Human Reproduction Update Advance Access originally published online on July 28, 2006
Human Reproduction Update 2006 12(6):769-784; doi:10.1093/humupd/dml029
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The classification, functions and clinical use of different isoforms of HCG
1 Department of Clinical Chemistry and 2 Department of Gynecology and Obstetrics, Helsinki University Central Hospital, Helsinki, Finland
3 To whom correspondence should be addressed at: Department of Clinical Chemistry, Helsinki University Central Hospital, Helsinki, FIN-00027 HUS, Finland. E-mail: ulf-hakan.stenman{at}hus.fi
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Abstract |
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HCG is composed of two subunits, HCG
and HCGß. During early pregnancy, HCG stimulates progesterone production in the corpus luteum, and injection of HCG is widely used to induce ovulation in assisted reproduction treatment (ART). Under experimental conditions, the free subunits have been shown to exert functions other than those of HCG, but the relevance of these remains to be determined. Intact HCG, free subunits and degraded forms of these occur in biological fluids, and determinations of these are important for diagnosis and monitoring of pregnancy, pregnancy-related disorders and several types of cancer. Development of optimal methods for the various forms has been hampered by lack of appropriate standards and expression of the concentrations of the various forms in units that are not comparable. Furthermore, the nomenclature for HCG assays is confusing and in some cases misleading. These problems can now be solved; a uniform nomenclature has been established, and new standards are available for HCG, its subunits HCG and HCGß, the partially degraded or nicked forms of HCG and HCGß, and the beta-core fragment. This review describes the biochemical and biological background for the clinical use of determinations of various forms of HCG. The clinical use of HCG and studies on HCG vaccines are briefly reviewed.
Key words: cancer / Down’s syndrome / HCG isoforms / pregnancy / trophoblastic disease
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Introduction |
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The first assay for HCG described in 1927 was based on the biological activity of HCG partially purified from urine (Ascheim and Zondek, 1927
). Various modifications and improvements of this assay were used to diagnose pregnancy until immunoassays gradually replaced bioassays (Wide and Gemzell, 1960; Vaitukaitis et al., 1972). The introduction of monoclonal antibodies facilitated establishment of specific assays for subunits and various degraded forms of HCG, and these have widened the applications of HCG assays for diagnosis of pregnancy, trophoblastic disease and non-trophoblastic cancers (Stenman et al., 2004). Thanks to the high concentrations of HCG in urine during pregnancy, urine has been used for the preparation of HCG for clinical use, and partially purified urinary HCG plays an important role in assisted reproduction treatment (ART). This use prompted establishment of standards for HCG, which were assigned values based on bioactivity. All the international standards (ISs) for HCG have been calibrated by bioassay (Storring et al., 1980). Although not ideally suited, these standards have also been used for standardization of immunoassays (Stenman et al., 1993). Furthermore, the nomenclature used to describe HCG assays is confusing and often misleading. To solve these problems, the International Federation of Clinical Chemistry (IFCC) established a working group in 1995 with the aim of improving standardization of HCG determinations; the task included establishment of a uniform nomenclature and preparation of new standards. At that time, the clinically important variants were HCG and its subunits HCG and HCGß, the partially degraded or nicked forms of HCG (HCGn) and HCGß (HCGßn), and the beta-core fragment (HCGßcf) (Stenman et al., 1993). This nomenclature has been adopted by IFCC and the aim is to get it accepted by the scientific community. In addition to these variants, recently described glycosylation variants are of potential clinical utility. This review describes the classification and functions of various forms of HCG and the clinical use of HCG assays for diagnosis of pregnancy, pregnancy-related disorders and gynaecological cancers. Recent developments in the therapeutic use of HCG and studies on HCG vaccines are also described. The aim has been to critically discuss how earlier findings can be interpreted in the light of recent advances in biology and assay technology. |
Biochemistry of HCG |
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HCG is a member of the glycoprotein hormone (GPH) family, which also comprises LH, FSH and TSH. All GPHs are heterodimers consisting of an
-subunit (GPH) and a ß-subunit. The -subunit, which contains 92 amino acids, is common to all GPHs. The ß-subunits confer biological activity and display various degrees of homology, which between HCG and LH is 
80%. LHß contains 121 amino acids whereas HCGß contains 145 amino acids, the difference being due to a 24-amino-acid extension, the so-called C-terminal peptide (CTP) (Pierce and Parsons, 1981).
One-third of the mass of HCG is made up by eight carbohydrate moieties, of which six are attached to HCGß and two to HCG. The N-linked carbohydrate chains on HCG are attached to Asn52 and Asn78 and those on HCGß to Asn13 and Asn30. Four O-linked oligosaccharides are attached to Ser121, Ser127, Ser132 and Ser 138 on the CTP of HCGß (Kessler et al., 1979a,b; Elliott et al., 1997). Owing to variation in the content of terminal sialic acid, HCG displays extensive charge heterogeneity with isoelectric point (pI) values ranging from 3 to 7. HCGß is more acidic (pI range 3–5) than HCG (pI range 5–8) (Graesslin et al., 1973; Birken et al., 2003). In pregnancy, the N-linked carbohydrates on HCG are mainly monoantennary and biantennary and those on HCGß are biantennary and to a lesser extent triantennary. The carbohydrates on the CTP are mainly monoantennary type 1 o-core oligosaccharides. In HCG produced by cancerous tissues, most of the N-linked carbohydrates are complex containing more triantennary moieties on HCGß (Mizuochi et al., 1983) and biantennary on HCG, whereas type 2 o-core oligosaccharides occur on CTP (Elliott et al., 1997; Birken, 2005). This so-called hyperglycosylated HCG (HCGh) is also a major form in early pregnancy (Kovalevskaya et al., 2002a). It is produced by cytotrophoblasts, which dominate in the early placenta, whereas syncytiotrophoblasts, which are the main trophoblasts later in pregnancy, produce ‘normally glycosylated’ HCG (Kovalevskaya et al., 2002b). In addition to these differences, HCG produced by trophoblastic cancers occasionally displays reduced content of sialic acid (Nishimura et al., 1981; Imamura et al., 1987), but variants with unusually low pI values indicating increased content of sialic acid have also been described (Yazaki et al., 1987). Thus, various forms of aberrant glycosylation are common in tumour-derived HCG (Kobata and Takeuchi, 1999).
Because of heterogeneity of the CHO moieties, the molecular weight (MW) displays a spectrum of values. The average MW of HCG determined by MALDI-TOF mass spectrometry is 37 500, that of HCG is 14 000 and that of HCGß 23 500 (Birken et al., 2003). The calculated mass of the peptide moiety of HCG is 10 206 and that of the glycosylated subunit containing two biantennary N-linked sialylated oligosaccharides is 14 165. The MW of the HCGß peptide is 15 532 and that containing two N-linked biantennary CHO chains and four type 1 o-core carbohydrates is 24 316. Thus, the calculated MW of a ‘typical’ HCG molecule would be 38 931. The difference between the calculated and measured average MW indicates that the CHO chains are on average smaller than those used to calculate the theoretical MW. The MW of HCG produced by trophoblastic cancer is higher than that of pregnancy HCG (Mann and Karl, 1983), which is explained by larger carbohydrate chains (Elliott et al., 1997; Birken, 2005).
Part of the heterodimeric HCG in urine is nicked (HCGn), i.e. the peptide chain is cleaved at various positions between amino acids 44 and 48. This nicked HCG may also occur in the serum of cancer patients (Cole et al., 1991; Jacoby et al., 2000). Part of the HCGß isolated from urine is also in the HCGßn form. HCG lacking the CTP may occur in urine of some cancer patients (Cole et al., 1982).
Most of the HCG immunoreactivity in urine from pregnant women (Matthies and Diczfalusy, 1971) and cancer patients (Papapetrou et al., 1980; Wehmann and Nisula, 1980) consists of the beta-core fragment, HCGßcf (Stenman et al., 1993), which has been shown to comprise amino acids 6–40 and 55–92 linked by disulphide bridges. The carbohydrate moieties on Asn 13 and 30 are smaller than in those on intact HCG (Birken et al., 1988).
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Genes |
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Six non-allelic genes clustered on chromosome 19q13.3 encode HCGß, and a seventh gene encodes LHß (Fiddes and Goodman, 1980
). Genes ß1 and ß2 are thought to be pseudogenes that are not expressed; ß4 encodes LH whereas ß7 and ß9 are alleles to ß6 and ß3, respectively. Type I genes (ß6/ß7) encode a protein with alanine at position 117 whereas HCGß encoded by type II genes (ß3/ß9, ß5 and ß8) contains aspartic acid at this position. This heterogeneity is not known to affect function or immunoreactivity. Type I genes are mainly expressed in benign non-trophoblastic tissues whereas type II genes are expressed by trophoblastic and malignant tissues (Bellet et al., 1997). A single gene on chromosome 12q21.1–23 encodes GPH (Fiddes and Goodman, 1981). |
Function |
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HCG mediates its action through the LH/HCG receptor, and its major function is to maintain the progesterone production of corpus luteum during early pregnancy. Various other tissues also express the LH/HCG receptor, and its presence in the vasculature of the uterus may indicate that HCG exerts a physiologically important function in this tissue. The receptor is also expressed in a large number of tissues other than the ovary, and thus HCG and LH may have hitherto unknown functions. Known and putative functions of HCG have recently been extensively reviewed (Filicori et al., 2005
).
HCGh is produced by cytotrophoblast during early pregnancy (Kovalevskaya et al., 2002b). Because these cells display invasive properties (Red-Horse et al., 2004), HCGh has also been called invasive trophoblast antigen (ITA) (Cole et al., 1999). However, there is so far no evidence for an invasive function and no receptor other than the LH/HCG receptor has been identified.
HCGß lacks HCG activity, but several lines of study indicate that it exerts growth-promoting activity. It enhances growth of bladder cancer cells, and antibodies to HCGß inhibit this effect (Gillott et al., 1996; Butler et al., 2003). In rat breast cancer cells, HCGß has been shown to induce apoptosis (Srivastava et al., 1997) and inhibition of HCGß expression with antisense messenger RNA suppresses cell proliferation and induces apoptosis in choriocarcinoma cells (Hamada et al., 2005). However, a mechanism mediating this activity has not been found, but based on structural similarity, it has been speculated that HCGß interferes with the growth-inhibiting effect of transforming growth factor (TGF)-ß, platelet-derived growth factor (PDGF)-B and nerve growth factor (Butler and Iles, 2004).
HCG (GPH) also does not exert HCG activity, but it has been shown to stimulate prolactin production in decidual cells (Blithe et al., 1991; Moy et al., 1996). Furthermore, endometrial cells induce dissociation of HCG into subunits, and together with progesterone, the released HCG may mediate decidualization of these cells (Nemansky et al., 1998).
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Metabolism |
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The clearance of HCG from circulation has been studied both after injection of purified HCG and after pregnancy. The half-life of injected HCG is biphasic; the rapid phase has a half-life of 5–6 h whereas that of the slower phase is 24–33 h (Rizkallah et al., 1969
; Wehmann and Nisula, 1981). Similar clearance rates have been observed after an abortion and term pregnancy, but the clearance is best described by a triphasic model with median half-lives of 3.6, 18 and 53 h (Korhonen et al., 1997). The half-life of purified HCGß injected into humans is 0.7 and 10 h, which is shorter than that of HCG (Wehmann and Nisula, 1979). However, after term pregnancy or an abortion, HCGß actually disappears more slowly than HCG with half-lives of 1, 23 and 194 h. Thus, the proportion of HCGß of total HCG immunoreactivity increases from 0.8% at term to 27% after 3 weeks. The half-life of HCG is shorter than that of HCGß, and after term pregnancy half-lives of 0.6, 6 and 22 h have been observed (Korhonen et al., 1997). These half-lives are longer than those observed after injection of purified HCG, i.e. 0.1–0.22 and 1.2–1.3 h (Wehmann and Nisula, 1979; Blithe and Nisula, 1987). The discrepancy between half-lives determined for injected and naturally occurring subunits may indicate that the purified forms have been partially denatured during purification, and they are therefore metabolized more rapidly. It is also possible that the slower metabolism of endogenous free subunits is explained by differences in glycosylation.
Most of the HCG in circulation is metabolized by the liver, whereas about 20% is excreted by the kidneys (Nisula et al., 1989). During excretion, a major part of HCG is degraded to subunits, nicked forms and especially HCGßcf (Wehmann and Nisula, 1980; Nisula et al., 1989). The proportion of HCGßcf in urine is low in early pregnancy and starts to exceed that of HCG at 5 weeks of pregnancy (Figure 1). In the second trimester, about 80% of the HCG immunoreactivity in urine consists of HCGßcf (Norman et al., 1987). During pregnancy, HCGßcf can be detected in plasma (Nisula et al., 1989; de Medeiros et al., 1992b), but the concentrations are only 0.01% of those of HCG (Alfthan and Stenman, 1990). After injection of urinary HCG, HCGß or recombinant HCG (rHCG), HCGßcf is detected in urine (Nisula et al., 1989), but peak concentrations occur 6 h after the HCG peak in urine (Norman et al., 2000). HCGßcf can also be detected in the pituitary (Hoermann et al., 1995) and in follicular fluid and trophoblast culture fluid (de Medeiros et al., 1992a), and some HCGßcf is present in the placenta (Udagawa et al., 1998). Thus, some of the HCGßcf in urine can be derived from metabolism in these tissues, but studies on the metabolic clearance rate of HCGßcf show that >99% is formed in the kidneys during renal excretion (Wehmann et al., 1989).
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Standards for HCG, its subunits and fragments |
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The World Health Organization (WHO) has issued several sets of standards for HCG, which have been calibrated in IU against the preceding set by bioassay. The presently used third and the identical fourth ISs were prepared in 1972. They have been available from NIH as CR119 and were initially issued by WHO as the 1st International Research Preparations (1st IRP) (Canfield and Ross, 1976
) and adopted as the third IS WHO standards in 1980 (Storring et al., 1980). They comprise standards for HCG (75/537), HCGß (75/551) and HCG (75/569). The HCG standard was calibrated by bioassay against the 2nd standard, and 1 µg of HCG in the third IS corresponds to 9.3 IU. Because the free subunits lack HCG activity, they were assigned values based on mass with 1 µg corresponding to 1 IU (Storring et al., 1980). Thus, the units are not comparable with those of HCG. Table I summarizes the relationship between the units for HCG and its subunits. When HCGß is measured by assays detecting HCG and HCGß together, the results are erroneously based on the IU for HCG.
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The preparations in the third IS have been found to be contaminated with partially degraded variants of HCG, which is a problem when they are used for standardization of immunoassays. To solve these problems, the IFCC founded a working group with the aim of improving standardization of immunoassays for HCG and related molecules (Stenman et al., 1993). As a result of this project, new standards for HCG and clinically important HCG-related molecules have been prepared and approved by the WHO as reference reagents for immunoassay (Table II). Purer preparations than those in third IS were produced by utilizing modern chromatographic techniques. However, WHO recommends that the third (and fourth) IS still should be used for calibration of immunoassays. The purification and value assignment of the reference reagents have been described (Birken et al., 2003; Bristow et al., 2005).
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Because immunoassays reflect molar concentrations of protein rather than bioactivity, the use of substance concentrations, i.e. mol/l is the most appropriate way of expressing the concentrations. This is especially important when two (or several) analytes of different MW are detected together or when their concentrations are compared (Stenman et al., 1993). This is a major reason why the reference reagents for HCG and related substances have been assigned values in molar concentrations based on amino acid analysis (Birken et al., 2003; Bristow et al., 2005). Furthermore, the carbohydrate composition has little effect on immunoreactivity, but it strongly affects bioactivity. Because the new reference preparations are more pure than the third IS, the bioactivity of the HCG preparation, about 11 000–16 000 IU/mg, is 20–70% higher than that of the third IS (Birken et al., 2003). Thus, it is obvious that value assignment on the basis of bioactivity would have resulted in a substantial change in immunoassay calibration. The higher bioactivity is explained by absence of biologically inactive HCGn and HCGßcf in the new HCG preparation. When the new reference reagents are used to calibrate immunoassays for HCG, HCGß, HCG and HCGßcf, the concentrations should be expressed in pmol/l.
The reference reagents comprise six forms of HCG which were considered clinically important when the project was initiated. Some of them, e.g. HCGn, are valuable for the characterization of antibody specificity. Antibodies that do not recognize HCGn will miss this variant, which occurs especially in urine but also in the serum of some cancer patients (Cole et al., 1991; Hoermann et al., 1994). HCGßn is likewise mainly of importance for the characterization of the epitope specificity of antibodies (Berger et al., 2002). HCGßcf is of potential value as a marker for cancer (Papapetrou et al., 1980) and pre-eclampsia (Bahado-Singh et al., 1998), but assays for this variant have not become clinically accepted. As HCGßcf represents a major part of the HCG immunoreactivity in urine of pregnant women, the standard is important for the characterization of pregnancy tests and other assays for HCG immunoreactivity in urine.
When the reference reagents were prepared, the potential utility of HCGh was not recognized and a standard for this variant was not prepared. If HCGh assays become clinically accepted, a standard will be desirable. However, identification of a suitable source is problematic; although HCGh occurs in urine during early pregnancy (Kovalevskaya et al., 1999), it is not yet known whether this HCGh is comparable with that occurring in patients with trophoblastic and other types of cancer. It is well known that the carbohydrate composition of HCG produced by tumours may vary considerably, and HCGh is therefore not a well-defined entity (Birken, 2005). Recently, HCGh has been named ITA (Cole et al., 1999), but so far there is no evidence for an invasive function and a separate name for this form is mainly motivated by commercial interests. This does not justify the introduction of a new name in the scientific literature.
rHCG is also available as a laboratory product and is a potential calibrator for immunoassay purposes. Part of the variation between different immunoassays is caused by the use of impure calibrators, and improved agreement may be obtained by replacing the calibrators in commercial assays by pure HCG preparations (Cole et al., 2004). However, this is only one cause of between-method discrepancies, careful selection of antibodies and assay design being equally important (Stenman et al., 1993; Berger et al., 2002). rHCG would not be useful for calibration of assays based on antibody B152, which recognizes HCGh.
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Therapeutic use of HCG |
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The midcycle LH surge is essential for normal oocyte maturation and ovulation. In ART, administration of partially purified urinary HCG preparations has been used for decades as a surrogate for LH to achieve final oocyte maturation and ovulation in controlled ovarian hyperstimulation (COH) protocols. This facilitates correct timing of oocyte retrieval in connection with IVF/ICSI treatments. Urinary HCG has been the drug of choice, but there are now other options, i.e. recombinant LH (rLH) and HCG (rHCG). rLH has been available for use in clinical trials for several years. A single dose of 15 000–30 000 IU of rLH gives the highest efficacy to safety ratio. Such a dose is comparable with 5000 IU of urinary HCG, and it effectively induces final follicular maturation and early luteinization in IVF embryo transfer patients (The European recombinant LH study group, 2001
). However, rLH has not been shown to be superior to HCG in clinical practice. Moreover, it has recently been suggested that replacement of urinary HCG by rLH in agonist cycles results in a significantly lower pregnancy rate (Aboulghar and Al-Inany, 2005).
For many years, the most widely used form of COH in IVF/ICSI protocols has been to use GnRH agonists to desensitize the pituitary and suppress gonadotrophin secretion, followed by ovarian stimulation with FSH and/or HMG. Presently, GnRH antagonists are an alternative in preventing premature LH surges in COH. Because the pituitary remains responsive to GnRH agonists, administration of a bolus of GnRH agonist induces an endogenous LH surge (Griesinger et al., 2006). This form of treatment has been suggested to prevent the ovarian hyperstimulation syndrome (OHSS) (Orvieto, 2005). In clinical studies, ovulation induction with the GnRH agonist buserelin resulted in significantly more mature oocytes, but significantly lower implantation and clinical pregnancy rates were obtained than those by conventional ovulation induction with urinary HCG (Humaidan et al., 2005). Moreover, the rate of early pregnancy loss was higher, probably due to luteal phase deficiency. This has been confirmed in other studies, and a lower probability of ongoing pregnancy was achieved with the GnRH agonist triptorelin than with urinary HCG (Kolibianakis et al., 2005). At present, HCG appears to be the most reliable way to trigger final oocyte maturation both in antagonist and in agonist cycles (Griesinger et al., 2006).
It seems that similar characteristics and dynamics of luteal phase estradiol (E2) and progesterone are obtained after ovarian stimulation for IVF using GnRH agonists or antagonists (Friedler et al., 2006), and thus luteal support is needed in both protocols. Luteal phase support after ART results in an increased pregnancy rate compared with placebo or no treatment. When HCG is compared with progesterone treatment, there is no significant difference in pregnancy rates but HCG is associated with a greater risk of OHSS (Daya and Gunby, 2004).
Urinary HCG can also be used for other clinical applications. Thus low-dose HCG has been used alone to complete controlled ovarian stimulation (Filicori et al., 2005). When used in the late stage of ovarian stimulation (after the follicles were 
12 mm), this reduces FSH/HMG consumption while fertilization outcome is comparable. Furthermore, HCG use is associated with a reduced number of small pre-ovulatory follicles, which could reduce the risk of OHSS (Filicori et al., 2005). Even more interesting is the effect of HCG on uterine receptivity. In a recent study, administration of HCG to oocyte recipients was shown to increase endometrial thickness on the day of embryo transfer and to improve the implantation rates (Tesarik et al., 2003). This suggests that HCG might affect endometrial function independently of ovarian function by stimulating endometrial growth and maturation and by enhancing endometrial angiogenesis, thereby extending the implantation window (Filicori et al., 2005).
Recently, HCG produced by recombinant techniques in Chinese hamster ovary cells has become commercially available. In ART, a dose of 250 µg of rHCG has been found to be equivalent (Chang et al., 2001) or at least as effective as 10 000 IU of urinary HCG in inducing final stages of oocyte maturation. Furthermore, the use of rHCG was associated with significantly better patient tolerance (Abdelmassih et al., 2005). The content of HCG in urinary preparations for therapeutic use is expressed in IU based on bioactivity whereas that of rHCG is expressed in mass units (Gervais et al., 2003), which complicates comparison of dosage. In animal and clinical studies, 250 µg of rHCG has been found to have the same biological activity as 5000 IU of urinary HCG (Gervais et al., 2003; Al-Inany et al., 2005). This would translate into a specific activity of 20 000 IU/mg, which is higher than the potency of 15 000 IU/mg of the most pure urinary HCG preparations presently available (Birken et al., 2003). However, the content of rHCG is based on the mass of the peptide moiety only (Gervais et al., 2003), and thus 250 µg corresponds to 360 µg of glycosylated HCG and a specific activity of 13 900 IU/mg. This is comparable with that of highly purified urinary HCG but, according to the earlier mentioned studies, the activity may be even higher. These calculations are based on the typical carbohydrate structure of urinary HCG, and the carbohydrates of rHCG are only slightly different; the N-linked carbohydrates are of the same type as those in urinary HCG, but part of the O-linked carbohydrate chains on the CTP is different (Gervais et al., 2003). So far, this has not been reported to cause any adverse effects indicating that the carbohydrate moieties are not immunogenic. The half-life of rHCG in circulation is similar to that of urinary HCG (Gervais et al., 2003).
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Immunological determination of HCG |
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 TOPAbstractIntroductionBiochemistry of HCGGenesFunctionMetabolismStandards for HCG, its...Therapeutic use of HCG Immunological determination of... Use of HCG assays...Use of HCG assays...ConclusionsReferences |
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Assay nomenclature
Because of the extensive homology between HCG and LH, the first radioimmunoassays (RIAs) for HCG based on polyclonal antisera also measured LH (Odell et al., 1967). A quite specific rabbit antiserum (SB6) to HCG was produced in 1972 by immunization with HCGß, and an RIA based on this antiserum detected HCG at concentrations down to 5 IU/l. (Vaitukaitis et al., 1972). This assay has been widely employed (Hussa, 1987), which probably explains why the expressions ‘ß-HCG assay’ or ‘HCG-beta assay’ have become commonly used. Initially, these names implied that the assays did not cross-react with LH, but presently, they are mostly used for assays measuring HCG and HCGß together. IFCC recommends that assays should be exactly defined according to what they measure, e.g. HCG and HCGß separately or together (Stenman et al., 1993). Thus, an HCGß assay should detect HCGß but not HCG.
Effect of antibody epitopes on assay specificity
Although specific assays for HCG and its various forms can be established with polyclonal antisera, virtually all presently used assays utilize monoclonal antibodies or a combination of a monoclonal antibody and a polyclonal antiserum. Monoclonal antibodies with known epitope specificity facilitate design of assays specific for each form of HCG (Bidart et al., 1985; Ehrlich et al., 1985; Norman et al., 1985; Schwartz et al., 1986; Alfthan et al., 1992a). The antigenic regions on HCG have been extensively defined; five epitopes can be discerned on HCG (1–5) and seven on HCGß in intact HCG (ß1–ß5, ß8–ß9). Two epitopes, ß8 and ß9, positioned on the CTP, are completely specific for HCG and HCGß, and antibodies recognizing these epitopes are therefore used in many commercial assays. Four epitopes (C1–C4) are specific for heterodimeric HCG. Two of these conformation-dependent epitopes, C1 and C2, are lost in HCGn (Hoermann et al., 1994). Two epitopes are specific for each free subunit, 6 and 7 for HCG and ß6 and ß7 for HCGß (Berger et al., 1996; Berger et al., 2002).
The epitopes of 28 antibodies from various manufacturers and research groups have been determined in a collaborative study. On the basis of this information, the specificity of an assay in which these antibodies are used can be deduced (Berger et al., 2002). Exact information on assay specificity is obtained by analysing the reference reagents with the final assay (Birken et al., 2003).
Most epitopes are not dependent on variation in carbohydrate composition (Schwartz et al., 1991; Lottersberger et al., 2003), but two monoclonal antibodies have been shown to recognize certain carbohydrate variants on HCG. Antibodies B152 and CTP 104, which were prepared against aberrantly glycosylated HCG isolated from the urine of a choriocarcinoma patient, recognize carbohydrate epitopes (Kovalevskaya et al., 1999). That of B152 comprises the biantennary core 2 o-glycan on Ser132 and adjacent peptide structures whereas CTP 104 reacts with a sialylated glycan on Ser138 (Birken, 2005). A number of recent studies suggest that assay of HCGß is clinically useful in certain clinical conditions. The expression ‘hyperglycosylated’ was initially used to denote HCG containing complex carbohydrates (Elliott et al., 1997), but it is presently mainly used to denote HCG determined by assays using antibody B152. This nomenclature is an oversimplification of the actual complexity of the carbohydrate heterogeneity of HCG (Birken, 2005).
Design of assays for various clinical purposes
Serum samples are preferred for quantitative HCG determinations whereas urine samples are mainly used for pregnancy tests. Because both HCG and HCGß may occur in serum, most serum assays are designed to measure these together. Presently, virtually all commercial assays are based on the sandwich principle (Cole et al., 1997), and some assays utilize an antibody to CTP in combination with another antibody to HCGß. Such assays show no cross-reactivity with LH, but because CTP antibodies tend to have only moderate affinity (Berger et al., 2002), these assays are not very sensitive. Furthermore, HCG lacking CTP, which may occur in cancer (Cole et al., 1982), is not detected. Some assays detect HCG and HCGß fairly equally, but there are still considerable differences in this respect (Cole et al., 2004). Assays specific for HCG can be designed by using one antibody to HCGß (usually to capture HCG) together with an antibody to HCG as a tracer. Some of the most sensitive assays for HCG are based on this principle (Pettersson et al., 1983; Alfthan et al., 1992a), and they are also easy to standardize (Stenman et al., 1993).
Assays detecting HCG, HCGß and HCGßcf together have advantages for the measurement of HCG immunoreactivity in urine (Cole and Butler, 2002; McChesney et al., 2005). Immunoassays based on the binding inhibition principle, i.e. classical RIAs, mostly recognize all these forms, but few commercial HCG assays based on the sandwich principle do so (Cole et al., 2001; Cole et al., 2004). Urine samples are mainly useful for the identification of false-positive results in serum samples (Stenman et al., 2004), but for instance in the UK, they are also used to detect a relapse after treatment of trophoblastic tumours in outpatients (Mitchell, 1999).
Specific and sensitive assays for HCGß can be developed by using monoclonal antibodies (Ozturk et al., 1987; Alfthan et al., 1988; Marcillac et al., 1992; de Medeiros et al., 1992a). Most commercially available HCGß assays are intended for maternal screening of Down’s syndrome, and being optimized for the high serum concentrations occurring in pregnancy, they are not well suited for the determination of the low levels of HCGß that typically occur in the serum of cancer patients (Stenman et al., 2004).
Many non-trophoblastic tumours produce HCGß, most of which is degraded to HCGßcf when excreted into urine (Alfthan et al., 1992b). Several assays for HCGßcf in urine have been described and shown to be useful especially for the detection of gynaecological cancers (Cole et al., 1988; O’Connor et al., 1988; Alfthan et al., 1992a; de Medeiros et al., 1992a; Neven et al., 1993). Some assays measure several degraded forms of HCG (de Medeiros et al., 1992a), which collectively have been called urinary gonadotrophin fragments (Cole et al., 1988) and urinary gonadotrophin peptides (Schwartz et al., 1996). The use of these assays for monitoring of cancer is hampered by large day-to-day variation in the urine concentrations of HCGßcf, which is not eliminated by normalization against urinary creatinine (Ngan et al., 1995). Because of these problems, commercial assays for HCGßcf are presently not widely available. In patients with non-trophoblastic cancers, the concentrations of HCGßcf in urine reflect those of HCGß in plasma (Alfthan et al., 1992b) and, when measured by a highly sensitive assay, HCGß in serum is the more accurate marker (Alfthan et al., 1992b).
The most common use of HCG determinations is the detection of pregnancy with a semi-quantitative pregnancy test, which mostly is performed on a urine sample, but serum, plasma or whole blood can also be used with some tests. The pregnancy test is considered one of the most useful and reliable laboratory tests available (Chard, 1992). The first immunological pregnancy test based on haemagglutination inhibition had a detection limit of 500 IU/l and took 1.5 h to perform (Wide and Gemzell, 1960). The agglutination assays were gradually replaced by more sensitive and rapid enzyme immunoassays, which facilitated detection of HCG at concentrations down to 25 IU/l in 5–10 min (Wide, 2005). Presently, most pregnancy tests are based on immunochromatography and have a claimed sensitivity of 25–50 IU/l. However, some are actually more sensitive, detecting HCG at concentrations <10 IU/l while the detection limit of other pregnancy tests is in the range 100–200 IU/l (Cole et al., 2005).
The optimal sensitivity of a pregnancy test is debated; the more sensitive a test is, the earlier it detects a pregnancy, but because HCG may occur in serum and urine at concentrations up to 10–15 IU/l in non-pregnant women, a detection limit around 25 IU/l is considered optimal (Chard, 1992; Stenman and Alfthan, 2003). Determination of the sensitivity of a pregnancy test depends on the standards used. Much of the HCG immunoreactivity in urine consists of HCGßcf, but during the 5–7 first weeks of pregnancy, i.e. when pregnancy tests are used, HCG is the dominating form (Figure 1). Free subunits are also of minor importance, but failure to recognize HCGh and degraded forms of HCG, e.g. HCGn, is a potential problem (Butler et al., 2001). However, nicking of HCG occurs mainly during storage of urine (Birken et al., 2001), which is not a problem in pregnancy testing. Some pregnancy tests have been shown to underestimate HCGh (Butler et al., 2001), but these studies were performed on spiked samples and it remains to be determined whether this is a problem in fresh urine samples. There is considerable variation in the performance of pregnancy tests sold to the general public (Cole et al., 2005), but little information is available on the performance of presently used pregnancy tests for professional use.
Serum from men and non-pregnant women contain low levels of HCG and HCGß that can be detected by sensitive assays. Pituitary GPH is produced in excess of the ß-subunits, relatively high levels occur normally, and they increase after the menopause. However, GPHa is not increased in patients with non-trophoblastic cancers that produce HCGß (Braunstein et al., 1979). Assay of GPH has been reported to be useful for the diagnosis of testicular cancer (Mann and Karl, 1983), but this application is unusual and generally applicable reference values have not been established. Suppression of gonadotrophin secretion with GnRH analogues causes a continuous increase in GPH when the pituitary gonadotrophin production decreases (Unkila-Kallio et al., 2000). Thus, the regulation of GPH expression in the pituitary is different from that of the GPHß subunits.
The HCG levels expressed in IU/l are about 3–10% of those of LH, and they increase after menopause in women and after age 60 in men (Alfthan et al., 1992a). The secretion of pituitary HCG is regulated by GnRH, and elevated levels in post-menopausal women are suppressed by estrogen treatment (Stenman et al., 1987). The serum concentrations fluctuate in a pattern similar to that of LH (Odell and Griffin, 1987). Taken together, these results indicate that most HCG in normal serum is derived from the pituitary (Stenman et al., 1987). Low-level expression of the genes for both HCG subunits occurs in the testis, breast, prostate and skeletal muscle (Bellet et al., 1997), but it is not known whether these tissues contribute to the levels of HCG in circulation.
The concentrations of HCGß in the serum of healthy men and non-pregnant women are low and measurable in only some of the samples even with the most sensitive assays available. The concentrations do not increase with age (Alfthan et al., 1992a). The genes for HCGß are expressed at very low levels in many tissues without concomitant expression of HCG, e.g. bladder, adrenal, colon, thyroid and uterus, but the source of the ‘normal’ levels of HCGß in serum is not known (Bellet et al., 1997).
Table III summarizes the reference values for HCG and HCGß in serum and urine. To facilitate comparison, we expressed the concentrations in substance concentrations (pmol/l) and for HCG also in IU/l. One IU corresponds to 0.11 µg and 2.9 pmol of HCG, and thus the values in pmol/l are roughly 3-fold those in IU/l. The conversion factors for HCGß are quite different, with 1 IU corresponding to 1 µg and 42.5 pmol. The concentrations of HCGß are actually seldom expressed in IU/l based on its own standard (the third IS) but rather in pmol/l (Stenman et al., 1993).
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The upper reference limits for HCG based on the 97.5 percentile are 3 and 5.4 IU/l in fertile and post-menopausal women, respectively, and those for men below and above 60 years of age are 0.7–2.1 IU/l, respectively (Table III). However, occasional values up to 8 IU/l are observed in women and up to 5 IU/l in men. These values have been determined by highly sensitive immunofluorometric assays (Alfthan et al., 1992a). Most commercially available assays are less sensitive and because of this and variation in assay calibration, higher upper reference limits need to be used for many assays. In a recent study on 720 women using two different assays, occasional values up to 14 IU/l were observed in post-menopausal women (Snyder et al., 2005). Many laboratories and textbooks recommend an upper reference limit of 5 IU/l, but it is important to recognize that higher levels may be observed especially in post-menopausal women. Because of differences in calibration and specificity between various methods, it is desirable that reference values are determined separately for each assay.
Reference values for various forms of HCG in urine of men and non-pregnant women are summarized in Table III. The concentrations of HCG and HCGß in urine are similar to or slightly higher than those in serum. It is notable that the concentrations of HCGßcf are similar to those of HCG, and thus the concentration of total HCG immunoreactivity is slightly higher in urine than in serum of women and about two-fold higher than in men. This has to be taken into account when assays for HCG that recognize HCG, HCGß and HCGßcf together are used to measure urine concentrations. These will give higher values than methods recognizing only HCG or HCG and HCGß together. However, reference values for such methods have not been established. The values in Table III have been determined by specific methods and are strictly valid only for these (Alfthan et al., 1992a). Other than the pregnancy test itself, urine measurements are seldom used for monitoring of pregnancy or cancer, but the reference limits are of value when urine samples are used to confirm false-positive results with serum assays.
Reference values during pregnancy
The serum concentrations of HCG start increasing 7–10 days after the LH peak or 4–7 days after implantation (Lenton et al., 1982). During early pregnancy, the HCG concentrations increase exponentially doubling on average every 1.5–2 days, but the rate of increase varies individually and maximum concentrations ranging from 20 000 to 100 000 IU/l are reached at 7–10 weeks of pregnancy. After this, the levels decrease levelling out at 13–15 weeks and increase moderately again until weeks 30–33, after which there is a moderate decrease towards term (Alfthan et al., 1988) (Figures 1–3).
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The serum concentrations of HCGß correlate strongly with those of HCG, but the proportion of HCGß changes during pregnancy. In early pregnancy, it may reach 4% but it drops rapidly to <1% (Figure 4). Accurate reference ranges for HCG and HCGß during pregnancy are especially important for the diagnosis of Down’s syndrome, and owing to the rapid changes during pregnancy, the values are expressed as multiples of the median for each week (or even day) of pregnancy (discussed below). Because of between-method differences in calibration, it is essential that the reference values be established separately for each assay.
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The HCG immunoreactivity in pregnancy urine is more heterogeneous than that in serum, and therefore the results are strongly dependent on the specificity of the assay used. When measured by specific assays, the HCG concentrations in urine correlate strongly with those in serum. In early pregnancy, the average urine concentrations are 50–70% of those in serum, but after the fifth week, the urine-to-serum ratio of HCG decreases and HCGßcf becomes the dominant form in urine (Figure 1) (Norman et al., 1987). In most studies, HCGß has been found to be a minor component in urine (Norman et al., 1987). The proportions of the various immunoreactive forms vary considerably from day to day, whereas the sum of the various forms shows less variation indicating that loss of HCG is reflected by increased levels of degraded forms such as HCGß and HCGßcf (McChesney et al., 2005) (Figure 1). The large day-to-day and within-day variation limits the clinical utility of quantitative urine assays. The variation in urinary HCG concentration can be reduced by normalizing against urine density or urine creatinine concentration. However, this eliminates only part of the variation and serum assays are therefore better suited for quantitation. Because quantitative urine assays are not used for monitoring of pregnancy, appropriate reference values have not been established.
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Use of HCG assays in pregnancy |
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Monitoring of pregnancy
Quantitative determinations of HCG are used to predict complications especially in early pregnancy, e.g. pregnancy loss and ectopic pregnancy. About 20–30% of all pregnancies end in an early pregnancy loss, which often takes place before the pregnancy is clinically recognized (Wilcox et al., 1988). This condition became generally recognized when rapid and sensitive HCG assays were introduced into clinical practice, but initially an elevated HCG value in young women without evidence of pregnancy was often classified as a false-positive test (Seppälä et al., 1980). Early pregnancy loss, which also is called ‘biochemical pregnancy’ (Walker et al., 1988), is now well recognized. This condition is associated with lower than expected HCG levels, and assay of HCG in serum is used to identify it especially in connection with ART (Figure 5).
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