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Human chorionic gonadotropin: Biochemistry and measurement in pregnancy and disease

Human chorionic gonadotropin: Biochemistry and measurement in pregnancy and disease
Literature review current through: Jan 2024.
This topic last updated: Jun 23, 2023.

INTRODUCTION — Human chorionic gonadotropin (hCG) is an important biomarker for detection of pregnancy and pregnancy-related disorders, as well as a useful tumor marker, particularly in the management of trophoblastic disease and germ cell neoplasias. It is also a component of some prenatal screening tests for Down syndrome.

In its regular "intact" heterodimeric form, it is produced almost exclusively by the trophoblast, most prodigiously by the cytotrophoblast and syncytiotrophoblast of the mature placenta and by gestational trophoblastic neoplasms.

This topic will review the biology of hCG and laboratory issues relevant to clinicians. The multiple clinical uses of this test are discussed separately:

Diagnosis of pregnancy (see "Clinical manifestations and diagnosis of early pregnancy")

Diagnosis of ectopic pregnancy (see "Ectopic pregnancy: Clinical manifestations and diagnosis" and "Ultrasonography of pregnancy of unknown location")

Screening for Down syndrome (see "Maternal serum marker screening for Down syndrome: Levels and laboratory issues" and "First-trimester combined test and integrated tests for screening for Down syndrome and trisomy 18")

Screening and follow-up of gestational trophoblastic disease:

(See "Hydatidiform mole: Epidemiology, clinical features, and diagnosis".)

(See "Initial management of low-risk gestational trophoblastic neoplasia".)

(See "Hydatidiform mole: Treatment and follow-up".)

BIOCHEMISTRY AND hCG ISOFORMS

Overview — hCG is part of the glycoprotein hormone family, together with the luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH). These hormones are heterodimers that share a common alpha-subunit and varying degrees of homology in their beta-subunits.

hCG, in its regular intact heterodimeric form, is produced almost exclusively by the trophoblast, most prodigiously by the cytotrophoblast and syncytiotrophoblast of the placenta, gestational trophoblastic neoplasms (GTN), and germ cell tumors (GCT) with trophoblastic elements. Approximately 15 to 20 percent of seminiferous testicular tumors and 40 to 50 percent of nonseminiferous testicular tumors secrete hCG [1,2]. Approximately 30 percent of nontrophoblastic tumors, as well as some normal tissues, produce low amounts of the free beta-subunit of hCG rather than the intact biologically active heterodimeric form [3].

The beta-subunit of hCG is very similar to that of LH, sharing 80 to 85 percent amino acid (AA) sequence identity. However, the final C-terminal peptide (CTP) of the beta-subunit of hCG, AA 121 to 145, is additional and unique to hCG [4-6]. The beta subunit of chorionic gonadotropin (CGB) is encoded by six highly homologous genes (CGB3, CGB5, CGB6, CGB7, CGB8, and CGB9) that are located together with the LH beta subunit gene (LHB, also known as CGB4) at chromosome 19q13.3. It is thought that the CBG genes arose from a singular ancestral LH beta gene by gene duplication and mutation [7]. Two further genes at this locus, CBG1 and CBG2, which share a high degree of sequence homology between them, have different mutational rearrangements and a separate evolutionary path from the remaining CBG genes [8]. CB1 and CB2 were previously thought to be transcriptionally silent so called pseudogenes, but they have since been shown to be expressed, albeit at a 1000-fold lower transcript level than the other CGB genes [9]. The common alpha-subunit is encoded by a single gene on chromosome 12q21.1-23 [9].

Both LH and hCG (in their heterodimeric forms) can bind to and activate the LH receptor (hence the term hCG/LH receptor) [10]. Because of the latter, exogenous hCG can be administered as a surrogate for LH to trigger ovulation in assisted reproductive technology.

Glycosylation — Posttranslational glycosylation of hCG occurs by sequential addition of carbohydrate side chains and is completed shortly before secretion of the assembled dimer [11-13]. hCG is more than 30 percent carbohydrate by mass, and glycosylation is of structural and functional importance, affecting both the rate of clearance from the circulation and biologic activity [14,15].

Glycosylation of hCG is variable, and a wide variety of glycoforms occurs throughout pregnancy. Pregnancy-derived free beta-hCG has six glycation sites: two N-linked (at positions 13 and 30) and four of the O-linked type (at positions 121, 127, 132, and 138). The glycosylation of tumor-derived hCG is also highly variable and differs from pregnancy hCG in having increased triantennary N-glycans and abnormal biantennary N-glycans [16-18].

The term "hyperglycosylated hCG" was initially used to describe hCG containing increased triantennary N-glycans and tetra-saccharide core O-glycans and to differentiate it from the predominant biantennary N-glycans and disaccharide core O-glycans of pregnancy-derived hCG [16]. However, the term was subsequently adopted to describe a specific hCG glycoform, often abbreviated hCG-h, that is recognized by a specific antibody designated as B152. This is discussed in more detail below. (See 'Hyperglycosylated hCG' below.)

Metabolism and hCG isoforms — Aside from regular intact hCG (alpha/beta heterodimer), other variant forms in pregnancy and disease also occur. Enzymic degradation of hCG by tissue or circulating proteases produces nicked forms, which, when affecting the intact heterodimer, also lead to more rapid dissociation to the free alpha- and free beta-hCG subunits. The dissociated and various nicked isoforms are more rapidly cleared from the circulation. Approximately 80 percent of hCG is metabolized by the liver and 20 percent by the kidney [1,3,19]. Degradation in the proximal renal tubule cells also results in formation of subunits, nicked forms, and the hCG beta-subunit core fragment (hCGbcf), which is a dominant form in urinary hCG. The most commonly occurring protein variant dissociated and degraded isoforms include:

Free beta-subunit (hCGb)

Free alpha-subunit

Nicked hCG (hCGn)

Nicked hCG-beta (hCGbn)

hCG beta-subunit core fragment (hCGbcf)

Sulfated hCG (hCG-S)

Other rarer isoforms and truncations of hCG also exist, such as hCG missing CTP [20], but these are not regularly measured either experimentally or clinically.

Variations in glycosylation give rise to further variant possibilities, described as glycoforms. These are more heterogeneous than the isoform variations of the protein backbone of hCG. A specific glycoform of hCG, misleadingly named hyperglycosylated hCG, or hCG-h, has been claimed as a marker of the invasive trophoblastic phenotype and trophoblastic neoplasia. (See 'Hyperglycosylated hCG' below.)

Intact hCG (alpha/beta heterodimer) and free beta-subunit of hCG

Pregnancy – During the first weeks of pregnancy, intact heterodimeric hCG is the predominant form of hCG in maternal blood, comprising >95 percent of total hCG in the maternal circulation. The free beta-subunit of hCG accounts for <10 percent of total hCG in maternal blood in very early pregnancy and decreases to approximately 0.5 to 2 percent after the eighth week [21]. In urine, however, the proportion of the free beta-subunit of hCG is high, constituting between 9 and 40 percent of total hCG [22]; the remainder is composed mostly of hCGbcf, the terminal metabolite of hCG degradation and the predominant form in the urine. (See 'hCG beta-subunit core fragment (hCGbcf)' below.)

The clearance of hCG after an abortion or a term pregnancy is best described by a triphasic model with median half-lives of 3.6, 18, and 53 hours [23]. This approximates a single half-life of two to three days. In normal pregnancy, values peak at around 20,000 to 100,000 international units/L reaching maximal levels between 8 and 11 weeks of gestation. Serum hCG is usually undetectable four to six weeks following pregnancy.

Males and nonpregnant females – Males and nonpregnant females have low levels of circulating hCG, which are undetectable by most routine hCG assays. The level of intact hCG increases with age, especially in females, and is higher than the free beta-subunit of hCG level, which does not increase with age and is often undetectable even using highly sensitive assays [2,24]. Low levels of hCG are fairly commonly detected in postmenopausal females [24]. (See 'Pituitary hCG' below.)

Tumor – Ectopic production of the free beta-subunit of hCG has been identified in nearly all common epithelial tumors studied to date, but the incidence is highly variable [24,25]. Consistent with an apparent correlation with tumor type and its aggressiveness within the spectrum of trophoblastic neoplasms, many reports demonstrate that either tissue expression or elevation of serum free beta-subunit of hCG is associated with adverse prognosis in most cancers [2,25]. The free beta-subunit of hCG does not activate the LHCGR receptor, but several lines of evidence indicate that it has a growth promoting activity in cancer [3,25].

Several studies have suggested that separate measurement of serum free beta-subunit of hCG and determination of the proportion of total hCG immunoreactivity (percentage free beta-subunit of hCG) in the management of GTN may provide additional information compared with measurement of total hCG alone. The exact proportion is dependent on the assays used, but, based on molar concentrations, a proportion approximating 3 to 5 percent is typical of low-risk postmolar invasive GTN. Patients with postgestational histologically confirmed choriocarcinoma have a higher proportion, at approximately 10 percent [3]. The potentially most aggressive form of GTN, placental site trophoblastic tumor, has the highest proportion of the free beta-subunit of hCG, approximately 20 percent, but is highly variable in the author's laboratory [4,5,26].

Free alpha-subunit of hCG

Pregnancy – The free alpha-subunit of hCG comprises <10 percent of total circulating hCG in the first trimester but increases throughout pregnancy, reaching 30 to 60 percent at term [27].

Tumor – The free alpha-subunit of hCG can also be detected in patients with GTN.

Compared with intact hCG or the free beta-subunit of hCG in pregnancy, GTN, and GCT, the low predictive value of the free alpha-subunit of hCG excludes its clinical use in these settings [28,29].

Nicked forms (hCGn, hCGbn) — Nicked forms of intact hCG (hCGn) and the free beta-subunit of hCG (hCGbn) arise by enzymatic cleavage of either the intact heterodimer or the free beta-subunit, respectively, by proteases secreted by the placenta or by proteinases released by activated leukocytes [30]. Degradation in the proximal renal tubule also results in hCGbn in urine [31,32]. The rate of enzymic nicking is an important determinant of hCG clearance from the circulation and, hence, directly affects serum and urine concentrations.

Nicking results in a more rapid dissociation of the heterodimer, and immunoreactivity for some immunoassays for the nicked forms is reduced or lost [33-35]. Although most of the commercially available total hCG assays are able to detect both hCGn and hCGbn, relative detection is variable and is a significant source of measurement discrepancy between assays [20,35-38]. Nicked forms are more rapidly cleared from the circulation than intact hCG and, due to their comparative abundance, more greatly affect urine than serum measurements.

Pregnancy – The molar ratio hCGn to hCG in urine increases from 8 to 31 percent during pregnancy [39], while that in serum is <10 percent [40]. The proportion of hCGn is increased in preeclampsia and Down syndrome.

Tumor – hCGn is a major form of hCG in urine following molar evacuation or during treatment of trophoblastic neoplasias [41-43]. The proportion of hCGn in urine is increased in testicular and bladder cancer, as well as trophoblastic disease. hCGn may also occur in the serum of some cancer patients [39,44,45].

hCG beta-subunit core fragment (hCGbcf) — hCGbcf is the final end product of hCG metabolism. It comprises amino acid (AA) residues of the free beta-subunit of hCG 6 to 40 and 55 to 92 linked by five disulfide bonds [46].

Pregnancy – hCGbcf is the predominant form of hCG in urine from the fifth week of pregnancy onward [46-48]. It can be detected in plasma [47,49,50], but the concentrations are only approximately 0.01 percent of those of hCG [51].

Tumor – Urine from patients with hCG-producing tumors also contains predominantly hCGbcf [52]. hCGbcf has been identified in the normal placenta, the pituitary, hydatidiform mole, choriocarcinoma tumor, and tissue from various epithelial carcinomas [47]. In nontrophoblastic cancers, urinary hCGbcf concentrations reflect those of the free beta-subunit of hCG in serum, but when measured by highly sensitive assays, the free beta-subunit of hCG in serum is a somewhat better marker [24].

Many commercially available total hCG assays do not recognize hCGbcf or other less well-defined degradation products including truncated hCG and the free beta-subunit of hCG missing the carboxyl terminal extension (hCG-CTP). Assays that do not measure hCGbcf are not suitable for urine hCG measurement [20,34,35,38,42]. (See 'Routine "total" hCG assays and measuring isoforms' below.)

Hyperglycosylated hCG — The term "hyperglycosylated hCG," while initially used to describe hCG containing increased complex carbohydrates [16], has increasingly come to mean hCG detected by assays employing antibody B152 (see 'Glycosylation' above). This is misleading as B152 specifically detects a single glycosylated epitope variant of hCG (Ser 132 is substituted by a "core-type 2" glycan side-chain), often abbreviated hCG-h, and belies the complexity and heterogeneity of hCG glycosylation [3,17]. Using B152-based assays, which are no longer commercially available, the following has been shown:

Pregnancy – hCG-h is the predominant form of hCG during the first four to five weeks of pregnancy in both serum and urine [3,53,54]. Low levels are associated with pregnancy loss [53,55,56] and preeclampsia [57-59]. High levels are also reportedly associated with Down syndrome pregnancies [60].

Strong expression of hCG-h by the invasive extravillous cytotrophoblasts [61], together with the association of low serum levels in early pregnancy loss and preeclampsia [59], suggests that hCG-h could be a biomarker for the presence of "normally" functioning invasive extravillous cytotrophoblast, which is present in early pregnancy [3,53].

In one study, the N-glycan repertoire of hCG was found to be more diverse than previously reported and included LewisX and bisected structures, the latter being more abundant in urine collected in late (20 to 21 weeks of gestation) compared with early (6 to 9 weeks of gestation) pregnancy [62]. This observation contradicts previous reports that hyperglycosylation in early pregnancy is associated with a higher abundance of multiantennary N-glycans compared with late pregnancy [15,16].

Molar pregnancy – hCG glycoforms in urine collected from patients with complete hydatidiform molar disease have a decreased abundance of bisected and increased sialylated structures compared with hCG glycoforms in normal pregnancy [15].

Trophoblastic neoplasia – Cole and associated researchers have reported that hCG-h can discriminate benign, spontaneously resolving trophoblastic disease from choriocarcinoma or malignant GTN, where it accounts for 7 to 100 percent of total hCG immunoreactivity, with 96 to 100 percent accuracy [26,61,63], but these findings have not been validated by other groups.

Other tumors – hCG-h is a major isoform in a variety of malignant conditions including ovarian, cervical [64], colon [65], bladder [66], lung [67], and GCT [53]. In vivo, the B152 antibody inhibits tumor growth in mice injected with a choriocarcinoma cell line, suggesting a role for hCG-h in promoting growth of transformed trophoblasts [4]. In vitro experiments have shown that the addition of hCG-h to choriocarcinoma cell lines promotes growth and invasion [26,57,68]. In addition, evidence for potent angiogenic actions of hCG [69,70], and more recently hCG-h, has also been published [70].

Despite the claims for the B152-derived test for hCG-h (also termed by some as invasive trophoblastic antigen [ITA] [60]), the test is not widely available, so many findings have not been reproduced or validated by independent groups. Interpreting measurements is problematic since B152 assays are difficult to calibrate (there is no appropriate standard) and reference ranges to ascertain positive results have not been derived. Serial measurements of hCG using regular tests to check if hCG levels persist or are rising remain the approach recommended by the International Federation of Gynecology and Obstetrics (FIGO) in the diagnosis of trophoblastic neoplasia. (See "Gestational trophoblastic neoplasia: Epidemiology, clinical features, diagnosis, staging, and risk stratification", section on 'Diagnosis'.)

Pituitary hCG — hCG isolated from the pituitary contains approximately one-half the sialic acid content of pregnancy hCG, and some of the glycans are sulphated (hCG-S) [71,72]. hCG-S has 50 to 65 percent of the biologic activity of hCG isolated from pregnancy urine [72]. The plasma concentration of hCG-S increases during menopause [71,73-75] and ranges, for the majority of patients, from 0.5 to 5 international units/L, although levels up to 10 to 15 international units/L are occasionally observed in hypogonadal states, particularly in menopause/perimenopause or following chemotherapy. It is important to recognize its presence, as the low levels can mistakenly be interpreted as indicating early pregnancy or malignant disease [24]. (See 'Nonpathologic causes of low-level hCG elevations' below.)

LABORATORY MEASUREMENT OF hCG

WHO standards for hCG isoforms — International reference reagents (IRR) for the most important forms of hCG, intact biologically active heterodimeric hCG (hCG), nicked hCG (hCGn), free alpha-subunit of hCG, free beta-subunit of hCG, nicked free beta-subunit of hCG (hCGbn), and hCG beta-subunit core fragment (hCGbcf), have been formulated. These standards were established by a working group of the International Federation of Clinical Chemistry (IFCC) and are available from the World Health Organization (WHO) [36,52,76,77]. They were principally formulated to aid development and standardization of hCG assays (which should improve comparability of measurements between different assays) but are also useful to characterize the reactivity of commercially available hCG tests [20,38].

No IRR standard for hyperglycosylated hCG (hCG-h) has been developed because the carbohydrate composition of hCG produced by tumors may vary considerably, so determining an appropriate source for such a standard is problematic [3,78].

The IFCC has adopted a nomenclature to describe the isoforms and recommends its use to help improve scientific communications and understanding (table 1) [3].

One of the longest-standing issues regarding communication of hCG measurements is the use of the misleading expressions "beta-hCG assay" or "hCG-beta assay" to describe routinely available pregnancy hCG tests. The misnomer arose following the development of the first specific assay for hCG by Vaitukaitis and colleagues, who produced a specific rabbit antiserum, "SB6," by immunizing the rabbit with the free beta-subunit of hCG [79]. On distribution, the antiserum was labelled "beta-subunit assay," which confused the recipients into thinking that only the free beta-subunit of hCG was detected. In reality, most serum hCG immunoassays detect both intact hCG, the free beta-subunit of hCG, and some of the variant forms. Diagnostic assays for serum hCG or pregnancy immunoassay tests are more clearly described as simply "hCG assays" or, for clarity, "total hCG assays" to prevent confusion and help distinguish them from assays that specifically measure the free beta-subunit of hCG alone.

Types of assays — All routine commercial immunoassays for quantitation of serum hCG are sandwich-type "immunometric" assays. These assays employ two antibodies: a solid phase antibody to capture hCG molecules from the sample (the capture antibody) and a second antibody labelled with an enzyme, fluorophor, or luminescent marker that detects captured hCG (the detector antibody). Each antibody recognizes different sites or "epitopes" on the target analyte, both of which must be present on the molecule to be detected. Since the antibodies can be used in large excess, these assays are faster and have lower detection limits than competitive assays, and their precision is generally higher. These immunoassays can typically measure levels of hCG in the serum as low as 1 to 2 international units/L. The sandwich principle is also utilized in qualitative home urine pregnancy test devices, but their sensitivity is lower, typically around 10 to 50 international units/L.

Radioimmunoassays (RIAs) are competitive format assays where the principle of measurement is competition between a fixed and known amount of radio-labeled analyte and sample analyte for a fixed, limited number of antibody binding sites [80]. Several trophoblastic centers are still using competitive RIAs, including that of the UK Trophoblastic Screening and Treatment Centre in London. Some of these assays, including that of the UK trophoblastic service, have the advantage of recognizing all of the major protein isoforms of relevance in both serum and urine [20,38].

Routine "total" hCG assays and measuring isoforms — Knowledge of the epitope specificity of monoclonal antibodies (mAbs) used in constructing commercially available routine pregnancy or so-called "total" hCG assays has facilitated improved design of hCG assays, including the design of assays specific for the individual isoforms [24,81]. It is possible to establish sandwich assays by choosing mAbs of appropriate affinity and epitope specificity to use in pairs for either broad spectrum total hCG measurement or selected isoform determination [21,24,51,82].

Quantitative serum "total hCG" immunoassays provided by medical laboratory services are used in pregnancy, pregnancy-related disease, and oncology applications.

For oncology purposes, broad and equimolar recognition of all isoforms is preferable in order to detect both intact hCG and the free beta-subunit of hCG, as well as nicked and fragment forms derived from the free beta-subunit of hCG. Marked under- or over-reactivity, especially to the free beta-subunit of hCG, is likely the greatest source of interassay variability in serum hCG measurements in oncology. Careful selection of antibodies by assay manufacturers should lead to improved between-laboratory comparability for "total hCG" immunoassays, as much of the discrepancy between such assays is due to variable isoform reactivity, especially of the free beta-subunit of hCG and hCGbcf forms [20,83]. (See "Hydatidiform mole: Treatment and follow-up", section on 'Postoperative monitoring' and "Initial management of low-risk gestational trophoblastic neoplasia", section on 'Monitoring during treatment' and "Initial management of high-risk gestational trophoblastic neoplasia", section on 'Monitoring during treatment'.)

For detection and monitoring of pregnancy purposes, a serum test that detects intact hCG and the free beta-subunit of hCG is adequate. Assays performed in urine should detect intact hCG, the free beta-subunit of hCG, and hCGbcf. The rate of rise of hCG over time and ultrasound imaging are the basis for detection of pregnancy of unknown location, ectopic pregnancy, and failing pregnancy (see "Ectopic pregnancy: Clinical manifestations and diagnosis" and "Ultrasonography of pregnancy of unknown location"). Measurement of hCG or the free beta-subunit of hCG is a component of some prenatal screening tests for Down syndrome. (See "Maternal serum marker screening for Down syndrome: Levels and laboratory issues" and "First-trimester combined test and integrated tests for screening for Down syndrome and trisomy 18".)

For detection of pregnancy, hCG is often measured in urine using dedicated semiquantitative home pregnancy testing devices [3]. These simple assays, which are exclusively based on the lateral flow principle, comprise a semiporous strip with a zone impregnated with hCG reactive "detection" antibodies that are coupled to colored latex beads, and a second zone where a second "capture" antibody is immobilized to a membrane. In the presence of an aqueous fluid containing hCG the color labelled "detection" antibodies are bound by the immobilized "capture" antibody and a visible signal is generated. Most urine home pregnancy tests perform as expected, but some assays are sensitive to a false depression of the results in the presence of excess hCGbcf. This is most likely to occur at 7 to 12 weeks pregnancy when urine hCGbcf levels may be in 2- to 20-fold excess and may saturate the binding capacity of either tracer or capture antibody leading to falsely negative test strip results [84]. (See 'High-dose hook or prozone effect' below.)

Discrepancies between assays — Between-method variation in total hCG assay results due to analyte heterogeneity and variation in assay specificity is a long-standing and widely recognized problem [35,85-87]. This is especially true for samples from patients with gestational trophoblastic disease, germ cell tumors, and other neoplasias, in which higher proportions of free subunits, partially degraded fragments, and abnormally glycosylated variants of hCG may be present [17,20,24,32,35,87-89].

Despite the epitope recommendations of the International Society of Oncology and Biomarkers Tissue Differentiation 7 (ISOBM TD-7) workshop [82,90] and the availability of the new WHO-defined standards, manufacturers do not commonly describe the epitope specificity of their assays in their instructions for use (IFU) documents. Studies of commonly used routine serum hCG assays have shown that variable recognition between assays is greatest for the free beta-subunit of hCG and hCGbcf [20,38]. For example, among five of the most popular commercially available sandwich assays presently in use, only the Siemens Immulite and Roche Elecsys assays detected hCGbcf, measuring 73 and 31 percent of the target value, respectively [20,38].

INTERPRETING LABORATORY FINDINGS

Reference values for serum and urine —  (table 2 and table 3)

Serum – The upper reference limit (URL) for serum hCG assays, typically derived using samples from nonpregnant female patients, is used as a cut-off to help identify pregnancy and pathologically increased hCG concentrations. It is also used as a decision limit for cessation of chemotherapy and for detection of relapse in the management of gestational trophoblastic neoplasia. Because of differences in calibration and specificity between various methods, it is desirable to determine reference values separately for each manufacturer's assay. Manufacturers usually provide URLs for their hCG assays in their instructions for use (IFU) documents. The URL defined by the International Federation of Clinical Chemistry (IFCC) is defined as the 97.5th percentile, but individual manufacturers may use a different definition of the reference limit. Furthermore, the URL is different for assays specific for intact hCG, free beta-subunit of hCG, and total hCG (ie, assays measuring both intact hCG and the free beta-subunit of hCG).

Specific reference ranges for intact hCG, free beta-subunit of hCG, and hCG beta-subunit core fragment (hCGbcf) in serum and urine for men and pre- and postmenopausal females have been determined using highly sensitive immunofluorometric assays specific for the separate forms. These reference values are strictly only valid for these assays but are provided here as they are highly informative (table 2 and table 3) [24]. They show that in men and nonpregnant females, the concentrations of the free beta-subunit of hCG are lower than those of intact hCG, and, unlike hCG, the concentrations do not increase with age. In females, postmenopausal levels of hCG may be up to 5 to 10 international units/L, which is 5 to 10 times higher than those of the free beta-subunit of hCG. (See 'Nonpathologic causes of low-level hCG elevations' below.)

Urine – The urine concentrations of intact hCG and the free beta-subunit of hCG are similar to those in serum. However, total hCG in urine also includes hCGbcf, which is often present in concentrations similar to those of intact hCG. As a result, total hCG in urine is often higher than in serum, which contains only minute levels of hCGbcf.

Urine total hCG concentrations, though strongly correlated to those in serum, vary considerably due to greater heterogeneity of isoforms present and the diluting effects of the kidneys. Hence, determination of urinary hCG does not reflect hCG-producing disease status as accurately as serum measurement [91].

Nonpathologic causes of low-level hCG elevations

Pituitary hCG in menopause/chemotherapy – Menopausal and perimenopausal patients sometimes have raised serum levels of hCG; levels up to 25 international units/L have been reported [74], but levels around 7 to 15 international units/L are more common. The source of the hCG is the pituitary, and secretion of postmenopausal hCG is pulsatile and mirrors that of luteinizing hormone (LH) [71]. hCG isolated from the pituitary contains approximately one-half the sialic acid content of pregnancy hCG, and some of the glycans are sulphated (hCG-S) [71,72].

Lack of gonadotropin-releasing hormone (GnRH) suppression when sex steroid levels fall in menopause and perimenopause leads to raised LH and follicle-stimulating hormone (FSH) levels, which are accompanied by the secretion of low levels of hCG. Since the hCG can be suppressed with estradiol and parallels serum levels of LH, these findings suggest that the hCG is derived from the pituitary (hCG-S) [3,92]. The mechanism is believed to be due to low-level transcription from genes encoding the beta-subunit of hCG that are located on the same chromosome and in close proximity to the LH gene. Hence, when transcription of LH is strongly upregulated due to the lack of GnRH feedback suppression by sex steroids in menopause or hypogonadal states, transcription of the adjacent beta-subunit of hCG is also partially induced [93]. Since production of the alpha-subunit of hCG is not regulated and it is produced in relative excess by the pituitary gonadotrope cells, any translated beta-subunit protein is always associated with the alpha-subunit and secreted as the intact heterodimeric form.

It is important to recognize the presence of pituitary hCG, as the low levels can be interpreted mistakenly as indicating early pregnancy or malignant disease. The problem can be easily identified by measuring LH in these patients, as in the assessment of menopause. Levels of LH raised close to or above the reference limit for menopause indicate that the source of hCG is likely to be the pituitary. If necessary, estradiol replacement can be given to suppress hCG and confirm the source of hCG as pituitary and not pathologic [3,94,95].

Chemotherapy also induces gonadal suppression, and pituitary hCG is occasionally seen immediately following treatment, when LH levels often rise to menopausal levels or higher. Transient postchemotherapy, low-level elevation of hCG following successful therapy of hCG-producing tumors may not, therefore, indicate resistant disease or relapse, and it is therefore particularly important to clarify the hCG source in these patients [94,95]. The mechanism for production of intact hCG by the pituitary is analogous to that in menopause, and the pituitary source can be confirmed using the same approach described above (ie, by measuring LH/FSH and confirming suppression by administering estradiol).

hCG injection/plasma exchange – Exogenously administered hCG will lead to elevated serum levels that may be misinterpreted as endogenous production. For example, athletes may use hCG injections to stimulate gonadal steroid production [74]. (See "Use of androgens and other hormones by athletes".)

hCG injections are used for controlled ovarian hyperstimulation in assisted reproductive technology, and this must be considered when monitoring the patient to see if pregnancy has been achieved [3]. hCG administration prior to oocyte retrieval results in serum hCG levels between 60 and 300 milli-international units/mL and is completely cleared within two weeks after administration. As oocyte retrieval is approximately 12 days after hCG administration, the levels should not interfere with properly timed pregnancy testing. (See "In vitro fertilization: Procedure", section on 'Triggers for ovulation'.)

Although rare, there are also reports of unexplained hCG elevations observed in patients receiving blood products from female donors of child-bearing age [96].

Quiescent gestational trophoblastic disease – Quiescent gestational trophoblastic disease is a purported variant of trophoblastic disease that is associated with persistent, low-level hCG following a previous episode(s) of trophoblastic disease [63]. However, the existence of quiescent gestational trophoblastic disease as a distinct disease entity has not been widely accepted and is difficult to verify because: (1) the lack of a commercial assay for hyperglycosylated hCG (hCG-h), its purported characteristic isoform, and (2) the characteristic absence of disease on magnetic resonance imaging. (See "Hydatidiform mole: Treatment and follow-up", section on 'Plateaued hCG levels'.)

Familial hCG – Familial hCG is a rare genetic condition (estimated prevalence 1:60,000) in which serum and urine hCG concentrations are persistently elevated for at least several years and probably the lifespan of affected individuals. The hCG concentrations are usually low (10 to 200 international units/L) but sufficiently high to cause suspicion of pregnancy or cancer. It is important to recognize this condition because it may lead to unnecessary treatment [97-99].

Familial hCG is a diagnosis of exclusion once other causes of a raised hCG have been ruled out. The diagnosis is confirmed by the finding of similarly raised serum hCG in first-degree relatives. In 10 reported families, all individual cases produced a combination of intact hCG, the free beta-subunit of hCG, or hCG-beta-CTP isoforms (the beta-subunit of hCG contains more amino acids than the beta-subunit of LH; the additional amino acid chain is called the C-terminal peptide [CTP]).

Analytical causes of erroneous results; antibody-related interference — Immunoassays, though generally robust, are susceptible to occasional analytical errors. False or inaccurate determinations of hCG by immunoassay can have serious adverse consequences, especially in cases where neoplasia is suspected [3,99,100]. The first line of defense in minimizing the risk of patient harm is to suspect results that do not fit the clinical picture and to contact the laboratory immediately [101].

Analytical errors arising not as a result of human or machine error (eg, mislabeling of patient samples or pipetting errors), but sporadically as a result of the properties of the specimen itself, are particularly hard to detect. Such patient- or sample-dependent errors in immunoassay measurements are caused by the sporadic occurrence within the sera of some patients of cross-reacting substances interacting and binding with either the assay's antibodies or to the target analyte itself. These substances are described as assay interferents, and they may be caused by a number of possible factors, most commonly by so-called "heterophilic" host antibodies that can bind to and crosslink the capture and detector antibodies of sandwich assays causing falsely raised or false-positive measurements. While most interferents lead to false-positive measurements, it is important to remember that interferents can interact with assay antibodies in different ways, and false depression, as well as elevation of results, is possible.

Interferents causing mostly false positive or falsely elevated results

Heterophilic antibodies – Heterophilic antibodies are naturally occurring antibodies with low affinity to many antigens, including animal antibodies (such as those used in immunoassays), in individuals without a known previous exposure to animal antibodies. Estimates of the frequency of patient sera antibodies with affinity to animal immunoglobulins vary widely from 0.1 to 4 percent [102] to up to 40 percent [103]. The presence of heterophilic antibodies in patient sera can cause erroneous measurements because they can directly bind (and hence interfere with) the capture and/or detector antibodies used in sandwich immunoassays. False-positive results due to heterophilic antibodies directly crosslinking capture and detector antibodies in sandwich assays are estimated to be the most frequently occurring type of immunoassay interference [101,104].

Species-specific human anti-animal antibodies are a subtype of heterophilic antibody. They are distinguished from polyspecific heterophilic antibodies because they are confirmed to have been induced by exposure to animal antigens and they are of higher affinity. Due to their greater affinity, they usually cause greater measurement error than other heterophilic antibodies [105]. Human anti-mouse antibodies (HAMA) often develop as a result of iatrogenic exposure to mouse antibodies, which are increasingly used in diagnostic imaging procedures and in recombinant antibody therapies [103,106]. Such recombinant antibodies are mostly either chimeric (mouse Fc-region replaced with a human Fc, suffixed -ximab) or have been humanized (>95 percent of the mouse sequence replaced with human sequence, suffix -zumab) to help reduce the risk of developing anti-mouse antibodies.

Rheumatoid factors are a defined subset of heterophilic antibodies (usually immunoglobulin M [IgM] but may be any subtype) that are autoantibodies with affinity to the Fc region of the patient's own IgG. They are found in 5 to 10 percent of the general population and in approximately 70 percent of patients diagnosed with rheumatoid arthritis [103,107].

In contemporary commercial immunoassays, manufacturers routinely add animal immunoglobulin to their assay buffers to minimize the risk of heterophilic antibody interference. HAMAs are specifically targeted by addition of excess nonimmune mouse IgG which act as decoy targets blocking the heterophilic antibodies. This has undoubtedly reduced the frequency of problematic interference, but in some cases, the blocking can be overwhelmed [108]. Alternatively, the use of Fab fragments in the immunoassays instead of the entire Ig molecule can reduce heterophile interference. Most interfering heterophilic antibodies have affinity to the Fc-region of mouse IgG1-antibodies (the most common isotype employed in immunoassays) but little or no affinity to F(Ab’)2-fragments, mouse antibodies of other idiotypes, or antibodies from other animals. Accordingly, modern immunoassays are usually designed using recombinant immunoglobulin fragments that lack the Fc-region. In a large study of over 11,000 serum samples measured using a sandwich assay constructed using mouse monoclonal antibodies lacking the Fc region of the capture antibody, the frequency of interference was reduced from 4 to 0.1 percent [109].

Anti-hCG antibodies – Anti-hCG antibodies are very unlikely to be a frequent source of measurement error. hCG autoantibodies have been reported in a female patient who had received hCG injections for treatment of infertility. The antibodies caused false-positive hCG results at a level of 40 international units/L due to macro-hCG (large macromolecular complexes of the autoantibody and hCG). The macro-hCG was identified by precipitation with polyethylene glycol and gel filtration on a Superdex column. The interfering anti-hCG antibody was shown to be IgM by immunoextraction [110].

Interferents causing mostly false negative or falsely low results

Complement/biotin/others – Various other proteins, including complement, lysozyme [111], and paraproteins [112], have been shown to interfere with the reagent antibody-analyte antigen reaction. Complement binds to the Fc fragment, especially of IgG2 subtype, reducing their binding capacity and thereby causing falsely low results in sandwich assays and falsely high results in competitive assays [113]. IgG2 subtype antibodies are therefore infrequently used by present day assay manufacturers. Biotin, which in doses up to 100 times the recommended daily intake is common in dietary supplements, can cause interference in assays utilizing biotinylated reagents and streptavidin [114].

While not an example of an interferent, another cause of a falsely low result is high-dose hook effect. (See 'High-dose hook or prozone effect' below.)

Falsely negative results may also be caused by the presence of hCG variants that cannot be detected by the assay. (See 'Discrepancies between assays' above.)

How to identify antibody interference — It is important for clinicians to be aware of the possibility of antibody-related interferences and the need to contact the laboratory if results do not fit the overall clinical picture [101]. There are several simple laboratory tests that can be used to confirm the presence of substances interfering in hCG immunoassays, but, as they all have drawbacks, a combination of approaches is recommended.

The four typical methods used to identify interference in immunoassay measurement are (1) use of an alternate assay, (2) paired urine and serum analysis, (3) performance of linearity studies, and (4) measurement of hCG before and after addition of a blocking reagent.

Analysis using a different immunoassay – The finding of a markedly different, often lower, result using a second different immunoassay is the simplest test for identifying interference. Ideally, the two assays used for investigating interference should both have similar isoform reactivity. Otherwise, markedly different reactivities, particularly regarding the free beta-subunit of hCG, may account for, and therefore mask rather than confirm, any assay interference.

This approach has the vulnerability that agreement between assays may, though rarely, be due to the interferent affecting both assays similarly [103,104,115]. Hence, if the assays agree but the suspicion of assay interference remains high, additional testing is recommended.

Repeat analysis using paired serum and urine samples – As interfering antibodies are too large to be filtered through the glomeruli into urine, a much lower result in a paired urine sample is an indicator of a false-positive result in serum. This approach, however, is not reliable in the case of a low serum hCG concentration of ≤50 international units/L since physiologic formation of a dilute urine could also produce a negative result [2].

Perform linearity and recovery studies – Dilution of a suspicious sample with the zero calibrator or kit-supplied diluent buffer that is nonlinear (ie, the result does not decrease according to the dilution factor) may indicate heterophilic interference. Using the dilution method, the presence of heterophilic antibodies often results in a dramatic reduction in apparent concentration (lower than expected result), as previously overwhelmed blocking agents in the assay solutions become effective when the heterophile is diluted out.

Perform recovery and/or blocking reagent experiments Recovery experiments are performed by adding a known amount of hCG analyte (eg, using the assay standard) to the suspected serum sample and reassaying. The presence of heterophilic-type interferents is confirmed by under-recovery (lower than expected result); over-recovery suggests either macro-hCG or matrix-type interference by other serum components. Alternatively, interference may be identified by (1) adding the patient sample to a heterophile blocking tube; measurements of the samples are then compared, or (2) the addition of a blocking reagent to the sample and the finding of a significantly different result. If the result remains unchanged, further addition of more blocking agent and the use of a combination of blockers from different animal sources may help avoid the possibility of the blocking agents being overwhelmed [116].

More complex investigations, including gel filtration chromatography, protein A/G/L immunoadsorption chromatography, or the use of specialist "non-sense" interference assays, can also be used to further clarify the nature of interferences but are beyond the scope of this review [101,106].

High-dose hook or prozone effect — The prozone effect or "high-dose hooking" occurs in sandwich-type immunometric assays when analyte concentrations are so high that saturation of antibody binding sites prevents formation of the Ab-hCG-Ab* sandwich. This problem is seen with hCG assays at concentrations exceeding 5000 to 20,000 international units/L, the precise cutoff varying according to assay.

Many automated quantitative serum analyzers recognize this and dilute the sample and repeat the analysis; however, hCG, especially in gestational trophoblastic disease, can reach such high levels that the preset automated dilution is insufficient. So-called one-step sandwich assays, in which there is no wash step between the addition of sample and the detection antibody, generally have lower high-dose hook thresholds than two-step assays, as washing after sample incubation with the capture antibody washes away excess-free antigen [84,117,118].

The problem of falsely negative results is worse in urine sample-based pregnancy dipstick tests, as not only is there no automated dilution or washing but also the predominant urinary form hCGbcf is sometimes not recognized by both antibodies used in the assay [84,117].

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Gestational trophoblastic disease".)

SUMMARY AND RECOMMENDATIONS

Overview

Human chorionic gonadotropin (hCG), luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone (TSH) are heterodimers that share a common alpha-subunit and varying degrees of homology in their beta-subunits. (See 'Overview' above.)

Heterodimeric hCG is produced almost exclusively by trophoblastic cells of the placenta, gestational trophoblastic neoplasms, and germ cell tumors with trophoblastic elements. Approximately 30 percent of nontrophoblastic tumors, as well as some normal tissues, produce low amounts of the free beta-subunit of hCG. (See 'Overview' above.)

Metabolism and hCG isoforms

hCG exists in a range of isoforms that mainly arise by dissociation or enzymic degradation of isoforms. (See 'Metabolism and hCG isoforms' above.)

Selected isoforms of hCG can also be determined by specialist laboratories (eg, free beta-subunit of hCG or hyperglycosylated hCG [hCG-h]), but they are not routinely available to oncologists, and their clinical value remains to be ascertained. Interpreting these results is problematic as reference ranges/diagnostic cutoffs have not been established/validated. Intact hCG and free beta-subunit of hCG assays are more readily available, as they are used in Down syndrome screening tests. (See 'Hyperglycosylated hCG' above.)

Laboratory measurement

All commercial immunoassays for quantitation of serum hCG are sandwich-type "immunometric" assays. Most routinely available pregnancy-type hCG assays provided by medical laboratories for serum determinations detect regular intact hCG, the free beta-subunit of hCG, and other variant forms and may be described as "total hCG" type assays. Between-method variation in total hCG assay results due to analyte heterogeneity and variation in assay specificity is a widely recognized problem. (See 'Laboratory measurement of hCG' above.)

Total hCG assays vary in their ability to measure the variant isoforms of hCG relative to their reactivity to regular intact hCG, particularly with regard to the free beta-subunit of hCG and hCG beta-subunit core fragment (hCGbcf) (table 1). Between-method variation in total hCG assay results due to analyte heterogeneity and variation in assay specificity is a long-standing and widely recognized problem.

As urine contains a high proportion of hCGbcf, assays that do not recognize this form are not suitable for use in urine. (See 'hCG beta-subunit core fragment (hCGbcf)' above.)

Urine measurement or urine dipstick tests are not as reliable as serum assays and are not recommended for use in oncology. Suspicious urine results obtained during testing for pregnancy purposes should be checked by measuring serum hCG. (See 'Routine "total" hCG assays and measuring isoforms' above.)

For oncology purposes, serum assays with broad isoform reactivity are preferred. In oncology testing applications, if results are suspiciously low and the reactivity of the assay is unknown, repeating the measurement using assays with known reactivity is recommended. (See 'Routine "total" hCG assays and measuring isoforms' above.)

Selected isoforms of hCG can also be determined by specialist laboratories (eg, free beta-subunit of hCG or hyperglycosylated hCG [hCG-h]), but they are not routinely available to oncologists, and their clinical value remains to be ascertained. Interpreting these results is problematic as reference ranges/diagnostic cutoffs have not been established/validated. (Intact hCG and free beta-subunit of hCG assays are more readily available, as they are used in Down syndrome screening tests.) (See 'Hyperglycosylated hCG' above.)

Interpreting laboratory findings Suspicious results that do not fit the clinical picture should always prompt the clinician to immediately contact the laboratory to discuss the result so that the laboratory may undertake further investigations to confirm the integrity of the measurement. (See 'Analytical causes of erroneous results; antibody-related interference' above.)

Low-level elevations – Some causes of low-level hCG elevations include menopause (table 2), response to or recovery from chemotherapy-induced gonadal suppression, exogenous hCG injection, quiescent gestational trophoblastic disease, and familial hCG. (See 'Nonpathologic causes of low-level hCG elevations' above.)

False-positive results – False-positive or falsely raised measurements can occur as a result of antibody-related interferences. Interference in immunoassays may also cause falsely low measurements, but these are rarer. False or inaccurate determinations of hCG by immunoassay can have serious adverse consequences, especially in cases where neoplasia is suspected. Causes include heterophilic antibodies, anti-hCG antibodies, human anti-animal antibodies, and other interfering substances. Interference can be excluded by use of an alternate assay, pairing urine and serum analysis, performing linearity studies, and measuring hCG before and after addition of a specific blocking reagent. (See 'Analytical causes of erroneous results; antibody-related interference' above.)

Falsely lowered results – The prozone effect or "high-dose hooking" occurs in sandwich-type immunometric assays when hCG concentrations are so high that saturation of antibody binding sites prevents formation of the Ab-hCG-Ab* sandwich. It is seen with hCG assays at concentrations exceeding 5000 to 20,000 international units/L, the precise cutoff varying according to assay. (See 'High-dose hook or prozone effect' above.)

ACKNOWLEDGMENT — The UpToDate editorial staff acknowledges John R Lurain, MD, and Kristina M Mori, MD, who contributed to previous versions of this topic review.

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Topic 3209 Version 23.0

References

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