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Technical Briefs |
Departments of
1
Obstetrics and Gynecology and
2
Clinical Immunology, The Edith Wolfson Medical Center, Holon, Holon 58100, and Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
3
Department of Obstetrics and Gynecology, Chieti University, 66031 Chieti, Italy
aaddress correspondence to this author at: Division of Obstetrics and Gynecology, The Edith Wolfson Medical Center, Holon 58100, Israel; fax 9723-502-8503, e-mail sigi-ro{at}zahav.net.il
ß-Core fragment (ßcf) is the main metabolic breakdown product of human chorionic gonadotropin (hCG) (1). This analyte appears in maternal urine during pregnancy, but is undetectable in serum. Recent studies (2)(3)(4) have shown that concentrations of hCGßcf are substantially increased in pregnancies affected by Down syndrome, suggesting the use of this analyte for midtrimester antenatal screening. However, various groups of investigators have reported large differences in the efficacy of screening by means of hCGßcf (5)(6)(7). Some of these inconsistencies have been attributed to conditions used for storage of urine specimens (8), as well as chance, assay method, and study design (7).
hCG serum concentrations show distinct diurnal variation (9). We investigated whether urine concentrations of hCGßcf, the terminal breakdown product of serum hCG, also show diurnal variation. Alterations in the time of day when urine specimens were collected in previously reported studies could potentially explain the inconsistent findings on the screening efficacy of this analyte for Down syndrome. We prospectively and longitudinally examined hCGßcf concentrations in urine from women with uncomplicated midtrimester pregnancies.
We prospectively studied 46 healthy pregnant women at 15–19 weeks of gestation. Pregnancies were dated by menstrual history and first-trimester ultrasonographic examination and followed until delivery. A detailed history was obtained from all volunteers to exclude preexisting medical or obstetric complications.
Midstream urine samples were collected at 0600, 1200, 1800, and 0000. Specimens were frozen within 4 h and stored at -20 °C until assayed for hCGßcf within 7 days of collection. hCGßcf concentrations were measured by use of a commercially available enzyme-immunoassay reagent set (Novamed Ltd.). The test was repeated at a different dilution if the measured concentration was outside the operating range. Measurements of hCGßcf were indexed to urine creatinine concentrations, as measured by an enzymatic creatinine assay. Fetal karyotypes obtained by amniocentesis were available for 14 participants and were normal. Fetuses without available karyotypes were normal at birth. Normalized hCGßcf concentrations were also expressed as multiples of the smoothed medians (MoMs) for the duration of gestation. Medians for the duration of gestation (per week) were obtained from the analysis of 674 urine specimens from pregnant women (15–19 weeks gestation) by use of the same laboratory methods. For these specimens, no attention was paid to the time of day of collection.
Ratios of urine hCGßcf/urine creatinine concentrations were statistically analyzed by one-way ANOVA (SPSS, Ver. 8.00) after natural log transformation. P <0.005 was defined as statistically significant.
Mean maternal age and gestational age in the study group were 29.8 years (SD, 3.94; range, 19–35 years) and 17.2 weeks (SD, 1.41 weeks; range, 15–20 weeks), respectively. Mean gravidity and parity were 1.82 (SD, 1.01; range, 1–5) and 0.56 (SD, 0.94; range, 0–4), respectively. Mean maternal weight was 58.3 ± 6.5 kg (range, 47–85). Measurements of hCGßcf and creatinine at the various sampling times are reported in Table 1
. Crude hCGßcf concentrations varied significantly during the day, with peak concentrations occurring at 0600 and the lowest concentrations at 1800; mean differences in measured concentrations were 225%. Creatinine concentrations also showed distinct diurnal variation, with concentrations highest at 0600 and lowest at 1200. Creatinine concentrations were used to normalize hCGßcf concentrations in urine specimens (Table 1
). The mean difference between creatinine-normalized peak and nadir urine hCGßcf concentrations was 211%. When hCGßcf/creatinine ratios were expressed as MoMs for 1 week of gestation, the difference between median peak and nadir concentrations was 0.8 MoM. Normalized hCGßcf concentrations were highest at 0600 and lowest at 1800 (Fig. 1
); ANOVA showed that the difference was statistically significant (P <0.0001). Compared with baseline values at 0, concentrations were significantly different at 0600 (P <0.005) and 1200 (P <0.005).
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The main finding of our study was that creatinine-normalized hCGßcf concentrations in urine showed significant diurnal variation. The difference between peak and nadir concentrations was equivalent to 0.8 MoM. One would expect, therefore, that calculations of the risk of Down syndrome must be substantially affected by the timing of urine specimen collection. In turn, these findings could explain some of the reported inconsistencies in reported screening efficacies.
Urine hCGßcf is the final metabolic breakdown product of the nicked hCG ß subunit. Increased nicking of the ß subunit in Down syndrome pregnancies was first demonstrated by Rotmensch and coworkers (10)(11), but the mechanism for this phenomenon remains unclear. Greater phagocytic activity against the placenta as a foreign semiallograft for the mother and increased nicking activity in the sera of pregnant women have been proposed.
Early studies found urine concentrations of hCGßcf to be five- to sixfold higher in Down syndrome than in normal pregnancies (12). However, as more studies have been published, the early results have been at the high end of the range. In fact, the variability of results has been unusually great; concentrations of hCGßcf between 1.3- and 6.1-fold higher than the unaffected median have been reported (12). This variability suggests that factors other than the biology of Down syndrome pregnancies affect the results. It is unclear, at present, whether dividing hCGßcf concentrations by creatinine concentrations properly normalizes for sample concentrations (13). It is conceivable that in very dilute urine, the relationship between creatinine and urine concentrations of hCGßcf is altered. Cole et al. (8) showed that urine concentrations of hCGßcf in Down syndrome pregnancies decrease over time in storage, possibly because of aggregation. This finding implied that the interval between sampling of urine and the assay could substantially affect hCGßcf concentrations. Cuckle et al. (7) considered the possibility that hCGßcf concentrations in urine may vary during the day, even after normalization with creatinine. As part of a large multicenter study on the screening efficacy of hCGßcf for Down syndrome, cross-sectional information on the time of voiding was obtained toward the end of patient recruitment. The data showed an impressive increase in urine hCGßcf concentrations, from 0.7 MoM at 0400 to 1.4 MoM at 0800. These findings appear to be consistent with our own data, showing a similar increase between 0000 and 0600. However, the study by Cuckle et al. (7) reported cross-sectional data between 0400 and 1600, whereas in our study, participants were followed up longitudinally for 24 h.
Our findings make it plausible that diurnal variation in urine concentrations of hCGßcf could affect the efficacy of screening for Down syndrome. On the one hand, there is no reason to believe that women with Down syndrome pregnancies and women with normal pregnancies have systematically collected samples at different times of the day in the previously reported studies. However, the number of Down syndrome pregnancies has been very small in most published studies. Therefore, we cannot exclude differences in timing of sampling for these few specimens, compared with the women with normal pregnancies, as a cause of the reported inconsistencies.
In summary, hCGßcf displays substantial diurnal variation. Assessment of the value of hCGßcf as a screening marker for Down syndrome should be reconsidered when data adjusted for time of sampling are available.
References
The following articles in journals at HighWire Press have cited this article:
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  S. F. de Medeiros and R. J. Norman Human choriogonadotrophin protein core and sugar branches heterogeneity: basic and clinical insights Hum. Reprod. Update, October 22, 2008; (2008) dmn036v1. [Abstract] [Full Text] [PDF]
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