In utero diethylstilbestrol (DES) exposure alters Hox gene expression in the developing müllerian system.

Diethylstilbestrol (DES) was widely used to treat pregnant women through 1971. The reproductive tracts of their female offspring exposed to DES in utero are characterized by anatomic abnormalities. Here we show that DES administered to mice in utero produces changes in the expression pattern of several Hox genes that are involved in patterning of the reproductive tract. DES produces posterior shifts in Hox gene expression and homeotic anterior transformations of the reproductive tract. In human uterine or cervical cell cultures, DES induces HOXA9 or HOXA10 gene expression, respectively, to levels approximately twofold that induced by estradiol. The DES-induced expression is not inhibited by cyclohexamide. Estrogens are novel morphogens that directly regulate the expression pattern of posterior Hox genes in a manner analogous to retinoic acid regulation of anterior Hox genes. Alterations in HOX gene expression are a molecular mechanism by which DES affects reproductive tract development. Changes in Hox gene expression are a potential marker for the effects of in utero drug use that may become apparent only at late stages of development.

Development and characterization of antibodies to a nicked and hyperglycosylated form of hCG from a choriocarcinoma patient: generation of antibodies that differentiate between pregnancy hCG and choriocarcinoma hCG.

Human chorionic gonadotropin (hCG) exists in blood and urine as a variety of isoforms one of which contains peptide bond cleavages within its beta-subunit loop 2 and is referred to as nicked hCG (hCGn). This hCG isoform appears to be more prevalent in the urine of patients with certain malignancies and possibly in some disorders of pregnancy. Until now, only indirect immunoassays could be used to quantify hCGn. We report the development of two monoclonal antibodies (MAbs) to a form of hCGn isolated from a choriocarcinoma patient. This hCG isoform was not only 100% nicked, but also contained 100% tetrasaccharide-core O-linked carbohydrate moieties in its beta COOH-terminal region. Two-site immunometric assays have been developed using these new antibodies, B151 and B152. The former exhibits good specificity for hCGn independent of the source of the hCGn, the form excreted by choriocarcinoma patients or the form of hCGn from normal pregnancies. The latter antibody, B152, is sensitive to the carbohydrate moieties and possibly other differences in hCG isoforms, but is not for nicking of the beta-subunit. These two immunometric assays provide potential novel diagnostic tools for direct measurement of hCG isoforms which could not be accurately quantified earlier before development of the assays using these newly generated antibodies.

HOX gene expression is altered in the endometrium of women with endometriosis.

HOXA10 and HOXA11 are homeobox genes that function as transcription factors essential to embryonic development. We have recently described a role for each of these two genes in regulating endometrial development in the adult during the course of a menstrual cycle. Both Hoxa10 and Hoxa11 are essential for implantation in the mouse and appear to play a similar role in women. To investigate the role of HOX genes in the endometrium of women with endometriosis, quantitative Northern blot analysis was performed on the endometrium of 40 normal cycling controls and 40 patients with documented endometriosis. Patients with endometriosis failed to show the expected mid-luteal rise in HOX gene expression as demonstrated in the controls. Aberrant HOX gene expression suggests that altered development of the endometrium at the molecular level may contribute to the aetiology of infertility in patients with endometriosis.

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.

The stability of hCG and free beta-subunit in serum samples.

Human chorionic gonadotropin (hCG) free beta-subunit measurement is used as a screening test for Down syndrome pregnancies. Use, shipping, and storage conditions have, however, to some extent been limited by a stability problem; the swamping of serum free beta-subunit levels by new molecules coming from the dissociation of hCG. We examined the stability of free beta-subunit levels in six fresh serum samples from the first trimester of pregnancy. The mean hCG level in the fresh serum samples was 3710 +/- 886 micrograms/l; this included a 3.0 +/- 0.39 per cent nicked hCG component. The mean free beta-subunit level was 0.27 +/- 0.04 per cent and the nicked free beta-subunit level was 0.10 +/- 0.04 per cent of the hCG concentration. Samples were incubated for 0, 0.5, 1, 2, and 4 weeks at 21 degrees C, with no additives. After half a week, the free beta-subunit level rose to 137 +/- 17 per cent, and after 4 weeks to 360 +/- 53 per cent of the starting level (ANOVA P < or = 0.05). Parallel, but greater, increases were observed in nicked hCG and nicked free beta-subunit levels. The experiment was repeated with the addition of penicillin-streptomycin-fungizone to fresh serum. After half a week, the free beta-subunit level increased to only 101 +/- 3.0 per cent (t-test, with/without additive, P < or = 0.05), and after 4 weeks to only 136 +/- 7.8 per cent (P < or = 0.05) of the starting level. At 4 weeks, nicked hCG production was reduced from 499 +/- 83 to 158 +/- 15 per cent of the starting level. We infer that nicking of hCG and dissociation of unstable nicked hCG may be pathways supplementing free beta-subunit in serum samples. We further infer that hCG nicking activity comes from microbes, and that free beta-subunit levels can be stabilized for shipping and longer storage by antibiotic/antimycotic additives.

Nicked free beta-subunit of human chorionic gonadotropin: a potential new marker for Down syndrome screening.

OBJECTIVE: Our purpose was to investigate maternal serum levels of nicked free beta-subunit in normal and Down syndrome pregnancies. STUDY DESIGN: Serum specimens were obtained from 64 karyotypically normal and 6 Down syndrome pregnancies before amniocentesis. Two immunoenzymometric assays for the beta- subunit of human chorionic gonadotropin were used to determine the level of free beta-subunits with peptide linkage breaks ("nicks") between residues 43 to 48. RESULTS: The mean level of nicked free beta-subunit in Down syndrome was 4.76 multiples of the median, which was significantly elevated compared with normal controls (1.07 multiples of the median, p<0.05). In 5 of 6 cases levels were > or = 2 multiples of the median, with a range of 1.96 to 15.43 multiples of the median, which was 2- to 3-fold higher than the regular free beta-subunit assay (mean level 1.53 multiples of the median, range 0.70 to 3.10 multiples of the median). In one case no nicked free-beta subunits were detectable. CONCLUSION: Our preliminary data indicate that nicked free beta-subunit of human chorionic gonadotropin might be a sensitive marker for Down syndrome screening.

Gonadotropin beta-subunit nicking enzyme (GBNE), a potential marker of early malignancies.

GBNE is an arginine-specific metalloprotease that cleaves human chorionic gonadotropin free beta-subunit in pregnancy serum. We tested GBNE activity in 539 serum samples, from individuals that were healthy, with benign diseases, or with a broad mixture of cancers. The mean GBNE activity in 130 samples from healthy individuals, 151 from those with benign disease, and 258 from cancer patients was 5.4 +/- 0.32, 5.8 +/- 0.20 and 16 +/- 1.2 units, respectively. ROC analysis indicated 86% discrimination between control samples and cancer. A cut-off of 12 units was selected. This was equaled or exceeded by 3.1% of samples from healthy individuals, 3.9% from those with benign disease, and 57% from those with cancer. There was no significant difference in detection of the different cancer primaries, breast (sensitivity 56%), gastrointestinal (53%), genitourinary (62%), gynecological (58%) and lung (55%) cancers. Sensitivity was highest for early stage, and lowest for advanced malignancies, for all cancer primaries. Taken together, sensitivity was 70, 44, 43 and 24%, for stages I, II, III and IV, respectively. GBNE is suggested to be a nonorgan site-restricted tumor marker with high sensitivity for early stage cancers.

Immunological recognition and clinical significance of nicked human chorionic gonadotropin in testicular cancer.

We studied the physical properties, immunological recognition, and clinical significance of nicked human chorionic gonadotropin (hCGn) in testicular cancer. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, the beta-subunit of hCGn (hCG beta n) dissociated into two peptides, and, by reversed-phase chromatography, hCG beta n was found to be less hydrophobic than hCG beta. Immunologically, hCGn lacked two epitopes specific for holo-hCG (c1, c2), whereas at least five hCG beta epitopes (beta 1-beta 5) were preserved, and, as a result, recognition of hCGn by different hCG assays varied widely. In 309 sera and 88 urine samples from patients with seminomatous or nonseminomatous testicular cancer, hCG-only, hCG+hCGn, and hCG+hCGn+hCG beta+hCG beta n+hCG beta core-fragment assays gave parallel results. hCGn was more abundant in urine than in serum samples. In conclusion, hCGn lacks two conformationally dependent epitopes of hCG, causing a change in hydrophobicity and explaining its failure to react in certain holo-hCG assays. Recognition of hCGn, however, does not seem to be crucial in the routine use of serum hCG as a tumor marker in patients with testicular cancer.

Human chorionic gonadotropin beta-subunit nicking enzymes in pregnancy and cancer patient serum.

hCG is a dimer composed of an alpha- and a beta-subunit, joined noncovalently. In addition to the hCG dimer, uncombined alpha- and beta-subunits (free beta) and nicked hCG and free beta molecules (cleaved at 44-45 or 47-48) can be detected in the circulation. Of these circulating molecules, only the intact hCG dimer fully expresses biological activity. The pathways that dissociate, nick, and degrade hCG and beta-subunit molecules in pregnancy are unknown and could have a major role in regulating hormone levels. Immunoassays for intact (nonnicked) hCG and intact (beta-subunit (with < 1% detection of nicked molecules) and a subtractive immunoassay system for measuring nicked hCG levels have been described previously. A multiantibody scavenger assay is described here for measuring nicked beta-subunit levels (< 6% detection of intact beta-subunit). In this report we use these four assays to assess conversion of intact hCG or beta-subunit to nicked forms over time (nicking enzyme activities) in control (healthy nonpregnant), pregnant, and cancer patient serum samples. Pools of pregnancy and control sera were supplemented with intact hCG and its dissociated beta-subunit and incubated at 37 C. Intact and nicked molecule measurements were made between 0-48 h. In two different pools of control sera, no loss of intact hCG or intact beta-subunit and no significant gain in nicked hCG or nicked beta-subunit were detected over 48 h. This indicated a lack of nicking enzyme activity in control serum. In two different pools of first trimester pregnancy sera, we found no obvious loss of intact hCG or gain of nicked hCG levels over 48 h. However, we found 70% and 62% losses (pools 1 and 2) of intact beta-subunit and 51% and 39% gains of nicked beta-subunit over the same time period. We inferred that an uncombined or free beta-subunit-modifying activity was present in pregnancy serum. We repeated the pregnancy serum experiment with six different concentrations of beta-subunit (0.62-29 mg/L). A linear relationship, percent nicking against time, existed for the six concentrations for up to 6 h at 37 C (r = 0.97); after that, the rate of nicking declined. A plot of rate against concentration against revealed a classical Michaelis-Menten enzyme relationship (logarithmic regression, r = 0.96). The pregnancy serum beta-subunit nicking activity was partially purified by gel filtration. A single peak of activity emerged, eluting between the 150,000-443,000 mol wt standards.(ABSTRACT TRUNCATED AT 400 WORDS)

[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.

Urine hCG beta-subunit core fragment, a sensitive test for ectopic pregnancy.

hCG is a glycoprotein hormone composed of 2 dissimilar subunits, alpha and beta, joined non-covalently. hCG and its free beta-subunit are the principal hCG beta immunoreactivities in pregnancy serum samples, and the same plus beta-core fragment (beta-subunit residues 6-40 disulfide-linked to residues 55-92) in urine samples. Ectopic or tubal pregnancy is difficult to diagnose in emergency rooms. With the objective of finding better hCG-related assays for differentiating tubal and normal pregnancies, we tested 2 hCG, 1 free beta-subunit and 2 beta-core fragment immunoassays. Twelve urine samples were collected in the emergency room from women later shown by surgery to have tubal pregnancy. All were 38 to 80 days since last period. A further 36 urine samples were collected from the same period from those with normal intrauterine pregnancies. Using the 2 hCG assays the median level in tubal pregnancy samples was 1/38th and 1/48th of normal pregnancy concentrations. With the free beta-subunit assay tubal pregnancy levels were 1/28th of normal levels. Using 2 beta-core fragment assays (Ciba-Triton UGP kit and B204-FBT11 scavenger test), however, tubal levels were most different from intrauterine pregnancy, 1/149th and 1/800th of normal levels. A cut-off level of 100 micrograms/L was considered for the B204-FBT11 beta-core fragment test, at which a predictive value of > 98% was suggested for ectopic pregnancy. In an additional patient, levels were measured 15 days prior to the diagnosis of tubal pregnancy. At this time, results from the 2 hCG tests were 1/97th and 1/126th, from the free beta-subunit test was 1/8th and the 2 beta-core assays were 1/413th and 1/240th of median normal intrauterine pregnancy levels. While hCG levels are reduced in tubal pregnancy, beta-core fragment are reduced much further. beta-core fragment measurements may offer a major improvement over hCG in diagnosing tubal pregnancy in the Emergency Room, and in screening for this life threatening disease.

Effect of peptide nicking in the human chorionic gonadotropin beta-subunit on stimulation of recombinant human thyroid-stimulating hormone receptors.

It is now generally accepted that human chorionic gonadotropin (hCG) has thyroid-stimulating activity. Heterologous forms of the hCG molecule occur in the purified preparations extracted from urine of pregnant women and patients with trophoblastic diseases. This work was undertaken to determine the effect of peptide nicking in the hCG-beta subunit on its thyrotropic potency. Using Chinese hamster ovary cells expressing functional human thyroid-stimulating hormone (TSH) receptors, we examined the effect of nicked hCG on cycliC AMP (cAMP) production and receptor binding. The effect of human leukocyte elastase (hLE), a nicking enzyme, on standard hCG also was examined in the cAMP assay and on receptor binding. We studied five hCG preparations extracted from the urine of normal pregnancy (CR-127 and P8) and trophoblastic diseases (C2, C5 and M4). Two preparations (C2, 96% nicked and M4, 100% nicked in the beta 44-49 region) showed about a 1.5-fold potency of standard hCG CR-127, which is also 20% nicked in the same region. Non-nicked hCG (P8) had the weakest potency among all of the samples tested. Treatment of standard hCG with hLE increased the cAMP response about two-fold. Dose-dependent displacement of bovine [125I]TSH by standard hCG and hLE increased the cAMP response about two-fold. Dose-dependent displacement of bovine [125I]TSH by standard hCG and hLE-digested hCG was observed and was almost identical. We have confirmed the increased in vitro thyrotropic activity of hCG nicked in the beta-intercysteine loop on recombinant human TSH receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Selecting human chorionic gonadotropin immunoassays: consideration of cross-reacting molecules in first-trimester pregnancy serum and urine.

OBJECTIVE: We investigated the variation in human chorionic gonadotropin results found with different commercial kits. Levels of human chorionic gonadotropin and related molecules were determined in pregnancy serum and urine and compared with the specificities of different laboratory, office, and home test kits. STUDY DESIGN: Total human chorionic gonadotropin (nicked+nonnicked), nonnicked human chorionic gonadotropin, free beta subunit, and beta core fragment were measured in 242 serum samples and 125 urine samples from early pregnancies. RESULTS: In serum, in the 2 weeks after the missed period when most pregnancy tests are performed, median levels of total, nonnicked, and beta human chorionic gonadotropin (total+free beta+beta core) were similar (< or = 12% difference). Individual values, however, varied significantly. For nonnicked human chorionic gonadotropin, values ranged from 41% to 145% and for beta from 101% to 145% of the total human chorionic gonadotropin level. In urine individual nonnicked values varied from < 1% to 148% and beta values from 102% to 547% of the total human chorionic gonadotropin level. A survey of 29 kits revealed that 10 were types detecting total human chorionic gonadotropin, five detecting nonnicked only, and 14 were beta assays. CONCLUSIONS: Results from total, nonnicked, and beta human chorionic gonadotropin kits are not necessarily interconvertible. Individual variations in levels of nicked human chorionic gonadotropin, free beta and beta core, and differences in their recognition by immunoassays causes discordant results.

Characterisation of UGP and its relationship with beta-core fragment.

Urinary gonadotrophin peptide (UGP) was originally identified by immunoassay in the urine of patients with various types of cancer and by immunohistochemistry in human cancers of various histological types. Extracts of normal adult male urine also contained UGP by immunoassay. Purified UGP from different starting material was subjected to high pressure liquid chromatography (HPLC) prior to defining amino acid sequences. Chromatographed UGP after HPLC showed three distinct fractions. The N-terminal sequence of peptide 2 was completely homologous with the beta-core fragment of human chorionic gonadotrophin (hCG) and this was found associated with two smaller peptides. The N-terminal sequence of peptide 1 has not been described previously whilst the N-terminus of peptide 3 that was sequenced showed complete homology with the N-terminal sequence of eosinophil derived neurotoxin and non-secretory ribonuclease. The monoclonal antibodies 2C2 and 6D3 only bind beta core-fragment (peptide 2) whilst the polyclonal (rabbit) antibody AK12 could bind all three peptides. The radioimmunoassay system using AK12 could be inhibited by all three peptides and the immunoradiometric assay although based on a capture antibody (2C2) that only bound peptide 2, had the potential to measure all three peptides (when bound together as UGP) at the second step when 125I-AK12 was introduced as the detector. A specific radioimmunoassay for peptide 3 was generated using 125I-peptide 3 and the AK12 antibody. Beta core-fragment on iso-electric focusing was found to have a pI > 9.5, peptide 3 showed two bands at pI = 3.5 and 3.8 whilst insufficient purified peptide 1 was available to determine its iso-electric point. Bioassay studies on UGP showed that any biological activity could be attributed to trace contamination with hCG.

The deactivation of hCG by nicking and dissociation.

Human chorionic gonadotropin (hCG) is composed of an alpha- and a beta-subunit, joined noncovalently. A proportion of hCG molecules in pregnancy serum and urine samples have nicks or a missing peptide linkage between either beta-subunit residues 44 and 45 or beta-subunit residues 47 and 48. These nicks ablate the steroidogenic activity of hCG. We examined the source of nicking, and the occurrence and stability of nicked hCG molecules produced during pregnancy. We investigated the source of nicking. Standard hCG was added to three samples of whole blood, and incubated 18 h at 37 C. No change in extent of nicking was detected. However, nicking of hCG beta-subunit was detected by gel electrophoresis (bands at M(r) = 17,000 and M(r) = 22,000, corresponding to the peptides beta 1-47(44) and beta 48(45)-145, respectively) in culture fluids from first trimester placental explants and from JAr malignant trophoblast cells. We inferred that nicking occurs before or immediately upon secretion by trophoblast tissue. We examined the occurrence of nicking. Levels of total hCG (nicked+nonnicked) and intact hCG (nonnicked) were determined in 233 serum and 168 urine samples from 4-40 weeks of pregnancy. From the two measurements the extent of nicking was estimated. A linear relationship was indicated between advancing weeks of gestation and increasing extent of nicking (regression analysis, months vs. percent nicked, 95% correlation). Minimum nicking was observed in serum from the first 2 months of pregnancy (mean = 9% of hCG molecules), increased nicking in the months after, and maximum nicking in samples from the last 2 months of pregnancy (mean = 21% of hCG molecules, t test first 2 months vs. last 2 months, P < 0.00005). Similar results were observed with urine samples (first 2 months mean = 9%, last 2 months = 27%, t test P < 0.00005). We concluded that nicking is more prevalent after the hCG peak (after 2 months of pregnancy). Finally, we examined the stabilities of nicked and intact hCG molecules. Standard hCG (batch CR127, 20% nicked) and hCG preparation C5 (100% nicked) were incubated for varying times in whole blood. C5 hCG dissociated rapidly into free alpha- and beta-subunits (dissociation half-life 22 +/- 5.2 h), over 30 times faster than standard hCG (dissociation half-life 700 h).(ABSTRACT TRUNCATED AT 400 WORDS)

Discordant results in human chorionic gonadotropin assays.

Discordance has been reported in human chorionic gonadotropin (hCG) concentrations measured by different immunoassay kits. We examined the results for 40 serum samples assayed with 10 different hCG immunoassay kits. Results varied considerably. Individual sample results varied by as much as 58-fold. Average results for different kits varied by as much as 1.4-fold for pregnancy (20 samples) and 2.2-fold for trophoblast disease (20 samples) serum. We investigated the causes of this discordance. hCG or hCG beta are general names for mixtures of hCG, hCG alpha, or hCG beta immunoreactive molecules in serum. These mixtures include regular hCG, nicked hCG (missing peptide linkages at beta 44-45 or beta 47-48), carbohydrate variants of hCG, hCG missing the beta-subunit C-terminal segment, free beta-subunit, beta-core fragment, and free alpha-subunit. We prepared standards for each of these major variants and measured their reactivities in the 10 hCG immunoassay kits. Free beta-subunit reactivity varied from nonrecognition (anti-beta:anti-alpha type kits; Hybritech Tandem-R and others) to overrecognition (one kit had five-fold greater affinity for free beta than for hCG). Kits with antibodies to beta-subunit C-terminal segment (Organon NML and others) failed to recognize hCG missing this segment, a component of serum hCG in trophoblast disease. Kits with anti-hCG antibodies (Serono MAIA-clone and others) had minimal recognition of nicked hCG (12%), a component of all serum hCG samples, and consistently gave the lowest values with all serum samples. We conclude that differences in recognition of nicked hCG, free beta, and these other hCG variants cause discordance in hCG immunoassay results.

Polypeptide nicks cause erroneous results in assays of human chorionic gonadotropin free beta-subunit.

Pregnancy and trophoblastic disease testing laboratories measure human chorionic gonadotropin (hCG; human choriogonadotropin) free beta-subunit to screen for Down syndrome and to diagnose persistent trophoblastic disease (invasive mole and choriocarcinoma). The results from various laboratories, however, vary widely for similar groups of patients. Average concentrations of free beta-subunit reported for persistent trophoblastic disease, for instance, range from 2.6% to 37% of total hCG. On hCG and its free beta-subunit, peptide bonds can be missing between beta-subunit residues 44 and 45 or between residues 47 and 48 (nicked molecules). To explore the possibility that the disparity in the reported concentrations of free beta-subunit was due to differences in recognition of nicked molecules by different antibodies, we generated 0% and 100% nicked beta-subunit standards and investigated their recognition in three separate immunoassays of free beta-subunit. The immunoassay with antibody 1E5 did not recognize nicked beta-subunit (4% cross-reactivity with nicked beta-subunit) and thus detected only intact beta-subunit. The immunoassay with antibody FBT11 gave similar results with nicked and nonnicked thus standards (96% cross-reactivity with nicked beta-subunit), as did the assay with antibody B204 (73% cross-reactivity with nicked beta-subunit). These two immunoassays thus measured total (nicked + nonnicked) beta-subunit. We used the three immunoassays to examine sera from normal pregnancy, Down syndrome pregnancy, hydatidiform mole, and persistent trophoblastic disease, all of which contain nicked and nonnicked beta-subunit molecules. The results obtained resembled the studies with nicked beta-subunit standards. The results from the FBT11 and B204 assays (total beta-subunit) were highest, results from the 1E5 assay (intact molecules only) being as much as 10 times lower. We conclude that nicks in beta-subunit and the extent of recognition of nicked molecules by different antibodies affect the concentrations reported for free beta-subunit.

The biological and clinical significance of nicks in human chorionic gonadotropin and its free beta-subunit.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone composed of two dissimilar subunits, alpha and beta. Nicks or missing peptide linkages have been found in the beta 44-52 region of the beta-subunit of hCG, whether from pregnancy or trophoblast disease. This article reviews recent reports about the location of nicks in hCG, their origin and occurrence, their effects on the steroidogenic and receptor-binding activities of hCG, and on the immunological activities of hCG and its free beta-subunit. Taken together, the reports show: (1) nicks occur primarily between beta 47 and beta 48, and to a lesser extent between beta 44 and beta 45; (2) the extent of nicking in hCG samples varies widely, from undetectable to 100 percent of molecules; (3) nicks greatly reduce the steroidogenic activity of hCG in vitro (nicked molecules have less than 20 percent of the activity of the intact hormone); (4) nicks may occur at the trophoblast-myometrial interface or in the circulation by the action of human leucocyte elastase or similar leucocytic protease; (5) hCG testing kits using dimer-specific antibodies may not detect nicked molecules and may give different results from those using other antibodies; (6) hCG international reference preparations and the CR series of hCG standards are variably nicked (10 percent to 20 percent), complicating the problem of discordant hCG results in nick-sensitive assays; (7) results from commonly used immunoassays for measurement of the hCG free beta-subunit vary by as much as tenfold because some of the antibodies employed do not detect nick free beta-subunit.

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). 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.