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Doping in Sport Symposium Proceedings |
Departments of
1 Clinical Chemistry and
2 Obstetrics and Gynecology, Helsinki University Central Hospital, Haartmaninkatu 2, FIN-00290 Helsinki, Finland.
a Author for correspondence. Fax +358-9-471 4804; e-mail ulf-hakan.stenman{at}hyks.mailnet.fi
Abstract
The pregnancy hormone human chorionic gonadotropin (hCG) is also present at low concentrations in plasma and urine of men and nonpregnant women. hCG immunoreactivity occurs in various molecular forms: Besides the intact hCG heterodimer, considerable amounts of proteolytically cleaved forms, free subunits, and fragments are found in plasma and urine. Especially in urine, proteolytic fragments constitute a major part of the hCG immunoreactivity. The different forms of hCG cross-react to various degrees in immunoassays and constitute a problem for standardization of specific hCG determinations. After injection of hCG (10 000 IU of Pregnyl®; Organon), above-normal concentrations of hCG can be detected in serum and urine for 7–11 days. Most immunoassays for hCG also measure hCGß. Quantitative hCG determinations are mainly performed on serum samples, and very few commercial hCG determinations have been validated for determination of urine samples. Considerable care must therefore be exercised when utilizing such assays to analyze urines for doping control.
Key Words: indexing terms: standardization • steroid hermones • urine • reference values • pregnancy • cancer • luteinizing hormone • estrogen
The gonadotropins luteinizing hormone (LH)1 and chorionic gonadotropin (hCG) are therapeutically used to stimulate gonadal steroid production, both in women and men. hCG is also used by some athletes to stimulate testosterone production and to normalize the testosterone/epitestosterone ratio during or after administration of testosterone and other anabolic steroids. The International Olympic Committee (IOC) imposed a ban on hCG administration in 1987 (1), but no official decision limit for hCG in urine has been defined. IOC prescribes that hCG should be determined by two different assays, but neither the decision limit nor the specificity of the assays has been defined. Assay of hCG in serum and urine can now be performed with extremely sensitive and specific methods, which can determine the low basal concentrations found in nonpregnant women and men. From the hCG concentrations in urine from 1400 men, Laidler et al. have recommended that a cutoff value 10 IU/L should be used for doping control (1). However, this recommendation may be considered valid only for the method used. Different assay methods for determining hCG in serum are poorly standardized—e.g., the results obtained by various methods may vary by a factor of five (2)—and different methods show large variation in reactivity with various forms of hCG (3). Furthermore, given the more extensive heterogeneity of hCG in urine than in serum, larger differences will be observed in urine. Therefore, the various forms of hCG in circulation and urine and the design of the assays used will affect the results. Little is known about the extent of this effect, because quantitative assays for hCG in urine have very little clinical use, and kit manufacturers have therefore not validated their methods for assay of urine. Thorough knowledge of the forms of hCG detected in urine by various methods under normal conditions and after hCG administration are prerequisites for the use of hCG determinations for doping control.
Molecular Forms of hCG and Its Subunits
The following nomenclature for well-characterized molecular forms of hCG has been suggested by the Working Group for standardization of hCG determinations appointed by the IFCC (2).
Human chorionic gonadotropin (hCG) is the intact heterodimer,
consisting of an 
- and a ß-subunit. hCG is the major form of hCG
immunoreactivity during pregnancy.
The free beta subunit (hCGß) consists of a 22-kDa polypeptide containing 145 amino acid residues, with two N-linked and four O-linked carbohydrate chains. hCGß is secreted by the placenta during pregnancy, and the serum concentrations are ~1% of those of hCG.
The free alpha subunit (hCG) consists of a glycosylated 14-kDa
polypeptide containing 92 amino acid residues and two N-linked
carbohydrate chains. This subunit is common to hCG, LH,
follicle-stimulating hormone (FSH), and thyroid-stimulating hormone
(TSH). hCG is produced in excess of the ß-subunits by both the
pituitary and the placenta (4)(5).
The core fragment of hCGß (hCGßcf) is a 10-kDa two-chain polypeptide lacking the amino acid sequences 1–5, 41–54, and 93–145. The two chains are held together by disulfide bonds. hCGßcf is probably formed from hCG and hCGß by proteolytic degradation in the kidneys.
Nicked hCG (hCGn) and nicked hCGß (hCGßn) are partially degraded forms of hCG and hCGß, in which the peptide chain of hCGß is cleaved in the region 43–49 (usually between amino acids 47 and 48 and less frequently between 44 and 45).
Assay Methods
Serum assays.
Specific determination of hCG in serum
from nonpregnant subjects was first performed by RIA with antiserum SB6
(6). This assay also measures hCGß. Specific measurement
of hCG in serum became possible with the development of ultrasensitive
immunometric assays that were based on monoclonal antibodies
(7)(8). This technology has also made it
possible to develop "sandwich"-type assays for measuring hCGß in
serum (9) and hCGßcf in urine
(10)(11). Only a few commercially available
methods are capable of measuring the physiological concentrations of
hCG in men and nonpregnant women, and the concentrations of hCGß and
hCGßcf can only be measured by some research methods.
Many hCG immunoassays are designed to measure both hCG and hCGß. Such
methods are based on the use of two or three antibodies specific for
hCGß or of a monoclonal antibody and polyclonal antiserum. A common
problem with these assays is that they tend to overestimate hCGß
(2). On the other hand, some assays based on a combination
of anti-hCG and anti-ß antibodies may underestimate hCGn
(3). In pregnancy, urine hCG may be extensively
"nicked," but whether this form occurs in urine of nonpregnant
subjects is not known. By utilizing one antibody to hCGß and another
one to hCG or the ß dimer, one can design highly specific
assays reacting with hCG but not with hCGß. Cross-reaction with LH,
which used to be a problem with conventional RIA methods, is usually
negligible. However, denaturation of LH during storage has been shown
to increase its cross-reactivity, even in highly specific assays based
on monoclonal antibodies (7).
Urine assays.
Commercial hCG assays have usually not
been validated for determination of hCG in urine. The reactivity with
hCGßcf may be a problem in assays designed to measure both hCG and
hCGß. Another potential problem is the cross-reaction with denatured
LH. The core fragment of LH (LHßcf) is the predominant form of LH
immunoreactivity in urine and may interfere in hCG assays. Assays for
total hCG that overestimate hCGß cannot be used to specifically
measure hCG in urine, which may contain much hCGß and hCGßcf.
Given the close structural similarity between hCGßcf and LHßcf, some assays for hCGßcf also measure LHßcf (12), which is present in urine from nonpregnant subjects at much higher concentrations than hCGßcf (13). However, assays based on two monoclonal antibodies with negligible cross-reaction with LHßcf (<2%) have been described (11).
Because of the large differences in assay design and specificity for different forms of hCG, the concentrations measured are highly dependent on the assay used (2)(3). The concentrations measured in quality-control samples may vary fivefold (2). The relative amounts of various hCG forms also depend on the clinical situation of the subject tested. All these factors contribute to the large variation in reported hCG concentrations.
hCG can be detected in urine by highly specific mass-spectrometric methods. However, there is still at least a 100-fold difference in detection limit between mass-spectrometric and immunological methods (14).
The concentrations of protein hormones in urine are usually corrected for variations in urine flow. Mostly the concentrations are normalized by dividing the hormone concentrations with the creatinine concentration, but correction to mean urine density is also used (15). In healthy subjects the mean density is 1.015 mg/L (Alfthan and Stenman, unpublished); however, normal urine may range from 10-fold more dilute to 3-fold more concentrated than average. Although creatinine correction is widely used, in our experience the concentrations of gonadotropins are not directly correlated to creatinine in very dilute or concentrated urines (16), and better ways of normalizing urine concentrations of protein hormones in urine need to be developed. This appears to be especially important in athletes, whose urine output after strenuous exercise may be very low, which may be expected to increase the concentrations of protein hormones.
Concentrations of hCG in Serum and Urine in Health and
Disease
in nonpregnant women and men
Serum.
The concentrations of hCG in serum of nonpregnant
women are measurable by sensitive assays. These concentrations are
dependent on gonadal function, showing a clear increase around the
menopause, when the upper reference limit increases from 3 to 5 IU/L
(from 8.6 to 15.5 pmol/L) (17). Concentrations up to 8–10
IU/L are occasionally observed in healthy women.
In men, the concentrations are lower but mostly are measurable by highly sensitive assays. In those <50 years old, the upper reference limit is 0.7 IU/L (2.1 pmol/L); in those >50, it is 2 IU/L (6.1 pmol/L) (17). Concentrations as great as 3–4 IU/L are occasionally observed in healthy men.
The concentrations of hCGß are lower than those of hCG. Only a slight increase is observed with age and the sex difference is small. The upper reference limit in women <50 years is 1.6 pmol/L and in men 1.9 pmol/L. The corresponding values for those >50 years are 2.0 and 2.1 pmol/L, respectively. The concentrations of hCGßcf in serum are below the detection limit of present assays (17).
Urine.
The median concentrations of hCG and hCGß are
similar in serum and urine, whereas those of hCGßcf are much higher
in urine (17). This suggests that, as in pregnancy,
hCGßcf is produced by proteolytic degradation of hCG and hCGß in
the kidneys. The concentrations of hCG and hCGß in urine of women and
men increase with age but not as sharply as those in serum. The upper
reference limits for hCG in women <50 and 
50 years are 3 and 4 IU/L
(8.8 and 11.5 pmol/L), respectively. The corresponding values for men
are 1 and 3 IU/L (2.9 and 8.4 pmol/L) (17).
The concentrations of hCGßcf in urine are similar in women and men
(17), increasing at age ~50. By our method, the upper
reference limits for pre- and postmenopausal women are 8.1 and 9.5
pmol/L, respectively, and those for men <50 and 50 are 6.7 and 8.5
pmol/L, respectively (17).
In nonpregnant subjects, the common glycoprotein hormone subunit
(hCG) is mainly derived from the pituitary, and its serum
concentrations do not reflect hCG production. The concentrations in
urine are three- to fivefold those in serum
(18)(19).
in women with normal pregnancy
The concentration of hCG in serum starts to increase 7–11 days
after ovulation, corresponding to 21–25 days after the last menstrual
period (20). The increase is nearly exponential during the
first 5 weeks after implantation (21). As measured by our
method, the concentrations peak at ~110 000 IU/L during week 8–10
after the last menstrual period (i.e., 5–7 weeks after implantation),
after which they decrease and reach a nadir of 36 000 IU/L at the
beginning of the second trimester (9). (These values are
method dependent (2)(3).) There is a small
increase before delivery (22), and the concentrations of
hCG and its subunits are higher in multiple pregnancies than in
singleton ones (23). Individual variation in hCG
concentrations is large (9).
After normal delivery, the hCG concentrations decrease with a half-time of about 24–32 h (24) and normalize within 1–3 weeks (25)(26). Serum hCG concentrations after a first-trimester abortion may take 4–5 weeks to return to normal values (27). Mean serum concentrations of hCGß during pregnancy vary from 0.5% to 1.6% (range 0.1–3.6%) of the hCG concentrations (9)(28).
hCGßcf can be detected in serum during pregnancy in quantities corresponding to 0.03% of the hCG content. The concentrations in urine are ~4000-fold those in serum (29). hCGßcf is the major form of hCG immunoreactivity in pregnancy urine except during the first month of pregnancy, when hCG predominates (30). The concentrations of hCG immunoreactivity in paired serum and urine samples are similar and correlate strongly (31). Total hCG immunoreactivity is higher in urine (20)(32) apparently attributable to hCGßcf.
in pregnancy-related disorders
Early fetal loss.
About 20–25% of all conceptions end
in an early spontaneous abortion (33)(34).
Often the only indication of pregnancy is an increased concentration of
hCG in serum or urine; hence, the condition is also called subclinical
abortion or biochemical pregnancy (35). Spontaneous
abortions occur most often during the first trimester. Initially, the
concentration of hCG in serum may increase normally before it starts
falling (36).
Ectopic pregnancy.
In ectopic pregnancy, the
concentrations of hCG in serum tend to be lower than during normal
pregnancy (36)(37), but they may also be
normal for a long time.
in malignant disease
Gestational trophoblastic disease.
The concentrations of
hCG in serum and urine are strongly increased in choriocarcinoma and
molar disease (38). In benign trophoblastic disease, the
concentrations of hCGß are usually <5% of those of hCG, but in
choriocarcinoma the proportion of hCGß is higher
(39)(40). The proportion of hCGn is higher in
serum of patients with choriocarcinoma than in pregnancy
(41).
Testicular cancer.
About 50% of the nonseminomatous
testicular cancers cause an increase of hCG in serum and urine, often
to very high concentrations. hCG is seldom increased in seminomas, but
these tumors relatively often produce hCGß (42).
Nontrophoblastic tumors.
hCG is very rarely produced by
nontrophoblastic tumors, but low-level expression of hCGß is quite
common. Because the serum concentrations of hCGß in cancer patients
are usually only moderately increased, hCGß is a useful marker only
if measured by an ultrasensitive assay (43). The strong
correlation between hCGß in serum and hCGßcf in urine suggests
that, when excreted into urine, hCGß is degraded to hCGßcf.
However, by immunohistochemistry staining with monoclonal hCGßcf
antibodies, positive staining has frequently been observed in
nontrophoblastic cancer cells, suggesting that some hCGß (and hCG)
may be degraded within the cell before secretion (44). The
relative contribution of the various sources of hCGßcf remains to be
determined.
Effect of Drugs
gonadotropin-releasing hormone and estrogens
In men and nonpregnant women, gonadotropin-releasing hormone
causes a threefold increase in the serum concentrations of hCG, whereas
treatment with estrogen and progestagen lowers the serum hCG by
~50%. These results suggest that hCG in serum is derived from the
pituitary (7).
hcg administration
Intramuscular injection of partially purified urinary hCG is used
to induce ovulation in hormone-stimulated menstrual cycles. We have
measured the concentrations of hCG in serum and urine and of hCGßcf
in urine after injection of 10 000 IU of Pregnyl® to
women during stimulated cycles. The assays used have been described in
detail (9)(11)(17). The procedures
followed were in accordance with the Helsinki Declaration of 1975,
revised in 1983. Peak serum hCG concentrations of 200–300 IU/L are
observed 1 day after injection, after which they decline with a mean
(±SD) half-time of 1.56 ± 0.12 days (Fig. 1
A). hCG concentrations in serum stay above the upper reference
limit for 7–11 days after injection and reach baseline values within
8–13 days. Similar results have been obtained in another study
(45).
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The hCG concentrations in urine correlate fairly closely with those in
serum, and there is very little delay in excretion. The concentrations
remain increased for the same time as those in serum, and their
elimination half-times are similar, 1.45 ± 0.10 day (Fig. 1A
).
The pattern of hCGßcf in urine is slightly different. Concomitant
with the hCG peak in serum is a sharp peak of hCGßcf in urine. The
hCGßcf concentration decreases rapidly within 1 day, after which it
falls at the same rate as the concentration of hCG. The urine
concentrations of hCGßcf drop below the upper reference limit 2–3
days earlier than does hCG (Fig. 1A
). Pregnyl contains equal amounts of
hCG and hCGßcf, and the first hCGßcf peak is explained by rapid
excretion of hCGßcf into urine (46).
In women who conceive after hCG stimulation, the hCG concentrations in
serum and urine start increasing again before the injected hCG has
reached baseline values in serum. The hCG concentrations in urine are
similar to those in serum, but the hCGßcf concentrations start
increasing after a delay of 3–4 days (Fig. 1B
). This delay is similar
to the delay in excretion of LHßcf after the midcycle LH surge
(13). On the basis of this, we expected to also see a
delayed excretion of hCGßcf into urine, but this was not the case.
The metabolism of purified hCG may therefore differ from that of
endogenous LH. Interestingly, after conception, the excretion of
hCGßcf is delayed (Fig. 1
) in a way similar to that of LHßcf
(13).
Conclusions
hCG is a normally occurring hormone, and its concentrations in serum and urine can be measured in men and in nonpregnant women by sensitive methods. The determination is complicated by the occurrence of many different forms of hCG immunoreactivity, which exert a variable response in different assays. The urine concentrations are also affected by variations in urinary flow rate. To be useful for doping control, the reference values for hCG need to be determined on a sufficiently large number of samples from women and men of the proper age group. Furthermore, the reference values need to be determined by the same method as used for doping control. The results obtained by various hCG assays vary by as much as fivefold because of differences in specificity and calibration. Therefore, some methods may give false-positive results whereas others may miss doping cases. It is also essential that the effect of exercise be determined.
The disappearance curves for hCG in serum and urine after injection of urinary hCG in combination with appropriately established reference values provide the basis for detection of hCG administration. Intramuscular administration of hCG causes a prolonged increase of serum hCG and of the urinary excretion of hCG and hCGßcf. The serum and urine concentrations decrease to less than the upper reference limit in 6–9 days. Generally, however, decision limits higher than the upper reference limit are used for doping control.
If appropriately standardized and validated methods are used, investigators should be able to detect self-administration of hCG in men as reliably as anabolic steroids and testosterone are now being detected by mass-spectrometric methods. However, although the methods for such determinations are available, very few have been appropriately validated. Therefore, the present recommendation by the IOC that hCG should be determined by two different immunoassays needs to be more specifically defined before an assay is used to sentence athletes.
Footnotes
1 Nonstandard abbreviations: hCG, human chorionic
gonadotropin; IOC, International Olympic Committee; hCGß, free beta
subunit of hCG; hCG, free alpha subunit of hCG; LH, luteinizing
hormone; hCGßcf, core fragment of hCGß; hCGn, "nicked" hCG;
hCGßn, "nicked" hCGß; LHßcf, core fragment of LHß. 
References
hCG and ßhCG as measured by specific monoclonal immunoradiometric assays. Endocrinology 1987;120:549-558.
The following articles in journals at HighWire Press have cited this article:
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  U.-H. Stenman, A. Tiitinen, H. Alfthan, and L. Valmu The classification, functions and clinical use of different isoforms of HCG Hum. Reprod. Update, November 1, 2006; 12(6): 769 - 784. [Abstract] [Full Text] [PDF]
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 L. A. Cole, S. Shahabi, S. A. Butler, H. Mitchell, E. S. Newlands, H. R. Behrman, and H. L. Verrill Utility of Commonly Used Commercial Human Chorionic Gonadotropin Immunoassays in the Diagnosis and Management of Trophoblastic Diseases Clin. Chem., February 1, 2001; 47(2): 308 - 315. [Abstract] [Full Text] [PDF]
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D. A. Cowan and A. T. Kicman Doping in Sport: Misuse, Analytical Tests, and Legal Aspects Clin. Chem., July 1, 1997; 43(7): 1110 - 1113. [Full Text] [PDF]
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