Amounts Of Human Chorionic Gonadotropin Research Articles

Trophoblast stem cell-based organoid models of the human placental barrier

Human placental villi have essential roles in producing hormones, mediating nutrient and waste exchange, and protecting the fetus from exposure to xenobiotics. Human trophoblast organoids that recapitulate the structure of villi could provide an important in vitro tool to understand placental development and the transplacental passage of xenobiotics. However, such organoids do not currently exist. Here we describe the generation of trophoblast organoids using human trophoblast stem (TS) cells. Following treatment with three kinds of culture medium, TS cells form spherical organoids with a single outer layer of syncytiotrophoblast (ST) cells that display a barrier function. Furthermore, we develop a column-type ST barrier model based on the culture condition of the trophoblast organoids. The bottom membrane of the column is almost entirely covered with syndecan 1-positive ST cells. The barrier integrity and maturation levels of the model are confirmed by measuring transepithelial/transendothelial electrical resistance (TEER) and the amount of human chorionic gonadotropin. Further analysis reveals that the model can be used to derive the apparent permeability coefficients of model compounds. In addition to providing a suite of tools for the study of placental development, our trophoblast models allow the evaluation of compound transfer and toxicity, which will facilitate drug development.

Gestational Trophoblastic Neoplasia with Hyperthyroidism

Gestational Trophoblastic Neoplasia (GTN) is a malignant lesion arising from placental villous and extra-villoustrophoblastand occurs in 1:40,000 pregnancies. Invasive mole and choriocarcinoma are the vast majority of GTN whichproduce substantial amounts of Human Chorionic Gonadotropin (hCG). Hyperthyroidism in GTN is due to the stimulation ofthe thyroid gland by hCG which has a similar structure with Thyroid-Stimulating Hormone (TSH). A 28-year-old female,suspected with choriocarcinoma and anemia, had a history of recurrent vaginal bleeding for eight months, accompaniedwith loss of appetite, weight loss, palpitation, and tremor. Physical examination such as pulse rate of 114x/minutes, the0 respiration rate of 26x/minutes, temperature 38 C, conjunctival anemia, and dyspnea were reported. In addition, laboratoryfindings such as anemia, leukocytosis, hypoalbuminemia, hypokalemia, increase of LDH, increase of hCG >1,500,000mIU/mL, T4 levels of 14.1 ug/dL (4.40-10.90 ug/dL), FT4 levels of 1.95 ng/dL (0.89-1.76 ng/dL), and decrease of TSH were alsoreported. Abdominal CT Scan suggested uterine mass suspected as malignancy infiltrating to the rectum with metastaticfeatures in the liver, base of left lung, spleen and left kidney. Increased CA-125, and metastatic features of lung rightparacardial and left suprahilar from Chest X-ray were found. Diagnostic criteria for gestational trophoblastic neoplasia are asfollows: increased hCG 4 x tests; increased hCG three weekly tests; histology diagnosis of choriocarcinoma; increased hCG> 20,000 more than four weeks post evacuation and the presence of metastasis. Hyperthyroidism in GTN is potentiallylife-threatening because of heart failure and thyroid storm. Hyperthyroidism increases morbidity and mortality in GTNpatient; therefore, periodic thyroid tests is essential to prevent further complication of hyperthyroidism.

Gestational Trophoblastic Neoplasia Series of 17 Cases

Gestational Trophoblastic Neoplasia Series of 17 Cases Gestational trophoblastic neoplasia comprises a unique group of human neoplastic diseases that derive from fetal trophoblastic tissues. They are proliferative as well as degenerative disorders of placental elements and include complete or partial hydatiform mole (90%), invasive mole (5-8%) which could also be metastatic, villous or a villous choriocarcinoma (1-2%), and placental site tumor (1-2%). During the study period of 5 years, from January 2009 to December 2013, there were 17 cases analyzed retrospectively. Invasive mole and choriocarcinoma, which make up the vast majority of these tumors, always produce substantial amounts of human chorionic gonadotropin (hCG)..

Effects of Pregnancy on Papillary Microcarcinomas of the Thyroid Re-Evaluated in the Entire Patient Series at Kuma Hospital.

Background: An active-surveillance clinical trial of low-risk papillary microcarcinoma (PMC) patients has been performed at the authors' institution, Kuma Hospital, since 1993. Favorable oncological results have been reported. During the trial, a few patients were encountered with PMC that showed enlargement during pregnancy, and these cases have been reported. During pregnancy, a large amount of human chorionic gonadotropin (hCG) having weak thyrotropin (TSH) activity is produced, possibly affecting the progression of PMC. This study investigated how pregnancy and delivery influenced the progression of PMC in the entire active surveillance PMC patient series.Methods: From 1993 to 2013, 1841 patients with low-risk PMC chose the active surveillance program. Fifty of the 1549 female PMC patients experienced 51 pregnancies/deliveries. To minimize observer variation, a single specialist sonographer re-evaluated the changes in the size of these 50 patients' PMCs before and after the pregnancies/deliveries.Results: Four patients (8%) showed enlargement of PMC by ≥3 mm; one patient (2%) showed a decrease by ≥3 mm, and the remaining 44 patients (45 events, 90%) showed stable disease. None of the patients had a novel appearance of lymph node metastases during pregnancy. Of the four patients with enlargement, two underwent surgery after delivery, and the other two continued the active surveillance, since their tumors did not grow after the delivery. After delivery, the PMC of one of these four patients remained stable, and another showed a decrease in PMC size. To date, six more PMC patients underwent surgery after delivery for reasons other than disease progression due to pregnancy and delivery: two opted out of active surveillance, two were identified with a nodal metastasis during active surveillance after delivery, one had Graves' disease, and one showed enlargement of nodules of the contralateral lobe.Conclusions: Pregnancy and delivery was associated with an increase in size of PMCs in only 8% of the 51 pregnancies/delivery cases. None of the patients developed nodal metastasis during pregnancy. Thus, a possible future pregnancy does not prevent such patients from undergoing active surveillance, although watchful observation during pregnancy is recommended.

Clinical effects of a natural extract of urinary human menopausal gonadotrophin in normogonadotropic infertile patients.

Purified human menopausal gonadotropin (HMG) is a natural product extracted from the urine of postmenopausal women that contains pituitary follicle-stimulating hormone (FSH), luteinizing hormone (LH), and a small amount of human chorionic gonadotropin (HCG). Here we retrospectively conducted a clinical pharmaceutical study on a cohort of normogonadotropic infertile patients addressed to long GnRH-agonist protocol with serum LH concentration ranging from 0.5 IU/L to 1.5 IU/L during the midfollicle phase, aiming at evaluating the effects of purified HMG supplementation during ovarian stimulation. There was no significant difference in either the basic clinical features of the patients or the pregnancy rate (71.4% versus 66.3%, P > 0.05) or other related indicators of pregnancy outcome. However, there was a higher level of serum oestradiol (E2) on the day of human chorionic gonadotropin (HCG) (1999.10 ± 860.50 IU/L versus 2883.29 ± 1427.382 IU/L, P = 0.000) but lower fertilization rate (89.1% versus 69.6%, P < 0.000) in patients getting HMG supplementation and a higher risk of developing ovarian hyperstimulation syndrome (OHSS). We suppose that exogenous LH supplementation is not needed when serum LH concentration of the midfollicle phase is around 0.5–1.5 IU/L during the long GnRH-agonist protocol. Adding exogenous HMG may decrease the fertilization rate and increase the risk of developing OHSS.

World Congress on the Insulin Resistance Syndrome, 2009

This is the fourth of four articles summarizing presentations at the seventh World Congress on the Insulin Resistance Syndrome, held in San Francisco, California, on 5–7 November 2009. This installment pertains to insulin resistance (IR) mechanisms, the brain, and IR in youth and in the polycystic ovary syndrome (PCOS). ### Hyperinsulinemia and insulin secretion abnormalities #### Hyperinsulinemia causing IR. Jesse Roth (New York, NY) suggested that, rather than IR, hyperinsulinemia should be considered the major mediator of adverse outcome, with hyperinsulinemia the underlying characteristic of all syndromes of IR (1). Recalling Edgar Allan Poe's story “The Purloined Letter,” Roth argued that the mediator of IR is insulin, which is “hidden in plain sight,” causing hormone-induced inhibition of hormone action. Similar phenomena are seen in choriocarcinoma, in which enormous amounts of human chorionic gonadotropin have no biological effect, in the divergence between the effect of gonadotropin-releasing hormone, which enhances fertility when given in a pulsatile fashion, while when administered continuously, it halts precocious puberty, or between the effect of parathormone given continuously, causing bone resorption, while bolus administration increases bone formation. High levels of insulin similarly decrease the effectiveness of insulin, and mice overexpressing the insulin gene, with doubling and quadrupling insulin levels, fail to show change in weight or fasting glucose, while developing IR, postprandial hyperglycemia, and decreased insulin receptor binding. Similar effects of hyperinsulinemia may be demonstrated in normal rats that are given increasing daily doses of insulin or that have hypothalamic damage, whereas ob/ob mice that are given alloxan or streptozotocin show improvement in insulin sensitivity. In humans, the Somogyi effect is of decreasing insulin doses improving glycemia, whereas patients with insulinoma develop IR, and pulsatile administration in a manner mimicking its physiologic release appears to have greater biologic effect than that of continuous infusion of a considerably greater amount of insulin (2). Basal insulin levels are twice …

Inhibitory Effect of Thyroid Blocking Antibody (TBAb) on the Thyroid Stimulatory Effect of Human Chorionic Gonadotropin (HCG ) and Equine Chorionic Gonadotropin (ECG)

We examined the inhibitory effect of thyroid blocking antibody (TBAb) on the thyroid stimulating activity of human chorionic gonadotropin (HCG) and equine CG (ECG). Five TBAb positive sera obtained from patients who had been hypothyroid but were currently on T4 treatment. The TSH binding inhibitory immunoglobulin (TBII) activities of the sera were 60-160 IU/L. Inhibition of TSH binding to the TSH receptor (TSHR) [TSH binding inhibition (TBI) activity] of HCG or ECG, and inhibition of TBAb on HCG or ECG-stimulated cAMP production were examined. Both HCG and ECG preparations showed weak TBI activity in the presence of small amounts of protein [bovine serum albumin (BSA)] but were negative in the presence of large amounts of protein [normal human serum (NHS) or BSA]. Four thousand IU/mL of HCG and ECG preparation caused cAMP production similar to 100 microU/mL of bovine (b) TSH. The inhibitory effect of TBAb on cAMP production by this amount of HCG or ECG was then examined. The inhibitory effect of TBAb on cAMP production by HCG and ECG was similar to bTSH, and TBAb positive sera with more than 40 IU/L TBII activity completely blocked cAMP production by HCG, ECG and bTSH. This suggests that common alpha -subunit of both HCG and TSH are involved in the inhibitory effect of TBAb. Previous reports demonstrated that the thyroid stimulating activity of thyroid stimulating antibody (TSAb) was blocked by deglycosylated HCG (competitive antagonist of TSH binding to TSHR). The fact and our present study suggest that TSH, HCG ECG, TSAb and TBAb have a similar binding site (alpha-subunit-mimicking binding site) on the TSH receptor.

The Role of Human Chorionic Gonadotropin as a Thyroid Stimulator in Normal Pregnancy

The first clinical reports of a placental thyroid stimulator arose from studies indicating that placental tumors that secrete very large amount of human chorionic gonadotropin (hCG), hydatidiform moles and choriocarcinomas, were associated with hyperthyroidism [summarized by Yoshimura and Hershman (1)]. In 1967, Burger reported that impure, commercial hCG had thyroid-stimulating activity in a mouse bioassay (2). Several years later my laboratory (3) and that of Nisula (4) reported that highly purified hCG had activity in this bioassay. We found that the molar TSH was hCG (4). Because hCG varies in its carbohydrate composition, there has been considerable controversy in regard to the potency of these variants. hCG lacking its C-terminal tail, therefore closely resembling LH, is a more potent TSH than native hCG (5). Desialylated basic variants of hCG are more potent in binding to the TSH receptor than fully sialylated acidic hCG (6). However, the acidic hCG has a longer half-life in the circulation than the more basic partially desialylated hCG, and this may contribute to greater thyrotropic potency in humans (7). These studies mainly arose from patients with placental tumors or from a few patients with gestational thyrotoxicosis associated with high hCG levels. The possibility that hCG plays a role in regulation of the thyroid gland during normal pregnancy was first considered when there were more sensitive immunoassays for TSH. Braunstein and Hershman (8) reported that there was an inverse relationship between TSH and hCG at about 10–12 wk of pregnancy, the time of peak hCG levels. Harada et al. (9) further advanced this concept by showing the inverse relationship more clearly and amassing data showing an increase of free T4 and free T3 associated with the peak hCG. We postulated that hCG caused an increase of free thyroid hormone levels, thus suppressing TSH, albeit within the physiological range of serum TSH. Glinoer et al. (10), in a landmark study using a more sensitive TSH measurement, further refined the mirror image temporal relationship between hCG and TSH in normal pregnancies. Other reports also contributed to the concept that hCG is a thyroid stimulator in the early part of pregnancy when hCG levels are very high (11). In this issue of the Journal, Haddow et al. (12) have done a complex analysis of the relationship between hCG, TSH, and free T4 during the first trimester (wk 11–13) and second trimester (wk 15–18) in 9562 women with singleton pregnancies. They found that higher hCG levels were associated with a lower body mass index. This fits with the observation of Goodwin et al. (13) that hCG is higher in women with nausea of pregnancy and hyperemesis gravidarum than in women who do not have any nausea, assuming that weight gain is restricted by nausea. More importantly, the data of Haddow et al. further strengthen the role of hCG as a thyroid stimulator in normal pregnancy. At wk 11–13, higher hCG correlates with higher free T4 levels, although the relationship is a shallow slope, confirming the data of Glinoer et al. (10) that in normal pregnancy hCG only modestly increases free T4. The relationship between hCG and TSH is more difficult to appreciate in the complex analysis by Haddow et al. (12). It shows clearly that low TSH is associated with high levels of hCG, but when TSH is in the higher deciles, there is no correlation with hCG. My interpretation is simplistic. Placental secretion of hCG is not regulated by thyroid hormones. However, when hCG levels are high, they increase thyroid hormone secretion, and the negative feedback at the level of the pituitary suppresses TSH release. When TSH levels are in the higher part of the normal range, or perhaps slightly elevated for pregnancy, the contribution of hCG to increase free T4 and T3, resulting in lower TSH levels, is not sufficient to lower serum TSH significantly. Perhaps evolution designed the hCG system as a backup for stimulating the thyroid gland to produce essential amounts of thyroid hormone, but this backup system did not fully evolve to perfection.

P-129: Leuprolide acetate 0.2 cc with human chorionic gonadotropin to trigger ovulation as an effective method to decrease ovarian hyperstimulation syndrome pain without affecting the implantation rate in donor oocyte cycles

Ovarian Hyperstimulation (OHSS) is a significant complication of gonadotropin therapy. Ovulation induction cycles should be highly individualized and should use the minimum effective dosage of Human Chorionic Gonadotropin (HCG) to significantly reduce the risk of OHSS. However, it is unclear if pregnancy rates are compromised by Leuprolide Acetate (LA) trigger alone. Oocyte donors with OHSS will frequently have to take time off from work and will refuse to do another Donor Egg/IVF cycle. The use of LA alone has been shown in several studies to trigger ovulation through an endogenous LH surge. This effect is more physiological than an exogenous HCG trigger, thus reducing the risk of pain and OHSS. This is a retrospective study of donor oocyte patients: Leuprolide Acetate (LA) 1 mg (o.2cc) subcutaneously was given synchronously with 1.25 - 5K HCG. Patients were evaluated for OHSS symptoms (pain 1 to 2 days after the oocyte retrieval), number of oocytes retrieved, peak estradiol levels, number of cryopreserved embryos, and implantation rate. Retrospective review of 33 donor oocyte retrievals from 1/2004 - 1/2006 were evaluated in patients who received LA 0.2cc trigger with HCG. These patients were categorized according to if they had pain versus no pain 1 to 2 days after the retrieval. Ages of the egg donors ranged from 20 to 34. Patients were selected to receive HCG plus LA if their estradiol level ranged from 1600-8800 and the number of follicles greater than 15 ranged from 5-12 A total of 33 donor oocyte patients received LA 0.2cc with HCG. There were 8 shared cycles for a total of 39 transfers. Patients who received LA 0.2cc with HCG had estradiol peak levels ranging from 1618 to 8806, total number of oocytes ranging from 13-68, implantation occurred in 29 recipients (16 singletons, 11 twins, and 2 triplets) with cryopreserved embryos ranging form 0-45. There were a total of 7 patients with complaints of mild to moderate pain and 2 OHSS patients. There was a low incidence of pain in the LA plus HCG donors even though their estradiol levels were high and high number of oocytes retrieved. Oocyte donor patients who receive LA with HCG may have a reduced risk of OHSS symptoms, however the amounts of HCG may be the significant factor. It is prudent for all oocyte donor patients to be assessed for OHSS. Lowering HCG dose and/or using LA may decrease the incidenceof OHSS in a donor oocyte program. Oocytes donors may have less morbidity and may be more likely to pursue additional donation cycles if the incidence of OHSS were lower. Larger studies are needed to compare LA with HCG with donor oocyte patients, as well as regular IVF patients. Other factors to consider are evaluating whether it is the effect of the Gonadotropin Releasing Hormone (GnRH) agonist, LA 0.2cc with HCG or is it the lower dosage of HCG that decreases the incidence of OHSS. Also another question to explore would be if lower doses of HCG would be as effective without LA?

Effect of Nickel (Ni2+) on Primary Human Ovarian Granulosa Cells In Vitro

Human ovarian granulosa cells obtained from women undergoing in vitro fertilization were exposed to 15.6, 31.25, 62.5, 125, 250, 500, 1000 μM Ni2+ for 48 h. To determine the site of action of Ni2+, the granulosa cells were stimulated to produce progesterone (P) by using maximally stimulating amounts of human chorionic gonadotropin (0.1 IU/ml hCG) or dibutyryl cyclic adenosine monophosphate (1 mM db-cAMP). The luteinizing hormone (LH) analog hCG was chosen because resultant P production requires an intact membrane receptor and db-cAMP was used to test for post LH receptor defects caused by Ni2+. Progesterone content of the culture medium was determined by radioimmunoassay (RIA), and viability of the cells was measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction test. Concentration-dependent depression in both hGC and db-cAMP stimulated P production was seen at 15.625 μM or higher concentration of Ni2+, which is not cytotoxic on human ovarian granulosa cells. The viability of cells was unaffected up to 31.25 μM and decreased significantly at 62.5 μM. Our results show a dose-related depression in stimulated P production of granulosa cells at a dose that does not induce significant cytotoxic action. These data indicate that the effect of Ni2+ on P production is not due to cytotoxicity, and the cellular site(s) of inhibitory action appears to be subsequent to the membrane receptor and production of cAMP.

Quantifying the syncytialisation of human placental trophoblast BeWo cells grown in vitro

We have generated lines of BeWo cells that constitutively and stably express either histone H2B tagged with the green fluorescent protein (GFP), or the mitochondrial targeting sequence of subunit VIII of cytochrome c oxidase fused with a red fluorescent protein; one line has nuclei that fluoresce green, the other mitochondria that fluoresce red. Expression of these tagged proteins has no effect on the rates of DNA, RNA and protein synthesis, or on the amounts of human chorionic gonadotropin (hCG) secreted after treatment with forskolin. We used fluorescence-activated cell sorting (FACS) to monitor the extent of cell fusion (syncytialisation) between these two lines; fused cells are readily and accurately detected by their green/red fluorescence. This assay should prove useful in the investigation of the molecular mechanisms involved in trophoblast syncytialisation.

ED27 Trophoblast-like Cells Isolated from First-trimester Chorionic Villi are Genetically Identical to HeLa Cells Yet Exhibit a Distinct Phenotype

ED27 trophoblast-like cells were prepared from human chorionic villus samples obtained at 9 weeks gestation and have been grown continuously in vitro without phenotypic drift for nearly a decade. These cells express many trophoblast markers, including cytokeratin, placental alkaline phosphatase (PLAP), secretion of 17β-estradiol, and a microvillous apical surface. The ED27 cell line is a useful model system for studies of placental cell biology and has been distributed to laboratories world-wide. However, experiments to investigate their relationship to primary villous cytotrophoblast have shown that these cells do not secrete detectable amounts of human chorionic gonadotropin in culture and, when digested with trypsin, disperse into individual cells. Furthermore, immunocytochemical studies demonstrated that, unlike villous cytotrophoblasts, ED27 cells were immunoreactive with monoclonal antibodies recognizing some HLA Class I antigens. This was not HLA-G, however, as would be expected if these cells originated from extravillous cytotrophoblasts, but rather classical HLA-A, B which is thought not to be expressed by any trophoblast subpopulations. These inconsistencies prompted us to question the authenticity of the continuous cell line as it now exists. Genetic haplotype analysis using the polymerase chain reaction (PCR) revealed that ED27 was genetically identically to the HeLa cell line. Inasmuch as HeLa cells have never been grown in the laboratory (DAK), the only possible origin of HeLa cell contamination of ED27 cells was the WISH cell line, and further PCR analysis revealed that this cell line was also genetically identical to HeLa. Like ED27 cells, HeLa cells and WISH cells synthesized small amounts of estrogen and were found to express PLAP and antigens recognized by the monoclonal antibodies ED822, directed against the syncytiotrophoblast, and J1B5 directed against villous cytotrophoblast. These results point out the need for adherence to rigorous and consistent quality control measures to assure the authenticity of cell lines used as in vitro model systems.

Chorioamnionitis reduces placental endocrine functions: the role of bacterial lipopolysaccharide and superoxide anion.

Chorioamnionitis has been shown to be one of the most important factors in inducing preterm delivery. The present study was undertaken to examine the effects of chorioamnionitis on placental endocrine functions. Preterm placentas with histologic chorioamnionitis produced smaller amounts of human chorionic gonadotropin (hCG) and human placental lactogen (hPL) than those without chorioamnionitis (P < 0.001). To examine the mechanism involved in the suppression of placental endocrine functions induced by chorioamnionitis, we initially confirmed the expression of lipopolysaccharide (LPS) receptor, i.e. the CD14 molecule, on trophoblasts by Northern blot analysis and immunohistochemistry. We then stimulated purified trophoblasts with LPS, which is the major agent which induces inflammatory responses in the host via the LPS receptor. The trophoblasts stimulated with LPS produced reduced amounts of hCG, hPL, and progesterone in a time- and dose-dependent fashion in spite of the induced manganese-superoxide dismutase (SOD) synthesis. Stimulation of trophoblasts with hypoxanthine and xanthine oxidase resulted in suppressed hCG production, while the simultaneous addition of SOD into the culture medium reversed the suppression of hCG production. LPS in the placenta with chorioamnionitis might directly stimulate trophoblasts through the LPS receptor (CD14), thus reducing placental endocrine functions. Superoxide anions which exogenously act on trophoblasts might be generated by simultaneous stimulation of neutrophils and monocytes at the feto-maternal interface by LPS, and additively reduce placental endocrine functions.

Effect of human chorionic gonadotrophin on chloride current in human syncytiotrophoblasts in culture

Human trophoblast differentiates in vivo and in vitro by the fusion of cytotrophoblastic cells to form syncytiotrophoblasts. A large amount of human chorionic gonadotrophin (hCG) is produced by the syncytiotrophoblasts, which express hCG luteinizing hormone (LH) receptors. Since recent investigations with electrophysiological techniques support the conclusion that hormonal effects can be mediated by modulations of the membrane ionic conductances of the cells, a perforated patch-clamp technique was used to investigate the possible presence of a chloride current evoked by hCG. The perifusion of hCG (500 mIU/ml) activated a time-independent current, which presents a linear current-voltage (I/V) relationship in symmetrical chloride concentrations. The reversal potential was − 1.8 mV with 142 mm Cl − external solution and 134 m m cl − internal solution. This reversal potential shifted with changes in the transmembrane Cl − gradient. Moreover, this hCG-induced current was sensitive to 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (50 μ m), to diphenylalamine-2-carboxylic acid (DPC) (0.5 m m) and to 9-AC (1 m m), three known chloride channel blockers. These results confirm the autocrine action of hCG in the physiology of the trophoblast.

A Baboon (Papio anubis) simulated-pregnant model: cell specific expression of insulin-like growth factor binding protein-1 (IGFBP-1), type I IGF receptor (IGF-1 R) and retinol binding protein (RBP) in the uterus

In order to test the hypothesis that the baboon conceptus/placenta regulates the synthesis of specific proteins in the endometrium, we developed a simulated-pregnant baboon model. Baboons (n=2-6/group) were treated with increasing amounts of human chorionic gonadotrophin (hCG) for 10 or 12 days beginning on day 6 or 7 PO. Uterine tissues were obtained at day 18 PO following 12 days of hCG treatment. Animals in the day 25 and 32 PO group were treated for 10 days with hCG. Following the hCG treatment, estradiol (E) and progesterone (P) implants were inserted subcutaneously. Control groups consisted of E and P treatment only (day 25 PO), or ovariectomy on day 6 or 7 PO followed by hCG plus E and P treatment (days 18 and 25 PO). Serum samples were obtained daily or once every 2 days and analysed for E and P by radioimmunoassay. hCG activity in serum was determined by a Leydig cell bioassay. Portions of the endometrial tissue were either subjected to organ explant culture, analysed by immunocytochemistry or extracted for RNA. Peripheral serum levels of hCG, E and P in the experimental groups fell within the 95% confidence interval limits of hormone concentrations achieved during pregnancy. The morphology of the endometrium in the hCG treated baboons and pregnant baboons was similar i.e., distended convoluted glands, many spiral artery beds, a loose vacuolized stroma, and increased collagen staining. However, in the absence of hCG (E+P treatment only) the glands tended to be straight rather than corkscrew-shaped, and decreased stromal vacuolization and collagen staining was evident.(35)S-methionine labeled proteins in explant culture conditioned media (TCM) were analysed by two-dimensional SDS-PAGE and fluorography. A comparable pattern of protein synthesis was apparent in all treatment groups except for a low molecular weight (27 000-30 000 daltons) group of polypetides which only was evident in TCM from the hCG treated baboons. A similar group of proteins are also secreted by the baboon endometrium during pregnancy. The immunocytochemical localization of estrogen (ER) and progesterone receptors (PR) was comparable to that observed in pregnant baboons. IGFBP-1 localization was confined to the glandular epithelium in the hCG treated groups (intact and ovariectomized) and was virtually undetectable in the E and P treated group. The intensity of IGFBP-1 staining was variable within each of the hCG treatment groups on days 18, 25 and 32 PO. This variability was also apparent by Western blot analysis, immunoassay of proteins in TCM and on Northern blots of total RNA from the same animals. In contrast, IGF-I R immunostaining was evident in both glandular and surface epithelium of all treatment groups. Expression of RBP was confined to the basal glands. The characteristic upregulation of RBP synthesis in the functionalis observed during early pregnancy was not apparent in any of the treatment groups. In summary, these studies indicate that exogenous hCG in conjunction with E and P, can induce the general morphological and biosynthetic changes the baboon endometrium undergoes during early pregnancy. In addition, this hormonal treatment is also capable of maintaining the epithelial expression of IGFBP-1, IGF-1 and RBP. However, other factors from the conceptus appear to be necessary to induce the cell specific changes in the expression of these three proteins that are observed during pregnancy.

Human blastocyst grading: an indicator of developmental potential?

We have observed marked variation in the in-vitro development of individual human embryos to the blastocyst stage which was poorly associated with the grade of the embryo on the day of transfer (day 2). Therefore we also graded embryos at the blastocyst stage to determine if their development potential can be better predicted at this stage. A total of 41 blastocysts were categorized into three grades depending on their morphology. Various parameters such as hatching, adherence, growth and amount of human chorionic gonadotrophin (HCG) secreted were documented. The number of nuclei in another 34 blastocysts belonging to the three grades were measured. Also morula stage embryos with large vacuoles but no morphological differentiation were included in a separate group called 'vacuolated morulae'. Overall, there was no significant difference between grade 1 and 2 blastocysts as assessed by their development to day 14, cumulative HCG secretion and nuclei counts. Grade 3 blastocysts reflected 'poor' morphology, secreted significantly lower levels of HCG and had significantly fewer nuclei than the other two grades. Vacuolated morulae did not resemble blastocysts in their morphological development, secreted no HCG and had significantly fewer nuclei than grade 1 and 2 blastocysts. This study illustrates the need for selecting blastocysts for transfer to the uterus or for cryopreservation to allow a better assessment of the success of these techniques. In addition, the data will be useful for evaluating the effects of techniques such as co-culture with feeder cells on the development of embryos to the blastocyst stage and beyond.

Pregnancy: Corpus luteum failure in ectopic pregnancy

The endocrinology of ectopic pregnancy was studied in order to investigate the origin of the discordance in the circulating amounts of human chorionic gonadotrophin (HCG) and those of oestradiol and progesterone. Serial maternal blood samples were obtained at 4-9 weeks gestation from 93 patients who became pregnant following in-vitro fertilization and embryo transfer including 10 ectopic, 21 anembryonic and 62 normal singleton pregnancies. The samples were analysed for HCG, Schwangerschaft protein-1 (SP-1), pregnancy-associated plasma protein-A (PAPP-A), progesterone and oestradiol. In ectopic pregnancies, concentrations of all substances analysed were significantly reduced compared to singleton pregnancies from 5 weeks gestation (P < 0.05-0.001) but they were not significantly different from those of anembryonic pregnancies. In ectopic pregnancies, associations were found between the concentration of both HCG and SP-1 and those of progesterone and oestradiol. No associations were found between PAPP-A and any other substances analysed. This may be due to insensitivity of the PAPP-A assay; alternatively PAPP-A concentrations may be differentially reduced in ectopic pregnancy. These findings suggest that progesterone and oestradiol are derived from the corpus luteum in early ectopic pregnancy but that the corpus luteum fails rapidly and the dominant source of both hormones becomes the trophoblast as early as 5 weeks.

Fertilization and early embryology: Human chorionic gonadotrophin: embryonic secretion is a time-dependent phenomenon

Of 48 spare human pre-embryos achieving the expanded blastocyst stage, 22 (45.6%) secreted significant amounts of human chorionic gonadotrophin (HCG) (> 5 IU/l/day). Of these, nine remained intrazonal, seven partially hatched and six fully hatched. Embryonic production of HCG in vitro appeared to be time-dependent, starting after a certain minimum time (approximately 160 h post-insemination) and rising exponentially, with maximal HCG production around day 10. Hatching was not a prerequisite for HCG secretion, since similar amounts were produced by intrazonal blastocysts. Blastocysts derived from abnormally fertilized oocytes also began secreting HCG exponentially but secretion was delayed and the upper limit of maximum HCG secretion rate was comparatively low. The actual amount of HCG is thought to reflect the number of viable trophectoderm cells producing the hormone. HCG doubling times for blastocysts in vitro were rapid when compared to implanting blastocysts of a similar age in vivo, with 19/22 (86.4%) blastocysts having a doubling time of < 10 h. Provided a pre-embryo can secrete HCG and maintain an adequate doubling time, sufficient HCG should be produced for initial stages of embryonic recognition in vivo. Since intrazonal blastocysts are capable of fulfilling both of these criteria, the limiting factor in realizing their full potential may be escaping from the zona pellucida.

Necrosis and inhibition of growth of human lung tumor by anti-alpha-human chorionic gonadotropin antibody.

Human lung tumor cells (ChaGo) established in culture from a bronchogenic squamous cell carcinoma synthesize and secrete large amounts of human chorionic gonadotropin (HCG), predominantly the alpha subunit of the glycoprotein hormone. ChaGo cells lose their transformation phenotypes following treatment with anti-alpha-HCG antibody or following inhibition of intracellular synthesis of alpha-HCG by the anti-sense RNA technique. We report that tumors induced by ChaGo cells in female athymic mice undergo necrotic degeneration following local or intraperitoneal administration of alpha-HCG-specific-antibody. The alpha-HCG antibody did not affect the growth of tumor induced by alpha-HCG nonproducing human tumor cells. Histopathological examinations of the anti-alpha-HCG antibody-treated tumor tissues showed active necrosis. When the antibody treatment was discontinued, the tumorigenesis process resumed. When anti-alpha-HCG antibody was administered simultaneously with ChaGo cells, a concentration-dependent inhibition of tumor growth in athymic mice was completely prevented at higher concentrations of the specific antibody.

Human trophectoderm biopsy and secretion of chorionic gonadotrohin

We have previously developed a technique of trophectoderm biopsy to obtain cells from human blastocysts for preimplantation genetic diagnosis. To determine whether this technique affects the subsequent development of the blastocyst, 45 manipulated blastocysts were observed from days 3 to 14 in culture, the amount of human chorionic gonadotrophin (HCG) secreted by each embryo was measured and these results were compared with those of 26 non-manipulated controls documented in a previous study. A slit was made in the zona pellucida opposite the inner cell mass in 18 of the 45 blastocysts. This increased the rate of hatching but the other morphological changes up to day 14 were similar to those seen in the non-manipulated controls. There was no difference in the mean cumulative HCG secretion by these zona-slit controls (149.8 +/- 45.7 mIU/ml) compared to the non-manipulated controls (146.2 +/- 23.7 mIU/ml). A slit was also made in the zona pellicida of the other 27 blastocysts. Approximately 12-18 h later, in 18 blastocysts a biopsy of the herniating trophectoderm cells (5-30) was performed. The rate of hatching and adherence to the culture dish was similar to the non-manipulated controls. The mean cumulative HCG secretion decreased significantly (57.5 +/- 16.2 mIU/ml, P less than 0.01) after the biopsy procedure. However, if a small biopsy was performed (less than 10 cells removed) the decrease in HCG secretion (87.6 +/- 24.8 mIU/ml) was not significant, whereas when a large biopsy was performed (greater than 10 cells), HCG levels fell to 19.9 +/- 9.1 mIU/ml.(ABSTRACT TRUNCATED AT 250 WORDS)