Lowered reference limits for hCG improve follow-up of patients with hCG-producing tumors.

BACKGROUND: Human Chorionic Gonadotropin (hCG) is produced by germ cell tumors, but can also be elevated in benign conditions such as primary hypogonadism, where hCG is produced by the pituitary gland. In our experience, the reference limits for hCG (Elecsys hCG+ß-assay, Roche Diagnostics), were unnecessarily high and did not reflect levels encountered in clinical practice. We wanted to establish new reference limits to increase the clinical utility of the hCG-assay. METHODS: We analysed hCG in serum samples from a healthy adult population and in a cohort of testicular cancer survivors. The gonadotropins LH and FSH were measured in the cohort and in a selection of the reference population to assess gonadal function. RESULTS: We found low hCG levels for all men and women <45years (97.5 percentiles 0.1 and 0.2IU/L, respectively) from the healthy population (n=795) having normal FSH and LH. Due to assay limitations, we suggest a common reference limit of <0.3IU/L. For the age group ≥45, the 97.5 percentiles in the healthy population were 0.5IU/L for men and 6.0IU/L for women. In all subjects from both the reference population and the cohort (n=732), hCG levels exceeding the reference limit could be fully explained by reduced gonadal function indicated by elevated LH and FSH levels. CONCLUSION: The Elecsys hCG+ß-assay should have lower reference limits than recommended by the manufacturer, with important implications for tumor follow-up. Elevated hCG is rare with intact gonadal function, both in a normal population and among survivors of testicular cancer, and should lead to further investigations when encountered in clinical practice.

The analytical specificity of human chorionic gonadotropin assays determined using WHO International Reference Reagents.

BACKGROUND: Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone with considerable molecular heterogeneity. There is uncertainty regarding which hCG variants are detected by different hCG assays. The analytical specificity of 8 hCG assays was investigated. METHODS: WHO International Reference Reagents for hCG, nicked hCG (hCGn), beta subunit (hCGbeta), nicked beta subunit (hCGbetan), and beta core fragment (hCGbetacf) were individually added to hCG-free human serum. Specimens were analyzed with 8 commercially available hCG assays. Equimolar detection of hCG variants was defined as a recovery of 90-110%. RESULTS: All assays detected hCG and hCGn with mean recoveries of 98.3 and 94.6%, respectively. Seven assays detected hCGbeta (mean recovery 103.8%) but with high variation, and equimolar detection was observed only in four. The mean recovery of hCGbetan was 85.5% but was highly variable with only two assays showing equimolar detection. With a mean recovery of 53.4%, two assays detected hCGbetacf and both underestimated it considerably. Information provided by the assay manufacturer regarding hCG variant analytical specificity was inadequate or unclear in 75% of the assays. CONCLUSIONS: hCG assays vary considerably in their ability to detect different hCG variants. Manufacturers of hCG assays should clearly indicate the hCG variant specificity of their reagent systems.

Cutoff value of human chorionic gonadotropin in relation to the number of methotrexate cycles in the successful treatment of ectopic pregnancy.

OBJECTIVE: To assign cutoff values for human chorionic gonadotropin (beta-hCG) in pretreatment and after one methotrexate (MTX) cycle and determine its correspondence to the number of MTX cycles in successfully treated ectopic pregnancy. DESIGN: Retrospective study. SETTING: Polish university hospital. PATIENT(S): 68 women with ectopic pregnancies who qualified for medical treatment. INTERVENTION(S): A single-dose of MTX (50 mg/m(2)) repeated every 7 days, plus laparoscopy in cases of tubal rupture or increased (>or=50% over 1 week) beta-hCG concentration. MAIN OUTCOME MEASURE(S): Resolution of serum beta-hCG without the necessity of laparoscopy. RESULT(S): Success rate was 78% (53 of 64 women). The medians of pretreatment beta-hCG levels in the groups treated successfully and unsuccessfully (943 vs. 3085 mIU/mL) and after the first dose of MTX (564 vs. 4049 mIU/mL) were statistically significantly different. The decrease in beta-hCG level after one MTX dose differed statistically significantly only in successfully treated women. The receiver operating characteristic (ROC) curve cutoff value in the success group indicated an initial beta-hCG level of 1790 and 1218 mIU/mL after one MTX cycle. The median of beta-hCG titer was not statistically different in patients requiring one or more treatment cycles. CONCLUSION(S): When the beta-hCG level is >1790 mIU/mL, the MTX treatment of ectopic pregnancy is at risk of failure. However, the initial beta-hCG titer is not a predictor of the number of MTX cycles that can guarantee a successful outcome.

Background hCG in non-pregnant individuals: need for more sensitive point-of-care and over-the-counter pregnancy tests.

BACKGROUND: Currently, 20 IU/L is the accepted sensitivity of over-the-counter (OTC) hCG pregnancy tests, and the only sensitivity permitted for point of care (POC) tests. These sensitivity limitations cause significant numbers of false-negative tests, which mislead women and cause them to avoid precautions needed to avoid fetotoxic drug or alcohol damaged pregnancies. A large clinical trial is needed examining background hCG (hCG levels in absence of pregnancy) and determining whether sensitivities can be improved to avoid false negative detection. METHODS: Daily urine samples were collected prospectively from 126 women over 1-7 non-conceptive menstrual cycles and from 68 women while achieving pregnancy. Additional urines were collected from women peri- and postmenopause. A total of 11,991 urines were collected and tested for total hCG and LH. RESULTS: The upper reference limit for background ranges in the field of clinical chemistry is the 97.5th percentile. Considering the menstrual cycle, the 97.5th percentile of background hCG results was 1.0 IU/L. Including cycles with early pregnancy losses raised this percentile to 1.1 IU/L. Considering also that 10% of tests involve peri-menopausal women, the 97.5th percentile of background results increased to 1.2 IU/L. CONCLUSIONS: The 97.5th percentile or background cut-off was 1.2 IU/L. As such a test with sensitivity 1.2-5 IU/L may be appropriate for urine pregnancy testing. An OTC or POC test with a sensitivity in this range will detect 98% of pregnancies close to the time of missing menses, helping prevent fetotoxic pregnancies or retarded babies.

Development of dual-enzyme-based simultaneous immunoassay for measurement of progesterone and human chorionic gonadotropin.

The development of a simultaneous multianalyte immunoassay for the detection of progesterone and human chorionic gonadotropin (hCG) in serum is described. In this simultaneous multianalyte assay, two different enzymes, viz. horse radish peroxidase (HRP) and alkaline phosphatase (ALP), were used as markers. To the simultaneous immobilized progesterone and hCG antibody microwells, 50 microL of different concentrations of combined standards or serum samples was added in duplicate and then 100 microL of combined conjugate reagent, composed of 17-alpha-OH-P-ALP and hCG-biotin was added to all the wells and incubated for 1h at 37 degrees C. After incubation, the contents of the wells were decanted and washed thoroughly with running tap water. After washing, 100 microL alkaline phosphatase substrate along with streptavidin-horseradish peroxidase was added to all the wells and incubated for 0.5 h at 37 degrees C. After incubation, the developed color was measured at 405 nm. The absorbency at this stage provides the result for the progesterone assay. The contents of the wells were decanted and washed. In the next step, 100 microL of tetramethylbenzidene/H2O2 reagent was added to all the wells. After 15 min of incubation, 100 microL of 0.5 M H2SO4 was added to all the wells and the color was read at 450 nm. The absorbency at this stage provides the result for the hCG assay. Sensitivity of the progesterone and hCG assays were 0.118 ng/ml and 0.124 IU/ml respectively. Intra- and inter assay percentage coefficients of variation ranged from 1.8 to 7.1 and 9.1 to 11.5 for progesterone and from 2.1 to 10.4 and 7.2 to 11.3 for hCG. There was good correlation between the discrete and the simultaneous assays. For progesterone assay, R2 was 0.99 and for hCG R2 was also 0.99. The developed dual assay for progesterone and hCG may be useful for the diagnosis of abnormal pregnancies such as miscarriages and ectopic pregnancies.

Establishment, value assignment, and characterization of new WHO reference reagents for six molecular forms of human chorionic gonadotropin.

BACKGROUND: The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) established a Working Group to investigate means of improving the comparability of immunoassays for human chorionic gonadotropin (hCG), which was selected as a prototype glycoprotein analyte. The Working Group identified development of unambiguous nomenclature and production of new highly purified International Reference Reagents calibrated in substance concentrations as its primary objectives. METHODS: Preparations of intact hCG, nicked hCG, hCG beta-subunit, nicked hCG beta-subunit, hCG alpha-subunit, and hCG beta-core fragment were purified from a crude urinary hCG preparation, ampouled, lyophilized, and assigned values in substance concentrations (mol/L). Value assignment and accelerated degradation studies were carried out in accordance with WHO protocols for International Reference Reagents. RESULTS: The ampouled standards were assigned final values based on the recovery of immunoreactive material after reconstitution. The degradation studies showed that the standards were highly stable. CONCLUSIONS: The nomenclature of hCG-related molecules and immunoassays has been adopted by the IFCC, and the standards prepared and characterized by the Working Group have been formally adopted by the WHO as the First International Reference Reagents for six hCG-related molecules. These developments will enable better understanding of what assays for hCG measure and should ultimately help to improve the clinical application of these assays.

Between-method variation in human chorionic gonadotropin test results.

BACKGROUND: Results on sera and calibrators vary 1.4- to 2.3-fold among commercial human chorionic gonadotropin (hCG) assays. The relative contributions of calibrators, standards, hCG charge isoforms, and major structural variants to this variation have not been quantified. METHODS: Purified hCG was separated by isoelectric focusing into four fractions with pI ranges of 3-4, 4-5, 5-6, and 6-7. These four fractions together with pure hCG, hyperglycosylated hCG, hCG beta-subunit (hCGb), nicked hCG, and hCGb core fragment (hCGbcf) were tested in nine commonly used commercial serum assays for hCG. The compositions of pure hCG preparations, standards, and commercial hCG preparations were determined by immunoassay. RESULTS: The three pure hCG preparations and the four hCG charge isoforms each showed parallel responses in the nine commercial hCG assays. Although wide variations were found in the detection of hCG structural variants by the nine assays (range for hyperglycosylated hCG, 468-1544 IU/L; for hCGb, 3187-5535 IU/L; for nicked hCG, 2736-4240 IU/L; and for hCGbcf, <2-130 IU/L), this did not correlate with the between-method variation observed in results for the three pure hCG preparations. Commercial preparations of hCG and calibrators showed great variation in their content of hCG structural variants (from 34% to 100% intact hCG). CONCLUSIONS: Intermethod differences in hCG results were not explained by changes in responses attributable to hCG charge isoforms or to hCG structural variants, but wide variation was observed in concentrations of hCG structural variants in calibrators and in detection of these structural variants. Differences in assay specificity and in composition of the calibrators are the most likely sources of between-method variation.

Measurement of inaccuracy and imprecision of HCG methods using dilutions of the WHO 4th IS-HCG standard and a pregnant patient's serum.

INTRODUCTION: Differences in human chorionic gonadotropin (hCG) results obtained by seven different methods were documented by analyzing dilutions of the WHO 4th International Standard (IS) and a pregnant patient's serum. MATERIALS AND METHODS: Biases of +30.9 to -37.5% and +36.8 to -36.1% from the target concentration were found for the WHO 4th IS and patient sample dilutions, respectively. RESULTS: Imprecision was calculated from replicate measurements of hCG on the different sample dilutions. Imprecision ranged from 1.0% to 18.9% and 1.1% to 5.3% for the WHO 4th IS and patient sample dilutions, respectively. Using a maximum allowable error of 12.5% for hCG measurements, we found that two instruments were so biased that their hCG measurements could not be interchanged with hCG values produced by any of the other systems. DISCUSSION: It is ideal to use only one hCG methodology for the serial monitoring of hCG; otherwise, hCG methods should be carefully chosen to minimize inter-method bias.

Preparation and characterization of new WHO reference reagents for human chorionic gonadotropin and metabolites.

BACKGROUND: The currently used standards for human chorionic gonadotropin (hCG) and its alpha and beta subunits (hCGalpha and hCGbeta) contain substantial amounts of contaminating variants of hCG and other impurities. Furthermore, some partially degraded forms of hCG and its subunits have become of potential clinical importance, e.g., "nicked" forms of hCG (hCGn) and hCGbeta (hCGbetan), which contain cuts in the peptide backbone between amino acids 44-45 or 47-48 in hCGbeta, and a fragment of hCGbeta (hCGbetacf) consisting of amino acids 6-40 and 55-92 bound together by disulfide bridges. The IFCC appointed a working group with the aim of preparing new standards for hCG and related substances to improve standardization of their immunoassays. METHODS: Large amounts of hCG and its subunits as well as of hCGn, hCGbetan, and hCGbetacf were prepared by previously developed purification methods in combination with hydrophobic interaction chromatography and reversed-phase HPLC. Each preparation was characterized on the basis of amino acid and sequence analyses, carbohydrate composition, and electrophoretic patterns. Immunoassays for relevant contaminating proteins were also performed. RESULTS: The major preparations were homogeneous and free of contaminating proteins. Concentrations of the final preparations were determined by amino acid analysis. CONCLUSIONS: Calibrated in substance concentrations (mol/L) based on amino acid analyses, these preparations will facilitate improved standardization of immunoassays for hCG and its metabolites. The six preparations have now been established by the WHO as new 1st Reference Reagents for immunoassays with the following codes: hCG 99/688, hCGbeta 99/650, hCGalpha 99/720, hCGn 99/642, hCGbetan 99/692, and hCGbetacf 99/708. In contrast to the 3rd International Standard (75/537), the clinically most important Reference Reagent for hCG (99/688) contains no hCGn and negligible amounts of free subunits.

'Curing' empty follicle syndrome.

We report a novel method of rescuing empty follicle syndrome (EFS) and provide evidence that it is a drug-related problem rather than a clinical dysfunction. In a preliminary study we established that in EFS the serum beta-human chorionic gonadotrophin (beta-HCG) concentrations 36 h after HCG administration never exceeded 10 mIU/ ml. beta-HCG concentrations were thus used to confirm EFS when oocytes were not retrieved from one ovary after controlled ovarian hyperstimulation. The procedure was suspended leaving intact all follicles in the second, ovary. After confirmation of EFS, a second HCG from a different batch was administered and 36 h later mature oocytes were retrieved from the intact ovary, suggesting a fault with the HCG previously administered. Three patients have been treated in this way. In the first case, four out of five mature eggs were fertilized after intracytoplasmic sperm injection (ICSI) resulting in the transfer of three top grade (grade 1) embryos. In the second case all seven mature oocytes fertilized after in-vitro fertilization (IVF) and three grade 1 embryos were transferred resulting in a twin pregnancy, now delivered. In the third case, five out of nine oocytes were fertilized after ICSI and one out of the three treated with high insemination concentration IVF fertilized, resulting in the transfer of three ICSI embryos.

Pathophysiological importance of various molecular forms of human choriogonadotropin.

Human chorionic gonadotropin (hCG), its subunits and fragments are widely used for diagnostic purposes. In addition to the diagnosis of pregnancy and pregnancy related disorders, hCG determinations are used for diagnosis of trophoblastic and recently also nontrophoblastic tumors. The use for diagnosis of nontrophoblastic tumors requires highly specific and ultrasensitive assays. With these, it is possible to measure the concentrations of both hCG, the free beta-subunits and the so called beta-core fragment in healthy subjects. Therefore it is important to establish reference values for these and also to be aware of the influence of physiological factors on the serum and urine concentrations. Improved standardization of the assay methods is also essential for these novel applications of hCG determinations to become useful.

Evaluation of different ovarian stimulation protocols for in vitro fertilization.

In this study we evaluated retrospectively the efficacy of five different ovarian stimulation protocols in an in vitro fertilization program, in which 512 women were involved. Ovulation was induced by the following protocols: group I (271 cycles): buserelin short protocol (1 mg/day, intranasally) with human menopausal gonadotropin/human chorionic gonadotropin (hMG/hCG); group II (45 cycles): buserelin (short protocol) with pure follicle stimulating hormone (p-FSH)/hMG/hCG; group III (24 cycles): clomiphene citrate (100 mg/day) with hMG/hCG; group IV (122 cycles): hMG (3 ampules/day) and hCG; group V (113 cycles): hMG/hCG and prednisolone (7.5 mg/day) after cycle programming with oral contraceptives. The lowest cancellation rate (3.3%) was noted in group I, followed by group V (9.7%). The highest number of follicles was observed in groups I (8.3 +/- 0.3; mean +/- SEM) and V (7.8 +/- 0.5). Also, more oocytes were retrieved in group I (7.2 +/- 0.3, p < 0.001), which were of good quality based on oocyte maturity as well as on the fertilization rate, and more embryos (4.5 +/- 0.3, p < 0.05) were developed. The correlation between estradiol and the total follicular volume on the day of hCG administration was also examined in the five groups. The best correlation (r = 0.6502) was found in group I, followed by group V (r = 0.5810). Significant differences were observed in the five groups with regard to the number of hMG ampules administered (p < 0.0001, F = 15.393) and the stimulation days (p < 0.0001, F = 35.32). Sixty-six clinical pregnancies were achieved: 37 (17.5%) in group I, seven (25.9%) in group II, one (10%) in group III, ten (15.6%) in group IV and 11 (15.5%) in group V (differences were not statistically significant). In conclusion, all five protocols were satisfactory in ovarian stimulation for in vitro fertilization, and gonadotropin releasing hormone (GnRH) analogs seemed to be more advantageous by reducing the cancellation rate, enhancing the number of oocytes retrieved and embryos developed and by improving the pregnancy rates.

[The human chorionic gonadotrophin reference standard (Control 941) of the National Institute of Health Sciences].

Raw human chorionic gonadotrophin material was examined for preparation of the "Human Chorionic Gonadotrophin Reference Standard (Control 941)". The candidate material was assayed against the 3rd International Standard by the rat ovarian weight method. The potency of the new standard was defined as 1180 international units per ampoule as the result of 18 assays in four collaborative laboratories.

The heterogeneity of human chorionic gonadotropin (hCG). II. Characteristics and origins of nicks in hCG reference standards.

hCG, the hormone produced by the trophoblast throughout pregnancy, has peptide bond cleavages, or nicks, in the beta-subunit. We sought to compare the nature of these nicks in standard reference preparations of hCG, to determine the enzymes that may be responsible for generating the peptide bond cleavages, and to devise means of separating nicked from intact hormone. The standard reference preparations of hCG, which are purified from a commercial product made from large pools of pregnancy urine, were found to have varying concentrations of nicked hormone. The preceding report showed that 11 of 13 hCG preparations isolated from individual pregnancy urine samples were nicked at the beta 47-48 bond, with 2 of 13 having a second nick at beta 44-45. As shown here, all of the hCG reference standards are nicked to similar extents at both the beta 47-48 bond and the beta 44-45 bond. The percentage of peptide bond nicking in the various hCG standard preparations ranged from 10-20% and appeared higher in the more recent preparations. We showed that human leukocyte elastase is capable of specifically cleaving the beta 44-45 bond, and in extended digests it can also cleave the beta 48-49 and beta 51-52 peptide bonds. Thus, human leukocyte elastase may be the origin of some of these cleavages in the individual samples and the reference standards. Furthermore, we report that a monoclonal antibody directed to hCG alpha-beta dimer binds preferentially to nonnicked hCG and much less to nicked hCG.

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)

Quantitation of whole molecule human chorionic gonadotropin and free beta-human chorionic gonadotropin subunit in the Abbott IMx analyser.

An assay for the quantitation of the serum levels of free and whole molecule-associated beta-subunit of human chorionic gonadotropin on the Abbott IMx analyzer is described. The assay detects human beta-chorionic gonadotropin with a sensitivity of approximately 0.5 IU/l while showing no cross-reactivity with follitropin (1000 IU/l) or thyrotropin (2.0 IU/l), and 0.02% cross-reactivity with lutropin (1000 IU/l). Haemoglobin (7.50 milligrams), bilirubin (0.50 milligrams), and triacylglycerols (10.6 milligrams) did not interfere with the assay. Pooled within-run, between-run, and total assay CVs were less than or equal to 5.2%, and less than or equal to 2.7%, and less than or equal to 6.3%, respectively. Values obtained with this assay correlated well (r = 0.98, n = 228) with those values obtained using the Hybritech TandemR-R HCG (Total Beta-HCG) IRMA. The normal range of the assay was found to be less than or equal to 5 IU/l (n = 311). The assay protocol provides results for up to 23 serum samples in approximately 47.5 minutes with the ability to report the human beta-chorionic gonadotropin concentrations of 5 specimens in approximately 15.5 minutes. We conclude that this is an acceptable assay for monitoring human beta-chorionic gonadotropin levels associated with normal pregnancy, reproductive pathology, and reproductive technology such as in vitro fertilization or embryo transfer.