pregnancy-associated plasma protein-a polymorphism and the risk of recurrent pregnancy loss

Journal of Reproductive Immunology70 (2006) 99–108

Pregnancy-associated plasma protein-Apolymorphism and the risk of recurrent

pregnancy loss

Kana Suzuki a, Fumihiro Sata a,∗, Hideto Yamada b, Yasuaki Saijo a,Noriko Tsuruga b, Hisanori Minakami b, Reiko Kishi a

a Department of Public Heath, Hokkaido University Graduate School of Medicine,Kita 15, Nishi 7, Kita-ku, Sapporo 060-8638, Japan

b Department of Obstetrics and Gynecology, Hokkaido University Graduate School of Medicine,Sapporo 060-8638, Japan

Received 15 March 2005; received in revised form 4 October 2005; accepted 18 November 2005

Abstract

Pregnancy-associated plasma protein-A (PAPP-A)/insulin-like growth factor-binding protein-4(IGFBP4) protease is a member of the metzincin family of metalloproteases, known as a sensi-tive biomarker of adverse pregnancy outcomes. Recently, a missense A/C (Tyr/Ser) polymorphism(dbSNP: rs7020782) in the PAPPA gene has been reported. To examine the association betweenrecurrent pregnancy loss (RPL) and this polymorphism, a case-control study of 215 cases with twoor more pregnancy losses (PLs) and 420 fertile controls was performed. Genotyping of the PAPPApolymorphism was determined by allelic discrimination using fluorogenic probes and the 5′ nucleaseassay. Sixty-nine cases (32.1%) were heterozygous and 11 cases (5.1%) were homozygous for theC allele of PAPPA; the respective figures were 127 (30.2%) and 11 (2.6%) in the controls. Womencarrying the C allele had a tendency to increased risk of RPL (AA genotype [reference]; AC genotype:odds ratio [OR], 1.17; 95% confidence interval [CI], 0.82–1.68; CC genotype: OR, 2.06; 95% CI,0.87–4.90), but it was not significant. Women with three or more PLs had a similar tendency (AAgenotype [reference]; AC genotype: OR, 1.04; 95% CI, 0.66–1.64; CC genotype: OR, 2.20; 95% CI,0.82–5.91). The risk of RPL with at least one PL after 9 weeks’ gestation significantly increased inwomen carrying the C allele (AA genotype [reference]; AC genotype: OR, 1.54; 95% CI, 0.95–2.49;CC genotype: OR, 2.83; 95% CI, 1.00–8.05; AC + CC genotypes: OR, 1.65; CI, 1.04–2.62). This is the

∗ Corresponding author. Tel.: +81 11 7065067; fax: +81 11 7067805.E-mail address: [email protected] (F. Sata).

0165-0378/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.jri.2005.11.004

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dx.doi.org/10.1016/j.jri.2005.11.004

100 K. Suzuki et al. / Journal of Reproductive Immunology 70 (2006) 99–108

first report on the PAPPA gene polymorphism in women with RPL, demonstrating some associationbetween the investigated polymorphism and the risk of RPL.© 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Genetic polymorphism; PAPP-A/IGFBP4 protease; Molecular epidemiology; Recurrent pregnancyloss

1. Introduction

In the Japanese population, as well as in Caucasians, about 10–14% of clinically recog-nized pregnancies end in pregnancy loss. The etiology of recurrent pregnancy loss (RPL)remains largely unclear (Stirrat, 1990; Parazzini et al., 1991; Cramer and Wise, 2000;Yamada et al., 2001). Epidemiological studies have suggested that the condition mightbe multifactorial with a possible genetic predisposition and involvement of environmentalfactors in its pathogenesis (Fenster et al., 1991; Parazzini et al., 1991; Cramer and Wise,2000).

Pregnancy-associated plasma protein-A (PAPP-A) is a trophoblast-derived glycoproteinand concentrations of PAPP-A in the maternal circulation increase as pregnancy advances(Folkersen et al., 1981; Johnson et al., 1993). On the other hand, insulin-like growth factors(IGFs), IGF-I and IGF-II, have a key role in regulating feto-placental growth throughoutgestation. These factors have metabolic, mitogenic, and differentiative actions in a widerange of fetal tissues, including the placenta (Jones and Clemmons, 1995). IGFs bind to sixIGF-binding proteins (IGFBPs), IGFBP-1 to -6, which are able to modulate IGF actions by avariety of mechanisms, including an increase in the half-life, transportation, and localizationof the IGFs in specific tissues (Nayak and Giudice, 2003). Lawrence et al. (1999) haveisolated an IGF-dependent IGFBP-4-specific protease from human fibroblast-conditionedmedia and have identified it as PAPP-A. Proteolytic cleavage of IGFBPs is a powerful meansof rapid structural and functional modification of these important growth-regulatory proteins(Lawrence et al., 1999; Laursen et al., 2001). PAPP-A/IGFBP-4 protease is a member ofthe metzincin family of metalloproteases.

Maternal serum levels of PAPP-A are known to be elevated in primigravid women andmultiple pregnancies, and to correlate positively with placental weights (Westergaard etal., 1983b; Pedersen et al., 1995; Kwik and Morris, 2003). A low PAPP-A concentration,in conjunction with age and free �HCG measurement, has been reported as a potentiallyuseful marker in antenatal screening for Down’s syndrome (Benn, 2002; Yaron et al., 2002).

Recent studies have demonstrated that PAPP-A values significantly influence the repro-ductive condition. Tul et al. (2003) reported that a low serum PAPP-A level is associatedwith the delivery of a small-for-gestational age baby and a high PAPP-A level with the deliv-ery of a large-for-gestational age baby. Kwik and Morris (2003) reported that low maternalserum levels of PAPP-A in the first trimester are associated with adverse fetal outcomes,including fetal death and intrauterine growth restriction. Conversely, Goetzl et al. (2004)reported that women with normal values of PAPP-A have a very low risk of pregnancy loss.

Recently, a missense A/C (Tyr/Ser) polymorphism (dbSNP: rs7020782) in exon 14 ofthe PAPPA gene was described (Frosk et al., 2002). However, no studies assessing the asso-

K. Suzuki et al. / Journal of Reproductive Immunology 70 (2006) 99–108 101

ciation between PAPPA gene polymorphism and reproductive failure, such as RPL, havebeen reported. The aim of this study was to investigate whether the maternal PAPPA poly-morphism could be associated with the risk of RPL in a case-control study, and furthermore,whether it could be a susceptible marker for RPL among non-pregnant women.

2. Materials and methods

2.1. Subjects

This case-control study was performed in the city of Sapporo, Japan, during the period1999–2005. A total of 215 patients, aged 20–46 years, with a history of RPL, and 420controls, aged 18–54 years, who were obstetrically managed in the Hokkaido UniversityHospital, were studied. All the patients with RPL and controls were resident in Sapporoin Japan and the surrounding areas; all the patients and the controls were native Japanesewomen. In recent years, this geographical region has had little population immigration bydifferent ethnic groups. The characteristics of the study groups are shown in Table 1. RPLwas defined as having a history of two or more consecutive pregnancy losses (PLs). Thesecriteria have been accepted as a broad definition of RPL and are used in Japan. The primaryRPL group comprised 178 women with a history of two or more PLs, but no live births.The 37 secondary RPL women experienced two or more PLs after at least one live birth.A total of 200 women with RPL experienced all their PLs in the first trimester. Out of200 women with RPL, 122 experienced all their PLs before 9 weeks’ gestation, but theother 78 women experienced at least one PL between 9 and 13 weeks’ gestation, and theother 15 women experienced at least one PL after 14 weeks’ gestation. All women withRPL were subjected to examination by ultrasound and hysterosalpingography for detectionof anatomical abnormalities of the genital tract, to chromosome karyotypic analyses ofperipheral blood, and to other RPL screening, excluding any biochemical tests in very

Table 1Characteristics of 215 cases of recurrent pregnancy loss (RPL) and 420 controls in a Japanese population

Cases Controls

Number % Number %

Age≤29 58 27.0 167 39.830–39 139 64.7 230 54.8≥40 18 8.4 23 5.5

Pregnancy loss2 92 42.8 – –3 74 34.4 – –≥4 49 22.8 – –Primary RPL 178 82.8 – –Secondary RPL 37 17.2 – –<9 weeks 122 56.7 – –9–13 weeks 78 36.3 – –≥14 weeks 15 7.0 – –


early pregnancy. Couples who had balanced chromosomal translocation and women with auterine conformational abnormality, such as septate uterus, were excluded from this study.The control women consisted of 420 volunteers who had experienced at least one live birth,no PL, and no history of infertility. The women who had RPL were significantly olderthan the control subjects (mean 32.5 years versus 31.0 years, p < 0.01). This study wasconducted with the informed consent of all the subjects and approved by the InstitutionalEthical Board for Human Gene and Genome Studies of Hokkaido University GraduateSchool of Medicine.

2.2. Determination of PAPPA polymorphism

Genomic DNA was extracted from lymphocytes of peripheral blood using the QIAampDNA blood kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions.Genotyping of the PAPPA polymorphism was determined by allelic discrimination usingfluorogenic probes and the 5′ nuclease (TaqMan) assay, as described previously (Ranade etal., 2001). To detect the PAPPA A/C (Tyr/Ser) polymorphism in exon 14, two MGB probeswere prepared: A allele-specific probe, 5′-VIC-AGC AAT TGA GAT AAG CAT-MGB-3′and C allele-specific probe, 5′-FAM-CAA TTG AGA GAA GCA T-MGB-3′. Each of thereporters was quenched by MGB, which was typically located at the 3′ end. The design ofprimers for polymerase chain reaction (PCR) of the coding region including the A/C poly-morphism of the PAPPA gene was as follows: forward, 5′-TCG CAT GTG AGA AAA CTGACT GT-3′; reverse, 5′-CAC TGG GCA CCG TGG TA-3′. The reaction mixture containedapproximately 40 ng of template DNA, 5.0 �l of Taqman Universal PCR master mixtureand 0.25 �l of 40× assay mixture in a volume of 10 �l. Real-time PCR was performedon an Applied Biosystems 7500 real-time PCR system (Applied Biosystems, Foster City,CA, USA) using a protocol consisting of 50 ◦C for 2 min and 95 ◦C for 10 min, followedby 40 cycles of denaturation at 92 ◦C for 15 s and annealing/extension at 60 ◦C for 1 min.VIC and FAM fluorescence levels of the PCR products were measured at 60 ◦C for 1 min,resulting in the clear identification of three PAPPA genotypes on a two-dimensional graph.

2.3. Statistical analysis

We calculated age-adjusted odds ratios (OR) and 95% confidence intervals (CI)associated with the PAPPA genotypes using unconditional logistic regression analysis.Hardy–Weinberg equilibrium analyses were performed to compare observed and expectedgenotype frequencies using a χ2 test. Furthermore, a gene dosage effect of the C allele wasassessed by modeling a linear effect on the log odds scale for each C allele in a logisticregression model. All analyses were conducted using SPSS software for Windows version13.0 (SPSS, Chicago, IL, USA).

3. Results

The frequencies of the PAPPA genotypes were compared between 215 patients withRPL and 420 controls in a Japanese population (Table 2). The distribution of genotypes


Table 2Distribution of PAPPA genotypes among 215 cases of women with RPL and 420 controls

Cases Controls OR (95% CI)a p value

Number % Number %

AA 135 62.8 282 67.1 1.00 (reference)AC 69 32.1 127 30.2 1.17 (0.82–1.68) 0.39CC 11 5.1 11 2.6 2.06 (0.87–4.90) 0.10

0.12 (p for trend)A allele 339 78.8 691 82.3C allele 91 21.2 149 17.7

a Age-adjusted logistic regression analysis.

in each group was in Hardy–Weinberg equilibrium. Sixty-nine (32.1%) cases were het-erozygous and 11 (5.1%) were homozygous for the C allele of PAPPA compared with127 (30.2%) and 11 (2.6%) of the controls, respectively. The adjusted OR for RPL riskin women with heterozygosity for the C allele was 1.17 (95% CI, 0.82–1.68) and the ORin women with homozygous C alleles was 2.06 (95% CI, 0.87–4.90), but this was notsignificant.

We evaluated the risk of pregnancy loss in the subgroups of patients according toage of gestation when PLs occurred, the number of PLs and the type of RPL (primaryor secondary). We found an increased risk of RPL with at least one PL after 9 weeks’gestation in women carrying the C allele (Table 3). The adjusted OR for that RPL riskin women with heterozygosity for the C allele was 1.54 (95% CI, 0.95–2.49) and inwomen with homozygous C alleles it was 2.83 (95% CI, 1.00–8.05) with a gene dosageeffect (p for trend = 0.02). The adjusted OR in women with heterozygous or homozy-gous C alleles was 1.65 (95% CI, 1.04–2.62). Regarding the risk of three or more PLs,the adjusted OR in women with heterozygosity for the C allele was 1.04 (95% CI,0.66–1.63), and in women with homozygous C alleles it was 2.20 (95% CI, 0.82–5.91).The ORs in women with homozygous C alleles for primary and secondary RPL were 2.03(95% CI, 0.81–5.05) and 2.31 (95% CI, 0.47–11.3), respectively. However, they were notsignificant.

Table 3Distribution of PAPPA genotypes among 93 cases in women who experienced at least one pregnancy loss after 9weeks’ and 420 controls

Cases Controls OR (95% CI)a p value

Number % Number %

AA 52 55.9 282 67.1 1.00 (reference)AC 35 37.6 127 30.2 1.54 (0.95–2.49) 0.08CC 6 6.5 11 2.6 2.83 (1.00–8.05) 0.05

0.02 (p for trend)AC + CC 41 44.1 138 32.9 1.65 (1.04–2.62) 0.03

a Age-adjusted logistic regression analysis.


4. Discussion

Recently, many investigations have demonstrated that maternal gene polymorphisms arerelated to risk of RPL without considering the liability of environmental factors (Yamadaet al., 2005). These genes included factor V Leiden and prothrombin mutations (Rey etal., 2003), plasminogen activation inhibitor I and factor XIII (Dossenbach-Glaninger et al.,2003), coagulation factors (Wramsby et al., 2000; Pihusch et al., 2001), methylenetetrahy-drofolate reductase (MTHFR) (Lissak et al., 1999), HLA-G (Aldrich et al., 2001; Pfeiffer etal., 2001), GSTM1 (Sata et al., 2003a), GSTP1 (Zusterzeel et al., 2000), IL-1 (Unfried et al.,2001; Wang et al., 2002; Karhukorpi et al., 2003), IL-6 (Saijo et al., 2004), CYP17 (Sata etal., 2003b), and NOS3 (Tempfer et al., 2001). This is the first report on the PAPPA gene poly-morphism in women with RPL, demonstrating an association between the polymorphisminvestigated and the risk of RPL.

Some studies demonstrated elevated risk of pregnancy loss in women with low serumPAPP-A levels during the first and early second trimesters (Westergaard et al., 1983a;Fialova and Malbohan, 2002; Yaron et al., 2002; Kwik and Morris, 2003; Goetzl et al., 2004;Santolaya-Forgas et al., 2004). Westergaard et al. (1985) reported that among pregnancy-related hormones and proteins, the low PAPP-A value was highly sensitive at predictingsubsequent pregnancy loss, while the fetus was still alive. Recently, an association betweenlow PAPP-A levels in early pregnancy and fetal growth restriction has also been reported(Smith et al., 2002a,b; Kwik and Morris, 2003; Bersinger and Odegard, 2004). In particu-lar, women with a PAPP-A level in the lowest fifth percentile at 8–14 weeks’ gestationwere at increased risk of intrauterine growth restriction (Smith et al., 2002a,b). Inter-estingly, it was recently reported that mice homozygous for targeted disruption of thePAPPA gene were viable, but were 60% the size of wild-type littermates at birth, pro-viding the first direct evidence that PAPPA was an essential growth regulatory factor in vivo(Conover et al., 2004). Santolaya-Forgas et al. (2004) suggested that low levels of circu-lating PAPP-A could lead to a downregulation of IGF-II availability early in placental andfetal development. The control of the IGF system in the first and early second trimester tro-phoblast and endometrial stroma may play a key role in determining subsequent pregnancyoutcome.

The successful establishment of pregnancy requires synchronous development and com-munication between the maternal endometrium and embryonic tissues (Nayak and Giudice,2003). PAPP-A is expressed in syncytiotrophoblasts, extravillous cytotrophoblasts anddecidualized endometrial stromal cells (Giudice et al., 2002; Nayak and Giudice, 2003).During pregnancy in the maternal circulation, IGFBP-4 proteolysis and elevated levels ofPAPP-A were detected early in gestation, although the identity and site of production of thecirculating IGFBP-4 protease had been unclear. Giudice et al. (2002) have identified the IGF-II-dependent IGFBP-4 protease in the trophoblast and decidualized endometrial stromalcells as being PAPP-A. Furthermore, the differential regulation of decidual PAPP-A supportsthe role of trophoblast-derived IGF-II as a paracrine regulator of these maternal decidualproducts that have the potential to regulate IGF-II bioavailability at the trophoblast–decidualinterface. Thus, maternal PAPP-A in the endometrial stroma as well as the trophoblast isbelieved to play a crucial role in placental and fetal growth and development during implan-tation and pregnancy.


The PAPPA gene, which is located on chromosome 9q33.1 and encodes PAPP-A, alsoknown IGFBP-4 protease, has a missense A/C (Tyr/Ser) polymorphism in exon 14 (Frosket al., 2002). Although a high concentration of PAPP-A was found in the serum of pregnantwomen, it was also detectable in non-pregnant women and men using a highly sensitiveimmunoassay. Lower levels of PAPP-A expression are also found in a variety of othertissues, including endometrium, ovaries, and breast. It is possible that the PAPPA genepolymorphism or serum PAPP-A levels in non-pregnant women may be a susceptible markerfor adverse pregnancy outcomes. Unfortunately, no association between the PAPPA genepolymorphism and serum levels of PAPP-A has been reported.

This PAPPA polymorphism is located in the Sushi domain. Sushi domains are alsoknown as complement control protein (CCP) modules, or short consensus repeats (SCR),and exist in a wide variety of complement and adhesion proteins (Norman et al., 1991). Thesedomains are characterized by a consensus sequence spanning approximately 60 residuesthat contains four invariant cysteine residues that are involved in intramolecular disulphidebonds, a highly conserved tryptophan, and conserved glycine, proline, and hydrophobicresidues (Henderson et al., 2001; Kirkitadze and Barlow, 2001). These domains are knownto be involved in protein–protein and protein–ligand interactions (Kirkitadze and Barlow,2001). It was possible that there might be functional differences between A and C alleles.As PAPP-A is a potential regulator of fetal growth, its dysfunction must result in PL. Wehypothesize that the C allele might keep its function to some extent to avoid death untilthe late stage of pregnancy or delivery under good environmental conditions, whereas itmight result in PL under severe conditions. In the present study, we found that the womencarrying homozygous C alleles had a 2.8-fold risk of RPL with at least one PL after 9weeks’ gestation. We also found that these women had a more than two-fold risk of atotal of three or more PLs, primary and secondary RPL, but this was not significant. Thesefindings provide a little evidence that a genetic factor related to the IGF systems such as thePAPPA polymorphism may affect the risk of some types of RPL.

A reduced level of PAPP-A in smokers has been reported (Spencer, 1999; De Graaf et al.,2000; Niemimaa et al., 2003). Smoking inhibits apoptosis of the syncytiotrophoblast andmay weaken feto-placental exchange (De Graaf et al., 2000). Smoking adversely affects theplacental vessels and nutrient supply to the fetus, affecting PAPP-A production and loweringIGFs, which may have synergistic adverse effects on fetal growth (Tul et al., 2003). Duringpregnancy, the burden of environmental factors, such as cigarette smoking, physical andmental stress in women who experienced live births, may differ from that in women whonever experienced live births, affecting genetic susceptibility to RPL.

There were a few limitations of the present study. Firstly, maternal serum PAPP-A levelsreflected those in the trophoblast and decidualized endometrial stroma. We were not able toobtain fetal blood samples in this study. We will examine them in other prospective cohortstudies performed simultaneously. Secondly, our sample size was not large enough exceptin the RPL subgroup, which at least had PL after 9 weeks’ gestation, because the frequencyof the CC genotype was relatively rare. According to the frequencies observed in this study,to detect statistical significance with 80% power (α = 0.05) using a χ2 test, about 930 casesand 930 controls would be needed for RPL in total, and 650 cases and 650 controls forthree or more PLs. Thirdly, the functional consequences of the Tyr/Ser mutation in PAPP-Aremain unknown. There have been few reports that this mutation could affect proteolytic


activity, stability or expression of PAPP-A. Further studies are also needed to elucidatewhether any functional consequence of this mutation could occur.

In conclusion, there was some association between the PAPPA polymorphism and therisk of RPL in the present study. To elucidate the role of maternal PAPP-A, the feto-maternalconnection with placental formation and fetal growth and gene–environmental interactionsduring pregnancy in relation to the pathogenesis of RPL, further molecular epidemiologicalstudies, with a larger population, need to be performed, with consideration of other maternaland fetal IGF-related genetic factors and the interaction of environmental factors, such ascigarette-smoking, caffeine intake, and physical or mental stress.

Acknowledgements

This work was supported in part by Grants-in-aid for Scientific Research from theJapanese Society for the Promotion of Science and the Japanese Ministry of Health, Labourand Welfare.

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pregnancy-associated plasma protein-a polymorphism and the risk of recurrent pregnancy loss

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