit in 4 patients with non-trophoblastic tumors were studied by gel HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences between white and Chinese populations in human leukocyte antigen sharing and gestational trophoblastic tumors.

The prevalence of gestational trophoblastic tumors varies widely among different populations: it is lowest in whites (3 to 6/100,000) and highest in Chinese (68 to 202/100,000). This observation suggests that the origin of the disease is different in the two populations. To test this hypothesis, we examined couples in whom the woman developed a gestational trophoblastic tumor in a white population (Pittsburgh) and a Chinese population (Taiwan) for sharing of human leukocyte A, B, DR, and DQ antigens, which we consider markers for sharing of major histocompatibility complex-linked recessive genes affecting both embryogenesis and carcinogenesis. No human leukocyte antigen sharing occurred between partners in Pittsburgh, but there was significant human leukocyte antigen sharing in Taiwan. The latter couples shared human leukocyte antigen B (p less than 0.04) and human leukocyte antigen DQ (p less than 0.007) and shared three or more human leukocyte A, B, DR, and DQ antigens (p less than 0.02) significantly more frequently than did normal couples. However, there was no increased sharing of any specific human leukocyte antigen allele. These findings support the hypothesis that gestational trophoblastic tumors occur on a sporadic basis in whites and on a genetic basis in Chinese.

Human chorionic gonadotropin and its subunits in pre-eclampsia.

The serum levels of hCG and its subunits were studied in 11 cases of pre-eclampsia (8 cases of severe pre-eclampsia and 3 cases of mild pre-eclampsia) and 72 normal pregnant women. The hCG (human chorionic gonadotropin) immunoactivity measured by H80 RIA (radioimmunoassay) and Sb6 RIA showed no significant difference between pre-eclampsia and normal pregnancy. This result suggests that no significant amount of free hCG-beta existed, since H80 RIA measured intact hCG but not hCG-beta, and Sb6 RIA measured both hCG and hCG-beta. Furthermore, from the result of elution behavior of gel HPLC (high performance liquid chromatography), hCG of pre-eclampsia and comparison of the Sb6 RIA/H80 RIA ratio between pre-eclampsia and normal pregnancy, it was evident that there was little amount of free hCG-beta secretion in pre-eclampsia as in normal pregnancy. However, the free hCG-alpha was significantly higher in severe pre-eclampsia, probably caused by placental dysfunction. In summary, free hCG-alpha is increased in pre-eclampsia, but its role as a marker of placental function in pre-eclampsia requires further investigation.