Urine pregnancy test (UPT) hCG negative molar pregnancy: a short report from Masanga/Sierra Leone.

In Masanga, Sierra Leone, a multigravid woman presented with a urine pregnancy test negative molar pregnancy. This can be explained by the 'hook-effect'. In resource-poor settings where quantitative serum hCG cannot be determined, it is of paramount importance to remain vigilant of the diagnosis of molar pregnancy. Clinical judgement and sonography remain key in diagnosing molar pregnancy in district hospitals in low- and middle-income countries (LMICs), especially since their occurrence is much more common in these countries.

Negative ßhCG and Molar Pregnancy: The Hook Effect.

Molar pregnancy, included in gestational trophoblastic disease, is a benign pathology with ability to metastasize, usually occurring with excessively high ßhCG levels. Clinical scenario is usually a woman in extremes of reproductive age presenting with amenorrhoea, pain and vaginal blood loss; signs derived from high ßhCG levels may be present (hyperthyroidism, hyperemesis). Diagnosis is based on a positive pregnancy test - usually a qualitative urinary test. The limitation of this test results from its inability to become positive in presence of markedly high levels of ßhCG, saturating the antigens used - known as the 'hook effect'. With the widespread use of gynaecological ultrasound cases of molar pregnancy have been diagnosed in timely fashion. We describe a case referred as a degenerating fibroid, with a negative urinary pregnancy test. Transvaginal ultrasound was highly suggestive of molar pregnancy, which was confirmed with a quantitative ßhCG test, allowing for timely treatment. The importance of a high index of suspicion for this pathology is tremendous to avoid the devastating consequences of a delayed diagnosis.

A gravidez molar, incluída na doença gestacional do trofoblasto, é uma patologia benigna com capacidade de metastização, cursando com níveis excessivamente elevados de ßhCG. O quadro clínico traduz-se por amenorreia, dor e perda de sangue vaginal numa mulher frequentemente no extremo da idade reprodutiva, podendo estar presentes sinais decorrentes dos níveis de ßhCG (hipertiroidismo, hiperemese). O diagnóstico é histológico, e suspeitado por um teste de gravidez positivo, sendo normalmente realizado um teste urinário qualitativo. A limitação deste advém da incapacidade de se tornar positivo na presença de níveis exageradamente altos de ßhCG, que satura os antigénios utilizados ­ 'efeito hook'. Com a ecografia ginecológica os casos de gravidez molar têm sido diagnosticados mais atempadamente. Descrevemos um caso referenciado como um mioma degenerescente, com teste de gravidez urinário negativo. A ecografia transvaginal realizada foi altamente sugestiva de gravidez molar, confirmada com um teste quantitativo de ßhCG e permitindo tratamento atempado. A importância de um elevado índice de suspeição para esta patologia é fulcral para evitar as consequências devastadoras de um diagnóstico tardio.

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.

Direct analysis of hCGßcf glycosylation in normal and aberrant pregnancy by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The analysis of human chorionic gonadotropin (hCG) in clinical chemistry laboratories by specific immunoassay is well established. However, changes in glycosylation are not as easily assayed and yet alterations in hCG glycosylation is associated with abnormal pregnancy. hCGß-core fragment (hCGßcf) was isolated from the urine of women, pregnant with normal, molar and hyperemesis gravidarum pregnancies. Each sample was subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis following dithiothreitol (DTT) reduction and fingerprint spectra of peptide hCGß 6-40 were analyzed. Samples were variably glycosylated, where most structures were small, core and largely mono-antennary. Larger single bi-antennary and mixtures of larger mono-antennary and bi-antennary moieties were also observed in some samples. Larger glycoforms were more abundant in the abnormal pregnancies and tri-antennary carbohydrate moieties were only observed in the samples from molar and hyperemesis gravidarum pregnancies. Given that such spectral profiling differences may be characteristic, development of small sample preparation for mass spectral analysis of hCG may lead to a simpler and faster approach to glycostructural analysis and potentially a novel clinical diagnostic test.

[Pregnancy tests: urine versus blood pregnancy tests]. / Zwangerschapstesten: urinetest versus bepaling in bloed.

In this article, we discuss possible explanations for the discrepancy in results between urine and blood pregnancy tests. The first patient, a 26-year-old woman, had breast tenderness, was tired and suffered from abdominal pain. A urine pregnancy test was negative, but blood human chorion gonadotropin (hCG) concentration was 455 U/l (reference value < 6 U/l). It was concluded that the patient was pregnant and she was followed in the outpatient clinic. Three days later she suffered blood loss and her hCG levels returned to normal. The diagnosis was a spontaneous abortion. The second patient, a 45-year-old woman, complained of abdominal pain, nausea and more blood loss than with a normal period. The urine pregnancy test was negative, but the hCG level in her blood was 470.000 U/l. Echography showed a thickened, irregular endometrium. A molar pregnancy was suspected and curettage was performed. The hCG level dropped initially but had increased at follow-up. Persistent trophoblastic disease was suspected and the patient underwent additional treatment.

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.

Qualitative beta-hCG urine assays may be misleading in the presence of molar pregnancy: a case report.

Molar pregnancy is a rare complication of pregnancy and the diagnosis is usually confirmed with a markedly elevated beta-hCG and a "snowstorm" appearance on pelvic ultrasound. Patients frequently present with a positive pregnancy test, vaginal bleeding, nausea and vomiting. A 23-year-old woman presented to our Emergency Department with a history of 7 weeks of intermittent vaginal bleeding and 1 h of peri-umbilical abdominal pain. She reported that 7 weeks before this visit she was diagnosed with a miscarriage. The bedside qualitative urine human chorionic gonadotropin (hCG) test that we performed was negative, but the quantitative serum hCG was markedly elevated. Ultrasound and operative findings confirmed the diagnosis of molar pregnancy. We conclude that rapid urine qualitative hCG assays may not be reliable in the presence of markedly elevated hCG levels found in molar pregnancy.

Gestational trophoblastic disease: is intensive follow up essential in all women?

OBJECTIVE: To determine the timescale of the registration process for gestational trophoblastic disease and its impact on hCG level at registration and subsequent need for chemotherapy. DESIGN: A prospective observational study using a standardised protocol for registration, assessment and treatment for molar pregnancy. SETTING: A supra-regional tertiary referral centre for gestational trophoblastic disease. PARTICIPANTS: A total of 2046 consecutive women registered between January 1994 and December 1998 with a diagnosis of molar pregnancy. METHODS: Data at and after registration, collected prospectively on a computerised database, were statistically analysed (by multiple logistic regression and ANOVA). MAIN OUTCOME MEASURES: Relationship between length of time to and hCG value at registration; also the subsequent need for chemotherapy. RESULTS: A total of 2046 women with a diagnosis of molar pregnancy were registered in the study period. The mean time interval between first evacuation and registration at the referral centre was 47 days (median 37, range 0-594). One hundred and five out of 2046 (5.1%) women needed chemotherapy. Sixty-three precent of the women (1296 out of 2046) had a normal level of urinary hCG (less than 40 IU/24 hours) at the time of registration and only one (0.08%) needed chemotherapy. Binary logistic regression analysis showed a statistically significant relationship between time to registration, hCG value, histology, pretreatment risk score and decision to administer chemotherapy. CONCLUSION: Women with gestational trophoblastic disease who were registered late were significantly more likely to have normal levels of hCG and were less likely to need chemotherapy. A less intensive follow up may be justified in women with gestational trophoblastic disease who are registered with a normal hCG level.

Persistent low-level "real" human chorionic gonadotropin: a clinical challenge and a therapeutic dilemma.

OBJECTIVE: The finding of persistent low-level human chorionic gonadotropin (hCG) with or without a preceding pregnancy event presents a rare but clinically important challenge and a therapeutic dilemma. These are patients with "real" hCG shown by the positive test in both serum and urine or by specialized testing. The problems associated with "phantom" hCG have been recognized and should now be clinically resolvable. Four cases of low-level "real" hCG are described to illustrate the problems encountered, the management, and the resolution achieved. METHODS: Two patients presented with persistent low-level hCG after hydatidiform mole pregnancy, one after an early pregnancy loss and one as amenorrhea and irregular bleeding. A detailed clinical description is provided to illustrate the difficulties encountered. RESULTS: All patients have real hCG. The hCG level of Patient 1 was responsive to hormonal contraception and disappeared with such medication. Over a period of 3 years hCG reappeared whenever estrogen was stopped. Patient 2 achieved two pregnancies and the hCG subsequently disappeared. The hCG in Patient 3 persisted over a period of 6 years although she is now menopausal. Patient 4 developed metastatic placental site trophoblastic tumor after 2 1/2 years of observation of low-level hCG. CONCLUSIONS: The finding of unexplainable low-level hCG in a patient without evidence of a uterine lesion or of trophoblastic metastases provides a therapeutic challenge. The administration of single-agent chemotherapy had no effect on the level of hCG in the three patients to whom it was administered. The administration of multiple-agent chemotherapy appears unjustified in the absence of a demonstrable trophoblastic tumor. A small number of trophoblastic cells must be providing this hCG and these cells may be quiescent for years. Nevertheless these cells may proliferate and manifest themselves as trophoblastic tumor. Continuing long-term surveillance of these patients is necessary.

Carbohydrate and peptide structure of the alpha- and beta-subunits of human chorionic gonadotropin from normal and aberrant pregnancy and choriocarcinoma.

Human chorionic gonadotropin (hCG), purified from the urine of 14 individuals with normal pregnancy, diabetic pregnancy, hydatidiform mole, or choriocarcinoma, plus two hCG standard preparations, was examined for concurrent peptide-sequence and asparagine (N)- and serine (O)-linked carbohydrate heterogeneity. Protein-sequence analysis was used to measure amino-terminal heterogeneity and the "nicking" of internal peptide bonds. The use of high-pH anion-exchange chromatography coupled with the increased sensitivity of pulsed amperometric detection (HPAE/PAD) revealed that distinct proportions of both hCG alpha- and beta-subunits from normal and aberrant pregnancy are hyperglycosylated, and that it is the extent of the specific subunit hyperglycosylation that significantly increases in malignant disease. Peptide-bond nicking was restricted to a single linkage (beta 47-48) in normal and diabetic pregnancy, but occurred at two sites in standard preparations, at three sites in hydatidiform mole, and at three sites in choriocarcinoma beta-subunit. In the carbohydrate moiety, alpha-subunit from normal pregnancy hCG contained nonfucosylated, mono- and biantennary N-linked structures (49.3 and 36.7%, means); fucosylated biantennary and triantennary oligosaccharides were also identified (7.3 and 6.9%). In choriocarcinoma alpha-subunit, the level of fucosylated biantennary increased, offset by a parallel decrease in the predominant biantennary structure of normal pregnancy (P < 0.0001). The beta-subunit from normal pregnancy hCG contained fucosylated and nonfucosylated biantennary N-linked structures; however, mono- and triantennary oligosaccharides were also identified (4.6 and 13.7%). For O-linked glycans, in beta-subunit from normal pregnancy, disaccharide-core structure predominated, whereas tetrasaccharide-core structure was also detected (15.6%). A trend was demonstrated in beta-subunit: the proportions of the nonpredominating N- and O-linked oligosaccharides increased stepwise from normal pregnancy to hydatidiform mole to choriocarcinoma. The increases were: for monoantennary oligosaccharide, 4.6 to 6.8 to 11.2%; for triantennary, 13.7 to 26.7 to 51.5% and, for O-linked tetrasaccharide-core structure, 15.6 to 23.0 to 74.8%. For hCG from individual diabetic pregnancy, the principal N-linked structure (34.7%) was consistent with a biantennary oligosaccharide previously reported only in carcinoma; and sialylation of both N- and O-linked antennae was significantly decreased compared to that of normal pregnancy. Taken collectively, the distinctive patterns of subunit-specific, predominant oligosaccharides appear to reflect the steric effect of local protein structure during glycosylation processes. The evidence of alternative or "hyperbranched" glycoforms on both alpha- and beta-subunits, seen at low levels in normal pregnancy and at increased or even predominant levels in malignant disease, suggests alternative substrate accessibility for Golgi processing enzymes, alpha 1,6 fucosyltransferase and N-acetylglucosaminyltransferase IV, in distinct proportions of subunit molecules.

[Urinary gonadotropin fragment measurement in the monitoring of trophoblastic disease].

Urinary gonadotropin fragment (UGF) is a small peptide which is present in the urine of pregnant women and of women with trophoblastic diseases as well as with certain nontrophoblastic malignancies. 275 samples each of urine and blood from 46 patients with trophoblastic diseases were taken for UGF and hCG measurements and compared. 24 samples from 12 healthy, nonpregnant women were taken as control. Cut-off values of UGF and hCG used for measuring the sensitivity of trophoblastic diseases were respectively > 0.2 microgram/L and above 20 micrograms/L. It was found that 64.0% of the urine samples gave UGF values > 0.2 microgram/L and 66.5% of the blood samples showed hCG levels above 20 micrograms/L (P > 0.1). No false-positive rate was observed in the control group. However, among patients who were found to have low or negative hCG values, 57.6% showed positive UGF levels. These findings suggest that in patients with positive levels of both UGF and hCG, the UGF measurement may not be necessary. But for patients with low or negative blood hCG values, certain percentage of urine UGF could still be detected.

The heterogeneity of human chorionic gonadotropin (hCG). I. Characterization of peptide heterogeneity in 13 individual preparations of hCG.

Peptide variations in the alpha-subunit (molecules starting at alpha 3 and alpha 4) and beta-subunit (missing linkages at beta 44-45 and beta 47-48) of hCG have been reported by several investigators. Studies, however, have been limited to standard hCG preparations (purified from large pools of urine) and other hCG samples from mixed urines. In this study we used chromatographic procedures to purify the total hCG content of 13 individual urines, 6 from patients with pregnancy and 7 from those with trophoblast disease (no hCG-containing fractions were excluded). Then, we examined for the first time the peptide variability among individual samples of hCG. We report 1) that individual hCG preparations have nicks (missing linkages) in the beta-subunit, primarily between residue 47-48 (11 of 13 samples) and, less commonly, at the linkage 44-45 or 46-47 (3 of 13 samples); 2) the extent of nicking varies greatly between individual preparations (range, 0-100% of molecules); 3) varying alpha-subunit N-terminal heterogeneity (N-terminus starting at alpha 3 or alpha 4) was also present (range, 0-28% of molecules), but was confined to preparations from individuals with trophoblast disease (6 of 7 samples from trophoblast disease urine, 0 of 6 from pregnancy urine); 4) hCG missing the beta-subunit C-terminal region was also detected (2 of 13 hCG preparations); and 5) 1 of 13 preparations was nicked on the hCG alpha-subunit, between residues 70 and 71. Thus, 12 of 13 individual hCG samples demonstrated at least 1 of 4 different forms of peptide heterogeneity. We conclude that individual hCG samples vary widely in the type and extent of peptide heterogeneity, an observation that is not appreciated when pools of hCG are studied.

The heterogeneity of human chorionic gonadotropin (hCG). III. The occurrence and biological and immunological activities of nicked hCG.

Nicks, or missing peptide linkages, have been found in hCG beta-subunit between residues 44 and 45 and between residues 47 and 48. We examined the occurrence and biological and immunological activities of nicked hCG. As shown by sequence analysis, CR127 standard hCG is approximately 20% nicked, half at beta 44-45 and half at beta 47-48. Treatment with human leukocyte elastase increased the extent of nicking of CR127 standard hCG. The longer the incubation of CR127 standard with human leukocyte elastase (0, 2, and 21 h), the greater the extent of nicked hCG (20%, 46%, and 89%). As the extent of nicking increased, the receptor-binding ability diminished, as did the ability to stimulate progesterone production by rat corpus luteal cells in vitro (0.9, 0.74, and 0.29 microgram/microgram hCG, respectively). In a regression analysis, a linear relationship was indicated between the extent of nicking and receptor binding values (97% correlation) and between the extent of nicking and steroidogenic activity in vitro (99% correlation). From the intercepts of the regression lines, it was estimated that nicks reduced receptor binding by 11-fold and reduced the steroidogenic activity of hCG by 5-fold. We examined eight individual hCG preparations, three purified from pregnancy urine, three from urine from patients with hydatidiform mole, and two from urine from women with choriocarcinoma. In descending order, the eight individual hCG preparations were 100%, 100%, 85%, 76%, 42%, 41%, 0%, and 0% intact. Although no correlation was observed between the percent intact and the ability of the eight individual samples to displace 50% [125I]hCG in binding CG/LH receptor (r less than 0.5), a close correlation was noted between the percent intact and the steroidogenic activity in vitro (98% correlation). This separated the effects of nicking on receptor binding and steroidogenic activities and indicated that while multiple factors influence receptor binding, only nicking suppresses the steroidogenic activity of bound hCG. We examined the recognition of nicked hCG molecules by different hCG immunoassays. The Hybritech Tandem assay measured total hCG and did not distinguish nicked and intact hCG molecules (in a regression analysis, immunoactivity vs. percent intact hCG, r less than 0.5). In contrast, the immunometric assay using B109 hCG dimer-specific monoclonal antibody and anti-beta-peroxidase only detected the intact component of hCG (in a regression analysis, immunoreactivity vs. percent intact hCG, 98% correlation). We used these assays together to estimate the percentage of intact hCG and to deduce the extent of nicking.(ABSTRACT TRUNCATED AT 400 WORDS)

Altered glycosylation of human chorionic gonadotropin decreases its hormonal activity as determined by cyclic-adenosine 3',5'-monophosphate production in MA-10 cells.

Human chorionic gonadotropin (hCG) purified from pooled urine of normal pregnant women contains four asparagine-linked sugar chains and four mucin-type sugar chains. The structures of asparagine-linked sugar chains of this hormone are constant and site-specific. hCGs obtained from the urine of patients with invasive mole or choriocarcinoma have quite different sets of oligosaccharides although the primary structures of the polypeptides and the numbers of the sugar chains are the same as those of normal hCG. In order to examine the biological activities of these hCGs with altered glycosylation, we measured the amount of cAMP produced in a murine Leydig tumour cell line, MA-10, after incubation with the hCG samples. The extent of sialylation of oligosaccharides in the three hCG samples used in this study were 88% in normal hCG, 82% in invasive mole hCG and 63% in choriocarcinoma hCG. The hormonal activity of invasive mole hCG was slightly lower while that of choriocarcinoma hCG was significantly (P less than 0.01) lower than that of normal hCG. Complete desialylation induced remarkable loss of full activities in all the samples. However, the hormonal activities of the three samples were different even after desialylation. The full activities of the desialylated samples of invasive mole hCG and choriocarcinoma hCG were 78 and 65% of that of desialylated normal hCG. These results indicated that the structures of the neutral oligosaccharide portion are also important for the expression of full hormonal activity.

[The serum concentration of specific beta 1-glycoprotein in trophoblastic disease]. / Prouchvaniia na serumnata kontsentratsiia na spetsifichniia beta 1-glikoprotein pri trofoblastna bolest.

Twelve women with hydatidiform mole were studied between 12 and 20 weeks-gestation. The level of SP1 was determined by radioimmunologic method. The author found statistically significant increase of SP1 over 90th percentile of gestational age (p less than 0.001). Examining the level of SP1 after evacuation of hydatidiform mole it was established that SP1 was eliminated more quickly than human chorionic gonadotropin (HCG) in urine during early pregnancy. Quite the contrary was discovered in women with advanced pregnancy, in whom the concentration of SP1 disappeared more slowly, while HCG was eliminated for a shorter time. The observed dissociation in elimination of SP1 and HCG showed that the combined examination of HCG and SP1 provided better information in the follow-up of women with trophoblastic disease. The author think that treatment of trophoblastic disease should continue till complete elimination both of HCG and SP1.

Immobilized Datura stramonium agglutinin column chromatography, a novel method to discriminate the urinary hCGs of patients with invasive mole and choriocarcinoma from those of normal pregnant women and patients with hydatidiform mole.

Datura stramonium agglutinin (DSA) binds the asparagine-linked sugar chains commonly found in the human chorionic gonadotropin (hCG) purified from urine of patients with invasive mole or choriocarcinoma, but not in that of normal pregnant women or patients with hydatidiform mole. With the use of an immobilized DSA column, a novel method to discriminate urinary hCGs from various trophoblastic diseases was developed.