Apolipoprotein E synthesis by cultured human ovarian granulosa cells: regulation by human chorionic gonadotropin and cholesterol.

OBJECTIVE: Apolipoprotein E (Apo E) is a surface component of several classes of plasma lipoproteins and functions as a receptor ligand for the low-density lipoprotein (LDL) (B/E) receptor, the hepatic E receptor, and the LDL receptor related protein. In continuing studies of Apo E synthesis by extrahepatic steroidogenic tissue, we have examined Apo E production and its control in cultured human granulosa cells (GC). DESIGN: Human GC obtained at the time of oocyte aspiration for in vitro fertilization were cultured, and the following parameters were measured: progesterone secretion into the medium, Apo E synthesis and secretion, and Apo E messenger ribonucleic acid (mRNA) content of the GC. RESULTS: Human chorionic gonadotropin induces a decrease in Apo E protein synthesis and Apo E mRNA content. The addition of aminoglutethimide (which blocks cholesterol conversion to pregnenolone) and/or LDL (which provides the cell with cholesterol), both result in an increase in Apo E protein synthesis and Apo E mRNA content. These latter findings are in agreement with studies in nonsteroidogenic cells, which show that Apo E production is positively correlated with cell cholesterol content.     

Human chorionic gonadotropin stimulation of relaxin secretion by luteinized human granulosa cells.

OBJECTIVE: To determine the effect of human chorionic gonadotropin (hCG) on relaxin secretion by long-term cultures of luteinized human granulosa cells (GC). DESIGN: Luteinized human GC were collected from 10 women undergoing in vitro fertilization (IVF) cycles. Luteinized human GC from each woman were plated in replicate wells at 1 x 10(5) cells/well and exposed to medium 199 (GIBCO, Grand Island, NY), medium 199 with 1 IU/mL hCG, and/or medium 199 with 100 IU hCG/mL. Luteinized human GC were maintained for up to 40 days in culture. Spent media were changed every 2 days and assayed for relaxin and progesterone (P) at the conclusion of each experiment. SETTING: Tertiary care center. PATIENTS, PARTICIPANTS: Luteinized human GC were obtained from women undergoing controlled ovarian hyperstimulation for IVF with one of the following regimens: (1) clomiphene citrate with human menopausal gonadotropins (hMG); (2) hMG alone; or (3) hMG with leuprolide acetate. All women were less than 40 years of age, in good health, and were not taking medications other than those used in the ovulation-induction regimen. MAIN OUTCOME MEASURES: Levels of P and relaxin in spent media. RESULTS: Relaxin secretion by luteinized human GC was dependent on hCG stimulation and was detected only after a time lag in culture. After relaxin secretion was detected, it was maintained throughout the culture period (10 to 22 days). Luteinized human GC produced P immediately under both basal and stimulated conditions. Progesterone production continued throughout the culture period with hCG-stimulated cells producing significantly greater P after 4 to 8 days in culture. CONCLUSIONS: Luteinized human GC obtained at the time of oocyte retrieval secrete relaxin in response to hCG stimulation and secrete P under both basal and hCG-stimulated conditions, thereby serving as a model to explore luteal function and control.     

Disappearance of human chorionic gonadotropin following removal of ectopic pregnancy

A study was undertaken to determine the length of time serum beta-subunit of human chorionic gonadotropin (beta-hCG) could be detected following removal of ectopic pregnancy. Seven patients underwent complete removal of trophoblastic tissue by either salpingectomy or partial resection of the involved fallopian tube. Nine other patients had conservative surgical treatment by either linear salpingostomy or fimbrial expression of the fallopian tube. Serum beta-hCG levels were determined serially in all these patients. The results demonstrate that the initial titer of hCG is a significant factor in determining the length of time that it can be detected in the serum postoperatively. In addition, decreasing titers, conforming to the disappearance curve of hCG, as constructed in this study, are a helpful aid in avoiding further surgery in the group of patients who had a conservative removal of the trophoblastic tissue. Finally, the serum clearance of hCG by radioimmunoassay may take at least up to 24 days after surgery.     

Discordant secretion of pregnancy specific beta 1-glycoprotein and human chorionic gonadotropin by human pre-embryos cultured in vitro.

OBJECTIVE: To study and compare the secretion of pregnancy specific beta 1-glycoprotein (SP1) and human chorionic gonadotropin (hCG) by human pre-embryos, cultured in vitro, with their respective morphological development. DESIGN: Spare human pre-embryos from randomly selected women participating in a program of in vitro fertilization (IVF) were studied prospectively. SETTING: Pre-embryos were cultured, and hormone release was determined in academic research laboratories. PATIENTS, PARTICIPANTS: Pre-embryos (n = 108) cultured for 14 days after fertilization in Ham's F-10 medium (GIBCO Ltd., Paisley, Scotland) were observed, and hCG and SP1 were measured in the culture media at regular intervals. MAIN OUTCOME MEASURES: Discordant secretion of SP1 and hCG. RESULTS: Of the 98 bipronucleate pre-embryos, 53.6% formed blastocysts, 17.3% of which hatched. Human chorionic gonadotropin was detected from day 7 after fertilization concomitantly with blastocyst formation, thereafter showing a logarithmic increase (maximum 10,650 mIU) until the first signs of embryonic disintegration. Pregnancy-specific beta 1-glycoprotein release started 3 to 4 days after fertilization independently of the morphological development and the future production of hCG, thereafter displaying a nonlogarithmic increase (maximum 41 ng). CONCLUSIONS: Hormone secretion and morphological development are unique for each pre-embryo. Human chorionic gonadotropin and SP1 seem to have different biochemical and physiological regulation.     

Effects of cyclic adenosine monophosphate on human chorionic gonadotropin and estradiol output by cultured human placental cells

We have previously shown that adenosine-3',5'-cyclic monophosphate (cAMP) inhibits basal estradiol output in human trophoblast cells. The objective of this study was to investigate further the effect of 8-bromo-cAMP on the conversion of C19 androgens to estradiol by placental cells. Trophoblast cells were prepared from human term placenta and maintained in monolayer culture. On days 3 and 4 of culture, these cells were treated with dehydroepiandrosterone sulfate, androstenedione, or testosterone with or without the concomitant presence of 8-Br-cAMP. 8-Br-cAMP markedly enhanced human chorionic gonadotropin secretion into the culture medium. On the other hand, the concomitant addition of 8-Br-cAMP with the androgen precursors led to an inhibition of estradiol output. The concentrations of androstenedione and dehydroepiandrosterone in the culture medium after treatment with dehydroepiandrosterone sulfate were elevated by the concomitant presence of 8-Br-cAMP. From these results we conclude that 8-Br-cAMP enhances human chorionic gonadotropin output in human term placental cells, whereas the presence of 8-Br-cAMP in cells given androgen precursors inhibits estradiol output, probably at the level of aromatization.     

Buserelin suppression of endogenous gonadotropin secretion in infertile women with ovarian feedback disorders given human menopausal/human chorionic gonadotropin treatment

Fifty infertile women with oligomenorrhea, anovulation, or luteal phase defects were selected for a combined therapy consisting of a gonadotropin-releasing hormone analog (Buserelin Hoechst AG, Frankfurt/Main, FRG) and human menopausal gonadotropin/human chorionic gonadotropin (hMG/hCG). Serving as their own controls, these women had been subjected to a total of 238 hMG/hCG treatment cycles with no pregnancy observed (average, 4.7 cycles; range 2 to 14). Of these 238 hMG/hCG cycles, only 98 (41.1%) appeared normal, while the others showed symptoms consistent with inadequate follicle maturation, luteal phase defects, and premature luteinization. In contrast, 89 cycles from 133 combined buserelin/hMG/hCG treatment cycles (66.9%) appeared to be normal, with no evidence of premature luteinization, and 21 patients became pregnant. These data indicate that the likelihood of group II World Health Organization (WHO) patients becoming pregnant with hMG/hCG therapy may be enhanced when endogenous gonadotropin secretion is suppressed at the same time.     

Effects of diacylglycerol and gonadotropin-releasing hormone on human chorionic gonadotropin release by cultured trophoblast cells.

The effect of activation of calcium- and phospholipid-dependent protein kinase (protein kinase C) on human chorionic gonadotropin (hCG) release by cultured trophoblast cells was studied and a role of protein kinase C in the GnRH-mediated hCG release was also evaluated. Both GnRH and 1-oleoyl-2-acetylglycerol (OAG), a protein kinase C activator, stimulated hCG release after 3 h incubation in a dose-dependent manner with ED50 of 55 nmol/l and 4.0 nmol/l, respectively. A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) also stimulated hCG release while two non-tumor-promoting compounds, phorbol and 4 alpha-phorbol, failed to stimulate hCG release. hCG release by maximal effective dose of GnRH (10 mumol/l) or OAG (1 mumol/l) was further stimulated when cells were incubated with same concentrations of GnRH and OAG. OAG-stimulated hCG release was completely inhibited by a protein kinase C inhibitor, H-7, with ID50 of 23 nmol/l while H-7 did not affect GnRH-mediated hCG release. These results indicate that GnRH-stimulated hCG release is not mediated by protein kinase C pathway, however, the secretion of hCG is also regulated by the mechanism that involves protein kinase C activation.     

Sex-dependent induction of human suppressor T cells by chorionic gonadotropin

Human chorionic gonadotropin (hCG) in physiological retroplacental concentration has been shown to induce human female lymphocytes which suppress the p     

Human chorionic gonadotropin in the ectopic gestation

The ectopic pregnancy is a relatively common condition in the south African black patients. The beta-specific subunit radioimmunoassay for human chorionic gonadotrophin (HCG) was utilized in procuring information in our series of 30 patients. Levels of the hormone were significantly lower when compared to normal gestation of similar duration, never exceeding 2000 mIU/ml. The clearance rate of HCG following normal vaginal delivery was about 24 h and less variable than that of ectopic gestation. The half-life clearance rate of HCG in the ectopics could be divided into three phases, suggestive of HCG compartmentalization. The possible buffering effect of this hormone in the maintenance of the receptor-saturated pregnancy is discussed. The possibility that the HCG produced by the normal pregnancy is dissimilar to that of ectopic is speculated upon.     

Serial human chorionic gonadotropin determinations by fluoroimmunoassay for differentiation between intrauterine and ectopic gestation

A new means for differentiation between ectopic and early intrauterine pregnancy--the human chorionic gonadotropin score--is described. The score relates the rate of serum hCG rise per day to the initial human chorionic gonadotropin level. The positive predictive value for ectopic pregnancy was 94.7%, based on human chorionic gonadotropin scores from 41 women with increasing human chorionic gonadotropin levels in the range of 10 to 4000 IU/L. The method may be useful for identification of ectopic pregnancy in a category of women in whom ultrasonography is of limited diagnostic value.     

Prednisolone promotes the secretion of progesterone and oestradiol by luteinised granulosa cells independent of human chorionic gonadotropin

An in vitro model based on luteinised granulosa cells gained during an in vitro fertilisation programme examines the question of whether prednisolone, and thus glucocorticoids in general, are capable of exerting an influence on the secretion of oestradiol and progesterone. It can be demonstrated that prednisolone leads to dosage-dependent increases in the concentration of both steroids, even independently of concurrent stimulation by human chorionic gonadotropin. A similar mechanism for aromatase activity of human adipose cells is suggested.     

A semiquantitative human chorionic gonadotropin assay for the detection of ectopic pregnancy

Human chorionic gonadotropin (hCG) was measured in 117 serum samples with known quantities of hCG after a dilutional modification of a reliable, simple, and inexpensive qualitative assay for hCG. The modification yielded a semiquantitative assay for hCG with a sensitivity of 5000 mIU/mL. At hCG concentrations below 4000 mIU/mL, the assay had no false-negative or false-positive results; above 6500 mIU/mL, there were also no false-negative or false-positive results. In the range of 4000-6500 mIU/mL, the clinical false-positive rate was 28%. Using the described dilutional modification of this qualitative hCG assay, the test is semiquantitative, and is useful in selecting the appropriate time to perform ultrasound and laparoscopy in women suspected of having an ectopic pregnancy.     

Response to human chorionic gonadotropin in patients with ovarian tumors

The results of hCG stimulation on peripheral levels of androstenedione (A), testosterone (T) and estrone (E1) were examined in 14 patients with ovarian tumors and in 9 tumor-free subjects, after the menopause. Following hCG injection, 9 postmenopausal patients with ovarian tumors showed a significant rise in A peripheral levels. The responsive subjects generally had significant increases in A baseline levels, too. The remaining 5 subjects with advanced or poorly differentiated ovarian cancer with no stroma were not responsive to hCG. Moreover, in the tumor group, 7 subjects had increased baseline T and/or E1 and in 3 of them an increase of these steroids was observed following hCG. In the absence of ovarian tumor, no subject in the control group was responsive to hCG administration. The results of the present investigation seem to confirm the in vivo responsiveness to hCG of ovarian tumors.     

Reorganization of human ovarian adenocarcinoma cells and endometrial adenocarcinoma cells cultured in double-layered floating collagen gels

Collagen gels have been used as the substratum for epithelial cell culture to maintain their differentiated states. In this report, we proposed a double-layered floating collagen gel method for the culture of human carcinoma cells to show their differentiated structures which closely resembled those in the tumors in vivo. On the 3rd week of the culture, OMC-1 cells (human ovarian mucinous adenocarcinoma) showed organized ductular structures. PAS-positive substances were seen along the surface of the ductules and also in cytoplasms. Electron microscopically, numerous microvilli were observed at the apical surfaces of the cells, with formation of rootlets. Ishikawa line cells (human well-differentiated endometrial adenocarcinoma) formed tubular structures with back-to-back arrangements. Electron microscopically, numerous microvilli, well-developed junctional complexes, and well-developed mitochondria and Golgi apparatus were observed, similarly to those in the cells grown in vivo. SNG-M cells (human moderately-differentiated endometrial adenocarcinoma) were arranged in sheets with occasional formation of tubular structures. Mitochondria, Golgi apparatus and desmosomes were poorly developed in general; however, the cells forming tubular structures showed signs of cellular differentiation. The floating and non-floating methods were compared with the result that the former method was seen to be far superior to the latter for expressing cellular differentiation.     

Diagnosis of ectopic pregnancy: value of the discriminatory human chorionic gonadotropin zone

A prospective study was conducted to test the hypothesis that the absence of an intrauterine gestational sac when the serum level of human chorionic gonadotropin (hCG) is above 6500 mIU/mL is indicative of ectopic pregnancy. A total of 383 patients who were clinically suspected to have ectopic pregnancies had pelvic ultrasound examinations with serum hCG determinations on the day of the scan. There were 217 (57%) intrauterine gestations, 104 (27%) ectopic pregnancies, and 62 (16%) spontaneous abortions. Forty-one percent of patients had an hCG level above 6500 mIU/mL. The absence of an intrauterine gestational sac at an hCG concentration above this level had a sensitivity of 100%, a specificity of 96%, a positive predictive value of 86%, a negative predictive value of 100%, and was 98% efficient, based on a 19.4% prevalence of ectopic pregnancies among this group.     

Beta human chorionic gonadotropin levels in cul-de-sac blood of patients with ectopic pregnancies

hCG levels in cul-de-sac and peripheral blood in patients with ectopic pregnancies were studied. hCG levels were significantly higher in the cul-de-sac blood than in peripheral blood (P less than 0.01). The high hCG level in the cul-de-sac blood was not due to interference by proteolytic enzymes in cul-de-sac blood since the protease inhibitors, aprotinin and phenylmethylsulfony fluoride, did not alter the RIA results from cul-de-sac samples, nor did Con-A chromatography of the samples. Assay of cul-de-sac blood samples for hCG may increase the sensitivity of diagnosing ectopic pregnancies.     

Prognostic value of human chorionic gonadotropin changes after methotrexate injection for ectopic pregnancy

Because early prediction of clinical outcome (one or more injections or surgery) of methotrexate treatment of ectopic pregnancy could ease the intensity of follow-up and patient compliance required, we studied the relationship between the change in hCG levels after methotrexate injection and outcome in 129 consecutive patients. A 20% decline in hCG levels between days 1 and 4 during methotrexate treatment has a positive predictive value of 97%.     

Immunocytochemistry of human chorionic gonadotropin in human chorionic villi

The immunocytochemical localization of human chorionic gonadotropin was investigated in chorionic villi from the seventh to twelfth week of gestation. By the light microscopic peroxidase-antiperoxidase technique, positive reactions of human chorionic gonadotropin were found exclusively in the syncytiotrophoblast. Immunoelectron microscopy by means of the protein A-gold technique reveals localization of the immunoreactive gold particles in two kinds of membrane-bound granular inclusions in this cell; one type is granules of 200 to 300 nm in diameter with moderate electron density and the other is large electron-dense bodies of 500 to 1000 nm. The former seems to be Golgi-derived secretory granules that play a role in the release of human chorionic gonadotropin from the syncytiotrophoblast. Although the origin of the latter is still uncertain, a certain amount of this hormone might be stored or treated by lysosomal digestion in the large bodies during these stages.