Human chorionic gonadotropin (hCG) testing – Part I

The first pregnancy test based on the biological activity of human chorionic gonadotropin (hCG) in partially purified urine was described in 1927. Various modifications of this assay were used until the introduction of immunoassays in 1960.¹ With the advent of monoclonal antibodies, new specific assays for the various subunits and degradation products of hCG were developed. In 1984, the two-antibody immunometric assays for hCG were developed²; during the same time period, antibody enzyme labeling and highly sensitive fluorimetric and chemiluminescent detection were developed — the basis of hCG tests used in all commercial labs today, and in point-of-care (POC) and over-the-counter (OTC) tests sold today. The introduction of efficient and sensitive hCG assays widened the application of hCG for diagnosis of pregnancy, trophoblastic disease, and non-trophoblastic neoplasms.³ It also led to the rise of a body of hCG research and the discovery of multiple hCG variants and their independent biological functions.

hCG biochemistry

hCG belongs to the glycoprotein-hormone family, along with luteinizing hormone (LH), folilcle stimulating hormone (FSH), and thyroid stimulating hormone (TSH). It is a heterodimer composed of two subunits a and b, joined non-covalently. The a-subunit composed of 92 amino acids linked by five disulfide bridges is common to all members of the glycoprotein-hormone family. It has two N-linked oligosaccharide side chains at amino-acid residues 52 and 78. The b-subunit is unique to hCG and confers its biological activity. It is composed of 145 amino acids linked by six disulfide bridges and contains two N-linked oligosaccharides, four O-linked oligosaccharide chains, and a proline and serine-rich C-terminal extension (C-terminal polypeptide). hCG displays extensive charge heterogeneity due to variation in its scialic-acid content. Because of heterogeneity of carbohydrate moieties of hCG, the molecular weight displays a range of values. The average molecular weight of hCG is 37500 Da, with the a-subunit being 14000 Da and the b-subunit 23500 Da.

Multiple variants of hCG that exhibit independent functions are currently recognized. Hyperglycosylated hCG, which is an hCG variant with additional carbohydrate residues that is produced by cytotrophoblast cells,⁴ is the principal molecule produced in early pregnancy. The free b-subunit of hCG, which is made by non-trophoblastic neoplasma, is the principal molecule produced in cancer. Therefore, the term hCG refers to a group of variants that have the same a– and/or b-subunit peptide structure but have separate biological functions. Regular hCG functions in maintaining pregnancy much more consistently with its concentration during the length of gestation. It mediates its action through the LH/hCG receptor by maintaining progesterone production early during pregnancy. Hyperglycosylated hCG produced in invasive trophoblastic disease, choriocarcinoma, and in early pregnancy displays a different structure from that of regular hCG.⁵ It was shown to predominate in early pregnancy-serum samples, to be directly associated with the implantation of pregnancy, to be associated with invasion in invasive mole, and gestation-trophoblastic neoplasms including choriocarcinoma.⁶ In addition, the free b-subunit (bhCG) is produced by many non-trophoblastic neoplasms. It has been detected in the media of several malignant cell lines such as cervical, breast, bladder, ovarian, brain, colorectal, uterine, and lung.⁷ The free b-subunit is not, however, a reliable diagnostic marker for specific malignancies but, rather, has potential as a marker of poor prognosis.⁷

A variety of dissociation and degradation products of hCG b-subunit are detected in serum and urine samples. The action of the enzyme leukocyte elastase-like protease on hCG generates nicked hCG, nicked hyperglycosylated hCG, and nicked free b-subunit. These molecules are further degraded by the action of proteases, including leukocyte elastase, via the cleavage of the b-subunit C-terminal segment (CTP; peptide resides 92-145), generating nicked hCG, nicked hyperglycosylated hCG and nicked free b-subunit missing the CTP. It is noteworthy that there are 10 common degradation variants of hCG detected in serum and/or urine samples (see Table 1). These are found in serum and urine samples in pregnancy, gestational-trophoblastic disease, and different maligncies. The variability in the forms of hCG found in various conditions must be considered when measuring total hCG in different stages of pregnancy and different cancer cases.

Uses of hCG testing

Pregnancy: The only FDA-approved use of hCG testing is for pregnancy detection. hCG tests are also often used as part of a medical examination to check for pregnancy. Serum and urine hCG in early pregnancy is primarily hyperglycosylated with mean proportions of hyperglycosylated hCG in serum at the third, fourth, fifth, and sixth weeks of gestation of 90%, 54%, 42%, and 29% of total hCG respectively.⁸ The predominant form of hCG in the second and third trimesters of pregnancy becomes regular hCG with a very minor portion (<2%) being hyperglycosylated hCG.⁷ The hCG b-core fragment is the predominant form of hCG in urine from seven weeks of pregnancy until term.⁹ In addition, the free b-subunit is also present in early pregnancy, becoming a minor component (<1%) of total hCG during the remainder of pregnancy.⁷

Failed pregnancies: Generally, a rapid increase in hCG follows an intrauterine pregnancy. The hCG-doubling test is used as an indicator of pregnancy failure, miscarriage, or ectopic pregnancy in the period between four and seven weeks of gestation.¹⁰ The hCG-doubling test involves determining if the hCG serum level has doubled after two serum hCG measurements obtained 48 hours apart. It is used in individuals with fear of miscarriage, previous history of miscarriage, or infertility problems. After the hCG-doubling test confirmation, methotrexate is used to destroy and abort the ectopic pregnancy or a salpingectomy is performed.

Screening for Down syndrome: hCG is used in combination with a-fetoprotein and unconjugated estriol as part of a triple test to screen for the risk of Down-syndrome pregnancy, or those warranting the risk of amniocentesis in the second trimester of pregnancy. Improvement in the sensitivity for detecting Down-syndrome pregnancy was achieved with the addition of dimeric inhibin A as a fourth marker in a quadruple screen.¹¹ The combination of ultrasound nuchal translucency with laboratory measurement of hCG and pregnancy-associated plasma protein (PAPP)-A became the standard to assess risk for Down-syndrome fetus. While a positive test with this combination of markers is the optimal prediction, it still only indicates approximately one in 20 chance of having a Down-syndrome fetus, therefore chorionic villous sampling or amniocentesis is warranted to give a definitive prediction.

Gestational trophoblastic disease: Gestational-trophoblastic diseases are a group of pregnancy disorders including a complete hydatidiform mole, a partial mole, and choriocarcinoma. The complete hydatidiform mole is a pregnancy comprised solely from placental tissue that takes on a hygromatous-cystic anatomy since there is no transfer of nutrients to a fetus. A partial mole is a combination of cystic-complete mole-like anatomy, some normal anatomy villous placenta plus some fetal components. The malignant villous placenta descending from a hydatidiform mole is considered an invasive mole, while choriocarcinoma is an extremely invasive malignancy comprising mostly cytotrophoblast cells of villous origin.

The use of total hCG measurement in gestational trophoblastic diseases is an example of a tumor marker with 100% sensitivity and 100% specificity for trophoblast-tissue mass, with the amount of tumor tissue or mole being directly proportional to the circulating concentration of total hCG. Hyperglycosylated hCG, on the other hand, is also a marker of invasion and malignancy in invasive mole and choriocarcinoma.⁷ Since multiple forms of hCG characterize gestational-trophoblastic diseases such as regular and hyperglycosylated hCG, it is important for physicians managing gestational trophoblastic disease cases to make sure that an appropriate total hCG test is being used by the testing laboratory.⁷

Other malignancies: Despite test manufacturers advising against the use of their tests for cancer cases, variants of hCG are used as markers of germ cell and other non-trophoblastic malignancies including testicular choriocarcinoma, testicular yolk-sac carcinoma, testicular embryonal carcinoma, and testicular teratoma. Free b-subunit in serum and the b-core fragment in urine could be measured as part of an annual physical examination to exclude cancer.⁷ It is common for macrophage enzymes in non-trophoblastic malignancies to degrade the molecules produced yielding multiple degradation products of free b-subunit. Thus, it is important for hCG assays to detect nicked molecules and hCG, and free b-subunit with cleaved C-terminal peptides. It is important that an appropriate total hCG test is being used for cancer management.

Pituitary production of hCG: Pituitary-hCG production defined as low levels of hCG in women around the time of menopause has been reported.¹² As part of a normal physiologic response, the mid-cycle pre-ovulatory surge of LH is accompanied by low levels of hCG. Due to the decreased production of estrogen and the suppression of progesterone, pituitary hCG is normally produced with increasing menopausal production of LH.¹² In a study of changes in hCG concentrations with age in non-pregnant women, it was reported that hCG increases with age, and that a cutoff of 14.0 IU/L should be used in women >55 years of age. In addition, pregnancy was deemed unlikely in women 41 to 55 years of age when the hCG levels were between 5.0 IU/L and 14.0 IU/L and the FSH level was > 20.0 IU/L.¹² A positive pregnancy test in these women may be part of normal physiology and not gestational-trophoblastic disease, or cancer. A study looking at the diagnostic utility of serum FSH to rule out the placental origin of hCG in perimenopausal women with hCG levels between 5.0 IU/L and14.0 IU/L suggested that women with greater than 45 mIU/mL FSH are most likely in perimenopause or menopause.¹³

hCG in doping: hCG and LH stimulate the production of testosterone in the testicles of males, and progesterone and estradiol in the female ovaries. hCG, and, therefore, can be used by male athletes to increase testosterone production and to normalize testicular-testosterone production that is suppressed during and after prolonged use of anabolic steroids. The use of hCG and LH is illegal in male but not in female athletes.¹⁴ Determination of hCG in urine is used to detect illegal use in male athletes. The World Anti-Doping Agency states that the hCG assay used should have a detection limit of at least 5 IU/L and that two different assays should be used. In addition, the World Anti-Doping Agency has undertaken efforts to stabilize human urine-doping samples for hCG measurements. Methods based on immunoextraction and mass spectrometry have been developed especially for doping control. These are intended for use as confirmatory tests but there are no reports on their actual use.

Note: Read Part II of “Human chorionic gonadotropin (hCG) testing” in MLO’s March 2011 issue.

Charbel Abou Diwan, PhD, is a Clinical Chemistry Post-Doctoral Fellow working in the Department of Pathology and Laboratory Medicine at Emory University School of Medicine in Atlanta, GA.

References

1. Wide L, Gemzell CA. An immunological pregnancy test. Acta Endocrinol (Copenh). 1960;35:261-267.

• Hussa RO, Hudson EN. A two-site immunometric assay in evaluation of low levels of serum hCG. Am Clin Prod Rev. 1984; 3(12):12-17.
• Stenman UH, Alfthan H, Hotakainen K. Human chorionic gonadotropin in cancer. Clin Biochem. 2004;37(7):549-561.
• Kovalevskaya G, Genbacev O, Fisher SJ, Caceres E, O’Connor JF. Trophoblast origin of hCG isoforms: cytotrophoblasts are the primary source of choriocarcinoma-like hCG. Mol Cell Endocrinol. 2002;194(1-2);147-155.
• Elliott MM, Kardana A, Lustbader JW, Cole LA. Carbohydrate and peptide structure of the alpha- and beta-subunits of human chorionic gonadotropin from normal and aberrant pregnancy and choriocarcinoma. Endocrine. 1997;7(1):15-32.
• Cole LA, Khanlian SA, Riley JM, Butler SA. Hyperglycosylated hCG in gestational implantation and in choriocarcinoma and testicular germ cell malignancy tumorigenesis. J Reprod Med. 2006;51(11):919-929.
• Cole LA. Human chorionic gonadotropin tests. Expert Rev Mol Diagn. 2009;9(7):721-747.
• Cole LA, Khanlian SA, Sutton JM, Davies S, Stephens ND. Hyperglycosylated hCG (invasive trophoblast antigen, ITA) a key antigen for early pregnancy detection. Clin Biochem. 2003;36(8):647-655.
• Cole LA. Immunoassay of human chorionic gonadotropin, its free subunits, and metabolites. Clin Chem.1997;43(12):2233-2243.
• Batzer FR, Schlaff S, Goldfarb AF, Corson SL. Serial beta-subunit human chorionic gonadotropin doubling time as a prognosticator of pregnancy outcome in an infertile population. Fertil Steril. 1981;35(3):307-312.
• Wald NJ, Densem JW, George L, Muttukrishna S, et al. Prenatal screening for Down’s syndrome using inhibin-A as a serum marker. Prenat Diagn.1996;16(2):143-153.
• Snyder JA, Haymond S, Parvin CA, Gronowski AM. Diagnostic considerations in the measurement of human chorionic gonadotropin in aging women. Clin Chem. 2005;51(10):1830-1805.
• Gronowski AM, Fantz CR, Parvin CA, Sokoll LJ, et al. Use of serum FSH to identify perimenopausal women with pituitary hCG. Clin Chem.2008;54(4):652-656.
• Stenman UH, Hotakainen K, Alfthan H. Gonadotropins in doping: pharmacological basis and detection of illicit use. Br J Pharmacol. 2008;154(3):569-583.