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Gene expression of human pregnancy-associated plasma protein-A in placenta from trisomic pregnancies
Placenta (1996), 17, 33-36
Gene Expression of Human Pregnancy-associated Plasma Protein-A in Placenta from Trisomic Pregnancies
M. L. B r i z o t a, J. A. H y e t t a, A. T. M c k i e b, N. A. B e r s i n g e r c, F. Farzaneh b and K. H. N i c o l a i d e s ~'d a Harris Birthright Research Centre for Fetal Medicine, King's College Hospital Medical School, London b Department of Molecular Medicine, King's College Hospital Medical School, London c Department of Obstetrics & Gynaecology, University of Berne, Switzerland Paper received 21 June 1995; accepted 30 August 1995
Placental pregnancy-associated plasma protein-A (PAPP-A) mRNA expression, placental PAPP-A protein concentration and maternal serum levels of PAPP-A were examined in pregnancies affected by trisomy 21 (n=8), trisomy 18 (n=7) and 15 normal controls at 12-15 weeks of gestation. The maternal serum concentration of PAPP-A in the trisomic group of pregnancies was significantly lower than in the normal controls. However there were no significant differences between the three groups in PAPP-A mRNA exprcssion or PAPP-A protein concentration in the placental tissues. There was no significant association between the level of placental mRNA and either placental protein or maternal serum PAPP-A concentrations in the normal or trisomic pregnancies. There was however a significant association between placental protein and maternal serum PAPP-A concentrations in the normal and trisomy 21 pregnancies but not in those affcctcd by trisomy 18. These findings suggest that the decrease in maternal serum PAPP-A in trisomic pregnancies is due to alterations in post-translational events such as protein stability, alterations in the release mechanism of the protein, impaired protein transport across the placenta or modified serum stability of PAPP-A. O 1996 W. B. Saunders Company Ltd Placenta (1996), 17, 33-36
INTRODUCTION
The cDNA and protein sequence encoding pregnancyassociated plasma protein-A (PAPP-A) has recently been characterized (Kristensen et al, 1994), allowing stud)" of the mechanisms involved in the production of this protein in normal and pathological pregnancies. The main source of PAPP-A in maternal serum is the placenta (Lin, Halbert and Kiefer, 1976) and during the first trimester fetal trisomies 21 and 18 are associated with decreased maternal serum levels (Wald et al, 1992; Brambati et al, 1993; Brizot et al, 1994). This study examines the possible causes of this decrease by investigating the relationship between placental mRaNA expression and the concentration of PAPP-A protein in both placental tissue and maternal serum in normal and trisomic pregnancies. SUBJECTS AND METHODS
Samples of placental tissue were collected at the time of surgical termination of pregnancy at 12-15 weeks of gestation in eight pregnancies with fetal trisomy 21, seven with trisomy 18 and 15 normal controls undergoing termination for psychosocial reasons. Maternal serum was obtained immediately a To whom correspondence should be addressed. 0143-4004/96/010033+04 S12.00/0
before the termination. The diagnosis of trisomy was made by chorion villus sampling in singleton pregnancies referred to our centre because of increased fetal nuchal translucency thickness detected at routine ultrasound examination (Nicolaides et al, 1994a, b). Gestational age was determined by ultrasound measurement of crown-rump length and in all cases a regular fetal heart rhythm was present at the time of pregnancy termination. Written informed consent was obtained from the patients. The study was approved by the local Research Ethics Committee. Tissue collection was made in accordance with the Polkinghorne guidelines on the research use of fetal material (Polkinghorne, 1989). Placental tissues were snap frozen in liquid nitrogen and kept at - 70~ until assayed. Similarly, maternal serum samples were kept at 30~ Total RNA was extracted from 200 mg of frozen placental tissue using a modified version of the single step protocol of Chomczynski and Sacchi (1987). For Northern blot and slot blot analysis, samples containing 30 lag of total RaNA were used as previously described (Brizot et al, 1995). RNA was then fixed to the membranes and hybridization was carried out using the specific DNA probe. PAPP-A DNA probe was amplified by polymerase chain reaction (PCR) with sequence specific primers for the respective placental gene. The probe used was a 1169bp (corresponding to nucleotides 3648--4792 of -
1996 W. II. Saundcrs Corot,any Ltd
Placenta (1996), Vol. 17
34
Table 1. Median and ranges of placental mRNA expression (NDS, normalized densitometrie score), placental protein and maternal serum concentrations of PAPP-A in normal, trisomy 21 and trisomy 18 groups Group
Placental mRNA expression (NDS)
Placental protein concentration (mIO/g)
Maternal serum concentration (mIU/mL)
Normal Trisomy 21 Trisomy 18
0.938 (0.184-5.959) 0.745 (0.228-4.556) 0.883 (0.403-1.680)
18.150 (4.84-33.91) 11.235 (4.63-29.79) 15.950 (11.66--24.45)
8.000 (2.30-14.70) 2.575 (1.12-6.72) ~ 0.710 (0.40-1.55) ~
a Significantly lower than normal P<0.005, using a Mann-Whitney Test.
40
4
35
I
25
%
E 14-
-
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16
3-
<,
20
9
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9
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~ 10
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5 I 0
T18
0
9
I
9
9
t
Control T21
•
N
tA
Control T21
th=
T18
o
Control T21
T18
F i g u r e 1. Individual values of PAPP-A placental mRNA (NDS, normalised densitometric score), PAPP-A placental protein and matemal serum levels of PAPP-A in controls (left), trisomy 21 (middle) and trisomy 18 (right). The horizontal lines indicate the median in each group.
the PAPP-A protein sequence described by Kristensen et al (1994)). The probe for ]3-aetin used as a control for loading and transfer, was a 1.1 kb pstl fragment of plasmid pAL41 as described by Alonso et al (1986). The PCR products were labelled with [3zp]dATP (Deoxyadenosine-5'-Triphosphate) according to the hexanucleotide priming method of Feinburg and Volgelstein (1983). The membranes were exposed at - 0~ on x-ray films for 2-20 h. Signals from slot blots were scanned using a densitometer (GDS 2000, Mitsubishi, Tokyo, Japan). The densitometric scores of PAPP-A were normalized to the signal obtained for [3-actin mRNA by dividing the densitometric values of the target gene by the actin value, thus correcting for any uneven loading of the RNA samples. Placental tissue (200-600 mg wet weight) was extracted with three volumes of ice-cold Tris-buffered saline (50 mM, pH 7.4) using a Polytron homogenizer. Homogenates were centrifuged at 2000 g for 20 min at 4"C. The pellets were discarded and the supernatants (extracts) stored at - 8 0 " C until assayed in one batch. This procedure was efficient for PAPP-A extraction from placental tissue (Bersinger, Schneider and Keller, 1986). PAPP-A protein was determined in maternal sera and in the extracts by a double-antibody microplate enzyme immunoassay (Bersinger et al, 1995). Maternal sera and serum-based assay standards were diluted 1:21 in phosphate-buffered saline containing non-fat milk proteins (0.5 per cent w/v, 'Blotto', Pierce, USA). Placental extracts were diluted 1:100 in the
same buffer containing 4.8 per cent (v/v) horse serum. The W H O reference preparation 78-610, designated to contain 100 m I U / m L was used as a calibrator. The significance of differences in placental mRNA expression, placental protein and maternal serum concentrations between trisomy 21, trisomy 18 and control groups were examined with the Mann-Whitney test. The relationship between PAPP-A RNA in the placenta, placental protein and maternal serum concentration of PAPP-A were examined with linear regression analysis.
R ES U LTS The maternal serum concentration of PAPP-A in trisomy 21 and 18 pregnancies was significantly lower than in the normal controls (Figure 1, trisomy 21: z= - 2.21, P<0.05; trisomy 18: z = - 4 . 0 4 , P<0.0001), but there were no significant differences between the groups in mRNA expression and protein concentration in the placental tissues (Figure 1, Table 1). There was no significant association between relative abundance of placental mRNA expression and placental protein in the normal (r=0.20) or trisomy 21 (r=0.45) or trisomy 18 ( r = - 0.04) pregnancies. Similarly, there was no significant association between relative abundance of placental mRNA expression and maternal serum PAPP-A concentrations in the
Brizot et al: Gene expression of PAPP-A in placenta from trisomlc pregnancies
35
normal (r=0.22) or trisomy 21 (r=0.62) or trisomy 18 ( r = - 0 . 3 9 ) pregnancies. The relationship between the amounts of PAPP-A protein present in the placenta and maternal serum (Figure 2) was significant in normal (r=0.81, P<0.0001 and trisomy 21 (r=0.69, P<0.05) pregnancies but not in those affected by trisomy 18 (r= - 0.04).
16
14
12
DISCUSSION The data presented confirm that in fetal trisomies 21 and 18 the maternal serum concentration of PAPP-A is decreased (Brizot et al, 1994). However, placental PAPP-A mRNA expression is not significantly different from normal and there is no significant association between maternal serum PAPP-A and placental mRNA. These findings suggest that the decrease in maternal serum PAPP-A in trisomy 21 and 18 pregnancies is not due to an alteration in placental mRNA expression and therefore not due to a change in transcription. Placental PAPP-A protein levels in trisomy 21 and trisomy 18 pregnancies are not significantly different from normal pregnancies, suggesting that low serum levels of PAPP-A may not be the consequence of decreased placental proteil{ synthesis. In normal pregnancies, there is a significant association between the concentration of PAPP-A in placental tissue and maternal serum. Such a relationship is also found in trisomy 21 pregnancies, but compared with the normals in trisomy 21 the maternal serum concentration of PAPP-A is lower for a given placental level. The lowest levels of maternal serum PAPP-A are observed in trisomy 18 pregnancies and in this condition the maternal serum concentration is not related to the placental level. These findings indicate that the decrease in maternal serum PAPP-A in trisomic pregnancies is due to alterations occurring post-translationally such as alterations in the release mechanism of the protein (impaired transport across the placenta), or in the stability of the protein in serum due to modified glycosylation and/or trisomy dependent serum protease levels or activity affecting PAPP-A degradation. Alternatively, in addition to the placenta there may be other important
10 <
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., 9
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9 9 9
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/
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0
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10 15 20 25 Placental PAPP-A (mlU/g)
I
30
35
Figure 2. Individual values for maternal serum PAPP-A in controls ( I ) , trisomy 21 (Ak) and trisomy 18 ( 0 ) in relation to placental PAPP-A protein and the best fit lines for these relations.
(maternal) sources of PAPP-A, which might in turn be affected by fetal trisomies. PAPP-A has been demonstrated in the decidua (Bischof et al, 1984). Fetal sources include liver, spleen, adrenal and stomach (Schindler and Bischof, 1984). However the contribution of these tissues to maternal serum PAPP-A levels in normal and trisomic pregnancies remains to be determined.
A C K N O W L E D G EM ENTS
M. L. Brizot was supported by a grant from CNPq (Conselho Nacional de Pesquisa e Desenvolvimento Cientifico e Tecnologico-Brazil).
REFERENCES
Alonso, S., Mint)', A., Bourlct, Y. & Buckingham, M. (1986) Comparison of three actin-coding sequences in the moose; evolutionary relationships between the actin genes of warm-blooded vertebrates. Journal of Molecular Evolution, 23, 11-22. Bersinger, N. A., Schneider, H. & Keller, P.J. (1986) Synthesis of placental proteins by the human placenta perfused in vitro. Gynecolog),and Obstetric hivestigation, 22, 47-51. Bersinger, N. A., Zakher, A., }tuber, U., Pescia, G. & Schneider, tt. (1995) A sensitive enzyme immunoassay for pregnancy-associated plasma protein A (PAPP-A): a possible first trimester method of screening for Down syndrome and other trisomles. Archires of Gynecology and Obstetrics, 256, 185-192.
Bischof, P., Duberg, S., Sizonenko, M. T., Schindlcr, A. M., Beguin, F., tterrmann, W. L. & Sizonenko, P. C. (1984) In vitro production of pregnancy-associated plasma protein A by human dccidua and trophoblast. American Journal of Obstetrics and Gynecology, 148, 13-18. Brambati, B., Macintosh, M. C. M., Teisner, B., Shrimanker, K., Lanzani, A., Maguiness, S., Bonacchi, I., Tului, L., Chard, T. & Grudzinskas, J. G. (1993) Low maternal serum level of pregnancy associated plasma protein (PAPP-A) in the first trimester in association with abnormal fetal karyotype. BritishJournal of Obstetrics and Gynaecology, 100, 324-326. Brizot, M. L., Snijders, R. J. M., Bersingcr, N. A., Knhn, P. & Nicolaides, K. It. (1994) Maternal serum pregnancy-associated plasma protein A and fetal nuchal translucency thickness for the prediction of fetal trisomies in early pregnancy. Obstetricsand Gynecology, 84, 918-922.
36 Brizot, M. L., Jauniaux, E., McKie, A. T., Farzaueli, F. & Nicolaides, K. tl. (1995) Placental expression of ~t and 13subunits of human chorionic gonadotrophin in early pregnancies xvith Down's syndrome, lluman Reproduction, 10, 2506-2509. Chomczynski, P. & Sacchl, N. (1987) Single-step method of RNA isolation by acid guanidium thiocyanate-phcnol-chloroform extraction. Annals of Biochemisto, , 162, 156-159. Feinburg, A. P. & Vogelstein, G. (1983) A technique for radiolabelling DNA restriction endonuclease fragments to a high specific activity. Annals of Biochemisto, , 132, 6--13. Kristensen, T., Oxvlg, C., Sand, O., Moiler, N. P. 1I. & Sottrup-Jensen, L. (1994) Amino acid sequence of human pregnancy-associated plasma protein A derived from cloned eDNA. Biochemisto', 33, 1592-1598. Lin, T. M., llalbert, S. P. & Kiefer, D. (1976) Quantitative Analysis of pregnancy-associated plasma proteins in human placenta. The Journal of Clinical bzvestigation, 57, 466-472.
Placenta (1996), Vol. 17 Nieolaides, K. II., Brizot, M. L., Patel, F. & Snijders, R. (1994a) Comparison of chorion villus sampling and amniocentesis for fetal karyotyping at 10-13 ~veeks gestation. Lancet, 344, 435-439. Nicolaldes, K. II., Brizot, M. L. & Snijders, R. (1994b) Fetal nuchal translucency: ultrasound screening for fetal trisomy in the first trimester of pregnancy. British Journal of Obstetrics and Gynaecolo~,, 101,782-786. Polklnghorne, J. (1989) Review of the guidance on the research use of fetuses and fetal material, tler Majesty's Stationery Office, London. Cm762. Schindler, A. M. & Bischof, P. (1984) ttistochemieal loealisation of pregnancy-associated plasma protein A in fetal, infant, and adult organs and comparison between antisera. Gynecolog), attd Obstetric Investigation, 18, 88-94. Wald, N., Stone, R., Cuekle, II. S., Grudzinskas, G., Barkal, G., Brambati, B., Teisner, B. & Fuhrmann, W. (1992) First trimester concentrations of pregnancy-associated plasma protein A and placental protein 14 in Down's syndrome. British Medical Journal, 305, 28.
Gene Expression of Human Pregnancy-associated Plasma Protein-A in Placenta from Trisomic Pregnancies
M. L. B r i z o t a, J. A. H y e t t a, A. T. M c k i e b, N. A. B e r s i n g e r c, F. Farzaneh b and K. H. N i c o l a i d e s ~'d a Harris Birthright Research Centre for Fetal Medicine, King's College Hospital Medical School, London b Department of Molecular Medicine, King's College Hospital Medical School, London c Department of Obstetrics & Gynaecology, University of Berne, Switzerland Paper received 21 June 1995; accepted 30 August 1995
Placental pregnancy-associated plasma protein-A (PAPP-A) mRNA expression, placental PAPP-A protein concentration and maternal serum levels of PAPP-A were examined in pregnancies affected by trisomy 21 (n=8), trisomy 18 (n=7) and 15 normal controls at 12-15 weeks of gestation. The maternal serum concentration of PAPP-A in the trisomic group of pregnancies was significantly lower than in the normal controls. However there were no significant differences between the three groups in PAPP-A mRNA exprcssion or PAPP-A protein concentration in the placental tissues. There was no significant association between the level of placental mRNA and either placental protein or maternal serum PAPP-A concentrations in the normal or trisomic pregnancies. There was however a significant association between placental protein and maternal serum PAPP-A concentrations in the normal and trisomy 21 pregnancies but not in those affcctcd by trisomy 18. These findings suggest that the decrease in maternal serum PAPP-A in trisomic pregnancies is due to alterations in post-translational events such as protein stability, alterations in the release mechanism of the protein, impaired protein transport across the placenta or modified serum stability of PAPP-A. O 1996 W. B. Saunders Company Ltd Placenta (1996), 17, 33-36
INTRODUCTION
The cDNA and protein sequence encoding pregnancyassociated plasma protein-A (PAPP-A) has recently been characterized (Kristensen et al, 1994), allowing stud)" of the mechanisms involved in the production of this protein in normal and pathological pregnancies. The main source of PAPP-A in maternal serum is the placenta (Lin, Halbert and Kiefer, 1976) and during the first trimester fetal trisomies 21 and 18 are associated with decreased maternal serum levels (Wald et al, 1992; Brambati et al, 1993; Brizot et al, 1994). This study examines the possible causes of this decrease by investigating the relationship between placental mRaNA expression and the concentration of PAPP-A protein in both placental tissue and maternal serum in normal and trisomic pregnancies. SUBJECTS AND METHODS
Samples of placental tissue were collected at the time of surgical termination of pregnancy at 12-15 weeks of gestation in eight pregnancies with fetal trisomy 21, seven with trisomy 18 and 15 normal controls undergoing termination for psychosocial reasons. Maternal serum was obtained immediately a To whom correspondence should be addressed. 0143-4004/96/010033+04 S12.00/0
before the termination. The diagnosis of trisomy was made by chorion villus sampling in singleton pregnancies referred to our centre because of increased fetal nuchal translucency thickness detected at routine ultrasound examination (Nicolaides et al, 1994a, b). Gestational age was determined by ultrasound measurement of crown-rump length and in all cases a regular fetal heart rhythm was present at the time of pregnancy termination. Written informed consent was obtained from the patients. The study was approved by the local Research Ethics Committee. Tissue collection was made in accordance with the Polkinghorne guidelines on the research use of fetal material (Polkinghorne, 1989). Placental tissues were snap frozen in liquid nitrogen and kept at - 70~ until assayed. Similarly, maternal serum samples were kept at 30~ Total RNA was extracted from 200 mg of frozen placental tissue using a modified version of the single step protocol of Chomczynski and Sacchi (1987). For Northern blot and slot blot analysis, samples containing 30 lag of total RaNA were used as previously described (Brizot et al, 1995). RNA was then fixed to the membranes and hybridization was carried out using the specific DNA probe. PAPP-A DNA probe was amplified by polymerase chain reaction (PCR) with sequence specific primers for the respective placental gene. The probe used was a 1169bp (corresponding to nucleotides 3648--4792 of -
1996 W. II. Saundcrs Corot,any Ltd
Placenta (1996), Vol. 17
34
Table 1. Median and ranges of placental mRNA expression (NDS, normalized densitometrie score), placental protein and maternal serum concentrations of PAPP-A in normal, trisomy 21 and trisomy 18 groups Group
Placental mRNA expression (NDS)
Placental protein concentration (mIO/g)
Maternal serum concentration (mIU/mL)
Normal Trisomy 21 Trisomy 18
0.938 (0.184-5.959) 0.745 (0.228-4.556) 0.883 (0.403-1.680)
18.150 (4.84-33.91) 11.235 (4.63-29.79) 15.950 (11.66--24.45)
8.000 (2.30-14.70) 2.575 (1.12-6.72) ~ 0.710 (0.40-1.55) ~
a Significantly lower than normal P<0.005, using a Mann-Whitney Test.
40
4
35
I
25
%
E 14-
-
Z E
16
3-
<,
20
9
T
< ~ lO-
9
I I
9
I
2I
E
1
_$_
~ 10
9
5 I 0
T18
0
9
I
9
9
t
Control T21
•
N
tA
Control T21
th=
T18
o
Control T21
T18
F i g u r e 1. Individual values of PAPP-A placental mRNA (NDS, normalised densitometric score), PAPP-A placental protein and matemal serum levels of PAPP-A in controls (left), trisomy 21 (middle) and trisomy 18 (right). The horizontal lines indicate the median in each group.
the PAPP-A protein sequence described by Kristensen et al (1994)). The probe for ]3-aetin used as a control for loading and transfer, was a 1.1 kb pstl fragment of plasmid pAL41 as described by Alonso et al (1986). The PCR products were labelled with [3zp]dATP (Deoxyadenosine-5'-Triphosphate) according to the hexanucleotide priming method of Feinburg and Volgelstein (1983). The membranes were exposed at - 0~ on x-ray films for 2-20 h. Signals from slot blots were scanned using a densitometer (GDS 2000, Mitsubishi, Tokyo, Japan). The densitometric scores of PAPP-A were normalized to the signal obtained for [3-actin mRNA by dividing the densitometric values of the target gene by the actin value, thus correcting for any uneven loading of the RNA samples. Placental tissue (200-600 mg wet weight) was extracted with three volumes of ice-cold Tris-buffered saline (50 mM, pH 7.4) using a Polytron homogenizer. Homogenates were centrifuged at 2000 g for 20 min at 4"C. The pellets were discarded and the supernatants (extracts) stored at - 8 0 " C until assayed in one batch. This procedure was efficient for PAPP-A extraction from placental tissue (Bersinger, Schneider and Keller, 1986). PAPP-A protein was determined in maternal sera and in the extracts by a double-antibody microplate enzyme immunoassay (Bersinger et al, 1995). Maternal sera and serum-based assay standards were diluted 1:21 in phosphate-buffered saline containing non-fat milk proteins (0.5 per cent w/v, 'Blotto', Pierce, USA). Placental extracts were diluted 1:100 in the
same buffer containing 4.8 per cent (v/v) horse serum. The W H O reference preparation 78-610, designated to contain 100 m I U / m L was used as a calibrator. The significance of differences in placental mRNA expression, placental protein and maternal serum concentrations between trisomy 21, trisomy 18 and control groups were examined with the Mann-Whitney test. The relationship between PAPP-A RNA in the placenta, placental protein and maternal serum concentration of PAPP-A were examined with linear regression analysis.
R ES U LTS The maternal serum concentration of PAPP-A in trisomy 21 and 18 pregnancies was significantly lower than in the normal controls (Figure 1, trisomy 21: z= - 2.21, P<0.05; trisomy 18: z = - 4 . 0 4 , P<0.0001), but there were no significant differences between the groups in mRNA expression and protein concentration in the placental tissues (Figure 1, Table 1). There was no significant association between relative abundance of placental mRNA expression and placental protein in the normal (r=0.20) or trisomy 21 (r=0.45) or trisomy 18 ( r = - 0.04) pregnancies. Similarly, there was no significant association between relative abundance of placental mRNA expression and maternal serum PAPP-A concentrations in the
Brizot et al: Gene expression of PAPP-A in placenta from trisomlc pregnancies
35
normal (r=0.22) or trisomy 21 (r=0.62) or trisomy 18 ( r = - 0 . 3 9 ) pregnancies. The relationship between the amounts of PAPP-A protein present in the placenta and maternal serum (Figure 2) was significant in normal (r=0.81, P<0.0001 and trisomy 21 (r=0.69, P<0.05) pregnancies but not in those affected by trisomy 18 (r= - 0.04).
16
14
12
DISCUSSION The data presented confirm that in fetal trisomies 21 and 18 the maternal serum concentration of PAPP-A is decreased (Brizot et al, 1994). However, placental PAPP-A mRNA expression is not significantly different from normal and there is no significant association between maternal serum PAPP-A and placental mRNA. These findings suggest that the decrease in maternal serum PAPP-A in trisomy 21 and 18 pregnancies is not due to an alteration in placental mRNA expression and therefore not due to a change in transcription. Placental PAPP-A protein levels in trisomy 21 and trisomy 18 pregnancies are not significantly different from normal pregnancies, suggesting that low serum levels of PAPP-A may not be the consequence of decreased placental proteil{ synthesis. In normal pregnancies, there is a significant association between the concentration of PAPP-A in placental tissue and maternal serum. Such a relationship is also found in trisomy 21 pregnancies, but compared with the normals in trisomy 21 the maternal serum concentration of PAPP-A is lower for a given placental level. The lowest levels of maternal serum PAPP-A are observed in trisomy 18 pregnancies and in this condition the maternal serum concentration is not related to the placental level. These findings indicate that the decrease in maternal serum PAPP-A in trisomic pregnancies is due to alterations occurring post-translationally such as alterations in the release mechanism of the protein (impaired transport across the placenta), or in the stability of the protein in serum due to modified glycosylation and/or trisomy dependent serum protease levels or activity affecting PAPP-A degradation. Alternatively, in addition to the placenta there may be other important
10 <
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., 9
~
9 9 9
/
/
9 /
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0
5
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/-
/
j//
/
/
[
I
W
10 15 20 25 Placental PAPP-A (mlU/g)
I
30
35
Figure 2. Individual values for maternal serum PAPP-A in controls ( I ) , trisomy 21 (Ak) and trisomy 18 ( 0 ) in relation to placental PAPP-A protein and the best fit lines for these relations.
(maternal) sources of PAPP-A, which might in turn be affected by fetal trisomies. PAPP-A has been demonstrated in the decidua (Bischof et al, 1984). Fetal sources include liver, spleen, adrenal and stomach (Schindler and Bischof, 1984). However the contribution of these tissues to maternal serum PAPP-A levels in normal and trisomic pregnancies remains to be determined.
A C K N O W L E D G EM ENTS
M. L. Brizot was supported by a grant from CNPq (Conselho Nacional de Pesquisa e Desenvolvimento Cientifico e Tecnologico-Brazil).
REFERENCES
Alonso, S., Mint)', A., Bourlct, Y. & Buckingham, M. (1986) Comparison of three actin-coding sequences in the moose; evolutionary relationships between the actin genes of warm-blooded vertebrates. Journal of Molecular Evolution, 23, 11-22. Bersinger, N. A., Schneider, H. & Keller, P.J. (1986) Synthesis of placental proteins by the human placenta perfused in vitro. Gynecolog),and Obstetric hivestigation, 22, 47-51. Bersinger, N. A., Zakher, A., }tuber, U., Pescia, G. & Schneider, tt. (1995) A sensitive enzyme immunoassay for pregnancy-associated plasma protein A (PAPP-A): a possible first trimester method of screening for Down syndrome and other trisomles. Archires of Gynecology and Obstetrics, 256, 185-192.
Bischof, P., Duberg, S., Sizonenko, M. T., Schindlcr, A. M., Beguin, F., tterrmann, W. L. & Sizonenko, P. C. (1984) In vitro production of pregnancy-associated plasma protein A by human dccidua and trophoblast. American Journal of Obstetrics and Gynecology, 148, 13-18. Brambati, B., Macintosh, M. C. M., Teisner, B., Shrimanker, K., Lanzani, A., Maguiness, S., Bonacchi, I., Tului, L., Chard, T. & Grudzinskas, J. G. (1993) Low maternal serum level of pregnancy associated plasma protein (PAPP-A) in the first trimester in association with abnormal fetal karyotype. BritishJournal of Obstetrics and Gynaecology, 100, 324-326. Brizot, M. L., Snijders, R. J. M., Bersingcr, N. A., Knhn, P. & Nicolaides, K. It. (1994) Maternal serum pregnancy-associated plasma protein A and fetal nuchal translucency thickness for the prediction of fetal trisomies in early pregnancy. Obstetricsand Gynecology, 84, 918-922.
36 Brizot, M. L., Jauniaux, E., McKie, A. T., Farzaueli, F. & Nicolaides, K. tl. (1995) Placental expression of ~t and 13subunits of human chorionic gonadotrophin in early pregnancies xvith Down's syndrome, lluman Reproduction, 10, 2506-2509. Chomczynski, P. & Sacchl, N. (1987) Single-step method of RNA isolation by acid guanidium thiocyanate-phcnol-chloroform extraction. Annals of Biochemisto, , 162, 156-159. Feinburg, A. P. & Vogelstein, G. (1983) A technique for radiolabelling DNA restriction endonuclease fragments to a high specific activity. Annals of Biochemisto, , 132, 6--13. Kristensen, T., Oxvlg, C., Sand, O., Moiler, N. P. 1I. & Sottrup-Jensen, L. (1994) Amino acid sequence of human pregnancy-associated plasma protein A derived from cloned eDNA. Biochemisto', 33, 1592-1598. Lin, T. M., llalbert, S. P. & Kiefer, D. (1976) Quantitative Analysis of pregnancy-associated plasma proteins in human placenta. The Journal of Clinical bzvestigation, 57, 466-472.
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