USA HCG REFERENCE SERVICE
HYPERGLYCOSYLATED HCG     

Click on link below to show the most recent publications-

 

NEW REVIEW

Cole LA, Hyperglycosylated hCG, Placenta, 2007; in press.

Cole LA, Dai D, Leslie KK, Butler SA, Kohorn EI. Gestational trophoblastic diseases: 1. Pathophysiology of hyperglycosylated hCG-regulated neoplasia. Gynecologic Oncology, in press, 2006

Cole LA, Khanlian SA, Riley JM, Butler SA. Hyperglycosylated hCG (hCG-H) in Gestational Implantation, and in Choriocarcinoma and Testicular Germ Cell Malignancy Tumorigenesis, J Reprod Med, in press 2006

 

Laurence A. Cole, Sarah A. Khanlian, Jaime M. Riley

and Stephen A. Butler

 

 

 

 Hyperglycosylated hCG Review

Cole LA and Khanlian SA
USA hCG Reference Service

          Hyperglycosylated human chorionic gonadotropin, acronym hCG-H, has been shown to be a completely independent molecule to regular hCG, produced by separate cells and having a totally different function (1-4). It is for this reason that we call it a biologically independent molecule to regular hCG. Its established functions are, however, autocrine rather than endocrine, more like those of a cytokines (1-8). Hyperglycosylated hCG has double size O-linked sugar structures and larger N-linked structures than hCG, changing its biological function (5-8). Here we review hCG-H biochemistry and clinical chemistry. We review research on the following topics: a. Structure of hCG-H; b. Source and Biological Function of hCG-H; c. Measurement of hCG-H; d. hCG-H in Pregnancy Detection; e. hCG-H Measurement in Detection of Pregnancy Outcome; f. hCG-H in Prediction of Gestation Down Syndrome; g. hCG-H in Management of Gestational Trophoblastic Diseases.  

 

a. Structure of hCG-H

hCG is a heterogeneous molecule produced by trophoblastic cells in pregnancy. Peptide variants are detectable in serum and urine samples during pregnancy and gestational trophoblastic diseases (9). Oligosaccharide variants originate from differences in the availability of sugars, variations in cellular metabolism, and the differential expression of different glycosyltransferases, the sugar-adding enzymes in cells (5, 8, 10). It has long been recognized that the hCG molecule produced in choriocarcinoma or cancer of the trophoblastic cell is a larger molecule than that produced through most of normal pregnancy [11-13]. In 1987-8, a significant and consistent difference was demonstrated between the 4 O-linked oligosaccharides on hCG from choriocarcinoma patients and those found on hCG from normal pregnancy (6, 7). This was later confirmed in a study with a larger number of pregnancy and choriocarcinoma cases in 1997 (5).

 While the principal O-linked oligosaccharide structure in all normal pregnancies is a trisaccharide with the structure NeuAc2-3Galß1-3GalNAc-O-Serine (where NeuAc is sialic acid, Gal galactose and GalNAc is N-acetylgalactosamine), the principal structure in all choriocarcinoma cases is a double size hexasaccharide with the structure NeuAc2-3Galß1-3(NeuAc2-3Galß1-4GlcNAcß1-6)GalNAc-O-Serine (where GlcNAc is N-acetylglucosamine) (5-7). In 1997 (5) we demonstrated that the difference in the 4 O-linked oligosaccharides is the principal difference between choriocarcinoma and pregnancy hCG. While first trimester normal pregnancy urine hCG contained 12.3 to 19% of the larger hexasaccharide side chains (n=6 individuals, mean = 15.6%), choriocarcinoma urine hCG contained 48 to 100% hexasaccharide side chains (n=6 individuals, mean = 74.2%).

Lesser differences have also been shown in the 4 N-linked oligosaccharide structures on pregnancy and choriocarcinoma molecules (5, 8, 10). While in normal pregnancy, sialyl-N-acetylactosamine biantennary N-linked oligosaccharides and fucosylated N-linked oligosaccharides predominate at the two sites on the ß-subunit of hCG in normal pregnancy, significantly larger fucosylated sialyl-N-acetylactosamine triantennary oligosaccharides predominate in choriocarcinoma cases (5,8). While in normal pregnancy sialyl-N-acetylactosamine biantennary N-linked oligosaccharides and biantennary oligosaccharides with the a1,3 antenna terminating in Mannose predominate at the two sites on the a-subunit of hCG in normal pregnancy, significantly larger fucosylated biantennary oligosaccharides predominate in choriocarcinoma cases together with abnormal triantennary oligosaccharides with the a1,3 antenna terminating in Mannose (5,6).

 The oligosaccharides on hCG in normal pregnancy contribute to the molecular weight of 36,700 daltons. Choriocarcinoma hCG, in contrast, with its large sugar side chains has a molecular weight of approximately 41,000 daltons (5). The name hyperglycosylated hCG (hCG-H) was formulated for this molecule with unduly large sugar side chains.

 

b. Source and Biological Function of hCG-H

hCG with larger O-linked oligosaccharides (hCG-H) characterizes the molecules produced in choriocarcinoma. A specific monoclonal antibody to hCG-H (antibody B152) was generated against the hCG with 100% hexasaccharide O-linked structures (100% hCG-H) produced by a single patient with choriocarcinoma (14). Using this monoclonal antibody specific sandwich assays have been established for detecting intact hCG-H (4, 15).

Multiple publications examining hCG carbohydrate structure shows that patients with choriocarcinoma and choriocarcinoma cell line produce primarily a molecule with primarily hexasaccharide O-linked oligosaccharide and an abundance of triantennary N-linked oligosaccharides, or hyperglycosylated hCG (5-8). This has been demonstrated now in large numbers of choriocarcinoma or gestational trophoblastic neoplasm (GTN) cases using the microtiter plate and commercial B152-based hCG-H assays (16, 17).

In 1998 O’Connor et al. used a B152-based microtiter plate assay to show that hCG-H is not only the principal form of hCG made in choriocarcinoma, but also the principal form of hCG made during the initiation of pregnancy, at the time of and in the 2 weeks following implantation of pregnancy (18).  This finding has now been confirmed by multiple investigators (3, 4, 14-20). Recent data on the proportion of total hCG accounted for by hCG-H in urine samples is presented in Table 1 (16-20).  A similarity has previously been suggested in the invasion processes of implantation and choriocarcinoma (21, 22)

          Cytotrophoblasts are phenotypically invasive cells. These are the principal cells in choriocarcinoma and GTN, the active cells in choriocarcinoma tumors, and the active invasive cells of blastocysts at the time of implantation (21-23). As published in multiple articles between 1999 and 2005, the cytotrophoblast cells of pregnancy trophoblast and of choriocarcinoma cell lines produce hCG-H, and the differentiated syncytiotrophoblast cells of pregnancy produce regular hCG (1, 3, 4). As such the high proportion of hyperglycosylated hCG of implantation and of choriocarcinoma is produced by cytotrophoblast cells. The hCG produced in pregnancy is made in syncytiotrophoblast or different cells.

        The primary function of hCG in pregnancy is to maintain progesterone production by corpus luteal cells (24). In contrast, hCG-H is only associated with invasive events, pregnancy implantation, and GTN or choriocarcinoma (5). The choriocarcinoma hCG immunoreactivity in the conditioned medium of JAR choriocarcinoma cells (invasive cytotrophoblast cells) is hCG-H (16). Studies by Lei et al. (2) in 1999, show that JAR cells are invasive in Matrigel basement membrane inserts (tumor invasion models) in vitro, and are rapidly tumorigenic when transplanted into athymic nude mice in vivo. Lei et al. (2) treated JAR cells with hCG a-subunit antisense cDNA. This blocked production of a-subunit and thus of hCG dimer in the choriocarcinoma cells (cells primarily produce hCG-H (1, 16), not specified by Lei et al.) and also blocked Matrigel membrane insert invasion in vitro and tumorigenesis in athymic nude mice in vivo. This data indicated that hCG-H has a separate role to hCG, a critical function in trophoblast cell invasion. Recently, we demonstrated that hCG-H has minimal activity at the corpus luteal hCG receptor (1). When isolated normal pregnancy cytotrophoblast cells and JEG-3 choriocarcinoma cells (100% cytotrophoblast) were cultured on Matrigel basement membrane inserts invasion occurred. In both cell sources, invasion was significantly promoted by hCG-H and not by hCG. JEG-3 choriocarcinoma cells are rapidly tumorigenic when transplanted into athymic nude mice in vivo (1). Cole et al. (1) treated transplanted mice with B152 antibody against hCG-H. Antibody therapy blocked tumor formation and tumor growth in vivo (1). These data suggest that hCG-H has a completely separate role to hCG. It has a critical function in cytotrophoblast invasion or implantation in pregnancy, and in tumor invasion or tumorigenesis in choriocarcinoma cases. That cytotrophoblast cells produce hCG-H, and that it acts directly on cytotrophoblast cells promoting invasion suggesting an autocrine function. That circulating antibody against hCG-H inhibits the invasive action indicates hCG-H has an autocrine action.

 In summary, hCG-H has a different structure to hCG, it is produced by separate cells to hCG, and acts on separate cells to hCG, with a very different tumorigenic function. As such, it needs to be considered as a completely independent molecule. It can be considered as a biologically independent molecule to regular hCG, yet it is not an endocrine or hormone per se.  It also is ineffective in promoting progesterone production, so is not a gonadotropin like hCG.

 

c. Measurement of hCG-H

A specific monoclonal antibody to hCG-H (antibody B152) was generated against the hCG with 100% hexasaccharide O-linked structures (100% hCG-H) produced by a single patient with choriocarcinoma (14). Using this monoclonal antibody a microtiter plate two antibody (B152 plus anti-ß tracer) assay was established for detecting intact hCG-H (4). Nichols Institute Diagnostics (San Clemente CA) now markets an automated chemiluminescence test using antibody B152 and an anti-ß tracer to detect hCG-H, for the Nichols Advantage immunoassay platform (15). This assay has been FDA approved for pregnancy-related applications.  As published, the microtiter plate assay with overnight or 4 hours incubation with antibody B152, and the automate test with 20 minutes incubation  have different specificity (4, 14, 16). The microtiter plate assay recognizes hCG-H (100% immunoreactivity), sialic acid deficient hCG-H (100%), hCG-H free ß-subunit (60%), pure CHO-cell recombinant hCG (<1%), and pure CHO (Chinese Hamster ovary) -cell recombinant hCG free ß-subunit (<1%). The automated Nichols Advantage assay has similar specificity but considerable lower affinity for hCG-H sialic acid deficient (~30%) and free ß-subunit (~20%) variants (4, 14, 16, 25). The difference may be due to the 20 minute incubation time in the automated assay, compared with 4 hrs in the microtiter plate test. Equilibrium has been demonstrated in the later test, but likely does not occur in the automated test.

As published (3, 4, 14-20),  the majority of total hCG immunoreactivity in serum and urine samples in early pregnancy samples is due to hCG-H. As shown in Table 1, hCG-H accounts for a 92% (median) of total hCG in the 3^rd complete week of gestation or the week following implantation, 73%  in the 4^th week or the week following missing menses when most pregnancy testing is done, and then declines rapidly thereafter. It accounts for 14% in the 7^th week of gestation and <2% in the second and third trimesters of pregnancy. Clearly a pregnancy test needs to appropriately detect hCG-H.  As shown in Table 2, only 3 of 14 professional laboratory serum pregnancy tests equally or near-equally detect hCG and hCG-H (16, 26). The majority (11 of 14 tests), under detect or very poorly detect (9 of 14 tests) hCG-H (16). Similarly, only 4 of 15 home pregnancy tests or over the counter tests equally detect hCG and hCG-H (16). None of the tested point of care or physician’s office tests equally detect hCG and hCG-H (16). Again, the vast majority of test (11 of 15 over the counter and 2 of 2 point of care) very poorly detect hCG-H (16, 20). Over the counter or point of care devices make claims like “99% accurate” or “100% accurate,” and “Use as Early as the First Day of Missed Period” or “Use Test Before You Expect Your Period.” Claims like these are concerning, especially when the device does not appropriately detect the principal hCG analyte in early pregnancy samples. One might ask with all these professional laboratory and front line pregnancy testing point of care and over the counter devices, are they really pregnancy tests if they poorly detect the principal analyte that marks early pregnancy?

          Today, 2005, 18 years since the literature first described hCG-H, and 7 years since publications started to show that this is the principal hCG immunoreactivity produced in early pregnancy, most manufacturers continue to ignore hCG-H and don’t make assays optimized for detecting hCG-H. This is primarily due to the non-availability of a formal standard or W.H.O standard. In recent years, several manufacturers have produced their own standard to optimize their assays. These standards have been prepared by partially purifying the hCG-H in JEG-3 choriocarcinoma cell line culture fluid (16). The hCG-H produced by JaR and JEG-3 (clones) culture fluids has been characterized and shown to be similar to choriocarcinoma patient hCG-H (16, 27). This culture fluid hCG-H has been calibrated using the Nichols Advantage hCG-H test (calibrated against a JEG-3 cell line standard). It appears that most manufacturer’s will follow FDA guideline of calibrating tests against hCG alone until either guidelines are changed or a WHO hCG-H standard is produced. Until that time mostly sub-optimal pregnancy tests with poor recognition of hCG-H may be continue to be produced.  

d. hCG-H in Pregnancy Detection

As indicated in Table 1, hCG-H is a marker of early pregnancy. Measurement of both hCG and hCG-H equally is optimal for hCG pregnancy testing. Between one third and one quarter of pregnancies may fail hours or days after implantation (18, 28). There are considered biochemical pregnancies or early pregnancy losses. When monitoring natural and in-vitro fertilized it is important to try and distinguish “clinical” pregnancies which may make it through to term from “biochemical” pregnancies which fail to thrive. As published (18, 28), all biochemical pregnancies fail to produce hCG-H.  The lack of hCG-H or the invasive stimulator could actually be the cause of failure. Ongoing studies in our laboratory, monitoring women eager to achieve pregnancy show that approximately 1 in 4 “biochemical” pregnancies produce no detectable hCG-H and 3 of 4 produce borderline detection amounts of hCG-H compared to “clinical” pregnancies. hCG-H may potentially be a better pregnancy test than hCG because it does not or poorly detects “biochemical” pregnancies (18, 28, ongoing studies in Cole laboratory).

Women are administered hCG to promote maturation of multiple follicles for collection for in- vitro fertilization. As such, the slow disappearance of exogenous hCG has to be demonstrated, over 2 weeks, before endogenous production from a pregnancy can be demonstrated. Women who pay for in-vitro fertilization, and go through the operative procedures are usually eager to learn about their success or failure in achieving pregnancy. Waiting for exogenous hCG to clear requires a two week delay. Detection of hCG-H instead of hCG can avoid this delay. A pregnancy test measuring hCG-H may have multiple advantages both in the earlier detection of pregnancies in assisted reproductive technologies, and in the avoidance of detecting “biochemical” pregnancies.

 

e. hCG-H Measurement in Prediction of Pregnancy Outcome

In 2002 Kovalevskaya et al., discovered unusually low proportions of hCG-H in pregnancies that spontaneously abort in the first trimester of pregnancy (28). The low proportions started when hCG was first detected and continue until the time of spontaneous abortion. While this was an important discovery no clinical application was indicated. Two years later these findings were confirmed by Sutton-Riley et al. (29) and Byrn et al. (30).

Sutton-Riley et al. (29) showed that a simple single point cut-off of 13 ng/ml hCG-H in serum could be used to differentiate a failure outcome (<13 ng/ml) from term outcome (>13 ng/ml) pregnancy between 4 and 7 weeks of pregnancy (29). As shown in Table 3, this 13ng/ml cut-off was pivotal in detecting 73% of failures (spontaneous abortion and ectopic pregnancy) at 5% error rate, compared to 42% detection at this same error rate for a comparable cut-off for hCG. A highly significant difference was observed between the area under the ROC curve results, or test accuracies using hCG and hCG-H (P<0.00005). The use of this 13 ng/ml cut-off for detecting failures in urine was also demonstrated, with the feasibility of a 13 ng/ml marked point-of-care test for physician’s office applications. Byrn et al. (30) showed that a hCG-H single test or multi-point doubling test offered great improvement over an hCG single point or doubling test in detecting failing pregnancies. They showed that the hCG-H test offered improvement in both the detection of spontaneous abortion and ectopic pregnancies.

All 3 independent studies, with different populations (28-30) supported each other in showing that unduly low hCG-H accompanies spontaneously aborting pregnancies, from time of first hCG detection onwards. As discussed earlier (1-2), hCG-H has a clear role in promoting the invasive process in implantation of pregnancies. It is speculated that low hCG-H production, or trophoblast differentiation of the hatched blastocyst at the time of implantation, may lead to inappropriate implantation, and may be a major cause of spontaneous abortion of pregnancy.

 

f. hCG-H in Prediction of Gestation Down Syndrome

The first application demonstrated for hCG-H was in screening for gestational Down syndrome. In 1997 a preponderance of hCG-H was demonstrated in structure studies in mid-trimester Down syndrome pregnancies, and then later confirmed by using the B152-based hCG-H immunoassay (31, 32). This was a surprising finding. Why Down syndrome pregnancies? This enigma has been explained in recent years by the finding of a defect in cytotrophoblast differentiation into syncytiotrophoblast cells in Down syndrome cases (33 -35), or a resultant accumulation of cytotrophoblasts, the hCG-H producing cells, in Down syndrome pregnancies.

          Clinical trials were initiated in 1997 at Yale University (New Haven, CT). Serum and urine samples were sought over a period of 2 years from all women waiting to have amniocentesis because of maternal age concerns (4). None had been pre-selected with biochemical methods (i.e. triple or quadruple screen). While all consenting women volunteered a urine sample (1526 total), only 34% of this group also agreed to phlebotomy or to providing a serum sample (512 total). Samples were collected between 14 and 22 weeks of gestation and tested blindly (before karyotype known). All urine samples were collected from the amniocentesis center daily stored in a refrigerator (no freezing) and tested once each week for hCG-H and creatinine. hCG-H was measured using the B152-based microtiter plates test for hCG-H (4, 36). Tests on spot urine samples were normalized to creatinine concentration.   

MoM (multiples of median) statistics were used to analyze data. The logarithmic regression equation relating the hCG-H results for 1487 normal karyotype patients with gestational age (ga) was median = 6050 x (0.71ga). The median MoM for normal karyotype patients was 1.0 (standard deviation 0.47), and the median MoM for the 39 later proven Down syndrome cases was 9.5 (4). These data indicated that Down syndrome samples had 9.5-fold higher hCG-H values than those with normal karyotype. In addition, it was observed that 31 of 39 Down syndrome cases had hCG-H results that exceeded the 95^th centile of normal karyotype cases. ROC analysis indicated that this single test would achieve 80% detection at a 5% false positive rate.  A triple test (hCG + unconjugated estriol + a-fetoprotein) was later performed on the 512 serum samples provided, 64% detection of Down syndrome cases was observed at 5% false positive rate. Multivariate analysis combining maternal age, urine hCG-H results, and serum triple test values yielded 96% detection with a 5% false positive rate and 94% detection at a 3% false positive rate.

          In an initial serum study, hCG-H was measured in serum samples from 10 gestational Down syndrome cases and 66 normal karyotype pregnancies at 15-22 weeks of gestation. A lesser elevation was noted with these serum samples. Down syndrome samples had a median of 3.9 MoM. Using serum hCG-H concentration alone, 60% detection was indicated at a 5% false positive rate (37). In an independent serum study evaluating 19 gestational Down syndrome cases and 82 controls, collected in multiple centers in Connecticut and blindly tested at Quest Diagnostics, 81% detection was observed with the hCG-H test at a 5% false positive rate. In a multivariate analysis, combining serum hCG-H with a quadruple test (hCG + unconjugated estriol + a-fetoprotein + inhibin), 95% detection was achieved at a 5% false positive rate or 92% detection at a 3% false positive rate (38).  In a further study, looking at 49 Down syndrome cases shipped at ambient temperature and stored at -20°C for 8 years, only 55% detection at 5% false positive was indicated (39).

          The high sensitivity of 80% and high 9.5 median MoM was reported with the multititer plate assay with long incubation with B152 (4). In similar size studies with serum and urine samples using the Nichols Advantage hCG-H tests yielded much poorer detection rates (50% detection at 5% false positive rate) and lower elevations in Down syndrome cases (3.5 MoM) (52-55). This is seeming due to the difference in specificity of the microtiter plate assay and the automated test with short incubation. The poor detection of the free ß-subunit of hCG-H continuously released from dissociating hCG-H, and the poor detection of the sialic deficient isoforms like explains this difference. This is probably particularly pertinent when testing frozen libraries of serum samples, and serum samples shipped by mail as investigated by Pandian et al (53-55) and by others (40, 42). Recent studies by Sutton et al (25) clearly demonstrate that the principal isoform of hCG-H elevated in Down syndrome cases, whether first trimester or second trimester is hCG-H deficient in sialic acid. A major isoform of hCG-H was poorly detected by the Nichols Advantage hCG-H test. This explains the poorer utility of the Nichols Advantage hCG-H test in Down syndrome screening. We eagerly await a new, fixed or corrected hCG-H test for this application.

          As indicated in Table 4 (4, 16, 20), individual hCG and hCG-H results vary widely throughout pregnancy. The biggest variation occurs at the end of the first trimester, when hCG and hCG-H concentrations rapidly decline following their peak at 8-12 weeks. This wide variation may be due to inaccuracies in determination of gestational age during a time when concentrations are plunging every day. More consistent results are observed in the second trimester (15-22 weeks) when concentrations are leveling. For this reason, neither hCG nor hCG-H can be expected to be as good of a screening test at 9-13 weeks as they are in the second trimester of pregnancy.

In 2000 Weinans and colleagues (40) blindly tested 136 urine samples from the 10^th and 11^th weeks of gestation, including 11 pregnancies shown by chorionic villous sampling to have Down syndrome fetuses. In that study with the hCG-H test, 37% detection was observed at 5% false positive rate. This is similar to the detection rates when using either Pregnancy-Associated Plasma Protein-A (PAPP-A) or hCG free ß-subunit at the same time of gestation (41). In 2001, Strom and colleagues (42) used a case control set of samples involving 4412 women undergoing chorionic villous sampling or early amniocentesis at multiple-centers. Parallel urine and serum samples were available from 17 Down syndrome cases from the first trimester of pregnancy, and from 5 normal karyotype controls for each Down syndrome case. While the combination of serum PAPP-A and hCG free ß-subunit measurements detected 60% of Down syndrome cases at a 5% false positive rate, the combination of urine hCG-H plus serum PAPP-A and hCG free ß-subunit detected 81% of Down syndrome cases at a 5% false positive rate, or 74% at a 3% false positive rate (32). A similar but much larger study was completed recently in Europe with near identical results (43). Clearly, there is also a role for hCG-H measurement in first trimester Down syndrome screening. Based upon the findings of Sutton et al (25), vastly superior results may be expected for hCG-H in Down syndrome screening one the specificity limitations of the assay are fixed, or with the introduction of a test specific for the sialic acid deficient isoform that marks Down syndrome.

 

g. hCG-H in the Management of Gestational Trophoblastic Diseases.

          Quiescent gestational trophoblastic disease is a benign or inactive form of GTN

or choriocarcinoma, marked by persistent low hCG results, pertsisting for periods ranging from 3 months to 16 years (45-51). It may also be considered as a pre-malignant state in that approximately 1 in 5 cases transform into GTN or choriocarcinoma (44-51). We have reviewed the histology slides from two cases undergoing surgery for this condition.  In both cases, intermediate and highly differentiated syncytiotrophoblast cells were observed, with a clear absence of cytotrophoblast cells that mark most cases of choriocarcinoma (45, 46). Differentiated syncytiotrophoblast cells can be slow growing cells, and not invasive cells. It is inferred that the presence of syncytiotrophoblast cells with the absence of invasive cytotrophoblast cells is the nature of quiescent gestational trophoblastic disease. In the absence of cytotrophoblast cells, the hCG-H producing cells, no hCG-H can be detected. That is, syncytiotrophoblast cells remaining after treatment of an ectopic pregnancy, or parturition or abortion, or following evacuation of a hydatidiform mole.  Alternatively, slow growing syncytiotrophoblast cells can remain after chemotherapeutic resolution of cytotrophoblasts or following treatment of GTN or choriocarcinoma, where these cells may persist.  

          The USA hCG Reference Service has consulted on 69 cases with persistent low “real” (not false positive) hCG results (44). In all cases the persistent low levels persisted for 3 month or greater with minimal fluctuation and no clear upward or downward hCG trend. hCG-H was measured using the Nichols Advantage test as a proportion of total hCG in the DPC Immulite test (% hCG-H) as a marker for invasive cytotrophoblast cells. In 67 of the remaining 69 cases, which we class as quiescent gestation trophoblastic disease (44-51), no hCG-H was detected whatsoever. In the two remaining cases, 10% and 12% hCG-H was detected.  In both, detectable hCG persisted for greater that 6 months with minimal fluctuation. Considering these histories, and that in these exceptional cases the proportion of hCG-H was below those of all 79 GTM or choriocarcinoma cases referred to the Service, the diagnosis of quiescent gestational trophoblastic disease was conferred. This data shows the utility of hCG-H as a marker for differentiating quiescent and invasive disease. Considering that hCG-H is only produced by cytotrophoblast cells (3-4), the absence of hCG-H in quiescent gestational trophoblast disease confirms its origin from syncytiotrophoblast in the absence of cytotrophoblast cells.

          Of the 69 identified cases, 33 followed evacuation of a hydatidiform mole, 15 followed successful treatment of GTM or choriocarcinoma, and 21 following a spontaneously aborted, ectopic or term pregnancy. We examined the medical records of these 69 identified quiescent gestational trophoblastic disease cases. While 42 had received chemotherapy, combination chemotherapy, hysterectomy or other surgery for assumed active disease, none fully responded to the therapy, including chemotherapy.  We have interpreted this to mean that theses cases have slowly growing dispersed syncytiotrophoblast cells, that are too slow growing to respond to chemotherapy.  In all treated cases, hysterectomy partially but never completely suppressed the hCG result. We have interpreted this to mean that syncytiotrophoblast cells commonly remain outside of the uterus after a gestational event or, alternatively, are transposed through the fallopian isthmus by an endometriosis-like mechanism. With a 100% summary of records indicating that therapy does not work, and similar findings by others (50,51), it is inferred that treating physicians should refrain from using chemotherapy or surgery in these cases.

Our history with quiescent gestational trophoblastic disease cases is like that with false positive hCG cases- too many people receiving needless therapy for a poorly understood and only recently fully recognized condition that in fact, requires no therapy. In one case, a patient was shown by the Service on two separate occasions during the course of one year to have quiescent disease.  Despite our recommendations, additional combinations of chemotherapeutic agents were given. This patient tragically died from complications of pulmonary fibrosis following bleomycin chemotherapy.  Nevertheless, this case is important because it emphasizes the importance of making an accurate diagnosis of GTN/choriocarcinoma versus quiescent disease before initiating cytotoxic chemotherapy.  This requires all medical and gynecologic oncologists, and to some extent, whomever manages patients who undergo hCG-H determinations, to be able to recognize and diagnose the phenomenon of quiescent gestational trophoblastic disease.  If the diagnosis is in doubt or suspect, these data indicate the importance of referring such patients to centers with experience with this condition before therapy is initiated. 

          Published data show the value of hCG-H not just in the detection of quiescent disease, but in the earlier detection and differention of recurrent GTN/choriocarcinoma and in the staging of GTN/choriocarcinoma (44).

 

Summary

          hCG-H is biologically independent molecule to regular hCG, produced independently by separate cells and with a completely independent function. hCG-H offers clinical chemistry a new and extended scope beyond hCG in management of pregnancy, predicting pregnancy outcome, management of gestational trophoblastic diseases, and in screening for Down syndrome pregnancies. 

 

Table 1. The proportion of total hCG immunoreactivity in urine samples due to hCG-H in early pregnancy (16, 20). Total hCG was measured in the DPC Immulite total hCG test, which detects hCG and hCG-H equally (26).  hCG-H was measured in the Nichols Advantage hCG-H test, and hCG-H ng/ml was expressed as a percentage of total hCG. Gestational age is presented as complete weeks, where the 3^rd complete weeks is 3 weeks 0 days to 3 weeks 6 days since last menstrual period, or in most cases, the week following implantation.

 

 

Gestational age	n	Median percentage hCG-H	Range of hCG-H
3rd complete week	75	92%	55 - 100%
4th complete week	63	73%	15 - 100%
5th complete week	45	50%	10 - 100%
6th complete week	23	26%	7.9 - 53%
7th complete week	22	14%	2.3 - 21%

 

 

Table 2.   Sensitivity of commercial hCG tests. Pure intact hCG (CHO cell recombinant hCG, 0% hCG-H) and pure hCG-H (Preparation C5, 0% hCG, (5)) were calibrated by absorbance at A278 (26). The concentrations were then assessed blindly in 14 Professional Laboratory Tests (16, 26). Percentages are the assay value divided by the absolute concentration. hCG-H sensitivity was the relative sensitivity for detecting hCG-H vs. hCG.  Point of care and over the counter pregnancy test devices were tested for sensitivity by evaluating using multiple concentrations of absolute calibrated hCG and hCG-H in at least sextuplicate (16, 20). Sensitivity results are presented. These are the lowest concentration at which all devices were positive. hCG-H sensitivity was the relative sensitivity for detecting hCG-H vs. hCG.

Test or Device	hCG only	hCG-H only	hCG-H sensitivity
A. PROFESSIONAL LABORATORY TESTS
Roche Elecsys	76%	167%	220%
Tosoh A1A600	95%	97%	102%
DPC Immulite	102%	99%	97%
Dade Stratus	89%	85%	96%
Ortho Vitros ECi	89%	80%	91%
Bayer Centaur	121%	107%	89%
Wako hCG-CTP	86%	75%	87%
Bayer ACS180	119%	102%	86%
London radioimmunoassay	11`2%	95%	85%
New Haven radioimmunoassay	107%	91%	85%
Abbott AxSym	110%	86%	79%
Dade Opus	90%	69%	68%
Dade Dimension	90%	50%	54%
Serono MAIAclone	94%	4%	4%
B. POINT OF CARE TESTS
Beckman Icon 25	25 IU/L	50 IU/L	50%
Quidel Quick Vue hCG Combo	25 IU/L	50 IU/L	50%
C. OVER THE COUNTER TESTS
Answer,	12.5 IU/L	12.6 IU/L	100%
Equate	12.5 IU/L	12.5 IU/L	100%
First response, Early Result	9.4 IU/L	9.4 IU/L	100%
K-Mart, American Fare, Easy to Read	25 IU/L	25 IU/L	100%
Clear Blue Easy, One Minute	12.5 IU/L	25 IU/L	50%
Clear Plan Easy	25 IU/L	50 IU/L	50%
E.P.T.	12.5 IU/L	25 IU/L	50%
Eckerd, One-Step	50 IU/L	100 IU/L	50%
Inverness Medical, Early	25 IU/L	50 IU/L	50%
Inverness Medical, Early, Cassette	12.5 IU/L	50 IU/L	50%
Save-on, Osco, One-Step	25 IU/L	50 IU/L	50%
Confirm	6.3 IU/L	25 IU/L	25%
Rite Aid, One-Step	25 IU/L	100 IU/L	25%
Target Brand, One-Step	25 IU/L	100 IU/L	25%
Walgreens, One Step	12.5IU/L	100 IU/L	13%

 

Table 3. Use of hCG (total hCG) and hCG-H for predicting pregnancy failure using 120 serum and 167 urine samples. Cut-off values were indicated by ROC analyses. False positive rates and detection rates at these cut-off values were calculated and predictive values determined. ROC statistics were calculated for serum and urine hCG and H-hCG independent of any cut-off values.

 

		Serum hCG-H	Serum hCG
Term outcome pregnancies		n = 87	n = 87
Cut-off concentration		13 µg/L	125 IU/L
Corresponding false positive rate		5%	5%
Detection Rates for Failures		n = 33	n = 33
a. All failures		24 of 33 (73%)	14 of 33 (42%)
b. Spontaneous abortions only		20 of 29 (71%)	12 of 29 (41%)
c. Ectopic pregnancy only		4 of 4 (100%)	2 of 4 (50%)
Predictive value positive		85%	76%
Area under ROC curve ± SE		0.88 ± 0.003 1	0.71 ± 0.006 1
ROC 95% confidence interval		0.83 - 0.99	0.78 - 0.97

 

¹ A significant difference observed between serum hCG and serum H-hCG area under the ROC curve results (P<0.00005).

 

Table 4. Median concentration and ranges of total hCG concentrations (all forms of hCG including hCG-H) as measured in the DPC Immulite test and hCG-H concentrations measured in the Nichols Diagnostics hCG-H assay,  in 512 urine samples during the course of gestation (4, 16, 20). The proportion of ITA was determined as the ITA result divided by the total hCG result.

Gestational age	n	total hCG		hCG-H		Proportion hCG-H
(weeks)		ng/ml	Range	ng/ml	Range	(%)	range
4	63	45	(2-465)	28	(0.5-751)	73%	(7%-100%)
5	45	162	(11-4580)	78	(3.5-1747)	50%	(10%-100%)
6	14	268	(18-2043)	48	(3.7-593)	26%	(7.9%-53%)
7	5	1665	(160-5927)	187	(33-1405)	13%	(2-24%)
8 – 11	20	2455	(553-10022)	200	(18-1315)	10%	(0.8%-55%)
12 – 15	128	899	(228-11000)	36	(3-821)	4%	(0.2%-64%)
16 – 19	114	637	(176-5264)	21	(1-140)	3%	(0.1%-31%)
20 – 23	25	300	(56-1606)	5.1	(1-41)	1.5%	(0.1%-12%)
24 – 27	14	567	(120-4784)	4.9	(1-47)	1.2%	(0.1%-7.5%)
28 – 31	12	526	(83-7014)	4.2	(1-44)	1.6%	(0.1%-3.3%)
32 – 35	20	563	(139-11000)	4.9	(1.7-113)	1.5%	(0.3%-8.7%)
36 – 39	18	469	(27-10250)	16	(1-128)	3.0%	(0.5%-14%)

 

REFERENCES

 

1. Cole LA, Dai D, Butler S, Muller CY, Leslie K, Kohorn E. Gestational Trophoblastic Diseases: 1. Pathophysiology of Hyperglycosylated hCG. Gynecol Oncol 2005; in press

 

2. Lei ZM, Taylor DD, Gercel-Taylor C, Rao CV. Human chorionic gonadotropin promotes tumorigenesis of choriocarcinoma JAR cells. Troph Res 1999;13:147-59.

 

3. Kovalevskaya G, Genbacev O, Fisher SJ, Caceres E, O’Connor JF. Trophoblast origin of hCG isoforms: cytotrophoblasts are the primary source or choriocarcinoma-like hCG. Mol Cell Endocrinol 2002;94:147-55.

 

4. Cole LA, Shahabi S, Oz UA, Bahado-Singh RO, Mahoney MJ. Hyperglycosylated human chorionic gonadotropin (invasive trophoblast antigen) immunoassay: A new basis for gestational Down syndrome screening. Clin Chem 1999;45:2109-19.

 

5. Elliott MM, Kardana A, Lustbader JW, Cole LA. Carbohydrate and peptide structure of the a- and b-subunits of human chorionic gonadotropin from normal and aberrant pregnancy and choriocarcinoma. Endocrine 1997;7:15-32.

 

6. Cole LA. The O-linked oligosaccharides are strikingly different on pregnancy and choriocarcinoma hCG. J Clin Endocrinol Metab 1987;65:811-13.

 

7. Amano J, Nishimura R, Mochizuki M, Kobata A. Comparative study of the mucin-type sugar chains of human chorionic gonadotropin present in the urine of patients with trophoblastic diseases and healthy pregnant women. J Biol Chem 1988;263:1157-65.

 

8. Kobata A, Takeuchi M. Structure, pathology and function of the N-linked sugar chains of human chorionic gonadotropin. Biochim Biophys Acta. 1999;1455:315-26

 

9. Cole LA. Immunoassay of human chorionic gonadotropin, its free subunits, and metabolites. Clin Chem 1997;43:2233-43.

10. Takamatsu S, Oguri S, Toba Minowa M, Yoshida A, Nakamura K, Takeuchi M, Kobata A.

Unusually High Expression of N-Acetylglucosaminyltransferase-IVa in Human Choriocarcinoma Cell Lines: A Possible Enzymatic Basis of the Formation of Abnormal Biantennary Sugar Chain

Cancer Res 1999;59:3949-3953.

 

11. Peters BP, Krzesicki RF, Hartle RJ, Perini F, Ruddon RW A kinetic comparison of the processing and secretion of the alpha beta dimer and the uncombined alpha and beta subunits of chorionic gonadotropin synthesized by human choriocarcinoma cells. J Biol Chem. 1984;259:15123-30.

 

12. Hussa RO. Immunologic and physical characterization of human chorionic gonadotropin and its subunits in cultures of human malignant trophoblast. J Clin Endocrinol Metab 1977;44:1154-62.

 

13. Mann K, Karl HJ. Molecular heterogeneity of human chorionic gonadotropin and its subunits in testicular cancer. Cancer 1983;52:654-60.

 

14. Birken S, Krichevsky A, O’Connor J, Schlatterer J, Cole LA, Kardana A, Canfield R. 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. Endocrine 1999;10:137-44.

 

15. Pandian R, Lu J, Ossolinska-Plewnia J. Fully automated chemiluminometric assay for hyperglycosylated human chorionic gonadotropin (invasive trophoblast antigen). Clin Chem 2003;49:808-10.

 

16. Cole LA, Khanlian SA, Sutton JM, Davies S, Stephens N. hCG-H (Invasive Trophoblast Antigen, hCG-H) a Key Antigen for Early Pregnancy Detection. Clin Biochem, 2003;36:647-655

 

17. Khanlian SA, Smith HO, Cole LA. Persistent Low Levels of hCG: A Pre-malignant gestational trophoblastic disease. Am J Obstet Gynecol, 188: 1254-1259, 2003.

 

18. O’Connor JF, Ellish N, Kakuma T, Schlatterer J, Kovalevskaya G. Differential urinary gonadotropin profiles in early pregnancy and early pregnancy loss. Prenat Diagn 1998;18:1232–40.

 

19. Butler SA, Khanlian SA, Cole LA. Detection of early pregnancy forms of human chorionic gonadotropin by home pregnancy test devices. Clin Chem 2001;47:2131-06.

 

20. Cole LA, Khanlian SA, Sutton JM, Davies S, Rayburn WF. Accuracy of home pregnancy tests at the time of missed menses. Am J Obstet Gynecol 2004;190:100-05.

 

21. Genbacev O. DiFederico E. McMaster M. Fisher SJ. Invasive cytotrophoblast apoptosis in pre-eclampsia. Human Reproduction. 1999;2:59-66.

 

22. Tarrade A. Goffin F. Munaut C. Lai-Kuen R. Tricottet V. Foidart JM. Vidaud M. Frankenne F. Evain-Brion D. Effect of matrigel on human extravillous trophoblasts differentiation: modulation of protease pattern gene expression. Biology of Reproduction. 2002;67:1628-37

 

23. Paradinas FJ, Sbire NJ, Rees HC. Pathology. In: Hancock BW, Newlands ES, Berkowitz RS, Cole LA eds. Gestational Trophoblastic Disease. Sheffield University, Sheffield UK,  2003, pp. 77-129.

 

24. Tarrade A. Goffin F. Munaut C. Lai-Kuen R. Tricottet V. Foidart JM. Vidaud M. Frankenne F. Evain-Brion D. Effect of matrigel on human extravillous trophoblasts differentiation: modulation of protease pattern gene expression. Biology of Reproduction. 2002;67:1628-37

 

25. Sutton JM, Cole LA. Sialic acid-deficient invasive trophoblast antigen (sd-hCG-H): a new urinary variant for gestational Down syndrome screening. Prenat Diagn. 2004; 24: 194-197 

 

26. Cole LA ,Sutton JM, Higgins TN, Cembrowski GS. Between-method variation in hCG test results, Clin Chem, 2004; 50:874-882.

27. Cole LA. O-Glycosylation of proteins in the normal and neoplastic trophoblast. Troph Res 1987;2:139-148.

 

28. Kovalevskaya G, Birken S, Kakuma T, Osaki N, Sauer M, Lindheim S, et al.  Differential expression of human chorionic gonadotropin (hCG) Glycosylation Isoforms in failing and continuing Pregnancies: Preliminary characterization of the hyperglycosylated hCG Epitope.  J Endocrinol 2002; 172: 497-506.

 

29. Sutton-Riley JM, Khanlian SA, Byrn FW, Cole LA. Hyperglycosylated hCG: A Single Serum Test for Measuring Early Pregnancy Outcome with High Predictive Value. Clin Biochem 2005; in press.

 

30. Byrn FW, Sutton-Riley JM, Cole LA. The Predictive Value of Hyperglycosylated Human Chorionic Gonadotropin (H-hCG) in Evaluating Pregnancy Outcome. Fertil Steril 2005; in press.

 

31.     Cole, L.A., Cermik D., Bahado-Singh, R. Oligosaccharide variants of hCG-related molecules: Potential screening markers for Down syndrome. Prenat. Diagn. 1997;17:1188-1190. 

 

32.     Cole, L.A., Omrani, A., Cermik, D., Bahado-Singh, R.O., and Mahoney, R.O. Hyperglycosylated hCG, a potential alternative to hCG in down syndrome screening. Prenat. Diagn., 1998;18:926-933.

 

33.     Massin N Frendo JL, Luton D, Govagrandi Y, Muller F, Vidaud M, Evain-Brion D. Defect of syncytiotrophoblast formation and hCG expression in Down’s syndrome. Placenta 2001;22:S93-97.

 

34.     Frendo JL, Vidaud M, Guibourdenche J, Luton D, Muller F, Belet D, Giovagrandi Y, Tarade A, Porquet D, Blot P, Evain-Brion D. Defect of villous cytotrophoblast differentiation in syncytiotrophoblast in Down’s syndrome. J Clin Endocr Metab 2000;85:3700-07,

 

35.     Evain-Brion D, Frendo JL, Vidaud M, Muller F,. Failure of differentiation of the trophoblast in trisomy 21. Bull Acad Natl Med 2000;184:1033-45.

 

36.     Cole, L.A. Shahabi, S., Rinne, K.M., Oz, U.A., Bahado-Singh, R.O., Mahoney, M.J. Urinary Screening Tests for Fetal Down Syndrome: II. Hyperglycosylated hCG. Prenat Diagn, 1999;19:351-359.

 

37.     Shahabi, S., Rinne, Oz, U.A., Bahado-Singh, R.O., Mahoney, M.J., Omrani, A., Baugarten, A., and Cole, L.A. Serum hyperglycosylated hCG a potential screening test for fetal Down syndrome. Prenat Diagn, 1999;19:488-489.

 

38.     Palomaki GE. Neveux LM. Knight GJ. Haddow JE. Pandian R. Maternal serum invasive trophoblast antigen (hyperglycosylated hCG) as a screening marker for Down syndrome during the second trimester. Clin Chem 2004; 50:1804-8.

 

39.     Cuckle, H.S., Shahabi, S., Sehmi, I., Jones, R., and Cole, L.A. Maternal urine hyperglycosylated hCG in pregnancies with Down’s Syndrome. Prenat Diagn 1999;19: 918-920.

 

40.     Weinans  JN, Butler SA, Mantingh A, Cole LA. Urinary hyperglycosylated hCG in first-trimester screening for chromosomal abnormalities. Prenat Diagn 2000;20:976-978

 

41. Reynolds TM. Down's syndrome screening: a controversial test, with more controversy to come! J Clin Path 2000;53:893-898

 

42. Strom CM, Palomaki GE,  Knight GJ, Cole LA, Pandian R. Maternal urine Invasive Trophoblast Antigen (ITA) is a useful marker for Down syndrome in the 1^st trimester. 51^st Ann Meet Am Soc Hum Gen, San Diego, 2001 (Abstract 2839).

 

43. Weinans MJ. Sancken U. Pandian R. van de Ouweland JM. de Bruijn HW. Holm JP. Mantingh A. Invasive trophoblast antigen (hyperglycosylated human chorionic gonadotropin) as a first-trimester serum marker for Down syndrome. 2005; Clin Chem 51:1276-9.

 

44. Cole LA, Butler SA, Khanlian SA, Giddings A, Seckl MJ,  Kohorn EI. Gestational Trophoblastic Diseases: 2. Hyperglycosylated hCG as a Reliable Marker of Active Neoplasia. Gyn Oncol 2005; in press.

 

45. Cole LA, Khanlian SA Inappropriate management of women with persistent low hCG results. J Reprod Med 2004; 49: 423-432.

 

46.  Khanlian SA, Smith HO, Cole LA. Persistent low levels of hCG: A Pre-malignant gestational trophoblastic disease. Am J Obstet Gynecol 2003; 188: 1254-1259.

 

47. Cole, LA, Sutton JM. hCG tests in the management of gestational trophoblastic diseases. Clin Obstet Gynecol 2003; 46: 533-540.

 

48. Cole LA. Use of hCG Tests for evaluating trophoblastic diseases: Choosing an appropriate hCG assay, false detection of hCG, unexplained elevated hCG, and quiescent trophoblastic disease. In: Gestational Trophoblastic Disease 2nd edition, eds. Hancock BW, Newlands ES, Berkowitz RS and Cole LA, Sheffield University Press, 130-155, 2003

 

49. Cole LA, Sutton JM: Selecting an Appropriate hCG test for management of gestational trophoblastic diseases and cancer cases. J Reprod Med, 2004; 49: 545-553.

 

50. Hancock BW, Tidy JA. Clinical management of persistent low level  hCGelevation. Trophobl Dis Upd 4: 5-6

 

51. Kohorn EI. Persistent low-level “real” human chorionic gonadotropin: a clinical challenge and a therapeutic dilemma. Gynecol Oncol, 2002; 85: 315-20

 

52. Pandian R. Cole LA. Palomaki GE. Second-trimester maternal serum invasive trophoblast antigen: a marker for Down syndrome screening. Clin Chem. 50:1433-5, 2004

 

53. Palomaki GE, Knight GJ, Neveux LM, Pandian R, Haddow JE. Maternal serum invasive trophoblast antigen and first-trimester Down syndrome screening. [Journal Article. Multicenter Study] Clinical Chemistry. 51:1499-504, 2005.

 

54. Weinans MJ, Sancken U, Pandian R, van de Ouweland JM, de Bruijn HW, Holm JP, Mantingh A. Invasive trophoblast antigen (hyperglycosylated human chorionic gonadotropin) as a first-trimester serum marker for Down syndrome. [Journal Article] Clinical Chemistry. 51:1276-9, 2005.

 

55. Palomaki GE, Neveux LM, Knight GJ, Haddow JE, Pandian R. Maternal serum invasive trophoblast antigen (hyperglycosylated hCG) as a screening marker for Down syndrome during the second trimester. [Journal Article] Clinical Chemistry. 50:1804-8, 2004.

 

Links to other pages on the USA hCG Reference Service Website

 

A. hCG Reference Service HOME PAGE

B. hyperglycosylated hCG

C. pituitary hCG

D. synthesis

E. heterophilic antibodies

F. For further information on Gestational Trophoblastic Disease (GTD) and on the worlds GTD experts

G. For the USA hCG Reference Service detailed protocol

1. False positive hCG

2. Active invasive gestational trophoblastic disease

3. Quiescent (inactive) gestational trophoblastic disease

4. Active testicular germ cell malignancies

5. PSTT (Placental site trophoblastic tumor)

6. Ovarian germ cell and other non-trophoblastic hCG-producing
malignancies

7. Pituitary origin hCG in peri- or post-menopausal women

8. Ectopic pregnancy or spontaneously-aborting pregnancy