Insights into the hyperglycosylation of human chorionic gonadotropin revealed by glycomics analysis.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone that is essential for the maintenance of pregnancy. Glycosylation of hCG is known to be essential for its biological activity. "Hyperglycosylated" variants secreted during early pregnancy have been proposed to be involved in initial implantation of the embryo and as a potential diagnostic marker for gestational diseases. However, what constitutes "hyperglycosylation" is not yet fully understood. In this study, we perform comparative N-glycomic analysis of hCG expressed in the same individuals during early and late pregnancy to help provide new insights into hCG function, reveal new targets for diagnostics and clarify the identity of hyperglycosylated hCG. hCG was isolated in urine collected from women at 7 weeks and 20 weeks' gestation. hCG was also isolated in urine from women diagnosed with gestational trophoblastic disease (GTD). We used glycomics methodologies including matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) and MS/MS methods to characterise the N-glycans associated with hCG purified from the individual samples. The structures identified on the early pregnancy (EP-hCG) and late pregnancy (LP-hCG) samples corresponded to mono-, bi-, tri-, and tetra-antennary N-glycans. A novel finding was the presence of substantial amounts of bisected type N-glycans in pregnancy hCG samples, which were present at much lower levels in GTD samples. A second novel observation was the presence of abundant LewisX antigens on the bisected N-glycans. GTD-hCG had fewer glycoforms which constituted a subset of those found in normal pregnancy. When compared to EP-hCG, GTD-hCG samples had decreased signals for tri- and tetra-antennary N-glycans. In terms of terminal epitopes, GTD-hCG had increased signals for sialylated structures, while LewisX antigens were of very minor abundance. hCG carries the same N-glycans throughout pregnancy but in different proportions. The N-glycan repertoire is more diverse than previously reported. Bisected and LewisX structures are potential targets for diagnostics. hCG isolated from pregnancy urine inhibits NK cell cytotoxicity in vitro at nanomolar levels and bisected type glycans have previously been implicated in the suppression of NK cell cytotoxicity, suggesting that hCG-related bisected type N-glycans may directly suppress NK cell cytotoxicity.

When to stop human chorionic gonadotrophin (hCG) surveillance after treatment with chemotherapy for gestational trophoblastic neoplasia (GTN): A national analysis on over 4,000 patients.

OBJECTIVE: To determine the optimal duration of human chorionic gonadotrophin (hCG) surveillance following treatment for low and high risk gestational trophoblastic neoplasia (GTN) and establish whether the current surveillance protocol that recommends life-long hCG monitoring requires revision. METHODS: A population-based cohort study was undertaken using a national registry, comprising patients from both tertiary trophoblastic disease treatment units in the UK (London and Sheffield). All patients who received chemotherapy for low or high risk GTN in the UK between 1958 and 2014 in London and 1973 and 2015 in Sheffield (n = 4201) were included in the study. Patients with placental site trophoblastic tumours and epithelioid trophoblastic tumours were excluded due to their distinct clinical behavior, treatment and follow-up requirements. The risk of recurrence with time following completion of chemotherapy for low or high risk GTN was measured. RESULTS: The overall risk of relapse in this national cohort of 4201 patients was 4.7% (198/4201) with a median time to recurrence of 117.5 days (range 9 days to 6.54 years). The greatest risk of recurrence occurred in the first year after completing treatment for either low or high risk GTN measuring 72.7% (n = 112) or 86.4% (n = 38), respectively. The subsequent recurrence risk reduced over time with none observed beyond 7 years. CONCLUSIONS: The absence of any recurrences beyond seven years following completion of chemotherapy for GTN indicates that the UK policy of life-long hCG surveillance is unnecessary. Our revised conservative protocol recommends stopping after 10 years.

Follow-Up After Molar Pregnancy Evacuation: Feasibility of Using Semi-Quantitative Urine Pregnancy Tests.

OBJECTIVE: To evaluate human chorionic gonadotropin (hCG) trends after evacuation of complete hydatidiform moles to determine if urinary semiquantitative pregnancy tests (SQPTs) could replace blood draws. while still detecting early postmolar gestational trophoblastic neoplasia. STUDY DESIGN: A retrospective review of complete hydatidiform moles at a safety-net hospital from 2003-2013 was performed. hCG curves were used to extrapolate expected SQPT results over timefor a resolving hydatidiform mole. RESULTS: Of 61 complete moles, 37 had an uncomplicated hCG decline and at least 4 serum hCG results. All of those patients had hCG < 10,000 mIU/mL within 15 days, < 2,000 within 64 days, < 500 within 70 days (92.2% within 1 month), < 100 within 89 days (90% within 2 months), and < 25 within 152 days (95.2% within 3 months). After reaching levels < 25, hCG rose only in cases of new pregnancies. CONCLUSION: Based on this retrospective analysis, SQPT monitoring could have avoided 90% of blood draws while still flagging all patients with subsequent postmolar GTN within 45 days by limiting blood draws to (1) patients with SQPT levels of > 10,000, > 500, and >100 mIU/mL at 15, 30, and 45 days, respectively, (2) hCG > 25 after 60 days, or (3) increasing SQPT levels.

Persistent low levels of serum hCG due to heterophilic mouse antibodies: an unrecognized pitfall in the diagnosis of trophoblastic disease.

Phantom hCG refers to persistent mild elevations of hCG, leading physicians to unnecessary treatments whereas neither a true hCG nor a trophoblastic disease is present. We report the case of a 23-year-old woman with persistent low levels of serum hCG detected one month after miscarriage. As choriocarcinoma was suspected, a chemotherapy trial of methotrexate was prescribed, without any hCG reduction. Subsequently, laparoscopy ruled out a trophoblastic residue and the patient was referred to the Endocrine Unit for further investigations. While low levels of hCG were still detected in serum, no hCG was detected in the urine. In addition, when serum was processed in a HBT tube for revealing heterophilic antibodies, hCG was no longer detected. Such finding indicated the presence of phantom hCG due to heterophilic mouse antibodies interaction. This case raises the need of clinico-biological discussion to avoid inappropriate therapeutic decisions. Based on this case experience and after review of the literature, we suggest that current gynecological protocols for the diagnosis and treatment of trophoblastic disease should consider the inclusion of urinary hCG and/or a test for serum heterophilic antibodies when appropriate.

Gestational Trophoblastic Disease Diagnosis Delayed by the Hook Effect.

BACKGROUND: A "hook effect" resulting from saturation of antibodies used in pregnancy tests can occur at human chorionic gonadotropin (hCG) levels above 500,000 milliinternational units/mL, resulting in falsely negative values. CASE: A 34-year-old woman, gravida 5 para 3, presented to the emergency department after heavy bleeding. Ultrasonogram revealed a uterine mass, urine pregnancy test result was negative, and endometrial biopsy inconclusive. The patient was discharged and presented 10 days later with recurrent bleeding. Urine pregnancy test result was again negative, but serum hCG was 581 milliinternational units/mL. Serial dilution revealed an actual hCG higher than 5 million milliinternational units/mL. She was diagnosed with gestational trophoblastic disease. CONCLUSION: Awareness of the risk of a false-negative pregnancy test result when hCG levels are extremely high may prevent delayed diagnosis of gestational trophoblastic disease.

Quantitative urine hCG and urine pregnancy test in gestational trophoblastic disease patients with low hCG titer.

OBJECTIVE: To study the correlation between serum and urine hCG levels in gestational trophoblastic disease (GTD) patients with low hCG level. The correlation between serum hCG and results from urine pregnancy tests are evaluated along with quantitative measurement. MATERIAL AND METHOD: In this prospective study, 86 cases of gestational trophoblastic disease patients with positive and low level of serum hCG (< 100 mIU/ml) were recruited. Quantitative serum hCG urine hCG and urine pregnancy test were performed. The correlation coefficients between serum and urine hCG were then analyzed by SPSS 16.0. Furthermore, the levels of serum hCG were compared to the results of the urine pregnancy test. RESULTS: From February 2006 to June 2008, 86 cases were recruited for this study. The correlation coefficient between serum and urine hCG levels in all cases was 0.44 (using Pearson correlation), p = 0.01. In subgroup analysis, the correlation coefficient between serum and urine hCG levels in chemosensitive gestational trophoblastic neoplasia (GTN) patients (n = 27) was 0.73, p ≤ 0.01. The correlation coefficient in chemoresistant GTN patients (n = 38) was 0.29, p = 0.07; and the correlation coefficient in hydatidiform mole patients (n = 21) was 0.47, p = 0.03. A urine pregnancy test was positive only in 10 of 86 specimens. CONCLUSION: The correlation coefficient between serum and urine hCG in GTD patients with low hCG level showed significant correlation. However, patients with chemoresistance had less correlation than those with chemosensitivity and hydatidiform mole. Urine pregnancy test had low correlation with urine hCG and was not useful in this group of patients.

Identification of patients with persistent trophoblastic disease after complete hydatidiform mole by using a normal 24-hour urine hCG regression curve.

OBJECTIVE: The aim of this study was to establish a reference 24-hour urine human chorionic gonadotropin (hCG) regression curve in patients with complete hydatidiform mole (CHM) as diagnostic tool in the prediction of persistent trophoblastic disease (PTD). METHODS: From 2004 to 2011, 312 cases suitable for this study were registered at the Hydatidiform Mole Registry of the Royal Women's Hospital Melbourne, Australia. hCG levels of 61 patients diagnosed as having PTD according to FIGO 2000 criteria were compared with the 95th-percentile (p95) of the normal regression curve derived from hCG levels of 251 cases of uneventful CHM. RESULTS: In the test group of 61 patients PTD was diagnosed by FIGO 2000 criteria after a mean (±SD, min.-max.) of 7.6 (±3.4, 3.0-16.7) weeks after evacuation of the mole while in the same group hCG values for the first time exceeded the upper limit of the 95th percentile significantly earlier after 4.5 (±1.9, 2.0-9.9) weeks (P<0.001). However, hCG levels of 14% of the cases of uneventful CHM at least once exceeded the upper limit of p95, showing that one single value above p95 is not accurate enough for the diagnosis of PTD. CONCLUSIONS: The normal 24-hour urine hCG regression curve may be used as a tool in the follow-up of an individual case of CHM after evacuation. At least one hCG level exceeding the upper limit of p95 within 11weeks after evacuation could be added to the current FIGO criteria, in order to diagnose PTD early, but the lack of it may also prevent unnecessary treatment.

Predicting gestational trophoblastic neoplasia (GTN): is urine hCG the answer?

BACKGROUND: Previous studies on the significance of hCG to predict gestational trophoblastic neoplasia (GTN) have been too small for robust conclusions to be reached. Our aim in this study was to analyse the significance of urine hCG in predicting GTN in a large population. METHODS: Details of 3926 patients were available for analysis. Information regarding age, dates of diagnosis and registration, urine hCG levels, antecedent pregnancy and chemotherapy were prospectively collected and used for analyses. Patients were stratified into different groups according to urine hCG level (IU/L); < 50, 50-99, 100-249, 250-499, 500-999, 1000-9999 and ≥10,000. Multivariate analyses were used to identify the prognostic indicators of GTN. RESULTS: Urine hCG and antecedent pregnancy were the most powerful indicators for developing GTN (P<0.01). None of the patients with partial mole and urine hCG <50 IU/L (Normal level=40 IU/L) developed GTN. The risk of GTN was >35% in all patients with urine hCG ≥500 IU/L. GTN developed in 70% of patients with complete mole and urine hCG ≥10,000 IU/L. CONCLUSION: Urine hCG is sensitive test for GTN. Urine hCG level is a powerful prognostic indicator for the GTN. Patients with partial mole could be safely discharged from the surveillance programme once their hCG have normalised. Patients with urine hCG ≥249 IU/L, whether partial or complete molar pregnancy, appear to benefit from intensive surveillance. Prophylactic chemotherapy could be considered when there are problems with surveillance.

USA hCG reference service, 10-year report.

INTRODUCTION: The USA hCG Reference Service has been dealing with cases of persistent low levels of hCG and gestational trophoblastic diseases for 10years. Here we present the complete experience. METHODS: Total hCG in serum and urine was measured using the Siemen's Immulite 1000 assay. Hyperglycosylated hCG, nicked hCG, free ss-subunit and ss-core fragment were measured using microtiterplate assays with antibodies B152, B151, FBT11 and B210, respectively. RESULTS: The USA hCG Reference Service has identified 83 cases of false-positive hCG, 71 cases of aggressive gestational trophoblastic disease (GTD), 52 cases of minimally invasive GTD, 168 cases of quiescent GTD and 22 cases of placenta site trophoblastic tumor (PSTT). In addition, 103 cases of pituitary hCG have been identified, 60 cases of nontrophoblastic tumor, 4 cases of inherited hCG and 2 cases of Munchausen's syndrome. This is 565 cases total. Multiple new methods are described and tested for diagnosing all of these disorders. CONCLUSIONS: The USA hCG Reference Service experience shows new methods for detecting multiple hCG-related disorders and recommends new approaches for detecting these hCG-related disorders.

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 alpha(2-3)sialic acid, respectively, preferentially bound pregnancy-derived hCG, whereas the lectin, wheat germ agglutinin, which binds sialic acid and beta(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.

The need for an hCG assay that appropriately detects trophoblastic disease and other hCG-producing cancers.

This report was conceived at the 13th World Congress of Gestational Trophoblastic Disease after multiple presentations indicated widespread discrepancies in human chorionic gonadotropin (hCG) use and results. There appears to be a need for a discussion to describe the advantages and limitations of commonly used hCG tests in the management of gestational trophoblastic disease, and to monitor testicular, germ cell and other hCG-producing malignancies. In most countries hCG tests are certified only for pregnancy testing. Use in managing gestational trophoblastic diseases and other malignancies is considered an "off-label" use. Tests are not optimized or calibrated for these applications, and their use and the results therefore have to be considered experimental. Widespread variations in results occur and may lead to needless or inappropriate therapy. It therefore seems important for laboratory directors and treating physicians to familiarize themselves with which hCG test their laboratory is using and, if necessary, to contract an external laboratory for measuring hCG in the management of gestational trophoblastic disease and cancer.

Gestation trophoblastic diseases: management of cases with persistent low human chorionic gonadotropin results.

The finding of persistent low levels of human chorionic gonadotropin (hCG) raises concern, regardless of the antecedent history, and often provokes the evaluating physician to embark on further work-up. The USA hCG Reference Service was started in 1998 to aid physicians with these persistent low hCG cases. This article reviews the observations of the USA hCG Reference Service for 134 cases with persistent low hCG results. Examination of these cases provides clear insight for the appropriate management of those presenting with persistent low levels of hCG. Three distinct sources are discussed for persistent low hCG results: quiescence gestational trophoblastic disease, false-positive hCG results, and pituitary hCG.