Second-trimester maternal urine human chorionic gonadotrophin ß-core fragment concentrations in Asian pregnancies with fetal chromosomal abnormalities

Jenn J. Hsu1,4, Kevin Spencer2, Tai H. Hung1, T'sang T. Hsieh1 and Yung K. Soong3

1 Department of Obstetrics and Gynaecology, Taipei Chang Gung Memorial Hospital, Taipei, 2 The Endocrine Unit, Department of Clinical Biochemistry, Harold Wood Hospital, Romford, Essex, UK and 3 Lin-Kou Chang Gung Memorial Hospital, Tau-Young, Taiwan, ROC


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of this study was to investigate the second trimester concentrations of maternal urine human chorionic gonadotrophin ß-core fragment (HCGßcf) in Asian pregnanci2es with fetal chromosomal abnormalities. HCGßcf concentrations were analysed from 34 urine samples in chromosomally abnormal pregnancies, including 28 cases of Down's syndrome, one case of trisomy 18, and five cases of other chromosomal abnormalities (one mosaic deletion and four translocations), and in a cohort of 268 normal pregnancies receiving second trimester amniocentesis. Results were normalized to urine creatinine (Cr) concentration and converted to the multiple of the median (MOM) concentration for the appropriate gestation. The median HCGßcf MOM concentrations of Down's syndrome pregnancies (12.89) was significantly higher than that of normal pregnancies (1.06) (P < 0.00001). Wide variations of HCGßcf concentrations were observed in other chromosomally abnormal pregnancies. There were 18 of 28 (64%) Down's syndrome cases but one of five (20%) other chromosomally abnormal cases with HCGßcf concentrations above the 95th centile of the control values (8.22 MOM cut-off). These findings suggest that HCGßcf could be a potential marker in urine screening for fetal Down's syndrome in Asians.

Key words: chromosomal abnormalities/Down's syndrome/human chorionic gonadotrophin ß-core fragment/prenatal screening/urine


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human chorionic gonadotrophin (HCG), a placenta-derived glycoprotein hormone, is composed of {alpha} and ß subunits bonding non-covalently. HCG or its ß-subunit (HCGß) has been widely used as the discriminatory serum marker of Down's syndrome either in Asians (Hsu et al., 1997Go) or Caucasians (Palomaki et al., 1997Go). Elevations of both serum HCG and HCGß were observed in Down's syndrome pregnancies; however, measurement of HCGß has been shown to be a superior discriminator than measurement of HCG (Spencer et al., 1992Go; Wald et al., 1993Go; Extermann et al., 1998Go). The HCG molecule is less stable in affected pregnancy due to the presence of `nicking' of the ß-subunit (Rotmensch et al., 1992Go). Therefore, if serum concentrations of HCGß are higher in Down's syndrome pregnancies, urine HCGßcf concentrations, a major metabolic product of HCG in pregnant urine, should be elevated more than the circulating molecules (Cuckle et al., 1994Go).

Recent reports have indicated that the urine HCGßcf could be an even better marker of fetal aneuploidy than the existing serum markers in the second trimester. They found at least four-fold elevation of HCGßcf in Down's syndrome pregnancies and achieved a 62–88% detection rate with a 5% false-positive rate (Cuckle et al., 1994Go, 1995Go; Canick et al., 1995Go; Iles, 1996Go; Cole et al., 1997aGo; Isozaki et al., 1997Go). However, some authors were still uncertain of the effectiveness of using HCGßcf for Down's syndrome screening in Asians (Hayashi and Kozu, 1995Go; Lam et al., 1997Go) and Caucasians (Spencer et al., 1996Go). Because of racial differences between Asians and Caucasians, it remains to be seen whether these urine screening strategies are equally applicable in Asians. The aim of the current study is to investigate whether maternal urine HCGßcf might be an even better marker for Asian pregnancies with fetal chromosomal abnormalities.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Urine samples were collected before genetic amniocentesis or at the time of termination of pregnancy at 14–25 weeks of gestation in 34 chromosomally abnormal pregnancies, including 28 cases of Down's syndrome, one case of trisomy 18, and five cases of other chromosomal abnormalities [one mosaic deletion: 46,XX/46,XX,del(6)(p21); two translocations: 45,XX,t(q15,q21),pat; and 46,XX,t(4;8)(q35;q22),mat; and two mosaic translocations: 46,XX/46,XX,t(4;5)(q12;q25); and 46,XY/46,XY,t(4;7)(q31.1;q22)] and in a cohort of 268 normal pregnancies, matched with maternal age, undergoing genetic amniocentesis between January 1996 and December 1997. Both Down's syndrome and control cases had been enrolled as part of a previous collaborative study (Hsu et al., 1999bGo). The gestational age of all cases was determined by ultrasound parameters. Ultrasound was performed before amniocentesis (15–20 weeks) or termination of pregnancy (19–25 weeks). There was no intrauterine growth retardation in our case of trisomy 18 where ultrasound dating was compatible with menstrual dating. After delivery of the fetus or newborn, we collected the cord blood and placental tissue or fetal skin for karyotyping. The fetal outcomes of both chromosomally abnormal and normal pregnancies were all confirmed by karyotyping. The indications for genetic amniocentesis in affected and unaffected pregnancies were advanced maternal age (57 versus 56%), positive results of Down's syndrome screening (36 versus 25%), abnormal ultrasound findings (4 versus 1%), previous family or personal history of congenital anomaly (3 versus 2%), and others (0 versus 16%). The latter category included anxiety or voluntary request, drug or X-ray exposure, early gestation virus infection and habitual abortion.

Urine samples were centrifuged and stored at –40°C prior to analysis with mean storage time of 55 and 51 weeks in Down's syndrome and normal pregnancies respectively. The HCGßcf concentrations were measured by using a commercial enzyme immunoassay kit (UGF-EIA Toa, Toagosei Co. Ltd, Tokyo, Japan) in which only the HCGßcf was specifically detected with cross-reactivity of 0.44% to the HCGß (Cole, 1995Go). All samples were diluted at 1:50 and 1:2500 prior to assay. To correct the variation of urine-concentrating effects, HCGßcf concentrations (ng/l) were corrected to a standard urine creatinine (Cr) concentration (mmol/l). Cr was measured with a standard Jaffe reaction procedure after a 1 in 20 urine dilution in 0.9% normal saline. Optimal smoothed gestation-specific medians of HCGßcf were calculated by weighted non-linear regression from the observed medians at each gestational week (Table IGo). Considering the gestational variation of creatinine, the results were expressed in multiples of the median (MOM) for the relevant gestational week.


View this table:
[in this window]
[in a new window]
 
Table I. The distributions of urine human chorionic gonadotrophin ß-core fragment (HCGßcf), creatinine (Cr) and HCGßcf/Cr in normal controls
 

    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The mean (SD) maternal age in Down's syndrome pregnancies was 34.7 (4.3) years (median 34.4, range 27.8–43.5) and in normal pregnancies 33.6 (3.9) years (median 34.5, range 22.9–42.7) (P = 1.17, Mann–Whitney U-test). There was no relationship between the urine HCGßcf log MOM concentrations and maternal age in either Down's syndrome or normal pregnancies [r (affected) = 0.30, P = 0.12; r (unaffected) = 0.08, P = 0.21; Pearson's correlation].

The median HCGßcf values were inversely related to the gestation in weeks in unaffected pregnancies (log median (HCGßcf) = –29.7697 + 85.3906xlog (GA) –72.6908xlog (GA)2 + 19.8169xlog (GA)3, where GA is the gestation in weeks). The median, 5th, 10th, 25th, 75th, 90th, and 95th centiles of normal controls were 1.06, 0.19, 0.28, 0.58, 3.05, 6.76, and 8.22 MOM respectively. There was a wide spread of values in the controls, exemplified by the five-fold range between the 25th and 75th centiles or by the 24-fold 10th and 90th centile range. Urine HCGßcf log MOM values fitted a Gaussian distribution in both Down's syndrome and normal pregnancies (D = 0.91, P = 0.39; and D = 1.02, P = 0.25; respectively; Kolmogorov–Smirnov test).

Urine HCGßcf MOM concentrations in each case of Down's syndrome pregnancies are shown in Figure 1Go. There were 27 of 28 [96%, 95% confidence interval (CI) 90–103%] Down's syndrome cases (median 12.89 MOM, 95% CI 4.75–35.75) with urine HCGßcf above the median of normal controls (1.06 MOM, 95% CI 0.91–1.27) and 18 of 28 (64%, 95% CI 47–82%) cases at or above the 95th centile of the control values (8.22 MOM cut-off). One case of trisomy 18 (6.71 MOM) and four of five (80%) other chromosomally abnormal cases (median 2.70 MOM) had elevation of urine HCGßcf above the median of normal controls and one of five (20%) other chromosomally abnormal cases with HCGßcf concentrations at or above the 95th centile of the control values (Figure 2Go).



View larger version (18K):
[in this window]
[in a new window]
 
Figure 1. Distribution of urine HCG ß-core fragment (HCGßcf) plotted as the multiple of the median (MOM) concentrations in Down's syndrome pregnancies.

 


View larger version (13K):
[in this window]
[in a new window]
 
Figure 2. Distribution of urine HCG ß-core fragment (HCGßcf) the multiple of the median (MOM) concentrations from the 20 cases of trisomy 18 pregnancies and 22 cases of other chromosomally abnormal pregnancies from this study (large solid squares with symbol) and the world literature.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Throughout most of pregnancy, HCGßcf accounts for two-thirds of HCGß-related molecules in second trimester pregnancy urine (Kato and Braunstein, 1988Go). Although the origin of urine HCGßcf is unclear, there are reports that it is a renal breakdown product of either HCG or HCGß (Akar et al., 1988Go). Therefore, the decline curve of urine HCGßcf median values is parallel with those of serum HCG and HCGß in relation to advancing gestational age (Hsu et al., 1995Go, 1998Go). Because of unstable nicked HCGß and its faster clearance rate, urine HCGßcf should be elevated in the same way as serum HCG and HCGß (Spencer et al., 1992Go; Hsu et al., 1996Go; Extermann et al., 1998Go), as well as urine HCGß (Spencer et al., 1996Go; Hsu et al., 1999aGo), in Down's syndrome pregnancies.

We confirm that urine HCGßcf concentrations of Down's syndrome pregnancies is elevated in Asians, which is similar with those findings of previous studies in Asians (Hayashi and Kozu, 1995Go; Lam et al., 1997Go) and Caucasians (Cuckle et al., 1994Go, 1995Go; Canick et al., 1995Go; Iles, 1996Go; Cole et al., 1997aGo; Isozaki et al., 1997Go; Kellner et al., 1997Go). The median HCGßcf MOM value of Down's syndrome pregnancies in this study (12.89) was markedly higher than those of previous studies (Table IIGo). At a fixed 5% false-positive rate, the observed detection rate in this study (64%) was higher than previously reported results (Hayashi and Kozu, 1995Go; Spencer et al., 1996Go; Lam et al., 1997Go) and in agreement with results of previous studies (58–71%) (Cuckle et al., 1994Go; Iles, 1996Go; Cole et al., 1997aGo; Isozaki et al., 1997Go; Kellner et al., 1997Go), but lower than others (Canick et al., 1995Go; Cuckle et al., 1995Go) (Table IIGo).


View this table:
[in this window]
[in a new window]
 
Table II. The median values and observed detection rates of urine human chorionic gonadotrophin ß-core fragment (HCGßcf) in Down's syndrome pregnancies from the world literature
 
These wide variations of HCGßcf in median value and observed detection rate may be due to the differences in enrolled race, measured reagent or analytic methodology. It has been reported (Cole et al., 1997bGo) that the choice of urine immunoassay may affect the quality of the results and the usefulness for Down's syndrome screening. The results demonstrated that assays which only recognize HCGßcf are optimal for urine Down's syndrome screening with higher median values and detection rates. Therefore, that is the reason why the Wakotest ß-core, measuring the HCGß and HCGßcf together, may achieve the worst performance of Down's syndrome screening (Hayashi and Kozu, 1995Go) (Table IIGo). The assay we used in this study (UGF-EIA Toa) has been shown to be specific for the ß-core fragment only and shown to give the highest median MOM and the highest projected detection rate in a small comparison of 12 different assays for detecting HCG related molecules in the urine of 14 Down's cases (Cole et al., 1997bGo). However, we confirmed that the UGF-EIA Toa assay could achieve the highest median MOM but not the highest detection rate in comparison with results of other previous studies (Table IIGo).

Figure 2Go shows the distribution of urine HCGßcf MOM concentrations from the 20 cases of trisomy 18 pregnancies in our study and the world literature. These reports confirm that urine HCGßcf concentrations were decreased in trisomy 18 pregnancies (median 0.32 MOM, range 0.02–2.19) (Canick et al., 1995Go; Cuckle et al., 1995Go; Hayashi and Kozu, 1995Go; Spencer et al., 1996Go; Isozaki et al., 1997Go) as with serum free ß-HCG (median 0.37 MOM, range 0.27–0.42) in trisomy 18 pregnancies (Spencer et al., 1993Go). Reduced MOM concentrations in trisomy 18 pregnancies were observed in a small number of studies with median values of 0.33 (n = 5) (Cuckle et al., 1995Go) and 0.15 (n = 5) (Spencer et al., 1996Go). The observations of elevated MOM value in our case (6.71) and normal values previously reported (Canick et al., 1995Go) (1.09 and 1.30) are different from the previous findings. However, there were too few affected cases to make any conclusion and more work is needed to clarify this discrepant finding.

Figure 2Go also demonstrates the urine HCGßcf MOM concentrations from the 22 pregnancies with chromosomal abnormalities other than trisomy 18 and 21 from our study and the world literature. There were three cases of Turner's syndrome, one case of 47,XXY, one case of 47,XXX (Cuckle et al., 1995Go), two cases of triploidy (Cuckle et al., 1994Go; Canick et al., 1995Go), one case of trisomy 9 (Canick et al., 1995Go), three cases of trisomy 13 (Canick et al., 1995Go; Isozaki et al., 1997Go), four cases of inversion 9, and two cases of marker chromosome (Hayashi and Kozu, 1995Go). These results show that urine HCGßcf concentrations were also slightly decreased in other chromosomally abnormal pregnancies (median 0.86 MOM, range 0.02–90.00). However, three of four (75%) translocation cases and one mosaic deletion case in this study had an elevated urine HCGßcf concentration. It seems that more work is needed to evaluate urine HCGßcf concentrations for their usefulness in the detection of fetal chromosomal abnormalities other than Down's syndrome.

In conclusion, the apparent advantages and encouraging results of urine HCGßcf has been thought to be a promising marker for future Down's syndrome screening. This study has demonstrated the feasibility of HCGßcf in urine screening for Down's syndrome in an Asian population. Although maternal serum screening programmes have been conducted in Taiwan or other western countries for several years, there is still a limitation in the detection rates of Down's syndrome screening. Urine screening could replace serum screening not only because of logistical advantages but increased sensitivity (Cuckle et al., 1995Go; Cole et al., 1997aGo). However, although the screening performance using urine markers will not surpass the 80–90% detection rate achievable in the first trimester using nuchal translucency and serum biochemical markers (Spencer et al., 1999Go), urine markers could be useful in the area where Down's syndrome screening is not available in the first trimester. Recently, urine HCGßcf has been reported in combination with other analytes and ultrasound to improve the performance of a screening test (Bahado-Singh et al., 1998Go, 1999Go). However, a large-scale study is needed to survey the performance of urine screening for Down's syndrome and other chromosomal abnormalities in an Asian population.


    Acknowledgments
 
This study was supported by a grant from the National Science Council, Executive Yuan, ROC (NSC-86–2314-B-182A-061). The authors are grateful to Dr Kevin Spencer, the Endocrine Unit, Department of Clinical Biochemistry, Harold Wood Hospital, Romford, Essex, UK for his kind assistance in analysing these urine samples.


    Notes
 
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, Chang Gung Memorial Hospital, 199, Tung-Hwa North Road, Taipei, Taiwan, Republic of China Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Akar, A.M., Wehmann, R.E., Blithe, D.L. et al. (1988) A radioimmunoassay for the core fragment of the human chorionic gonadotropin ß-subunit. J. Clin. Endocrinol. Metab., 66, 538–545.[Abstract]

Bahado-Singh, R.O., Acuna, E., Cermik, D. et al. (1998) A new screening protocol combining urine ß-core fragment and ultrasonography for Down syndrome detection. Am. J. Obstet. Gynecol., 178, 779–782.[ISI][Medline]

Bahado-Singh, R.O., Oz, U., Kovanci, E. et al. (1999) A high-sensitivity alternative to `routine' genetic amniocentesis: multiple urinary analytes, nuchal thickness, and age. Am. J. Obstet. Gynecol., 180, 169–173.[ISI][Medline]

Canick, J.A., Kellner, L.I.E., Sailer, D.N. et al. (1995) Second trimester levels of maternal urinary gonadotropin peptide in Down syndrome pregnancy. Prenat. Diagn., 15, 739 744.

Cole, L.A. (1995) Down's syndrome screening using urine ß-core fragment test: choice of immunoassay. Prenat. Diagn., 15, 679–680.[ISI][Medline]

Cole, L.A., Acuna, E., Isozaki, T. et al. (1997a) Combining ß-core fragment and total estriol measurements to test for Down syndrome pregnancies. Prenat. Diagn., 17, 1125–1133.[ISI][Medline]

Cole, L.A., Kellner, L.H., Isozaki, T. et al. (1997b) Comparison of 12 assays for detecting HCG and related molecules in urine samples from Down syndrome pregnancies. Prenat. Diagn., 17, 607–614.[ISI][Medline]

Cuckle, H.S., Iles, R.K. and Chard, T. (1994) Urinary ß-core human chorionic gonadotrophin: a new approach to Down's syndrome screening. Prenat. Diagn., 14, 953–958.[ISI][Medline]

Cuckle, H.S., Iles, R.K., Sehmi, I.K. et al. (1995) Urinary multiple marker screening for Down's syndrome. Prenat. Diagn., 15, 745–751.[ISI][Medline]

Extermann, P., Bischof, P., Marguerat, P. and Mermillod, B. (1998) Second-trimester maternal serum screening for Down's syndrome: free beta-human chorionic gonadotrophin (HCG) and alpha-fetoprotein with or without unconjugated oestriol, compared with total HCG, alpha-fetoprotein and unconjugated oestriol. Hum. Reprod., 13, 220–223.[Abstract]

Hayashi, M. and Kozu, H. (1995) Maternal urinary beta-core fragment of HCG/creatinine ratios and fetal chromosomal abnormalities in the second trimester of pregnancy. Prenat. Diagn., 15, 11–16.[ISI][Medline]

Hsu, J.J., Hsieh, T.T., Lo, L.M. and Soong, Y.K. (1995) Midtrimester human chorionic gonadotropin levels: normal references values in Chinese pregnant women. Chang Gung Med. J., 18, 240–247.

Hsu, J.J., Hsieh, T.T. and Hsieh, F.J. (1996) Down syndrome screening in an Asian population using alpha-fetoprotein and free ß-HCG: a report of the Taiwan Down Syndrome Screening Group. Obstet. Gynecol., 87, 943–947.[Abstract/Free Full Text]

Hsu, J.J., Hsieh, T.T., Soong, Y.K. and Spencer, K. (1997) Comparison of Down's syndrome screening strategies in Asians combining serum free beta-HCG and alpha-fetoprotein with maternal age. Prenat. Diagn., 17, 707–716.[ISI][Medline]

Hsu, J.J., Hsieh, T.T., Hung, T.H. and Chiang, C.H. (1998) Midtrimester maternal serum free beta-human chorionic gonadotropin levels: normal reference values of Taiwanese pregnant women. Chang Gung Med. J., 21, 277–282.

Hsu, J.J., Hsu, T.Y., Hsieh, T.T. et al. (1999a) Urine free ß-HCG and total estriol for Down syndrome screening during the second trimester in an Asian population. Obstet. Gynecol., in press.

Hsu, J.J., Spencer, K., Aitken, D.A. et al. (1999b) Urinary free beta hCG, beta core fragment and total oestriol as markers. Down syndrome in the second trimester of pregnancy. Prenat. Diagn., 19, 146–158.[ISI][Medline]

Iles, R.K. (1996) Urinary analysis for Down's syndrome: is the measurement of urinary ß-core the future of biochemical screening for Down's syndrome. Early Hum. Dev., 47, 41–45.

Isozaki, T., Palomaki, G.E., Bahado-Singh, R.O. and Cole, L.A. (1997) Screening for Down syndrome pregnancy using ß-core fragment: prospective study. Prenat. Diagn., 17, 407–413.[ISI][Medline]

Kato, Y. and Braunstein, G.D. (1988) Beta-core fragment is a major form of immunoreactive urinary chorionic gonadotropin in human pregnancy. J. Clin. Endocrinol. Metab., 66, 1197–1201.[Abstract]

Kellner, L.H., Canick, J.A., Palomaki, G.E. et al. (1997) Levels of urinary ß-core fragment, total estriol and the ratio of the two in second trimester screening for Down syndrome. Prenat. Diagn., 17, 1135–1141.[ISI][Medline]

Lam, Y.I.E., Tang, M.H.Y., Tang, L.C.H. et al. (1997) Second trimester maternal urinary gonadotrophin peptide screening for fetal Down syndrome in Asian women. Prenat. Diagn., 17, 1101–1106.[ISI][Medline]

Palomaki, G.E., Knight, G.J., McCarthy, J. et al. (1997) Maternal serum screening for fetal Down syndrome in the United States: a 1995 survey. Am. J. Obstet. Gynecol., 176, 1046–1151.[ISI][Medline]

Rotmensch, S., Liberatum, M., Kardana, A. et al. (1992) Peptide heterogeneity of human chorionic gonadotropin (HCG) and its ß-subunit in Down's syndrome pregnancies. Am. J. Obstet. Gynecol., 169, 1558–1562.

Spencer, K., Coombes, E.J., Mallard, A.S. and Ward, A.M. (1992) Free beta human choriogonadotropin in Down syndrome screening: a multicentre study of its role compared with other biochemical markers. Ann. Clin. Biochem., 29, 506–518.[ISI][Medline]

Spencer, K., Mallard, A.S., Coombes, E.J. and Macri, J.N. (1993) Prenatal screening for trisomy 18 with free ß human chorionic gonadotrophin as a marker. Br. Med. J., 307, 1455–1458.[ISI][Medline]

Spencer, K., Aitken, D.A., Macri, J.N. and Buchanan, P.D. (1996) Urine free ß-HCG and ß-core in pregnancies affected by Down's syndrome. Prenat. Diagn., 16, 605–613.[ISI][Medline]

Spencer, K., Souter, V., Tul, N. et al. (1999) A screening program for trisomy 21 at 10–14 weeks using fetal nuchal translucency, maternal serum free ß-human chorionic gonadotrophin and pregnancy-associated plasma protein-A. Ultrasound Obstet. Gynecol., 13, 1–7.[ISI][Medline]

Wald, N., Densem, J., Stone, R. and Cheng, R. (1993) The use of free ß-HCG in antenatal screening for Down's syndrome. Br. J. Obstet. Gynecol., 100, 550–557.[ISI][Medline]

Submitted on February 2, 1999; accepted on April 23, 1999.





This Article
Abstract
FREE Full Text (PDF )
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Request Permissions
Google Scholar
Articles by Hsu, J. J.
Articles by Soong, Y. K.
PubMed
PubMed Citation
Articles by Hsu, J. J.
Articles by Soong, Y. K.