Introduction

Human chorionic gonadotropin (hCG) is a glycoprotein composed of two dissimilar subunits, a- and P-subunit, held together by charge interactions. hCG is produced by trophoblastic cells of the placenta in both pregnancy and gestational trophoblastic diseases. It is a remarkable glycoprotein in that up to 35% of the molecular weight (MW) is from oligosaccharide side chains. hCG is sometimes considered a mucopolysaccharide, like collagen, because of the large carbohydrate component. There is wide variation in hCG structure throughout normal and abnormal pregnancies, and in gestational trophoblastic diseases. In addition to "regular" hCG (hCG with intact subunits and the midtrimester pregnancy-like complement of oligosaccharides), at least six other key variants are present in serum samples: hyperglycosylated hCG, nicked hCG, hCG missing the P-subunit C-terminal peptide, free P-subunit, hyperglycosylated free P-subunit, and nicked free p-subunit as well as multiple combinations of these variants (i.e., nicked hyperglycosylated hCG missing the P-subunit C-terminal peptide). The same seven molecules plus the p-core fragment can be detected in urine samples (1-9). Table 1 and Fig. 1 summarize the structures of these key hCG-related molecules, which vary in size from p-core fragment (MW 9000-10,000) to hyperglycosylated hCG (MW 38,00042,000). hCG and related molecules may vary widely in charge because of differences in sialic acid content. As shown in Fig. 2, multiple charge isoforms in the range pi 3-7 are found in serum and urine samples, and in the range of pI 3-8 in hyperglycosylated hCG.

From: Current Clinical Pathology: Handbook of Clinical Laboratory Testing During Pregnancy Edited by: A. M. Gronowski © Humana Press, Totowa, NJ

Structure of hCG-Related Molecules Detected in Serum and Urine Samples in Normal and Abnormal Pregnancies and Trophoblastic Diseases (1—9)

Molecule a-subunit structure fi-subunit structure

Occurrence

Regular hCG (MW 37,000)

92 amino acids, no cleavages mono- and biantennary ^-linked oligosaccharides

145 amino acids, no cleavages biantennary ± fucose ^-linked, and mostly tri- and tetrasaccharide O-linked oligosaccharides

Principal form of phCG in serum, 6-40 wk of gestation

Hyperglycosylated hCG (MW 41,000)

Nicked hCG1 (MW 36,000)

92 amino acids, no cleavages mono- and biantennary + fucose ^-linked oligosaccharides

92 amino acids, no cleavages mono- and bi-antennary ^-linked oligosaccharides

145 amino acids, no cleavages bi-, short tri-, and triantennary ± fucose ^-linked and hexasaccharide O-linked oligosaccharides

145 amino acids, cleaved at p47-48, p43-44 or P44-45 biantennary ± fucose ^-linked, and mostly tri- and tetrasaccharide O-linked oligosac-charides

Principal phCG form produced at 3-5 wk of gestation, and in choriocarcinoma

Low concentrations in pregnancy, major hCG in weeks following termination or parturition hCG missing p-subunit C-terminal peptide1 cancer

Free p-subunit1 (MW 22,000)

92 amino acids, no cleavages mono- (8 sugars) and biantennary

^-linked oligosaccharides

No a-subunit

Residues 1-92, C-terminal peptide absent biantennary ± fucose

^-linked, and mostly tri- and tetrasaccharide O-linked oligosaccharides

145 amino acids, no cleavages biantennary ± fucose ^-linked, and mostly tri- and tetrasaccha-ride O-linked oligosaccharides

Primarily detected in gestational trophoblastic diseases and

Dissociation product of nicked hCG. Low levels in serum and urine in pregnancy continued

Table 1 (continued)

Molecule a-subunit structure

ß-subunit structure

Occurrence

Hyperglycosylated free No a-subunit

Nicked free ß-subunit1 (MW 22,000)

No a-subunit

145 amino acids, no cleavages bi-, short tri- and triantennary ± fucose ^-linked and hexasaccharide O-linked oligosaccharides

145 amino acids, cleaved at ß47-48, ß43-44 or ß44-45 biantennary ± fucose ^-linked, and mostly tri- and tetrasac-charide O-linked oligosaccharides

Significant production at 3-5 wk of gestation, and in choriocarcinoma

Most free p-subunit is nicked in serum or is dissociated from nicked hCG

Urine ß-core fragment (MW 10,000)

No a-subunit

Two peptides ß-subunit residues 6-40 linked to 55-92 degraded biantennary ^-linked and no O-linked side chains

Cleared from circulation rapidly. Principal of p-hCG in urine from 7-40 wk of gestation

Variations of these hCG variants are present in serum and urine, such as hyperglycosylated nicked hCG, nicked hCG missing ß-subunit C-terminal peptide, nicked hyperglycosylated free ß-subunit, nicked free ß-subunit missing the C-terminal peptide, and others.

Human Chorionic Gonadotropin Nicked

Fig. 1. Two-dimensional representation of structure and molecular weights of hCG forms produced by the placenta and present in serum and urine samples. Dark black lines represent peptide with numbers indicating N- and C-terminal residues. The letters N and O indicate sites of N- and O-linked oligosaccharides, and the thin lines indicate sites of disulfide linkages. Representations are based on the |3core fragment sequence as shown by Birken et al. (40), amino acid sequences as reported by Morgan et al. (41), disulphide bonds as shown by Lapthorn et al. (42), and the nicking sites as indicated by Elliott et al. (7).

Fig. 1. Two-dimensional representation of structure and molecular weights of hCG forms produced by the placenta and present in serum and urine samples. Dark black lines represent peptide with numbers indicating N- and C-terminal residues. The letters N and O indicate sites of N- and O-linked oligosaccharides, and the thin lines indicate sites of disulfide linkages. Representations are based on the |3core fragment sequence as shown by Birken et al. (40), amino acid sequences as reported by Morgan et al. (41), disulphide bonds as shown by Lapthorn et al. (42), and the nicking sites as indicated by Elliott et al. (7).

Fig. 2. Charge isoforms of hCG and hyperglycosylated hCG. Pregnancy urine samples were separated into 20 fractions by preparative isoelectric focusing using a Bio-Rad. Panel A shows total hCG isoforms in six samples (Rotofor DPC Immulite hCG assay); Panel B shows hyperglycosylated hCG isoforms in eight further samples (Nichols Advantage hyperglycosylated hCG [ITA] test). All results are distribution of isoforms (% ± standard error).

Fig. 2. Charge isoforms of hCG and hyperglycosylated hCG. Pregnancy urine samples were separated into 20 fractions by preparative isoelectric focusing using a Bio-Rad. Panel A shows total hCG isoforms in six samples (Rotofor DPC Immulite hCG assay); Panel B shows hyperglycosylated hCG isoforms in eight further samples (Nichols Advantage hyperglycosylated hCG [ITA] test). All results are distribution of isoforms (% ± standard error).

hCG PRODUCTION DURING PREGNANCY

hCG production does not begin until the developing blastocyst implants into the uterus. Circulating hCG can first be detected (following implantation of the fertilized egg) in serum and urine, with an ultrasensitive hCG test (detection limit 0.13 mlU/mL) as early as day 21 of the menstrual cycle (approx 7 d after fertilization) (10). Total hCG concentrations in serum and urine samples increase exponentially, doubling approximately every 40-48 h, and reach a peak between 8 and 12 wk of pregnancy (Fig. 3) (10a,10b). Total hCG production decreases between the 10th and 20th wk of pregnancy to between one-fifth and one-twentieth of peak hCG concentrations and then plateaus close to this concentration until term. Pregnancy hCG concentrations vary widely among individuals, probably more than any other hormone measurement. This is complicated further by interassay variation in quantitative serum hCG tests (see "Interassay Variation"). Table 2 illustrates the range of serum hCG results we have observed with the DPC Immulite (Diagnostic Products Corp., Los Angeles, CA) test in 300 serum samples. Table 2 is limited to hCG measurements over 4-wk periods and to ranges of concentrations; therefore, it is used only to illustrate the wide range of hCG results during pregnancy and not as a reference interval for different hCG tests or for patient care. As shown in Table 2, for any 4-wk period of pregnancy there is anywhere from a 10- to 200,000-fold variation in interindividual serum hCG concentrations.

Table 3 illustrates median concentrations and ranges for urine total P-hCG concentrations throughout the course of pregnancy, as determined using the DPC Immulite test. In our experience, first morning urine total P-hCG concentrations (including hyperglycosylated hCG and P-core fragment) are approximately one-half of serum total P-hCG concentrations. As shown in Table 3, for any one period of pregnancy there is approximately a 10- to 4000-fold variation in interindividual urine hCG concentrations.

Fig. 3. Concentration of chorionic gonadotropin in maternal serum as a function of gestational age. Lines represent the 2nd, 50th, and 97th percentiles. The maternal serum values from 14 to 25 wk are medians calculated from 24,229 pregnancies from testing performed at ARUP Laboratories, Inc., from January to October 1997. Evaluating health and maturation of the unborn: the role of the clinical laboratory. Clin Chem, 1992;38:1523-1529. Reprinted with permission from W.B. Saunders Co. Aswood, ER. Clinical chemistry of pregnancy. In Burtis CA, Ashwood, ER, eds. Tietz Textbook of Clinical Chemistry. 3rd ed. Philadelphia: W.B. Saunders, 1998, pp. 1736-1775. (Redrawn from ref. 10a: Reprinted with permission from W.B. Saunders [10b].)

Fig. 3. Concentration of chorionic gonadotropin in maternal serum as a function of gestational age. Lines represent the 2nd, 50th, and 97th percentiles. The maternal serum values from 14 to 25 wk are medians calculated from 24,229 pregnancies from testing performed at ARUP Laboratories, Inc., from January to October 1997. Evaluating health and maturation of the unborn: the role of the clinical laboratory. Clin Chem, 1992;38:1523-1529. Reprinted with permission from W.B. Saunders Co. Aswood, ER. Clinical chemistry of pregnancy. In Burtis CA, Ashwood, ER, eds. Tietz Textbook of Clinical Chemistry. 3rd ed. Philadelphia: W.B. Saunders, 1998, pp. 1736-1775. (Redrawn from ref. 10a: Reprinted with permission from W.B. Saunders [10b].)

Table 2

Range of Total p-hCG Concentrations in 300 Serum Samples Collected During the Course of Pregnancy, as Determined in the DPC Immulite hCG Assay

Table 2

Range of Total p-hCG Concentrations in 300 Serum Samples Collected During the Course of Pregnancy, as Determined in the DPC Immulite hCG Assay

Gestational age (wk)

Total ß-hCG range (mIU/mL)

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