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Is prostate-specific antigen a marker for pregnancies affected by down syndrome?

Prenatal screening for trisomy 21 (Down syndrome) based on the analysis of biochemical markers in maternal serum during the second trimester of pregnancy is becoming an established part of obstetric practice (1, 2). Several markers have been investigated in the second trimester, and of these, a combination of [alpha]-fetoprotein and free [beta]-human chorionic gonadotropin (hCG) has been shown in retrospective (3) and prospective studies (4, 5) to achieve detection rates of 65-75% (with a 5% false-positive rate) in routine practice. Similarly, combinations including [alpha]-fetoprotein, total hCG, and unconjugated estriol have been shown in prospective studies to perform almost as well, with detection rates on average of 65% (6).

Recent developments in this area of prenatal screening have focused on three specific areas. The first area is that of refinements to the risk algorithm, leading to improved detection efficiency by taking into account a variety of factors that influence the analyte concentrations, for example, multiple pregnancies (7), maternal weight (8-10), insulin-dependent diabetes mellitus (11,12), gravidity (13), ethnicity (9), previous pregnancy results (14,15), and smoking (16). Correction for these factors seeks to reduce the between-patient variance, leading to reductions in the false-positive rate. The second area involves the investigation of possible new markers of trisomy 21, such as dimeric inhibin A, which has been reported to add anything from 3% (17) to 22% (18) to the detection rate using existing double or triple marker approaches. In addition, beta core in urine (19) and free [beta]-hCG in urine (20) have both been proposed as possible markers of trisomy 21. Neither the urine approach nor the addition of dimeric inhibin A have yet been shown conclusively to be better than existing procedures, and indeed their use is still somewhat controversial. The third area of investigation is the development of screening programs in the first trimester, in which a combination of ultrasound (nuchal translucency) and the biochemical markers pregnancy-associated plasma protein A and free [beta]-hCG enabled detection rates of ~90% (21).

Recently, Lambert-Messerlian et al. (22) in this Journal reported increased concentrations of prostate-specific antigen (PSA) in the maternal serum of second trimester pregnancies affected by trisomy 21. Although PSA was originally thought to be a marker specific to the prostate, a variety of biological fluids and extracts of female tissues has now been shown to contain and produce a material that reacts positively in sensitive PSA immunoassays. The original observation leading to the study of PSA in cases of trisomy 21 was a study in amniotic fluid (AF) that observed low values of PSA in four of six cases (23).

In this study, we endeavored to reproduce these initial observations, using a similarly sensitive commercially available PSA assay (24), and to investigate the potential value of a sensitive PSA assay in screening for trisomy 21 in the second trimester of pregnancy.

The study population consisted of cases of trisomy 21 in women who presented through the Neural Tube Defect and Trisomy 21 screening program in this laboratory during the period 1994-1998. The serum collected from each woman was stored as aliquots at -20[degrees]C after being analyzed in routine screening with assays for [alpha]-fetoprotein and free [beta]-hCG. Our local ethics committee approved the use of stored serum banks for research. The samples plus analytical and clinical data were collated on the basis of abnormal birth outcome, with karyotyping or cytogenetic confirmation after midtrimester amniocentesis. A total of 43 singleton pregnancies associated with trisomy 21 were identified with maternal serum samples taken between 14 and 17 weeks of gestation. Each case of trisomy 21 was matched for gestational age, length of storage ([+ or -]14 days), and number of freeze-thaw cycles with five samples from unaffected singleton pregnancies (total, 215 controls).

Of the cases of trisomy 21, 32 of 43 were identified by the screening program to have a trisomy 21 term risk of <1 in 250, and 11 of 43 were identified as a result of a live-born trisomy 21 baby. The mean maternal ages were 28.4 years (range, 17-43 years) for the controls and 34.2 years (range, 18-43 years) for the trisomy 21 cases. The median gestational age was 115 days (range, 98-125 days) for the controls and 108 (range, 98-120 days) for the trisomy 21 cases. The mean storage time was 30.2 months (range, 4-49 months) for samples from controls and 30.0 months (range, 4-49 months) for samples from trisomy 21 cases.

AF samples were identified from those in a previous study (25, 26). A total of 38 AF samples from cases of trisomy 21 with gestational ages from 16 to 20 weeks were available for analysis. A total of 110 control samples with gestational ages from 16 to 20 weeks were identified as matched for the number of freeze-thaw cycles and length of storage ([+ or -] 1 month).

PSA concentrations were measured with an Immulite automated third generation ultrasensitive immunochemiluminescent assay (Diagnostic Products Corp., Los Angeles, CA). The assay has been shown by Ferguson et al. (24) to have comparable sensitivity and clinical performance to the assay used in the initial studies with cases of trisomy 21 (22, 23). The detection limit of the assay in our hands analyzing 30 replicates of the assay zero diluent was 2 ng/L (+2 SD of the measurement of the zero diluent). The between-run imprecision (CV) was 5.5% at 7786 ng/L, 2.6% at 2704 ng/L, and 3.5% at 32.4 ng/L. The precision and sensitivity are similar to those observed by Ferguson et al. (24).

To assess the suitability of using the Immulite third generation PSA assay to measure PSA concentrations in AF, which has ~1/10 of the protein concentration of serum, a series of dilution experiments were carried out in which five male serum samples were diluted in a both zero diluent and phosphate-buffered saline (to simulate the low protein concentrations in AF). The results of the assay validation showed parallel and almost identical 100% recovery in the buffer and protein matrix and indicated no problem with matrix effects when measuring samples with low protein concentrations.

Results for each analyte were expressed in multiples of the median (MoM) for unaffected pregnancies of the same gestational age, derived from the regressed [log.sub.10] medians for the analyte when appropriate and from the overall control population median for analytes with concentrations unrelated to gestational age. Statistical analysis of data was performed using Astute, a statistical software add-in for Microsoft Exce15 (DDU Software, University of Leeds, UK)

Table 1 shows the observed and regressed median data for AF PSA and the observed medians for maternal serum PSA across the second trimester period. For AF, the median values showed a statistically significant increase (P <0.01, Mann-Whitney U-test) across the second trimester period, whereas for maternal serum there was no significant change (P >0.05) across this period. The overall control population median in maternal serum was 9 ng/L, with PSA 25- to 100-fold higher in AF. The median concentrations in AF are comparable with those observed by Melegos et al. (23). Lambert-Messerlian et al. (22) stated that the median concentrations in maternal serum varied with gestational age (by ~20%); however, they showed no data. Our study cannot confirm a variation of maternal serum PSA with gestational age.

In maternal serum, the median PSA MoM in the trisomy 21 group was 0.89 (95% confidence interval, 0.672.56); this was not significantly different from the controls (P = 0.2543, Mann-Whitney U-test). In the control group the 10th and 90th centiles were 0.22 and 4.56, respectively, with a 5th to 95th centile of 0.11-13.0. In the trisomy 21 group the 10th and 90th centiles were 0.22 and 7.22, respectively. The distribution of PSA MoM in the trisomy 21 group with gestational age is shown in Fig. 1B. The distribution of PSA MoM fitted a gaussian distribution after [log.sub.10] transformation in the control group and the trisomy 21 group. The parameters of the distribution are mean [log.sub.10] (controls = -0.003; trisomy 21 group = -0.104) and [log.sub.10] SD (controls = 0.5635; trisomy 21 group = 0.5573).

In AF the median PSA MoM in the trisomy 21 group was 0.80 (95% confidence interval, 0.49-1.68); this was not significantly different from the control group (P = 0.4117, Mann-Whitney U-test). In the control group the 10th and 90th centiles were 0.23 and 4.15, respectively, with a 5th to 95th centile of 0.19-5.40. In the trisomy 21 group the 10th and 90th centiles were 0.16 and 7.18, respectively. The distribution of PSA MoM in the trisomy 21 group with gestational age is shown in Fig. 1A. The distribution of PSA MoM fitted a gaussian distribution after [log.sub.10] transformation in both the trisomy 21 group and the controls. The parameters of the distribution are mean [log.sub.10] (controls = 0.000; trisomy 21 group = -0.112) and [log.sub.10] SD (controls = 0.4424; trisomy 21 group = 0.6722).

When the AF data in the trisomy 21 group were analyzed by fetal sex, the median Molls were 0.81 and 0.73 for female and male fetuses, respectively (P = 0.3954, Mann-Whitney U-test). No difference could be established in the control group.

In conclusion, we have been unable to confirm the previous report of Lambert-Messerlian et al. (22) that maternal serum PSA is increased in pregnancies with trisomy 21 fetuses. PSA concentrations in the previous study (22) were less than the detection limit of the assay in 22% of the control cases, whereas in our study only 10% of control cases were below the detection limit. It is possible, therefore, that the study design and this higher percentage of controls with undetectable PSA concentrations deflated the median value in the earlier study (22), resulting in the trisomy 21 Molls being overestimated.

[FIGURE 1 OMITTED]

Previously, Melegos et al. (23) from the same group showed low concentrations of PSA in the AF of four of six cases of trisomy 21, and in our study we can confirm that AF contains PSA and that PSA concentrations on average tend to be lower in cases of trisomy 21. Moreover, AF PSA changes significantly with gestational age. The lowering of AF PSA in our study is mirrored by a lowering of maternal serum PSA in the trisomy 21 group. However, the amount of the lowering of maternal serum PSA and the width of the distribution are likely to mitigate against PSA being of value in a screening context.

We thank the DPL Division of Euro/Diagnostics Products Corporation, Ltd., Llanberis, United Kingdom for providing the kits to carry out this work.

References

(1.) Cuckle HS, Ellis AR, Seth J. Provision of screening for Down's syndrome. Br Med J 1995;311:512.

(2.) Palomaki GE, Knight GJ, McCarthy JE, Haddow JE, Donhowe JM. Maternal serum screening for Down syndrome in the United States: a 1995 survey. Am J Obstet Gynecol 1997;176:1046-51.

(3.) Spencer K, Coombes EJ, Mallard AS, Milford Ward A. Free beta human chorionic gonadotropin in Down's syndrome screening: a multicentre study of its role compared with other biochemical markers. Ann Clin Biochem 1992;29:506-18.

(4.) Spencer K. Despistage de la trisomie 21 a I'aide de la beta hCG libre: notre experience sur trois ans. Med Foetale Echographie Gynecol 1994;20:67-9.

(5.) Macri JN, Spencer K, Garver K, Buchanan PD, Say B, Carpenter NJ, et al. Maternal serum free beta hCG screening: results of studies including 480 cases of Down syndrome. Prenat Diagn 1994;14:97-108.

(6.) Macri JN, Spencer K. Towards the optimal protocol for Down's syndrome screening. Am J Obstet Gynecol 1996;174:1668-9.

(7.) Spencer K, Salonen R, Muller F. Down's syndrome screening in multiple pregnancies using alpha fetoprotein and free beta hCG. Prenat Diagn 1994;14:537-42.

(8.) Reynolds TM, Penney MD, Hughes MD, John R. The effect of weight correction on risk calculations for Down's syndrome screening. Ann Clin Biochem 1991;28:245-9.

(9.) Spencer K. Screening for Down's syndrome. The role of intact hCG and free subunit measurements. Scand J Clin Lab Investig 1993;53(Suppl 216):7996.

(10.) Neveux LM, Palomaki GE, Larrivee DA, Knight GJ, Haddow JE. Refinements in managing maternal weight adjustment for interpreting prenatal screening results. Prenat Diagn 1196;16:1115-20.

(11.) Wald NJ, Cuckle HS, Densem JW, Stone RW. Maternal serum unconjugated oestriol and human chorionic gonadotrophin in pregnancies with insulin-dependent diabetes: implications for Down's syndrome screening. Br J Obstet Gynaecol 1992;99:51-3.

(12.) Palomaki GE, Knight GJ, Haddow JE. Human chorionic gonadotrophin and unconjugated oestriol measurements in insulin-dependent diabetic pregnant women being screened for fetal Down syndrome. Prenat Diagn 1994; 14:65-8.

(13.) Spencer K. The influence of gravidity on Down's syndrome screening with free beta hCG. Prenat Diagn 1995;15:87-9.

(14.) Dar H, Merksamer R, Berdichevsky D, David M. Maternal serum marker levels in consecutive pregnancies: a possible genetic predisposition to abnormal levels. Am J Med Genet 1996;61:154-7.

(15.) Spencer K. Between pregnancy biological variability of maternal serum alpha fetoprotein and free beta hCG: implications for Down syndrome screening in subsequent pregnancies. Prenat Diagn 1997;17:39-45.

(16.) Spencer K. The influence of smoking on maternal serum AFP and free beta hCG levels and the impact on screening for Down syndrome. Prenat Diagn 1998;18:225-34.

(17.) Spencer K, Wallace E, Ritoe S. Second trimester dimeric inhibin A in pregnancies affected by Down's syndrome. Prenat Diagn 1996;16:110110.

(18.) Aitken DA, Wallace EM, Crossley J, Swanston IA, Paren Y, Maarle M, et al. Dimeric inhibin A as a marker for Down's syndrome in early pregnancy. N Engl J Med 1996;334:1231-6.

(19.) Cuckle HS, Iles RK, Chard T. Urinary beta core human chorionic gonadotrophin: a new approach to Down's syndrome screening. Prenat Diagn 1994; 14:953-8.

(20.) Spencer K, Aitken DA, Macri JN, Buchanan PD. Urine free beta hCG and beta core in pregnancies affected by Down's syndrome. Prenat Diagn 1996;16: 605-13.

(21.) Spencer K, Souter V, Nicolaides KH. A rapid first trimester screening program for Down syndrome using nuchal translucency, free beta hCG and PAPP-A. Ultrasound Obstet Gynaecol (in press).

(22.) Lambert-Messerlian GM, Canick JA, Melegos DN, Diamandis EP. Increased concentrations of prostate-specific antigen in maternal serum from pregnancies affected by fetal Down syndrome. Clin Chem 1998;44:205-8.

(23.) Melegos DN, Yu H, Allen L, Diamandis EP. Prostate specific antigen in amniotic fluid of normal and abnormal pregnancies. Clin Biochem 1996;29: 552-62.

(24.) Ferguson RA, Yu H, Zammit S, Diamandis EP. Ultrasensitive detection of prostate-specific antigen by a time resolved immunofluorometric assay and the Immulite immunochemiluminescent third generation assay: potential applications in prostate and breast cancers. Clin Chem 1996;42:675-84.

(25.) Spencer K, Muller F, Aitken DA. Biochemical markers of trisomy 21 in amniotic fluid. Prenat Diagn 1997;17:31-7.

(26.) Spencer K, Muller F, Aitken DA. Amniotic fluid and maternal serum levels of CA125 in pregnancies affected by Down syndrome: a re-evaluation of the role of CA125 in Down syndrome screening. Prenat Diagn 1997;17:701-6.

Kevin Spencer * and Paul Carpenter (Endocrine Unit, Clinical Biochemistry Department, Harold Wood Hospital, Gubbins Lane, Romford, Essex, RM3 OBE United Kingdom; * author for correspondence: fax 44 (0) 1708381486, e-mail Kevin_Spencer@Compuserve.com)
Table 1. Median AF and maternal serum PSA
concentrations in the second trimester.

 Observed
Gestation, Observed maternal serum
weeks No. AF PSA, Regressed No. PSA, ng/L
 ng/L

14 50 8.5
15 90 11
16 30 209 227 60 8.5
17 22 343 318 15 7.5
18 20 498 446
19 20 556 625
20 18 891 877
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Title Annotation:Technical Briefs
Author:Spencer, Kevin; Carpenter, Paul
Publication:Clinical Chemistry
Date:Nov 1, 1998
Words:2627
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