Determination of hyperglycosylated human chorionic gonadotropin produced by malignant gestational trophoblastic neoplasias and male germ cell tumors using a lectin-based immunoassay and surface plasmon resonance

The ability to reliably detect aberrant glycosylation of human chorionic gonadotropin (hCG) may have profound implications for the diagnosis and monitoring of malignant gestational trophoblastic neoplasia, germ cell tumors, other malignancies, and pregnancy complications. To become a clinically useful assay, however, this discrimination of glycoforms should be possible on minimally treated biological specimens. Towards this end, we have developed a lectin-based sandwich-type immunoassay to compare the glycosylation patterns of hCG among urine specimens from patients presenting with a normal pregnancy, invasive mole, choriocarcinoma, and male germ cell tumors using carbohydrate-free antibody fragments as capture reagents and a panel of eight lectins, five recognizing neutral sugars and three recognizing sialic acid. There was no significant difference in the binding of any of the lectins to hCG in the urine of women over the gestational range of 6–38 weeks. Three lectins, however, exhibited differential binding to urinary hCG derived from these normal pregnant controls and that from patients with malignant forms of gestational trophoblastic disease and male germ cell tumors. Galanthus nivalis agglutinin and Maackia amurensis lectin, which bind terminal mannose and α(2-3)sialic acid, respectively, preferentially bound pregnancy-derived hCG, whereas the lectin, wheat germ agglutinin, which binds sialic acid and β(1-4)N-acetylglucosamine, exhibited decreased binding to pregnancy-derived hCG compared to that from patients with male germ cell tumors and malignant gestational trophoblastic neoplasia. The differential binding observed with these three promising lectins is most encouraging and warrants further examination. The experimental paradigm also holds promise for the development of comparable assays for other glycosylated tumor markers.     

Amniotic fluid levels of dimeric inhibins, pro-αC inhibin, activin A and follistatin in Down''s syndrome

OBJECTIVE: In the second trimester of pregnancy, inhibin A is significantly increased in maternal serum and decreased in amniotic fluid in Down's syndrome pregnancies compared to normal. We wished to further evaluate the levels of inhibin A, inhibin B, pro-alpha C inhibin, activin A and the binding protein follistatin in amniotic fluid in Down's syndrome and control pregnancies. DESIGN: Case-matched control study. PATIENTS: 29 Down's syndrome and 290 chromosomally normal control pregnancies were identified from records and amniotic fluid, collected at second trimester amniocentesis, retrieved from routine storage for analysis. MEASUREMENTS: Inhibin A, inhibin B, pro-alpha C inhibin, total activin A and follistatin were measured using sensitive and specific enzyme linked immunosorbent assays. RESULTS: The median (10th-90th percentiles) amniotic fluid inhibin A level in the control pregnancies increased from 334 (122-553) ng/l at 14 weeks' to 695 (316-1475) ng/l at 19 weeks' gestation. The corresponding figures for inhibin B and the alpha-subunit precursor inhibin pro-alpha C were 632 (185-1354) and 2062 (1237-3381) ng/l, respectively at 14 weeks' and 2439 (748-5307) and 3115 (2021-6567) ng/l, respectively at 19 weeks' gestation. Total activin A increased from 3795 (1554-5296) at 14 weeks' to 5086 (3059-8224) at 18 weeks' gestation. Expressed as multiples of the median (MoM) the median (95% CI) amniotic fluid levels of inhibin A, inhibin B, pro-alpha C inhibin and acitivin A in the Down's syndrome samples were 0.77 (0.59-0.85), 0.94 (0.63-1.23), 0.77 (0.49-0.84) and 0.77 (0.53-0.87), respectively. Compared to controls the levels of inhibin A, pro-alpha C inhibin and activin A were significantly lower in Down's syndrome pregnancies (P < 0.01, Mann-Whitney U test). Follistatin levels in the controls declined slightly from 2106 (1421-3538) ng/l at 14 weeks' to 1600 (1281-2543) ng/l at 18 weeks' gestation. Levels in the Downs' syndrome pregnancies were similar to controls. CONCLUSIONS: The data suggest that the production, secretion or metabolism of the inhibin alpha- and beta A-subunits is altered in Down's syndrome pregnancies in the second trimester.     

Hyperglycosylated HCG expression in pregnancy: Cellular origin and clinical applications

Employing a monoclonal antibody (B152) specific for a carbohydrate epitope found on a choriocarcinoma derived hCG, it was discovered that a similar hCG isoform is expressed during early pregnancy. This form differs from later pregnancy hCG in carbohydrate moieties. Profiling of these two hCG isoforms throughout pregnancy utilized two IRMA's: B152–B207 (“hyperglycosylated hCG”-specific assay) and B109–B108 (an IRMA for standard intact hCG isoforms in the WHO hCG reference preparation). The WHO hCG standard was used in both assays. Values were presented as a ratio of hCG isoform concentrations (B152/B109 ratio). In early pregnancy urine concentrations of B152 hCG were significantly higher in normal pregnancy (NP) compared to early pregnancy loss (EPL). Matched serum–urine samples from the first and third trimesters revealed that the B152 hCG form is predominant in both serum and urine in the first trimester compared with the third trimester. The proportion of the B152 hCG (HhCG) form is higher in urine than in matched serum. There was a significant difference in the B152/B109 ratio between days 5 and 20 from time of embryo transfer in normally developing pregnancy versus EPL in the urine of IVF patients. In spontaneous abortion (SA) the level of B109 hCG remained higher in NP compared with SA. However, the B152/B109 ratio declined with gestational age faster in SA than in NP suggesting perhaps a different loss mechanism in SA versus EPL. The cellular origin of the different hCG glycoforms was identified by assay of cell media from cytotrophoblasts (CTBs) and syncytiotrophoblasts (STBs). Isolated CTBs expressed predominantly HhCG. The level of expression was the highest in the first trimester. STBs were the source of the less glycosylated B109 hCG isoform. Analysis of hCG glycoforms during early pregnancy can distinguish pregnancies that will fail from those that will proceed normally. Since the B152 assay does not effectively discriminate between intact HhCG and free beta HhCG (HhCGβ), a new HhCGβ assay was developed. This assay recognizes the HhCGβ which is produced by CTBs. We hypothesize that the measurement of HhCGβ may have a potential use in screening for Down syndrome and perhaps other pregnancy disorders and certain types of cancer.     

Serum levels of human chorionic gonadotropin in nonpregnant women and men are modulated by gonadotropin-releasing hormone and sex steroids

Serum hCG levels were measured in apparently healthy nonpregnant women and men using a highly sensitive and specific time-resolved immunofluorometric assay. The sensitivity of the assay was 0.03 IU/L. The levels were low in women of fertile age (median, 0.05 IU/L) and in men less than 60 yr of age (median, 0.04 IU/L). In women the median level increased to 1.1 IU/L after the menopause (range, 0.17-4.8 IU/L), and a similar but smaller increase occurred in men after 60 yr of age (median, 0.20 IU/L; range, less than 0.03-2.3). Stimulation with GnRH caused a 2- to 3-fold increase in the hCG level in both men and women. Chronic treatment of postmenopausal women with a combination of estrogen and progestagen lowered their serum hCG levels to about 50% of the pretreatment values. The hCG in serum could be separated from LH by gel chromatography, and the hCG immunoreactivity measured by direct assay of serum corresponded to the immunoreactivity eluted in the hCG fractions after gel chromatography. Thus, the results were not due to cross-reaction with LH. We conclude that serum of nonpregnant women and men contains hCG-like material, whose production is modulated by GnRH and sex steroids.     

Hormonal regulation of pituitary FSH sialylation in male rats

Sialic acid content in FSH is modulated by GnRH and sexual steroids. Galβ1,3GlcNAcα2,3-sialyltransferase (ST3Gal III) and Galβ1,4GlcNAcα2,6-sialyltransferase (ST6Gal I) incorporate sialic acid residues into FSH oligosaccharides. The aim of the present study was to assess pituitary FSH molecular microheterogeneity and ST3Gal III/ST6Gal I expression during sexual development and after castration in male rats. Preparative isoelectric focusing and lectin chromatography were used to isolate FSH glycosylation variants according to charge and complexity of their oligosaccharides; RT-PCR and immunohistochemistry were employed to analyse sialyltransferase expression. Sexual development was associated with a progressive shift towards more acidic/sialylated FSH glycoforms concomitantly with an increment in ST6Gal I gene and protein expression. After castration, a transient decrease followed by a marked increase in ST6Gal I expression were observed. Less acidic/sialylated FSH glycoforms bearing incomplete oligosaccharides increased after castration, despite high ST6Gal I expression. ST3Gal III expression remained unchanged in all the experimental conditions examined. These results show that the synthesis of FSH isoforms possessing α2,6-linked sialic acid is hormonally regulated in male rats.     

The nature of human chorionic gonadotrophin glycoforms in gestational thyrotoxicosis

OBJECTIVE: We examined the charge heterogeneity and carbohydrate complexity of hCG in healthy pregnant Asian subjects and Asian women with gestational thyrotoxicosis to assess whether particular glycoforms of hCG are associated with the thyrotoxicosis of pregnancy. DESIGN: Blood was taken at 8-16 weeks of gestation from five pregnant Asian women with clinical symptoms of gestational thyrotoxicosis and biochemical indicators of hyperthyroidism and from six age-matched healthy pregnant women. hCG charge heterogeneity and carbohydrate complexity were assessed by fast protein liquid chromatography chromatofocusing and concanavalin A affinity chromatography, respectively. The degree of terminal sialylation of the different glycoforms was measured by ricin affinity chromatography, which detects exposed galactose residues following desialylation. RESULTS: Free T3, free T4 and hCG levels were elevated and TSH suppressed in the thyrotoxic subjects compared to controls (P < 0.007). Overall, the glycoform distribution of the hCG in the blood from the control and thyrotoxic groups was similar, with a median pI of 4.34 (median and range: 3.78-4.68) and pI 4.23 (3.98-4.38). Free T4 (P < 0.028) and free T3 (P < 0.03) levels were positively correlated to both the absolute hCG concentration and the percentage of hCG forms between pI 3.36-4.0. The distribution of simple (73-88.6%), intermediate (9.7-26.4%) and complex (0.1-7.3%) branching oligosaccharide forms was similar in both groups, as was the percent hCG which bound to ricin (< 3.2%). CONCLUSION: We conclude that excessive thyroid stimulation in the thyrotoxic patients is associated both with the absolute concentration of hCG and the relative proportion of acidic glycoforms between pI 3.36 and 4.0.     

Human chorionic gonadotropin and the thyroid: hyperemesis gravidarum and trophoblastic tumors.

There is abundant evidence that human chorionic gonadotropin (hCG) is a weak thyrotropin (TSH) agonist. In FRTL-5 rat thyroid cells, hCG increases cyclic adenosine monophosphate (cAMP), iodide transport, and cell growth. hCG has thyroid-stimulating activity in bioassays in mice and in clinical studies in man. In cultured cells transfected with the human TSH receptor, hCG increases generation of cAMP. Molecular variants of hCG with increased thyrotropic potency include basic molecules with reduced sialic acid content, truncated molecules lacking the C-terminal tail, or molecules in which the 47-48 peptide bond in the beta-subunit loop is nicked. In normal pregnancy, when hCG levels are highest at 10 to 12 weeks gestation, there is suppression of serum TSH levels, presumably due to slight increases in free thyroxine (T4) concentration. In twin pregnancies, hCG levels tend to be higher and suppressed TSH levels are more frequent. Hyperemesis gravidarum, defined as severe vomiting in early pregnancy that causes 5% weight loss and ketonuria, is usually associated with increased hCG concentration. A high proportion of patients with hyperemesis gravidarum, about one-third to two-thirds in different series, have evidence of increased thyroid function. Only a small proportion of these patients have clinical hyperthyroidism, termed gestational thyrotoxicosis. These patients probably secrete a variant of hCG with increased thyroid-stimulating activity. Trophoblastic tumors, hydatidiform mole, and choriocarcinoma often cause hyperthyroidism because they secrete very large amounts of hCG. When the serum hCG exceeds about 200 IU/mL, hyperthyroidism is likely to be found. There is a correlation between the biochemical severity of hyperthyroidism and the serum hCG in these patients. Removal of the mole or effective chemotherapy of the choriocarcinoma cures the hyperthyroidism. In conclusion, hCG has thyroid-stimulating activity that influences thyroid function early in pregnancy when hCG levels are high. Excessive hCG secretion may cause hyperthyroidism in patients with hyperemesis gravidarum or trophoblastic tumors.     

Acidic isoforms of chorionic gonadotrophin in European and Samoan women are associated with hyperemesis gravidarum and may be thyrotrophic

OBJECTIVE: There is conflicting evidence concerning the role of human chorionic gonadotrophin (hCG) in the aetiology of hyperemesis gravidarum (HG); particular isoforms of hCG may be the critical factor. Ethnic differences in HG prevalence and putative thyrotrophic effects of hCG may also relate to differences in hCG isoform profiles. To address these issues we examined the relationship of hCG isoforms to HG and thyroid function tests in two groups of women from ethnic backgrounds with significantly different HG prevalence rates. PATIENTS AND DESIGN: We enrolled 10 European and 10 Samoan women with HG and an equally sized non-hyperemetic, gestational stage matched control group. MEASUREMENTS: We administered a questionnaire, generated serum hCG charge-isoform profiles by chromatofocusing and measured the serum concentrations of total hCG, oestradiol (E2), thyrotrophin (TSH) and free thyroxine (FT4). RESULTS: The mean serum total hCG levels were highest in the Samoan hyperemetics (176,268 IU/l), and overall higher in hyperemetics compared with controls (159,770 IU/l vs. 86,420 IU/l, P < 0.001). When compared with controls, hyperemetics displayed increased hCG concentrations in the more acidic half (pH < 4) of the chromatofocusing pH range (89,843 IU/l vs. 41,146 IU/l, P < 0.003). Serum E2 levels did not differ between the four groups, but correlated with the hCG concentration between pH 5.2 and 4.01. Mean serum TSH levels were significantly lower in hyperemetics than in controls (0.33 mIU/l vs. 1.19 mIU/l, P < 0.001) and correlated with the hCG concentration between pH 4.6 and 2.8, while serum FT4 correlated with the hCG concentration below pH 4.0. CONCLUSIONS: Acidic isoforms of hCG may play a role in the aetiology of HG and gestational thyrotoxicosis. Minor ethnic differences in hCG isoform profiles were observed, but the relationship of acidic hCG isoforms to HG and serum thyroid hormone levels was largely independent of the patients' ethnicity. The mechanisms by which acidic isoforms might provoke nausea remain uncertain, but do not seem to involve E2, while the longer half-life of acidic hCG isoforms may result in increased in vivo TSH receptor cross-talk with resultant thyrotrophic effects.     

Characterization of antisera distinguishing carbohydrate structures in the beta-carboxyl-terminal region of human chorionic gonadotropin

The beta-COOH-terminal peptide region (beta CTP) of hCG is an important immunochemical domain that is specific to hCG but absent from homologous hLH. Three types of polyclonal antibodies can be elicited to the beta CTP portion of hCG. The first and most common recognizes the beta CTP amino acid sequence independent of its carbohydrate content (carbohydrate-oblivious). It can be elicited against synthetic beta CTP as well as against native or asialo beta CTP. The second type binds best to desialylated beta CTP, (galactose-requiring). The third type binds well only to sialylated beta CTP (sialic acid-requiring). All three types of antisera recognize the sialylated beta CTP as equivalent to the whole hormone on a molar basis, indicating that this peptide region of the whole hormone is neither sequestered nor conformationally altered. We have characterized the epitopes for the sialic acid-requiring and galactose-requiring beta CTP antisera. Both require elements of the primary amino acid sequence of beta hCG as well as specific elements of the carbohydrate side-chains and, therefore, are not directed solely to either peptide or carbohydrate determinants. The galactose-requiring beta CTP antiserum was generated to an ovalbumin conjugate of a desialylated form of beta CTP, beta 123-145. It binds desialylated forms 1000 times better than it binds native hCG, and binds neither synthetic beta CTP nor asialo-agalacto beta 123-141. The antiserum requires residues 123-141 and a portion of an O-serine-linked oligosaccharide chain. Its binding requirements are, thus, very different from those of the carbohydrate-oblivious beta CTP antiserum, which requires the last two residues of the hCG beta polypeptide chain and is not sensitive to the presence or absence of carbohydrate. The sialic acid-requiring beta CTP antiserum, which was generated to sialylated beta 123-145-thyroglobulin conjugate, binds well to sialylated beta CTP, but poorly to asialo beta CTP and not at all to the carbohydrate-free synthetic peptide. This antiserum requires the entire beta 123-145 sequence as well as sialic acid-terminated oligosaccharide side-chains. Since the sialylated peptide beta 115-141 binds poorly to it, this antiserum resembles the carbohydrate-oblivious anti-beta CTP; both require the COOH-terminal amino acids of hCG beta. However, the former requires intact O-linked carbohydrate moieties for binding. These antisera can be used to assess the primary protein and carbohydrate structures of beta CTP at low concentrations in urine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential expression of human chorionic gonadotropin (hCG) glycosylation isoforms in failing and continuing pregnancies: preliminary characterization of the hyperglycosylated hCG epitope

Human chorionic gonadotropin (hCG) glycoforms change as pregnancy progresses. We have developed an antibody (B152) which can measure a hyperglycosylated early pregnancy isoform of hCG. This putative hyperglycosylated form of hCG arises very early in pregnancies and is rapidly replaced by an isoform that predominates for the remainder of the pregnancy. The profiles of these hCG glycoforms are measured as a ratio of values of two immunometric assays. The profiles of these ratios differ between pregnancies which persist and those which will experience early failure. In this report, daily urine hCG isoform ratios from donor eggs (no exogenous hCG pretreatment), in vitro fertilization pregnancies were profiled and analyzed from the first day following embryo transfer (ET). Significant differences were found between continuing pregnancy and pregnancy loss throughout days 5-20 post-ET. When hCG isoform ratios were analyzed from the first day of detectable hCG, pregnancy loss could be predicted in the case of a single fetus both during the 5- to 10-day time segment (P=0.018) and the 10- to 15-day time segment (P=0.045). When single and multiple fetus pregnancies were analyzed together significance was approached in the 10- to 15-day time period (P=0.058). In a second population of pregnant women who conceived naturally, in whom urine samples were collected at approximately weekly intervals to either term birth or clinical spontaneous abortion, the ratio could discriminate between miscarriages and normal term pregnancies (P=0.043). In later pregnancy, the ratio of hCG isoforms declined more rapidly in miscarriages than in term pregnancy. Antibody B152 was produced using a choriocarcinoma-derived hCG (C5), which was hyperglycosylated at both N- and O-linked sites and was 100% nicked at position beta(47-48). Western blot analyses supported the assay results showing that early pregnancy urine does not contain nicked C5-like hCG. Also, the early pregnancy hCG appeared to be the same size as later pregnancy hCG as judged by SDS gel electrophoresis. A series of Western blot analyses and immunoassays conducted with the samples either non-reduced or reduced showed that B152 is directed to a linear epitope located in the COOH-terminal peptide region of the beta subunit. This indicated that only the O-glycan groups and not the N-linked glycans are part of the antibody epitope.     

hCGbeta core fragment is a metabolite of hCG: evidence from infusion of recombinant hCG

The availability of recombinant human chorionic gonadotrophin (r-hCG) has allowed us to measure its metabolic and renal clearance rates and to study the origin of the beta core fragment of hCG (hCGbetacf). Serum and urine samples were collected from six subjects, after an intravenous injection of 2 mg (equivalent to 44 000 IU Urinary hCG) r-hCG, and assayed for hCG and the beta subunit (hCGbeta). Urine from four of the subjects was also subjected to gel chromatography and assayed for hCGbetacf and hCG. r-hCG, administered as an intravenous dose, was distributed, initially in a volume of 3.4+/-0.7 l (mean+/-s.d.) and then in 6.5+/-1.15 l at steady-state. The disappearance of r-hCG from serum was bi-exponential, with an initial half-life of 4.5+/-0.7 h and a terminal half-life of 29.0+/-4.6 h. The mean residence time was 28. 6+/- 3.6 h and the total systemic clearance rate of r-hCG was 226+/-18 ml/h. The renal clearance rate was 28.75+/-6.2 ml/h (mean+/-s.d). hCGbetacf was detected in all urine samples collected at 6 h intervals. Over the 138 h period of urine collection, 12.9% (range 10.1-17.3% ) of r-hCG injected was recovered as the intact molecule and 1.7% (range 0.8-2.9%) was recovered as the hCGbetacf, in 4 subjects. The molar ratio of hCGbetacf to hCG in urine increased from 3.1+/-1.7%, on day 1, to 76+/-34.3% (mean+/-s.e.m.) on day 5, after r-hCG infusion, suggesting that hCGbetacf is a metabolic product of the infused r-hCG.     

Expression of pro-EPIL peptides encoded by the insulin-like 4 (INSL4) gene in chromosomally abnormal pregnancies

The recent development of a specific immunoassay based on monoclonal antibodies directed to chain C and chain A of early placenta insulin-like peptide (EPIL) encoded by the INSL4 gene, has made it possible to demonstrate pro-EPIL peptide expression during normal pregnancy. In the present study, we report on the expression of pro-EPIL peptides in chromosomally abnormal pregnancies, namely trisomy 21 and 18. EPIL peptide levels were measured in amniotic fluid (AF) and maternal serum (MS) from pregnancies with trisomy 21 (n=16) or 18 (n=14) and compared to levels detected in AF and MS from 33 chromosomally normal pregnancies between 12 and 32 weeks of gestation. Pro-EPIL peptide levels were significantly higher in amniotic fluids from T21 than in AF from chromosomally normal pregnancies (mean pro-EPIL levels +/- SEM, 449+/-129.2 ng/mL vs 137+/-29.6 ng/mL, P = 0.0195), whereas there was only a trend towards an increase in pro-EPIL peptide levels in maternal serum. In a limited matched gestational age range (15 to 17 weeks), it was confirmed that pro-EPIL peptide levels were significantly higher in AF from T21 pregnancies (644.0+/-155.9 ng/mL, n = 11) than in AF from normal pregnancies (177.8+/-39.0 ng/mL, n = 12; P < 0.0001). Interestingly, the expression patterns of pro-EPIL peptides, human chorionic gonadotropin (hCG) and its free subunits were parallel in T21 pregnancies as recently observed in normal pregnancies. These results are in line with previous observations suggesting that the biosynthesis of both hCG and EPIL follows common regulation pathways.     

Specific enzyme immunoassay for human chorionic gonadotrophin

A sandwich-type enzyme immunoassay system specific for human chorionic gonadotrophin (hCG) was prepared with the antibody Fab'-beta-D-galactosidase complex and the antibody F(ab')2-immobilized silicone rubber solid phase by using a purified antibody to beta subunit of hCG (hCG beta). The assay system cross-reacted less than 4% with human luteinizing hormone (hLH) and human follicular stimulating hormone (hFSH), and proved to be highly sensitive with hCG measurable at levels as low as 0.3 mIU per assay tube. Using 50 microliter of serum sample, 6-600 mIU/ml of hCG in serum could be determined specifically with the same degree of precision as in radioimmunoassay but without sample interference with the assay. The coefficients of variation within-run and between-run were 8.6-8.9%, and 4.9-10.7%, respectively. Values obtained with the enzyme immunoassay correlated well with those of radioimmunoassay (r = 0.98, slope = 0.94, y-intercept = 10.2 mIU/ml for 75 serum samples). Results of the immunoassay of hCG levels in serial samples of serum from healthy women and patients with choriocarcinoma show that this method is useful in the clinical diagnosis of trophoblastic disease.     

妊娠一过性甲状腺毒症的临床研究

目的 调查沈阳地区既往健康的妇女妊娠早期妊娠一过性甲状腺毒症(GTT)的患病率及其病因.方法 对来自沈阳地区10家医院的534例妊娠早期妇女进行问卷调查、体格检查、血清促甲状腺素(TSH)、游离T4(FT4)、游离T3(FT3)、甲状腺过氧化物酶抗体(TPOAb)、促甲状腺素受体抗体(TRAb)和人绒毛膜促性腺激素(hCG)水平的检测.结果 (1)妊娠早期甲状腺毒症总患病率为9.75%,GTT的患病率为7.86%,占甲状腺毒症的80.77%;88.89%临床GTT表现为单纯FT3升高.(2)妊娠6、8～10和12周孕妇血清hCG水平逐渐升高,中位数分别为25300、85220和81780IU/L(P=0.000).血清TSH中位数依次降低(P＜0.01),分别为1.45、1.10和0.84mIU/L.(3)当妊娠妇女血清hCG水平＞50 000 IU/L时,GTT的比例明显升高;当血清hCG水平在80 000～110000IU/L时发生亚临床GTT的比例明显升高;当血清hCG水平＞110 000IU/L时发生临床CTT的比例明显升高.相关分析结果 显示,妊娠早期血清hCG与TSH显著负相关(r=-0.402,P=0.000),与FT3正相关(r=0.165,P=0.000),而与FT4无相关.结论 GTT是妊娠早期甲状腺功能亢进症的首要病因,占妊娠早期甲状腺毒症的80.77%,其血清学特点主要表现为血清FTT升高.妊娠早期血清hCG水平与GTT严重程度相关.     

Diagnosis of hydatidiform mole and persistent trophoblastic disease: diagnostic accuracy of total human chorionic gonadotropin (hCG), free hCG {alpha}- and {beta}-subunits, and their ratios

OBJECTIVE: Human chorionic gonadotropin (hCG) is widely used in the management of hydatidiform mole and persistent trophoblastic disease (PTD). Predicting PTD after molar pregnancy might be beneficial since prophylactic chemotherapy reduces the incidence of PTD. DESIGN: A retrospective study based on blood specimens collected in the Dutch Registry for Hydatidiform Moles. A group of 165 patients with complete moles (of which 43 had PTD) and 39 patients with partial moles (of which 7 had PTD) were compared with 27 pregnant women with uneventful pregnancy. METHODS: Serum samples from patients with hydatidiform mole with or without PTD were assayed using specific (radio) immunoassays for free alpha-subunit (hCGalpha), free beta-subunit (hCGbeta) and 'total' hCG (hCG + hCGbeta). In addition, we calculated the ratios hCGalpha/hCG + hCGbeta, hCGbeta/hCG + hCGbeta, and hCGalpha/hCGbeta. Specificity and sensitivity were calculated and paired in receiver-operating characteristic (ROC) curve analysis, resulting in areas under the curves (AUCs). RESULTS: hCGbeta, hCGbeta/hCG + hCGbeta and hCGalpha/hCGbeta show AUCs ranging between 0.922 and 0.999 and, therefore, are excellent diagnostic tests to distinguish complete and partial moles from normal pregnancy. To distinguish partial from complete moles the analytes hCGbeta, hCG + hCGbeta and the ratio hCGalpha/hCGbeta have AUCs between 0.7 and 0.8. Although hCGalpha, hCGbeta and hCG + hCGbeta concentrations are significantly elevated in patients who will develop PTD compared with patients with spontaneous regression after evacuation of their moles, in predicting PTD, these analytes and parameters have AUCs <0.7. CONCLUSIONS: Distinction between hydatidiform mole and normal pregnancy is best shown by a single blood specimen with hCGbeta, but hCGbeta/hCG + hCGbeta and hCGalpha/hCGbeta are also excellent diagnostic parameters. To predict PTD, hCGalpha, hCGbeta, hCG + hCGbeta and hCGalpha/hCGbeta are moderately accurate tests, although they are not accurate enough to justify prophylactic chemotherapy treatment for prevention of PTD.     

THYROID FUNCTION IN CHORIOCARCINOMA: DEMONSTRATION OF A THYROID STIMULATING ACTIVITY IN SERUM USING FRTL-5 and HUMAN THYROID CELLS

Hyperthyroidism is a well recognized complication of gestational trophoblastic tumours (GTT) and may be due to high circulating concentrations of human chorionic gonadotrophin (hCG) or its variants. We have studied 24 clinically euthyroid women with GTT. Eight were biochemically hyperthyroid with low or undetectable serum thyrotrophin (TSH) and had a mean serum hCG of 361.2 x 10(3) IU/l compared to 76.2 x 10(3) IU/l in the other patients (P less than 0.01). Purified hCG stimulated iodide uptake into FRTL-5 cells with 25 x 10(3) IU/l being equivalent in potency to 1 mU/l of thyrotrophin (TSH). Sixteen out of the 24 sera (67%) stimulated iodide uptake when applied to the cells at a 1:10 dilution. Sera from all eight hyperthyroid patients contained thyroid stimulating activity. The mean hCG concentration in the 16 stimulatory sera was 238.2 x 10(3) IU/l compared to 37.1 x 10(3) IU/l in the other eight sera (P less than 0.01). Six men with hCG-secreting testicular tumours were biochemically euthyroid although three of their sera stimulated iodide uptake into FRTL-5 cells. In human thyroid cells the mean cAMP production over 4 h with sera from five healthy controls was 54.2 +/- 1.81 pmol/mg cell protein compared to 67.0 +/- 3.8 pmol/mg protein with sera from five choriocarcinoma patients (P less than 0.02). Serum from patients with gestational trophoblastic tumours contains a thyroid stimulating activity which may be hCG and whose presence correlates with hyperthyroidism.     

Glycosylation changes in human chorionic gonadotropin and free alpha subunit as gestation progresses.

Carbohydrate is important to the structure, function, and circulatory survival of the glycoprotein hormones. Human CG (hCG) and the related free alpha-molecule of pregnancy contain four and two asparagine-linked oligosaccharides, respectively. The present study analyzes changes in the glycosylation patterns of hCG and free alpha in early vs. late gestation. Five volunteers provided 24-h urine samples, weekly, throughout their pregnancies. Extracts of early pregnancy (weeks 7-12) and late pregnancy (weeks 28-32) urines were pooled. Early and late samples from each patient were subjected to gel filtration to separate hCG and free alpha, and the populations thus obtained were analyzed by lectin affinity chromatography on Concanavalin A-Sepharose (Con A) and Lens culinaris-agarose (Lch). Using Con A, free alpha and hCG were separated into an unbound fraction (eluted with Con A buffer), a weakly bound fraction (eluted with 10 mmol alpha-methyl-D-glucoside) and a tightly bound fraction (eluted with 500 mmol alpha-methyl-D-mannoside). For free alpha-molecule, a significant decrease in tightly bound Con A forms, was noted from early to late pregnancy with a mean difference of 17.0 +/- 2.4% (P less than 0.01). Concomitantly, in late pregnancy, an increase in Con A unbound forms of free alpha was noted with mean difference of 12.5 +/- 1.7% (P less than 0.01). These changes indicate the presence of more highly branched oligosaccharides on free alpha as gestation advances. No changes were noted in the Con A binding of intact hCG; nearly all hCG bound in both early and late pregnancy. Using Lch, free alpha and hCG were separated into an unbound fraction (eluted with Lch buffer) and a bound fraction (eluted with 500 mmol alpha-methyl-D-mannose). Both free alpha and intact hCG in late pregnancy exhibited increased binding to Lch, with mean differences from early to late pregnancy of 30.2 +/- 4.8% (P less than 0.01) and 11.4 +/- 4.5% (P less than 0.05), respectively. These data indicate increased incorporation of fucose into the carbohydrate moieties in late pregnancy. Taken together, these data derived by analysis using lectin specificity imply the presence of more highly branched, fucosylated oligosaccharides as gestation progresses.     

Identification of post-translational modifications resulting from LHbeta polymorphisms by matrix-assisted laser desorption time-of-flight mass spectrometric analysis of pituitary LHbeta core fragment

Metabolism of the human chorionic gonadotrophin (hCG)- and LHbeta-subunits (hCGbeta, LHbeta) terminates with the urinary excretion of core fragment (hCGbetacf, LHbetacf) molecules that retain antigenic shape and constituent N-linked carbohydrate moieties. We have previously demonstrated the resolved mass spectra of hCGbetacf, from which the carbohydrate moieties present at two N-linked glycosylation sites were identified. LHbetacf was subjected to the same mass spectrometric analysis. As LHbeta shares 82% homology with hCGbeta but possesses only one glycosylation consensus site a simpler spectral fingerprint of LHbetacf glycoforms was expected. LHbetacf was reduced with dithiothreitol and analysed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Glycoforms were predicted by subtracting the peptide mass from the m/z values of the observed peaks and then sequentially subtracting the masses of the monosaccharide residues of hCGbeta N-linked carbohydrates reported in the literature. The mass spectra of LHbetacf revealed a broad single peak ranging from m/z 8700 to 10 700. Following reduction, this peak was replaced by a set of partially resolved peaks between m/z 4130 and 5205 corresponding to glycosylated forms of the peptide LHbeta6-40. A peak at m/z 4252.2 corresponded to the non-glycosylated peptide LHbeta55-93. Remaining peaks indicated that the pooled sample comprised a wide set of glycoforms, contained LHbetacf with two N-linked carbohydrate moieties and indicated evidence of further glycosylation due to amino acid substitution in polymorphic variants. This is evidence that a single nucleotide polymorphism alters the post-translational modification of a protein and hence its structural phenotype.