PRENATAL DIAGNOSISPrenat Diagn 2011; 31: 1101–1103.Published online 29 July 2011 in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/pd.2830

RESEARCH LETTER

Amniotic fluid angiogenin levels are decreased in pregnancieswith fetal trisomy 21

Ahmet Demir1*, Serkan Guclu1, Ozgur Bige1, Ahmet Solak2 and Namik Demir1

1Department of Obstetrics and Gynecology, Dokuz Eylul University School of Medicine, Izmir, Turkey2Department of Biochemistry, Dokuz Eylul University School of Medicine, Izmir, Turkey

KEY WORDS: trisomy 21; angiogenin; amniotic fluid

Trisomy 21 is the most common chromosomalabnormality among newborns, and it is one of the mostimportant causes of mental retardation in the population(Goshen, 1999). Angiogenin is a 14-kDa, nonglycolizedpolypeptide with angiogenetic and ribonucleic activitiesand plays a key role in the complex process of angiogen-esis (Shapiro et al., 1986). Large amount of angiogenesisis required for the growth of placenta and developmentof vascular structures. Angiogenin expression, placentalsecretion and maternal plasma levels increase betweenthe first trimester and birth. The existence of angio-genin in placental villi and increase in angiogenin lev-els toward birth point out that its production dependson the developmental stage (Rajeshekhar et al., 2002).Thus, changes in the level of angiogenin during placen-tal development raise the probability of its possible rolein the angiogenetic and morphological changes that arerequired in the placenta for a healthy gestation.

Syncytiotrophoblast is a major component of thehuman placenta, and it plays a leading role in fetal–maternal exchanges and the secretion of pregnancy-specific hormones. In the normal placenta, multinucle-ated syncytiotrophoblasts are formed by the fusion ofmononuclear cytotrophoblasts (Roberts et al., 2000). Adefect exists in the formation of syncytiotrophoblastsin the placentas of fetuses with trisomy 21: histopatho-logic analysis of placentas of trisomy 21 fetusesshows trophoblastic hypoplasia and villus hypovascu-larity throughout pregnancy (Qureshi et al., 1997). Thismay lead to a decrease in the production of pregnancy-specific hormones (Massin et al., 2001).

Considering the possible role of angiogenin on placen-tal characteristics, the aim of the present study was toexplore whether an association existed between Downsyndrome and this angiogenetic factor. Therefore, wecompared amniotic fluid angiogenin levels of fetuseswith normal karyotype and those with trisomy 21.

In the Perinatology Division of Department of Obstet-rics and Gynecology Clinic at Dokuz Eylul University,

*Correspondence to: Ahmet Demir, Department of Obstetrics andGynecology, Dokuz Eylul University School of Medicine, 35330Inciralti, Izmır, Turkey. E-mail: ahmet demir77@hotmail.com

we store approximately 2 mL of amniotic fluid sam-ples for research purposes from pregnant women whoundergo mid-trimester amniocentesis for various indi-cations after their informed consent is obtained. Amnio-centeses are performed under ultrasound guidance trans-abdominally in a sterile manner. The 2 mL of amni-otic fluids are centrifuged for 5 min at 3000 cycle/min.The supernatants are stored at −80 ◦C for futureinvestigations.

We retrospectively identified 15 fetuses with Downsyndrome, and we randomly selected 48 fetuses withnormal karyotype from our perinatology record databaseas controls, and we analyzed their amniotic fluid angio-genin levels using enzyme-linked immunosorbent assay(ELISA) (R&D Systems, Minneapolis, MN, USA). TheELISA was validated for amniotic fluid, and sampleswere assayed in duplicate. The ELISA sensitivity foramniotic fluid was 0.026 ng/mL, and the interassay andintraassay coefficients of variation were 4.6 and 2.9%,respectively. The ELISA used is specific for angio-genin and does not cross-react or interact with humaninterleukin-lc, interleukin-lβ, interleukin-2, interleukin-3, interleukin-4, interleukin-6, interleukin-7, interleukin-8 or tumor necrosis factor α. The results were analyzedby Mann–Whitney U test using Statistical Package forthe Social Sciences version 13 for Windows (SPSS Inc.,Chicago, IL, USA). A value of p < 0.05 was consideredto be significant.

Clinical characteristics of the two groups are shown inTable 1. There were no significant differences in mater-nal age, weight and gestational age at amniotic fluidsampling, gravidity and parity between the two groups.All women in the study were non-smokers of Turkishethnicity. Indications for amniocentesis were advancedmaternal age, increased risk for Down syndrome at firstor second-trimester screening tests, fetal anomalies orfamily history of genetic conditions (such as previouschild with trisomy 21).

Angiogenin levels were significantly lower in amni-otic fluid samples of fetuses with trisomy 21 (p = 0.04)(Figure 1). Median level of amniotic fluid angio-genin was 11.24 ng/mL (range, 6.13–17.91) for tri-somy 21 fetuses and 17.69 ng/mL (range, 5.69–45.02)for euploid fetuses. There were no significant changes

Copyright 2011 John Wiley & Sons, Ltd. Received: 11 April 2011Revised: 25 June 2011

Accepted: 25 June 2011Published online: 29 July 2011

1102 A. DEMIR et al.

Table 1—Clinical characteristics in relation to fetalkaryotype

Trisomy 21 Euploid p value∗

Maternal age(years)

36 (30–39) 34 (29–41) 0.06

Maternal weight(kg)

57 (48–69) 60 (47–74) 0.43

Gestational ageat amniocentesis(weeks)

17 (15–19) 16 (15–18) 0.05

Gravidity 2 (1–8) 2 (1–5) 0.38Parity 1 (0–5) 1 (0–2) 0.96

Data are given as median (range).∗ Mann–Whitney U test

4815N =

normaltrisomy 21

Ang

ioge

nin

leve

l (ng

/ml)

50

40

30

20

10

0

Figure 1—Box-plot graph of amniotic fluid angiogenin levels infetuses with normal karyotype and with trisomy 21

in angiogenin levels over the gestational age period(15–18 weeks) in euploid pregnancies.

Our novel finding that second-trimester amniotic fluidangiogenin levels are significantly lower in fetuses withtrisomy 21 when compared with euploid fetuses suggeststhat angiogenesis is impaired within the fetoplacentalcompartment in trisomy 21 pregnancies. Several linesof evidence support such hypothesis.

Massin et al. (2001) studied the formation and func-tion of the syncytiotrophoblast in the normal placentaand the placenta affected by trisomy 21. Cytotrophoblastcells were isolated from first-trimester, second-trimesterand term placentas. In vitro, cytotrophoblast cells iso-lated from normal placenta fused to form syncytiotro-phoblast, whereas they observed defective syncytiotro-phoblast formation in placentas affected with trisomy21. These results are in agreement with previous macro-scopic and histological observations of trisomy 21-affected placenta that revealed delayed maturation ofchorionic villi and syncytiotrophoblastic hypoplasia with

a resistant cytotrophoblastic layer in the third trimester.Normal angiogenesis is regulated by a series of factorsas well as the partial pressure of oxygen in the feto-placental vessels. Angiogenin is one of the most potentinducers of neovascularization. The origin of amnioticfluid angiogenin is unknown; however, fetoplacental unitis thought to be the origin of amniotic fluid angiogenin(Spong et al., 1997a). Rajeshekhar et al. (2002) showedthat angiogenin was expressed in all placental villi fromfirst trimester to term. The available evidence suggestsan imbalance in the production of angiogenin in earlypregnancy, which may lead to altered placental devel-opment and placental function.

As a screening modality, alpha-fetoprotein (AFP),human chorionic gonadotrophin (hCG) and estriol areanalyzed in triple-marker screening. AFP levels are rec-ognized to be lower (0.7 MoM), hCG levels to be higher(2.04 MoM) and estriol levels to be lower (0.79 MoM) infetuses with trisomy 21 (New England Regional Genet-ics Group, 1989). The cause of the elevated hCG levelsis not clear. Spong et al. (1998) found that second-trimester amniotic fluid levels of angiogenin in patientswith elevated maternal serum hCG are significantlylower than that in patients with normal maternal serumhCG at triple screen, suggesting inadequate angiogene-sis. Although none of their cases had trisomy 21, theirresult is compatible with our findings and may providean explanation for the high maternal serum hCG levelsfound in trisomy 21 gestations.

In a different study, Spong et al. (1997b) testedangiogenin in mid-trimester amniotic fluids of patientswith elevated maternal serum AFP, and they onlyincluded subjects with normal chromosome results. Theyfound mid-trimester amniotic fluid angiogenin levels tobe significantly elevated in such patients, suggesting thatelevation in amniotic fluid angiogenin is not specificto trisomy 21 but rather to any condition affectingfetoplacental growth and development.

Our study has the limitation of examining the levels ofangiogenin only in the amniotic fluid, as merely samplesof amniotic fluids of women undergoing amniocentesiswere stored. It would have been interesting if we had thematching serum samples to compare both the amnioticfluid and serum levels together. If angiogenin is alsodecreased in the maternal serum, it might be consideredas screening marker for fetal Down syndrome.

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Massin N, Frendo JL, Guibourdenche J, et al. 2001. Defect of syncytiotro-phoblast formation and human chorionic gonadotropin expression in Down’ssyndrome. Placenta 22(Suppl A): S93–S97.

New England Regional Genetics Group Prenatal Collaborative Study ofDown Syndrome Screening. 1989. Combining maternal serum α-fetoproteinmeasurements and age to screen for down syndrome in pregnant womenunder age 35. Am J Obstet Gynecol 160: 575–581.

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Copyright 2011 John Wiley & Sons, Ltd. Prenat Diagn 2011; 31: 1101–1103.DOI: 10.1002/pd

ANGIOGENIN LEVELS DECREASE IN TRISOMY 21 1103

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Copyright 2011 John Wiley & Sons, Ltd. Prenat Diagn 2011; 31: 1101–1103.DOI: 10.1002/pd