1. Early Pregnancy Human Chorionic Gonadotropin (HCG) Isoforms

Early pregnancy human chorionic gonadotropin (hCG) isoformsmeasured by an immunometric assay for choriocarcinoma-like hCG

G Kovalevskaya1, S Birken2, T Kakuma3 and J F OConnor1,4

1Irving Center for Clinical Research, Columbia University College of Physicians and Surgeons, 630 W 168th St., New York 10032, New York, USA2Department of Medicine, Columbia University College of Physicians and Surgeons, 630 W 168th St., New York 10032, New York, USA3New York Hospital-Cornell Medical Center, White Plains, New York 10605, New York, USA4Department of Pathology, Columbia University College of Physicians and Surgeons, 630 W 168th St., New York 10032, New York, USA

(Requests for offprints should be addressed to J F OConnor, Department of Pathology and Irving Center for Clinical Research, Columbia University College ofPhysicians and Surgeons, 630 W 168th St., PH10305, New York 10032, New York, USA)

Abstract

Human chorionic gonadotropin (hCG) exhibits molecularheterogeneity in both its protein and carbohydratemoieties. This communication describes changes in hCGisoforms detected directly in clinical samples. These iso-forms, quantified in blood or urine specimens, show aprogression of change throughout normal pregnancy. Earlypregnancy produces a type of hCG that resembles, interms of immunoreactivity, a major form of hCG excretedin choriocarcinoma. The isoforms predominate for the first56 weeks of gestation and then diminish, being replacedwith the hCG isoforms which predominate throughoutthe remainder of pregnancy. The alteration in hCGisoform content occurs in both blood and urine. Theprogression of isoforms is best delineated by calculating the

change in the ratio of the two forms, as many hCG assayseither do not detect or fail to discriminate among theseisoforms. An analogous pattern of hCG isoforms wasobserved in patients with in vitro fertilization pregnancies.hCG isolated from the pituitary displayed binding charac-teristics similar to those of the hCG derived from normalpregnancy urine. The early pregnancy hCG isoformsappear to have a diVerential expression in normal preg-nancy as opposed to pregnancies which will not carry toterm, suggesting that a determination of the relativebalance of hCG isoforms may have diagnostic applicationin predicting pregnancy outcome.Journal of Endocrinology (1999) 161, 99106

Introduction

Human chorionic gonadotropin (hCG) is produced by theplacental trophoblast of normal pregnancy, and in ges-tational trophoblastic disease (hydatidiform mole and chorio-carcinoma) (Hussa 1987). It is also produced in much smallerquantities by the pituitary (Birken et al. 1996) in both pre-and postmenopausal women and in men (Odell et al. 1990)and also in many non-gestational malignant tumors (Hussa1987).

Structural variations in both the protein and carbo-hydrate portions of the hCG molecule have been wellestablished. They may occur as a concomitant of its origin(Wide & Hobson 1987, Birken et al. 1996), or metabolictransformation (Nisula et al. 1989). The most prominentdiVerences documented to date are between the hCGproduced by choriocarcinoma and that of mid-first tri-mester normal pregnancy, with choriocarcinoma-derivedhCG having a generally higher content of more highlybranched oligosaccharides (Cole 1987, Elliott et al. 1997).

Even in normal pregnancy, multiple isoforms of hCGexist and their relative amounts vary as pregnancy

progresses (Wide & Hobson 1987, Skarulis et al. 1992,Diaz-Cueto et al. 1994, 1996, Wide et al. 1994). Many ofthese determinations are based on isoelectric focusing(IEF) separation of the isoforms. This technique separatesmolecules based on their net charge, which in the case ofglycoprotein hormones only reflects the degree of terminalcarbohydrate sialylation (or less commonly, sulfation), andnot major structural diVerences (Ulloa-Aguirre et al. 1990,Berger et al. 1993, Diaz-Cueto et al. 1994). However,these studies of microheterogeneity (by microhetero-geneity, we refer to the permutations displayed by thecarbohydrate groups) have not proven to have clinicalutility, since they are both too expensive and laborintensive to perform routinely. Other studies have dem-onstrated, by analysis of carbohydrate structure and sugarcontent, diVerences in the carbohydrate moiety of normalpregnancy hCG as it progresses to term (Skarulis et al.1992, Nemansky et al. 1998).

This report describes the observation of the progressivechanges of hCG isoforms throughout pregnancy by directassay of clinical samples of urine or serum, using acombination of two IRMAs. One of these, a new IRMA,

99

Journal of Endocrinology (1999) 161, 9910600220795/99/01610099 ? 1999 Society for Endocrinology Printed in Great Britain

Online version via http://www.endocrinology.org

is unique in that it is sensitive to changes in carbohydratestructure and can measure very early isoforms of hCG(OConnor et al. 1998), whereas the second assay, acommonly used format, detects isoforms of hCG whichpredominate later in pregnancy. This observation takes onan added significance because of reports that certainpregnancy disorders (e.g. Down syndrome, pre-eclampsia,early pregnancy loss) are characterized by abnormal hCGlevels (Bogart et al. 1987, Heinonen et al. 1996, Hurleyet al. 1996, OLeary et al. 1996, Onderoglu & Kabukcu1997, Haddow et al. 1998). We have evidence thatpregnancies which will not carry to term have an easilyrecognized diVerence in production of hCG isoforms(OConnor et al. 1998). Thus, hCG isoform productionand distribution in pregnancy may prove to have adiagnostic role in the evaluation of pregnancy status. Themethod of direct IRMA analysis of early pregnancyisoforms described in this report is suitable for large scalestudies using either serum or urine.

Materials and Methods

Hormones

The non-nicked hCG isolated from the CR127 prep-aration of hCG was used as a standard in both assays(Birken et al. 1993). The pituitary hCG was isolated asdescribed (Birken et al. 1996). C5, a 100% nicked hCGhaving extra sugars on both N- and O-linked carbo-hydrate moieties, purified from the urine of a chorio-carcinoma patient (Elliott et al. 1997), was supplied by DrL Cole (Yale University School of Medicine). Althoughthe C5 immunogen used in the development of B152antibody was 100% nicked hCG isoform (i.e. had cleav-ages in the peptide backbone of loop 2 of the subunit)the antibody did not discriminate nicked from non-nickedhCG (OConnor et al. 1998).

The same serial dilutions of non-nicked hCG, pituitaryhCG and C5 were used for binding characterization inhCG assays. Hormone concentrations of initial stockstandard solutions were determined by amino acid analysis.

IRMAs

The methodology used in the construction and validationof the B109B108* assay has been fully described else-where (OConnor et al. 1988); the first antibody is thecapture antibody, the second, with an asterisk, is aradioiodinated detection antibody. The B152B207* assayhas also been characterized (OConnor et al. 1998). Bothassays were performed with a slight modification of thepublished procedure: the capture antibody was adsorbedonto the wells of microtiter plates (Immulon IV,Dynatech, Chantilly, VA, USA) by incubating a 5 g/mlsolution (B109B108* assay) or 25 g/ml solution (B152

B207* assay) in coating buVer (02 M bicarbonate, pH 95)overnight at 4 )C. The coating antibody solution wasaspirated, the plates washed (wash solution: 09% NaCl,005% Tween 20) and blocked with a 1% solution of BSAin PBS with 01% sodium azide. Following incubationwith the BSA solution (minimum 3 h at room tempera-ture), the blocking solution was removed, the wells againwashed with wash solution and 200 l/well of the appro-priate hCG standards were added in phosphate buVer B(PBS with 01% bovine gamma globulin and 01% sodiumazide). After overnight incubation at 4 )C, the plates wereagain aspirated and washed. The 200 l of 125I-labeledantibody (50 000100 000 c.p.m.) were added to thewells, which were again incubated for 24 h at 4 )C. Thetracer was aspirated, the plates washed with wash solution,the individual wells placed in glass tubes and the radio-activity determined in a Packard Cobra gamma counter(Downers Grove, IL, USA). Doses were determined byinterpolation from a smoothed spline transformation of thedata points.

All samples were stored frozen at"20 )C prior to assay.Because extreme values of sample pH may interfere withantibody binding, the urine pH was adjusted with 10 MTris (pH 90), 50 l/ml urine, prior to assay, so that thefinal pH was in the range 7074 (OConnor et al. 1988).Intra-assay variation was 6% for both assays, inter-assayvariation was 12% for B109B108* and 13% for B152B207* assays. Sensitivity (least detectable dose) defined as+2s.d. from the zero calibrator, was 10 fmol/ml for theB109B108* assay and 22 fmol/ml for the B152B207*assay.

The only mapping of the epitope sites thus far hasestablished that the B108 and B207 tracer binding sitecorresponds to site II, the same site recognized by the SB6antiserum (Vaitukaitis et al. 1972). The B152 capture siteinvolves the carboxy-terminal portion of the hCG subunit, since B152 will not bind simultaneously with acarboxyterminal peptide-specific monoclonal antibody. Inthe B109B108* assay, B109 requires that the and subunits both be present in the hCG molecule, whereasB152 will bind either the intact, heterodimeric hCGmolecule or its free subunit (OConnor et al. 1994,Birken et al., unpublished observations).

Patient samples

Urine samples from in vitro fertilization (IVF) patients werea gift from Dr L Cole. They included spontaneous abortion(n=14, range of gestational age 1841 weeks fromembryo transfer (ET)), ectopic pregnancies (n=7, ges-tational age 2340 weeks) and normal pregnancy controls(n=65, encompassing the range 0654 weeks from ET).Some of the normal pregnancy urine samples throughoutthe pregnancies were also obtained from Dr Cole. Otherswere obtained from the clinical practice of collaboratingphysicians at Columbia Presbyterian Medical Center

G KOVALEVSKAYA and others Early pregnancy hCG100

Journal of Endocrinology (1999) 161, 99106

(CPMC) (total n=103). Matched serum/urine samplesfrom the first (n=12) and the third (n=11) trimesters wereprovided by Dr A Kelly at CPMC. Trophoblast diseaseserum (n=17) and urine (n=28) samples were obtainedfrom Dr Cole, but had been collected by Dr E Newlands(Charing Cross Hospital, London, UK). All specimencollection protocols were approved by the appropriateInstitutional Review Board.

Statistical analysis

Polynomial regression models of log-transformed hormoneratios were used to describe the relationship between thechange in ratio as a function of gestational age in normalpregnancy. A paired t-test was used to evaluate therelationship between matched serum and urine hormoneratios. Analysis of covariance (ANCOVA) was used todescribe the time-adjusted relationship of hormone valuesin ectopic pregnancy and spontaneous abortion to those ofnormal pregnancy.

Results

Immunoreactivity of diVerent forms of hCG in the twoIRMAs

The relative binding of three diVerent forms of hCG(urinary hCG, pituitary hCG and choriocarcinoma hCGC5) has been characterized in the two hCG assays (Fig. 1).Urinary non-nicked hCG and pituitary hCG are recog-nized nearly equally well by the two IRMAs, while C5recognition is quite diVerent. The B152B207* assay ismore sensitive to C5, which is to be expected becauseB152 antibody was developed and selected on the basis ofhigher aYnity to C5. Urinary non-nicked hCG is purifiedfrom the CR127 preparation of pooled normal pregnancyhCG. Conversely, C5 is recognized with lower aYnity bythe B109B108* assay, which has primary specificity forthe hCG isoforms of later pregnancy.

The B152/B109 ratio measured in urine samples throughoutthe pregnancy

The relative concentrations of hCG isoforms in 103normal pregnancy urine samples (539 weeks post lastmenstrual period (LMP)) were determined by the twoIRMAs (B152B207* and B109B108*). Both becauseof the wide range of hCG concentrations in diVerentsamples, even at the same gestational age, and becauseneither of the assays is totally specific for the two (or more)families of hCG isoforms present, we find that presentingthe data as a ratio of the observed two isoform groups moreclearly delineates the change in isoform content as preg-nancy progresses. This calculated ratio is shown in Fig. 2.In weeks 58 of pregnancy, the ratio of B152/B109

isoforms ranged from 62 to 13, indicating a predomi-nance of the B152 isoform(s) in early pregnancy. Duringthe 1012 week period, the ratio ranged from 10 to 02,indicating that an inversion in hCG isoform content isoccurring as pregnancy progresses. This decline in theratio continues, ranging from 054 to 008 in the 1518week period and reaching an inflection point at 29 weeks.

Figure 1 Binding curves for three hCG types in the B152B207*assay (upper panel) and the B109B108* assay (lower panel).

Early pregnancy hCG G KOVALEVSKAYA and others 101


At that time, the ratio reached a value of around 006, afterwhich the ratio displayed a rise to a range from 020 to007 in the 37395 weeks of gestation time period.

Statistical analysis involved fitting the log-transformedratio data to second and third order polynomial regressionmodels. Since the third order term was not significant(likelihood ratio 2(1)=132, P=025), the second ordermodel was used (r2=0793). The log B152/B109 ratioreached an inflection point at LMP=29 weeks, based onthis model.

The B152B207* values reflect a measurement of theB152 isoform in terms of later pregnancy hCG equiva-lents, not in absolute quantities. It must be emphasizedthat the absolute concentrations measured in the B152assay cannot be compared with the results of the B109assay on an equimolar basis, since the potency of thehyperglycosylated isoform is much higher in the B152assay vis-a`-vis the standard, i.e. normal later first trimesterpregnancy hCG. The actual molar values of this isoformare of the order of tenfold less than those recorded in theassay. For this reason, we have chosen not to analyzeabsolute molar quantities of the two analytes, but only theratio of the two measurements.

The B152/B109 ratio in matched serum/urine samples in thefirst and third trimesters of pregnancy compared with hCGfrom JAR cells

The B152/B109 ratio in serum is analogous to that foundin matched urine samples and undergoes a similar change

as pregnancy progresses (Fig. 3). The B152/B109 ratio inthe cell supernatant from JAR cells (a choriocarcinoma-derived cell line) was similar to that of early pregnancy.

The B152/B109 ratios of both serum and urine hCGconcentrations are significantly higher in the first trimesteras compared with the third trimester of normal preg-nancies (Table 1). Significant diVerences between serumand urine hCG concentration ratios as well as log-transformed ratios in early (56 weeks) and late (3639weeks) gestation were evaluated by paired t-tests (Table2). In both the first and third trimesters, urinary B152/B109 ratios were significantly higher than serum ratios,indicating that there was a preferential clearance of theB152-recognized isoform into urine, regardless of therelative concentrations of the two isoforms.

The B152/B109 ratio in urine samples from IVF patients

In urine samples from IVF patients (14 weeks post ET)the B152/B109 ratio was again between 2 and 8 anddecreased as pregnancy progressed (Fig. 4), similar to thatobserved in natural conceptions. The eVect of pregnancyduration with respect to outcome variables could best berepresented by a linear or quadratic function. ANCOVAmodels including the second order week were fitted to thegeneral equation: Outcome=(eVect of time postET)+(eVect of diagnosis). After an appropriate ANCOVAmodel was determined, the least square means (adjustedfor week post ET eVect) were compared among the

Figure 2 Ratio of hCG isoforms measured by the B152B207*and B109B108* assays in normal pregnancy urine (n=103) atdifferent gestational ages. (Regression curve and 95% confidenceintervals are shown, r2=079.) An inflection point in the curveoccurs at approximately 29 weeks.

Figure 3 Box plot of the B152/B109 ratio for pregnancy matchedserum/urine at 56 weeks of gestational age (n=12), at 3639weeks of gestational age (n=11) and in JAR cell supernatant.Boxes extend to the 25th and 75th percentile. The upper andlower symbols indicate the 90th and 10th percentile respectively.A solid line inside the box marks the value of the 50th percentile.

Table 1 Analysis of the B152/B109 ratio in serum and in urine inthe first vs third trimesters of pregnancy

t-test=(df) P

MeasureSerum, ratio B152/B109 t(11) =665 00001Serum, log(ratio B152/B109) t(23) =2161 00000Urine, ratio B152/B109 t(11) =464 00007Urine, log(ratio B152/B109) t(157)=1685 00001



normal pregnancy, ectopic pregnancy and spontaneousabortion populations (Table 3). The log-transformed val-ues of both B109B108*- and B152B207*-measuredhCG forms discriminated both ectopic pregnancy andspontaneous abortions from normal pregnancy (P=00001). The ratio of the log-transformed values discrimi-nated abortion from normal pregnancy (P=0016). How-ever, neither the ratio of B152/B109 nor the log of thatratio discriminated either of the pregnancy disorders fromnormal pregnancy.

hCG analysis of trophoblastic disease samples

Trophoblast disease serum (17 samples) and urine(28 samples) were obtained from patients post therapy andhence contained low hCG levels. Due to limited amountsof sample, all of these specimens were run at a 1:10 initialdilution. hCG levels in serum were low. The highest hCG

concentration in serum was 202 fmol/ml in the B152B207* assay, with a corresponding value of 148 fmol/mlin the B109B108* determination. Six of seventeensamples in serum had detectable levels, with four out of sixhaving a higher value in the B152B207* assay. Of the15 out of 28 positive urine samples, however, 14 outof 15 had higher levels in the B152B207* assay thanin the B109B108* assay, with the highest hCG valuebeing 20 000 fmol/ml in the B152B207* assay and18 715 fmol/ml in the corresponding B109B108* assay.Due to the small sample size, no statistical treatment wasperformed on these data, but even in these post-treatmentpatients the B152/B109 ratio was >1, which correspondsto the early pregnancy hCG isoform ratio.

Discussion

We have developed a method to directly profile changesof hCG isoforms in serum or urine throughout pregnancy.Two IRMAs for hCG are employed, each based onmonoclonal antibodies to diVerent hCG epitopes. TheB109B108* assay is a commonly used intact hCG assayto the heterodimeric-dependent epitope. A new assay,B152B207*, is most likely sensitive to the carbohydrateportion of hCG carboxy-terminal peptide. The samestandard non-nicked hCG was used in both assays. Non-nicked hCG was employed, since the B109 assay reactspoorly with nicked forms of hCG, while the B152 assaydoes not discriminate between nicked and non-nickedforms of the hormone. The B152 assay detected withgreatly enhanced sensitivity hCG isoforms which appearearlier in pregnancy than isoforms measured by the B109assay (OConnor et al. 1998). Prior to development of thenew IRMA system described in this report, it was notpossible to readily discern the changes in hCG isoformsfrom very early pregnancy to mid pregnancy. The onlyavailable procedure for examining these changes was IEFof every patient specimen followed by immunoassay ofevery focused fraction (Ulloa-Aguirre et al. 1990, Bergeret al. 1993). The IEF pattern reflects the heterogeneity ofthe charged sugar, sialic acid, which varies with themulti-antennary structures of the carbohydrate moieties inwhich sialic acid is the terminal sugar. Although we do notyet know the precise nature of the isoform epitopes being

Figure 4 Ratio of hCG isoforms measured by the B152B207*and B109B108* assays in the urine of IVF patients (n=65).(Regression curve and 95% confidence intervals are shown,r2=059.)

Table 2 Analysis of the B152/B109 ratio in serum vs urine in the first and third trimestersof pregnancy

Measure Paired t (df) P

Gestational age (weeks)56 Ratio B152/B109 t(11)=325 00077

Log(ratio B152/B109) t(11)=625 00001

3639 Ratio B152/B109 t(10)=547 00003Log(ratio B152/B109) t(10)=714 00001



measured, the evidence for carbohydrate discrimination isbased upon the hyperglycosylated structure of the im-munogen, C5, used to develop the B152 monoclonalantibody and the antibodys reactivity with the hCGisoforms found in the JAR choriocarcinoma cell line.C5 hCG was isolated from a choriocarcinoma patient andhas been thoroughly characterized as to its protein andcarbohydrate content and structure (Elliott et al. 1997). Ithas been shown that C5, and hCG from other choriocar-cinoma subjects, diVer in the protein moiety mainly by thepresence of an increased number of nicked sites and byincreased glycosylation relative to the hCG of normalpregnancy. In comparison with the hCG of normalpregnancy, choriocarcinoma-derived hCG has increasedfucosylation of the N-linked biantennary oligosaccharidesin the subunit. In addition, the O-linked oligosac-charides in preparation C5 (a form of hCG produced froma single patient with choriocarcinoma) has a 100% tetra-saccharide core on the carboxy-terminal region of the subunit. Normal mid pregnancy hCG has only 1020% ofthis structure (Elliott et al. 1997). These observations, plusour own determination that the hCG synthesized by theJAR choriocarcinoma cell line provides a B152/B109isoform ratio similar to that observed in early pregnancy,lead us to the conclusion that, in very early pregnancy, thedeveloping trophoblast secretes an isoform of hCG whichresembles that produced in choriocarcinoma.

We have also tested recognition of pituitary hCG, sinceits N-Asn carbohydrates diVer somewhat from those ofplacental hCG, bearing a closer resemblance to those ofhuman luteinizing hormone, which have both sialic acidand sulfate groups (Birken et al. 1996). The carbohydratestructure of the carboxy-terminal portion of pituitaryhCG is not yet known. Since B152 did not recognize any

substantial diVerences between pituitary and placentalhCG (Fig. 1), diVerences in N-Asn recognition areunlikely. In terms of the carboxy-terminal carbohydrates,it appears that pituitary and placental hCG (mid pregnancyisoforms) may be similar, assuming the O-linked carbo-hydrate on the C5 antigen is part of the epitope of B152.

Even in normal pregnancy, the hCG values obtainedvary widely according to the characteristics of the im-munological reagents employed (Cole & Kardana 1992,Cole et al. 1993). We hypothesize that the two assaysdescribed in this report primarily detect hCG isoforms atopposing ends of this spectrum, each primarily recognizinga subset of closely related molecules in the continuum ofearly to later pregnancy hCG molecular forms.

We have retained the use of normal pregnancy hCG asthe standard in the B152B207* assay, despite its de-creased aYnity in this antibody configuration. The reasonsfor this include the limited and unrenewable supply of C5(which was isolated from the urine of a single patient) andthe variability in data which would result from investi-gations using diVerent standards. The consequences of thischoice are that the early pregnancy hCG isoforms havemarkedly increased immunopotency over that of normalpregnancy and hence their molar quantities are overesti-mated in this assay. We use this diVerence in aYnity to ouradvantage by employing a ratio of the molar results of twoassays (B152 and B109). Either assay taken alone obscuresthis change due to the wide excursion of hCG valueswhich occur in normal pregnancy.

Others have documented progressive changes in hCGisoforms throughout pregnancy. Skarulis et al. (1992)found that the fucose content of both intact hCG and alsoits free subunit increased as pregnancy progressed.Diaz-Cueto et al. (1996), investigating the IEF pattern of

Table 3 IVF patients: analysis of covariance of hCG isoforms among normal pregnancy,ectopic pregnancy and spontaneous abortion as a function of gestational age

Adjusted r2c Diagnosis

Fe P

Pairwise differenced

Outcome

Log(ratio B152/B109)a 051 089 041 None

Log(B109B108*)a 056 2133 00001Normal pregnancy vs

abortion and ectopic

Log(B152)/log(B109)b 045 434 0016Normal pregnancy vs

abortion

Log(B152B207*)b 050 2694 00001Normal pregnancy vs

abortion and ectopic

aANCOVA model with 2nd order polynomial coefficient (or parameter).bANCOVA model with only 1st order (linear) coefficient.cAdjusted r2 is an r2 adjusting number of coefficients on the ANCOVA model so that comparisons oftwo r2 with different ANOVA models with different numbers of coefficients are meaningful.dPairwise difference is based on t-test comparing the least-square means of outcome variables (afteradjusting effect of week of ET).eDegrees of freedom (dfl, df2) for F-test are 2,82) for a model with only linear coefficient and (2,81) fora model with both linear and 2nd order coefficient.



circulating hCG throughout pregnancy, found that inearly pregnancy more than 80% of the hCG isoforms wereacidic. This fraction decreased to less than half (47%) latein the third trimester (Diaz-Cueto et al. 1996). In contrast,Wide & Hobson (1987) found that the hCG of earlypregnancy was more choriocarcinoma-like by virtue of itsgreater biological activity than the hCG of normal preg-nancy. Fein et al. (1980), in a study which employed gelfiltration, determined that first trimester hCG was a largersize than that of the third trimester. Treatment withexoglycosidases eliminated the size diVerential, indicatingthat the first trimester hCG was more highly glycosylated(Fein et al. 1980).

A significant number of spontaneous abortions andectopic pregnancies occur in IVF pregnancies. We did notfind a diVerence in the ratio of the isoforms between eitherof these two categories as compared with normal controls,possibly a consequence of low statistical power. However,a significant diVerence was found between the B152 hCGisoforms levels in normal pregnancy and spontaneousabortion. This supports our previous finding in earlypregnancy loss, where diminished or absent levels of theB152 isoforms characterized an early pregnancy loss(OConnor et al. 1998).

The specimen limitations discussed above precludeour reaching any definitive conclusion on the analysis oftrophoblastic disease samples. However, it appears, asmight be anticipated, that the B152 assay is more sensitivethan the B109 assay in detecting hCG immunoreactivityin the blood and in the urine of trophoblastic diseasepatients, even after treatment.

Acknowledgements

The authors express their appreciation to Dr LaurenceCole for providing the patient samples and choriocarci-noma hCG (C5) and Dr Amalia Kelly for providing someclinical specimens at CPMC. We also thank Dr MarkSauer for his insight into the clinical applications of thisassay system for programs of assisted reproduction. Thiswork was made possible by Grant R01 ES07589 fromthe National Institute of Environmental Health Sciences,and the OYce of Research on Womens Health, NIH.Additional support was provided to S B by NIH grantsHD 15454 and AG 13783

References

Berger P, Schwarz S, Spottl G, Wick G & Mann K 1993 Variants ofhuman chorionic gonadotropin from pregnant women and tumorpatients recognized by monoclonal antibodies. Journal of ClinicalEndocrinology and Metabolism 77 347351.

Birken S, Chen Y, Gawinowicz MA, Lustbader JW, Pollak S, AgostoG, Buck R & OConnor J 1993 Separation of nicked humanchorionic gonadotropin (hCG), intact hCG, and hCG beta fragmentfrom standard reference preparations and raw urine samples.Endocrinology 133 13901397.

Birken S, Maydelman Y, Gawinowicz MA, Pound A, Liu Y &Hartree AS 1996 Isolation and characterization of human pituitarychorionic gonadotropin. Endocrinology 137 14021411.

Bogart MH, Pandian MR & Jones OW 1987 Abnormal maternalserum chorionic gonadotropin levels in pregnancies with fetalchromosome abnormalities. Prenatal Diagnosis 7 623630.

Cole LA 1987 The O-linked oligosaccharide structures are strikinglydiVerent on pregnancy and choriocarcinoma hCG. Journal of ClinicalEndocrinology and Metabolism 65 811813.

Cole LA & Kardana A 1992 Discordant results in human chorionicgonadotropin assays. Clinical Chemistry 38 263270.

Cole LA, Seifer DB, Kardana A & Braunstein GD 1993 Selectinghuman chorionic gonadotropin immunoassays: consideration ofcross-reacting molecules in first-trimester pregnancy serum andurine. American Journal of Obstetrics and Gynecology 16815801586.

Diaz-Cueto L, Mendez JP, Barrios-de-Tomasi J, Lee JY, Wide L,Veldhuis JD & Ulloa-Aguirre A 1994 Amplitude regulation ofepisodic release, in vitro biological to immunological ratio,and median charge of human chorionic gonadotropin inpregnancy. Journal of Clinical Endocrinology and Metabolism 78890897.

Diaz-Cueto L, Barrios-de-Tomasi J, Timossi C, Mendez JP &Ulloa-Aguirre A 1996 More in vivo bioactive, shorter-lived humanchorionic gonadotrophin charge isoforms increase at the end of thefirst and during the third trimesters of gestation. Molecular HumanReproduction 2 643650.

Elliott MM, Kardana A, Lustbader JW & Cole LA 1997 Carbohydrateand peptide structure of the alpha and beta subunits of chorionicgonadotropin (hCG): characteristics and variants in 32 subunitpreparations from normal and aberrant pregnancy andchoriocarcinoma. Endocrine 7 1532.

Fein HG, Rosen SW & Weintraub BD 1980 Increased glycosylationof serum human chorionic gonadotropin and subunits fromeutopic and ectopic sources: comparison with placental andurinary forms. Journal of Clinical Endocrinology and Metabolism 5011111120.

Haddow JE, Palomaki GE, Knight GJ, Williams J, Miller WA &Johnson DO 1998 Screening of maternal serum for fetal Downssyndrome in the first trimester. New England Journal of Medicine 338955961.

Heinonen S, Ryynanen M, Kirkinen P & Saarikoski S 1996 Elevatedmidtrimester maternal serum hCG in chromosomally normalpregnancies is associated with preeclampsia and velamentousumbilical cord insertion. American Journal of Perinatology 13437441.

Hurley TJ, Miller C, OBrien TJ, Blacklaw M & Quirk JG Jr 1996Maternal serum human chorionic gonadotropin as a marker for thedelivery of low-birth-weight infants in women with unexplainedelevations in maternal serum alpha-fetoprotein. Journal ofMaternalFetal Medicine 5 340344.

Hussa RO 1987 The Clinical Marker hCG. New York: Praeger.Nemansky M, Thotakura NR, Lyons CD, Reinhold BB, Reinhold

VN & Blithe DL 1998 Developmental changes in theglycosylation of glycoprotein hormone free alpha subunitduring pregnancy. Journal of Biological Chemistry 151206812076.

Nisula BC, Blithe DL, Akar A, Lefort G & Wehmann RE 1989Metabolic fate of human choriogonadotropin. Journal of SteroidBiochemistry 33 733737.

OConnor JF, Schlatterer JP, Birken S, Krichevsky A, Armstrong EG,McMahon D & Canfield RE 1988 Development of highly sensitiveimmunoassays to measure human chorionic gonadotropin, its beta-subunit, and beta core fragment in the urine: application tomalignancies. Cancer Research 48 13611366.

OConnor JF, Birken S, Lustbader JW, Krichevsky A, Chen Y &Canfield RE 1994 Recent advances in the chemistry and



immunochemistry of human chorionic gonadotropin: impact onclinical measurements. Endocrine Reviews 15 650683.

OConnor JF, Ellish N, Kakuma T, Schlatterer J & Kovalevskaya G1998 DiVerential urinary gonadotropin profiles in early pregnancyand early pregnancy loss. Prenatal Diagnosis 18 12321240.

Odell WD, GriYn J & Sawitzke A 1990 Chorionic gonadotropinsecretion in normal, nonpregnant humans. Trends in Endocrinologyand Metabolism 1 418421.

OLeary P, Nichols C, Feddema P, Lam T & Aitken M 1996 Serumprogesterone and human chorionic gonadotrophin measurements inthe evaluation of ectopic pregnancy. Australian and New ZealandJournal of Obstetrics and Gynaecology 36 319323.

Onderoglu LS & Kabukcu A 1997 Elevated second trimester humanchorionic gonadotropin level associated with adverse pregnancyoutcome. International Journal of Gynaecology and Obstetrics 56 245249.

Skarulis MC, Wehmann RE, Nisula BC & Blithe DL 1992Glycosylation changes in human chorionic gonadotropin and freealpha subunit as gestation progresses. Journal of Clinical Endocrinologyand Metabolism 75 9196.

Ulloa-Aguirre A, Mendez JP, Cravioto A, Grotjan E, Damian-Matsumura P & Espinoza R 1990 Studies on the microheterogeneityof chorionic gonadotrophin secreted by the human cytotrophoblast inculture. Human Reproduction 5 661669.

Vaitukaitis JL, Braunstein GD & Ross GT 1972 A radioimmunoassaywhich specifically measures human chorionic gonadotropin in thepresence of human luteinizing hormone. American Journal ofObstetrics and Gynecology 113 751758.

Wide L & Hobson B 1987 Some qualitative diVerences of hCG inserum from early and late pregnancies and trophoblastic diseases.Acta Endocrinologica 116 465472.

Wide L, Lee JY & Rasmussen C 1994 A change in the isoforms ofhuman chorionic gonadotropin occurs around the 13th week ofgestation. Journal of Clinical Endocrinology and Metabolism 7814191423.

Received 6 July 1998Revised manuscript received 9 October 1998Accepted 24 November 1998



Documents

1. Early Pregnancy Human Chorionic Gonadotropin (HCG) Isoforms