An improved latex agglutination-inhibition test for the detection of human chorionic gonadotropin in urine

A commercially available slide latex agglutination-inhibition test for pregnancy was used in a tube modification for the detection of human chorionic gonadotropin (hCG) in urine. The tube method used 100 microliters sample and small quantities of reagents which were mixed continuously for 30 min. The results obtained, based on flocculation, were 15-30-fold better than those obtained by slide agglutination using spiked samples or urine samples from pregnant women. The lowest concentration of hCG detected in the spiked samples was 31 IU/l. When 43 clinical specimens were examined, the tube method detected four more samples than the 18 detected by the slide method. A good correlation (r = 0.96) was observed between the two methods in the determination of the hCG content in the positive samples.     

Analysis of chorionic gonadotrophin secreted by cultured human blastocysts

Embryos produced in an in-vitro fertilization (IVF) programme, but which were unsuitable for transfer to patients because they originated from one (1PN) or three pronuclear (3PN) oocytes or because they originated from two pronuclear (2PN) oocytes but cleaved normally, were maintained in tissue culture. The embryos that progressed to blastocytes were cultured to day 14 of development in order to study their daily output of human chorionic gonadotrophin (HCG). Blastocysts that released large amounts of immunoreactive HCG, which continued to increase daily during the study period, provided the culture supernatants used in the present studies. The heterogeneity of HCG released by blastocyst tissues on days 11 and 14 of development was studied by a chromatofocusing method which separates the isoforms of the gonadotrophin based on differences in their isoelectric points. It was found that the secreted HCG was composed of several molecular forms and that this heterogeneity changed from day 11 to 14 of development. The early blastocyst tissues produced more acidic HCG isoforms than the more advanced embryonic tissues. Differences in the apparent ploidy of the blastocyst tissues studied did not affect significantly the distribution of the HCG isoforms secreted either on day 11 or day 14 of development. These results suggest that the bioactivity of the HCG secreted by blastocytes may change with time and with differentiation of the trophectoderm. In addition, the results suggest that the ploidy of early blastocytes does not influence the nature of the HCG secreted.      

Suppression of mixed lymphocyte reactivity by human chorionic gonadotrophin

Highly purified human chorionic gonadotrophin inhibits the response of lymphocytes from both male and female subjects to allogeneic cells in mixed lymphocyte culture. Human chorionic gonadotrophin is not cytotoxic for human lymphocytes.     

Effect of insulin on human chorionic gonadotrophin secretion by placental explants

The effect of physiological concentrations of insulin (5–50µU/ml) was tested on human chorionic gonadotrophin (HCG)secretion by first trimester (7–9 weeks) and term placentalexplants using both static and dynamic culture models. In staticcultures, insulin exerted a significant biphasic inhibitoryeffect (80% at 5 µU/ml and 40% at 50 µU/ml) on HCGsecretion by placental explants. At approximate fasting plasmalevels, 25 µU/ml insulin added to superfused explantsfor 8 min also had a rapid inhibitory effect. A delayed inhibitoryeffect on HCG pulsatility was also observed using 25 µU/mlinsulin, with a 2-fold decrease in HCG pulse amplitude and a4-fold decrease in the area under the curve following overnightpre-incubation (P < 0.01). Insulin had no effect in staticcultures at term. The effect of insulin-like growth factor (IGF-I)and fibroblast growth factor (bFGF) on HCG secretion in staticcultures was not statistically significant. In conclusion, physiologicalconcentrations of insulin inhibit HCG secretion in first trimesterplacenta in vitro. This effect is gestational age dependentand specific since it is not mimicked by IGF-I or bFGF. Thus,insulin may be an important modulator of trophoblastic HCG secretionduring early pregnancy.     

Effect of beta-endorphin on human chorionic gonadotrophin secretion by placental explants.

In the present study the effect of physiological concentrations of beta-endorphin was examined upon human chorionic gonadotrophin (HCG) secretion by first trimester placental explants. Results show that at 7-9 weeks of gestation, beta-endorphin inhibited HCG secretion; a maximal suppression of 60% was noted at 5 x 10(-10) M concentrations, while fivefold lower or higher doses were less effective. This inhibitory effect was completely reversed by naloxone, an opiate receptor antagonist, indicating involvement of an opiate receptor in the action of beta-endorphin. The opioid peptide specificity was demonstrated by the failure of N-acetyl-beta-endorphin, a non-opiate analogue used at the same concentration range, to affect HCG secretion. Following the HCG peak, at 11 weeks however, the effect of beta-endorphin was stimulatory on HCG secretion, which suggests a gestational age-dependent effect of the opioid peptide. In conclusion, these data indicate that beta-endorphin, a mu and delta opioid receptor ligand, has a modulatory effect on HCG secretion in vitro in the young placenta.     

Use of microtiter plates for latex agglutination immunoassay of serum alpha 2-macroglobulin

A simple, sensitive turbidimetric immunoassay is described for analysis of alpha 2-macroglobulin (AMG) in serum, using microtiter plates and a commercial kit that features a sensitive latex reagent. Using microwells as reaction vessels and the latex reagent made it possible to minimize the sample size and the amounts of reagents. The procedure involves two pipetting steps and a brief incubation (10 min) at room temperature; the assay measures AMG concentrations ranging from 0.12 to 5 g/L and is unaffected by icterus, hemolysis, lipemia, or rheumatoid factor, even at high concentrations. Recovery of AMG added to three pooled serum samples averaged 95-105%. CVs for replicate analyses ranged from 1.9 to 4.8% (within-run) and 6.1 to 9.3% (between-run). Based on paired analyses of 70 serum specimens, AMG concentrations obtained by this turbidimetric microtiter immunoassay were in close agreement (correlation coefficient = 0.99) with results obtained by an enzyme-linked immunosorbent assay (ELISA). Serum AMG concentrations in 48 healthy adults (mean +/- 2SD) were 1.87 +/- 1.00 g/L.     

Studies on the microheterogeneity of chorionic gonadotrophin secreted by the human cytotrophoblast in culture.

In the present study, we investigated the biological characteristics of different molecular forms of chorionic gonadotrophin (HCG) secreted by the human cytotrophoblast during its morphological and functional differentiation in culture. Highly purified cytotrophoblasts were prepared from term placentae and cultured for 24 to 96 h in the absence or presence of 8-bromo-3',5'-cAMP. Media were collected at 24 h intervals and the secreted isoforms of HCG were then separated by polyacrylamide gel isoelectric focusing (pH range 8.0-3.0) and quantified by radioimmunoassay. The secretion of HCG was significantly increased by 8-bromo-cAMP (from 23.5 +/- 6.3 ng/ml at 24 h to 1619 +/- 835.8 ng/ml at 96 h; controls, 9.3 +/- 0.1 ng/ml at 24 h and 26.6 +/- 3.5 ng/ml at 96 h, mean +/- SD). Analysis of media concentrates from cAMP-stimulated cultures by isoelectric focusing revealed the presence of several distinct peaks of HCG within the pH range of 7.3-4.8; major peaks consistently exhibited isoelectric points (pI) of 7.3-7.0 (peak 1), 5.6-5.4 (peak 2) and 5.1-4.8 (peak 3). The relative HCG content of the most acidic peak (as % of total on gel) progressively increased with time of exposure to the cAMP analogue (from 19.8 +/- 1.6% at 24 h to 34.4 +/- 4.3% at 96 h, mean +/- SEM, P less than 0.01). HCG recovered from peak 1 exhibited the highest receptor-binding capacity and in-vitro biological potency.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of macrophage migration inhibitory factor in human ovary by human chorionic gonadotrophin

The role of macrophage migration inhibitory factor (MIF) in human ovarian function remains obscure. The aim of this study was to investigate how MIF was related to ovulation by quantitative analysis of serum, follicular fluid and culture medium of granulosa cells obtained from in-vitro fertilization (IVF) and embryo transfer patients. Serum MIF concentrations in ovarian stimulation cycles for IVF-embryo transfer were higher at day 1 (median 92.6 ng/ml), which took place 35 h after human chorionic gonadotrophin (HCG) administration and just before the retrieval of oocytes, than those before day -6 (12.1 ng/ml), at day -5 to about day 0 (17.5 ng/ml) or at day 2 to about day 14 (8.2 ng/ml). MIF concentrations in the follicular fluid (113.4 ng/ml) obtained in ovarian stimulation cycles for IVF-embryo transfer were significantly higher than in serum (72.0 ng/ml) collected at the same time. MIF concentrations in the follicular fluid in natural cycles were higher in the ovulatory phase (51.6 ng/ml) than in the late follicular phase (13.8 ng/ml). MIF concentrations in the culture media of granulosa cells increased from 3.2 ng/ml to 7.2 ng/ml with HCG stimulation, and decreased from 2.4 ng/ml to 1.2 ng/ml when stimulation was withheld. These results indicate that HCG can induce the elevation of serum and follicular fluid MIF concentrations through the stimulation of ovarian cells, and that MIF is probably involved in the mechanism of ovulation.     

Monoclonal antibodies to the β-subunit of human chorionic gonadotrophin

Research Institute of Human Morphology, Academy of Medical Sciences of the USSR. Research Institute of Clinical Oncology, All-Union Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 106, No. 12, pp. 693–695, December, 1988.     

Production of human chorionic gonadotrophin by a hepatoblastoma resulting in precocious puberty.

A two year old boy presented with precocious puberty associated with hepatoblastoma. Serum concentrations of beta human chorionic gonadotrophin and alpha-fetoprotein were raised. An aggressive chemotherapeutic regimen resulted in useful palliation and interesting changes in the beta human chorionic gonadotrophin and alpha-fetoprotein concentrations. In contrast to a previous report, the ultrastructure of the tumour showed frequent Golgi apparatus but no other electron dense membrane bound vesicles.     

Modulation of human chorionic gonadotrophin bioactivity during the first trimester of pregnancy

The objective of this study was to evaluate the bioactivity of human chorionic gonadotrophin (HCG) during first trimester pregnancy. This was done by means of a retrospective analysis of sera from patients with first trimester normal intrauterine and ectopic pregnancies. Serum samples were obtained from 38 women with amenorrhoea of <10 weeks. From these, 19 had a normal intrauterine pregnancy (IUP) and 19 an ectopic pregnancy (EP). Cases were allocated to either low serum immunoreactive HCG (HCGi), intermediate HCGi or high HCGi concentrations (HCGi <5000 mUI/ml, between 5000 and 40,000 mIU/ml and >40,000 mIU/ml respectively). HCGi and oestradiol were measured by enzyme immunoassays and bioactive HCG by the mouse Leydig cell bioassay. All results were analysed by analysis of variance and unpaired Student's t-test. There was a significant difference between bioactive to immunoreactive HCG ratios (b/i ratio) between the subgroups of low, intermediate and high HCGi concentrations. Lower b/i ratios were found when HCGi concentrations were high (HCG b/i mean +/- SEM: high subgroup, 0.33 +/- 0.07 versus low subgroup: 1.50 +/- 0.12; P < 0.0001). Furthermore, the b/i ratios were inversely correlated with oestradiol (P < 0.0001) and HCGi (P < 0.0001) concentrations but not with gestational age. There was no difference in the b/i ratios when comparing IUP with EP. It is concluded that, in first trimester pregnancies, there is a likely modulation of HCG bioactivity which is inversely correlated with HCGi and oestradiol concentration. The underlying mechanisms and their physiological relevance remain to be elucidated.      

人绒毛膜促性腺激素单克隆抗体的研制

目的获得针对人绒毛膜促性腺激素单克隆抗体。方法hCG蛋白免疫Balb／c小鼠,取其脾细胞与同系小鼠骨髓瘤细胞NS-1按8：1比例融合,间接ELISA法筛选阳性克隆,有限稀释法进行克隆化培养;制备腹水抗体;采用间接ELISA法鉴定抗体亚型和测定抗体效价。结果得到6株能稳定分泌单克隆抗体的杂交瘤细胞株;抗体经鉴定均为IgG1、κ型,效价均达10^-5以上。结论所获得的6株杂交瘤细胞株均有较强稳定分泌抗-hCG单克隆抗体的能力。这为有关hCG的检测、hCG本身相关研究以及避孕疫苗的研制打下了基础。     

A method for the determination of human chorionic gonadotrophin in urine extracts

A method is described for the preparation in rabbits of a specific antiserum to human chorionic gonadotrophin. The ability of this antiserum to fix complement in the presence of urinary human chorionic gonadotrophin is demonstrated. The standardization of antiserum against the international standard preparation of human chorionic gonadotrophin, the determination of antiserum specificity using appropriate controls, and the value of antiserum in the diagnosis of pregnancy and conditions associated with high urinary levels of human chorionic gonadotrophin is emphasized.     

A pilot study of the human chorionic gonadotrophin test for ovarian hyperandrogenism

A controlled clinical study was designed to investigate the value of human chorionic gonadotrophin (HCG) challenge as a test for functional ovarian hyperandrogenism. Dexamethasone administration was followed by 5000 IU HCG and blood samples for steroid hormone assay were obtained 0, 8, 16, and 24 h thereafter. Study subjects were normal women (n = 13); women with functional ovarian hyperandrogenism, defined by androgen excess, amenorrhoea and an increased 17-hydroxyprogesterone response to nafarelin (n = 6); and normal men (n = 4). The responses of 17-hydroxyprogesterone, androstenedione and testosterone to HCG in women with functional ovarian hyperandrogenism were significantly greater than in normal women. However, the 17-hydroxyprogesterone response to HCG in functional ovarian hyperandrogenism was significantly lower after HCG than after nafarelin. The oestradiol response was also significantly lower after HCG than nafarelin, although oestradiol concentration more than doubled in normal women as well as in women with functional ovarian hyperandrogenism. The responses to HCG confirm that functional ovarian hyperandrogenism abnormalities are luteinizing hormone (LH)-dependent. Therefore, the 17- hydroxyprogesterone response to HCG could represent a useful test for the diagnosis of ovarian hyperandrogenism. The lower 17- hydroxyprogesterone response to HCG than to nafarelin in functional ovarian hyperandrogenism suggests that a follicle-stimulating hormone (FSH)-responsive factor modulates thecal 17-hydroxyprogesterone secretion. The oestradiol response to HCG is consistent with HCG directly stimulating the oestradiol secretion by thecal cells.      

Endometrial stromal cell decidualization inhibits human chorionic gonadotrophin and human placental lactogen secretion by co-cultured trophoblasts

We have developed an in-vitro co-culture system to examine theinteraction between purified first trimester cytotrophoblastsand purified non-pregnant human endometrial stromal cells (ESC).ESC decidualization is an important step in endometrial maturationand may modulate embryo implantation. In order to investigatethe effects of ESC decidualization on trophoblast function,we examined human chorionic gonadotrophin (HCG), human placentallactogen (HPL), progesterone and oestrogen secretion by trophoblastsco-cultured in contact with ESC, either with or without decidualizationinduced by progesterone. Decidualized ESC inhibited basal HCGand HPL secretion for 3 days during the culture for HCG, andfor 5 days during the culture for HPL (P < 0.01 and P <0.03 respectively). After 5 days of co-culture, decidual transformationof ESC as indicated by prolactin production occurred in thecontrol cultures due to progesterone and oestradiol secretionby the co-cultured trophoblasts, but no significant differencesin HCG or HPL secretion were observed between the two groups.Although the type of trophoblast used in the present study isfar from implantation, our results clearly demonstrated thatHCG and HPL secretion by trophoblasts was inhibited by the presenceof co-cultured decidualized ESC, and suggested that ESC decidualizationmay regulate trophoblast function at the human fetal-maternalinterface.     

Predictive value of plasma human chorionic gonadotrophin following assisted conception treatment

A total of 429 pregnancies after assisted conception treatment was analysed, using receiver operator characteristic curves. The best balance between sensitivity and specificity for predicting viable (single and multiple births) and non-viable (fetal heart positive abortions, ectopic and biochemical pregnancies) outcomes was human chorionic gonadotrophin (HCG) 50 IU/l on day 14 and 200 IU/l on day 21 after treatment. Utilizing these indices all pregnancies could be classified into one of four groups. In group A (day 14 HCG <50 IU/l and day 21 <200 IU/l), the probability of a birth was 0%, pregnancy loss 72% and ectopic pregnancy 28%. Conversely for group D (day 14 HCG >50 IU/l and day 21 >1000 IU/l), the likelihood of a birth was 90%, pregnancy loss 8% and ectopic pregnancy only 1%. Between groups A and D there was, as expected, a gradually shifting balance in favour of a reduction in ectopic (28, 13, 3, 1%) and biochemical pregnancies (70, 36, 33, 2%) and an increase in fetal heart positive pregnancy losses (2, 6, 13, 7%) and births (0, 44, 50, 90%). The majority of multiple pregnancies (98%) occurred in group D. Two accurately linked HCG measurements allowed a greater predictive accuracy of pregnancy outcome than could be obtained using either alone.     

Characterization of a monoclonal antibody inhibiting the biological activity of human chorionic gonadotrophin

A mouse monoclonal antibody (MCA), raised against human chorionicgonadotrophin (HCG) and coded 130A, was characterized with severaltypes of immunoassay and with in-vitro and in-vivo bioassays.MCA 130A binds strongly to the intact HCG molecule but lessso to either -or -subunit; The dissociation constant of theMCA-HCG complex was found to be in the order of 70 pmol/l. MCA130A inhibits the biological activity of HCG very effectively,both in vitro and in vivo. In a competitive immunoassay MCA130A binds 30 x better to HCG than to human luteinizing hormone(HLH). The selectivity in the bioassays was much higher, e.g.in vitro, 50% inhibition of HLH-stimulated testosterone productionrequires 2600 x as much MCA as is needed for inhibiting HCG-stimulatedproduction. Possible reasons for this difference in selectivityare discussed     

Effect of chorionic gonadotrophin on hematopoietic stem cells

Department of Biological Chemistry, Perm Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR N. V. Vasil'ev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 109, No. 1, pp. 62–64, January, 1990.