Alterations of maternal serum and placental follistatin-like 3 and myostatin in pre-eclampsia

Alterations of maternal serum and placentalfollistatin-like 3 and myostatin in pre-eclampsiajog_1823 988..996

Jing Guo1*, Ting Tian1, Donghong Lu1, Guiyu Xia2, Hanzhi Wang1 and Minyue Dong1,2,3

1Women’s Hospital, School of Medicine, Zhejiang University, 2The Key Laboratory of Reproductive Health of ZhejiangProvince, and 3The Key Laboratory of Reproductive Genetics, Ministry of Education, Hangzhou, China

Abstract

Aim: To clarify the alterations of myostatin, a member of the transforming growth factor-b superfamily, andfollistatin-like 3 (FSTL3), a binding protein for myostatin, in pre-eclamptic women.Methods: Samples of blood and placenta were collected from 40 pre-eclamptic women and 40 controls. Theserum level and placental expression of FSTL3 and myostatin were determined with enzyme-linked immun-osorbent assay, real-time polymerized chain reaction and western blotting.Results: The serum levels of myostatin and FSTL3 were significantly higher in pre-eclamptic women than inthe controls (P < 0.001 for both). Placental expression of myostatin and FSTL3 were also significantly increasedin the pre-eclamptic placenta compared with that of the controls (P < 0.001 for both); however, there were nosignificant differences in myostatin or FSTL3 in either the maternal serum or the placenta in women with mildor severe pre-eclampsia (P > 0.05 for both).Conclusion: The serum levels and placental expression of myostatin and FSTL3 are elevated in pre-eclampsia,suggesting the role of myostatin and its binding protein in pre-eclampsia.Key words: follistatin-like 3, myostatin, placenta, pre-eclampsia, serum.

Introduction

Pre-eclampsia, potentially one of the most devastatingcomplications of pregnancy, is characterized by theonset of hypertension and proteinuria in the secondhalf of pregnancy, labor and/or early in the postpar-tum period.1,2 This condition is not a simple complica-tion of pregnancy, but a syndrome of multiple organdamage involving the liver, kidney, lung and neuro-logical systems, in addition to abnormalities in coagu-lation, immune response and vascular endothelialintegrity.1–3

The etiology and pathology of pre-eclampsia remainlargely unknown, but seem to be multifactorial; mucheffort has therefore been made to elucidate the molecu-

lar mechanisms underlying pre-eclampsia.1,2,4,5 Theactivin pathway is a promising area of research in thefield of the etiology and pathology of pre-eclampsia.6–11

Activin and inhibin are glycoproteins belonging to thetransforming growth factor-b (TGF-b) superfamily, andseveral lines of evidence have documented a widerange of biological roles for the activins and relatedproteins in cell growth and differentiation, in regulat-ing embryonic development and in maintaining tissuehomeostasis in adult animals.12,13 Follistatin, a bindingprotein for TGF-b superfamily members, regulates bio-logical activity of TGF-b superfamily members.7,12

It has been well evidenced that the placental expres-sion and maternal serum levels of activin A andinhibin A are elevated in pre-eclamptic women.6–12

Received: June 17 2011.Accepted: October 29 2011.Reprint request to: Dr Minyue Dong, Women’s Hospital, School of Medicine, Zhejiang University, 1 Xueshi Rd, Hangzhou,Zhejiang Province 310006, China. Email: [email protected]*Currently working at the Third Affiliated Hospital of Zhengzhou University.

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doi:10.1111/j.1447-0756.2011.01823.x J. Obstet. Gynaecol. Res. Vol. 38, No. 7: 988–996, July 2012

988 © 2012 The AuthorsJournal of Obstetrics and Gynaecology Research © 2012 Japan Society of Obstetrics and Gynecology

However, the changes in circulating follistatin arecontroversial. Reports of unchanged and enhanced fol-listatin in maternal serum were also available.7,14,15

Myostatin is a relatively new member of TGF-b super-family,16 and follistatin-like 3 (FSTL3), also namedfollistatin-related gene (FLRG), is a new member ofbinding protein for the TGF-b superfamily members.17 Itwas recently reported that placental expression ofFSTL3 at messenger RNA and protein and the maternalserum concentration of FSTL3 were significantlyelevated in pre-eclampsia compared with normal preg-nancy.18 However, the alterations of placental and circu-lating myostatin, the ligand of FSTL3, in pre-eclampsiahave not yet been investigated.

Based on the previous publications of TGF-b super-family members and their binding proteins in normalpregnancy and pre-eclampsia, we hypothesized thatmaternal circulating levels and placental expressionof myostatin and its major binding protein, FSTL3,were significantly different in pre-eclampsia and nor-motensive pregnancies. To verify this hypothesis, wedetermined maternal circulating levels and placentalexpression of myostatin and FSTL3 in pregnant womenwith and without pre-eclampsia.

Patients and MethodsPatients

Cross-sectional investigations were conducted at theWomen’s Hospital, School of Medicine, Zhejiang Uni-versity, with the approval from the Institutional ReviewBoard. Informed consent was obtained from all partici-pants. First, we determined the concentrations ofFSTL3 and myostatin in the serum of pre-eclampticwomen and pregnant controls. Second, to explore thepossible origin of altered circulating FSTL3 and myo-statin, we assessed their expression in the placentas ofpregnant women with and without pre-eclamptic.

Forty women with pre-eclampsia were recruited; 16were diagnosed with mild and 24 with severe pre-eclampsia. Seven women with pre-eclampsia had fetalgrowth restriction. Pre-eclampsia was diagnosed andclassified according to strict criteria recommended bythe American Congress of Obstetricians and Gynecolo-gists:19 a systolic blood pressure of �140 mmHg or adiastolic blood pressure of �90 mmHg on two occa-sions at least six hours apart occurring after 20 weeksof gestation in a pregnant woman with previouslynormal blood pressure, and detectable urinary protein(�1+ by urine dipstick or �0.3 g/24 h). Severe pre-eclampsia was defined as blood pressure �160/

110 mmHg with either a urine dipstick showing 3–4+in a random urine sample or proteinuria >5.0 g/24 h.Other evidence of severe disease included elevatedserum creatinine, eclampsia, pulmonary edema, olig-uria (less than 500 mL per 24 h), fetal growth restric-tion, oligohydramnios and symptoms suggestingsignificant end-organ involvement (headache, visualdisturbances, or epigastric or right upper quadrantpain). Women who met the criteria of pre-eclampsiabut not severe pre-eclampsia were diagnosed withmild pre-eclampsia. Fetal growth restriction wasdefined as a birth weight less than the 10th centile forthe gestational age.

Exclusion criteria included multiple gestation, diabe-tes mellitus, chronic hypertension, infectious diseasesrecognized in pregnancy, premature rupture of mem-branes, active labor, polyhydramnios and signs ofother concurrent medical complications. Gestationaldiabetes mellitus (GDM) was excluded after a 50 gglucose challenge test (GCT) during the period of24–28 weeks’ gestation.

For the comparison of maternal serum FSTL3 andmyostatin, 40 maternal and gestational age-matchednormally pregnant women were recruited (controlgroup 1). Since the majority of women with pre-eclampsia delivered before term, to exclude the pos-sible effects of preterm delivery-related conditions onplacental gene expression, 40 age-matched pregnantwomen who delivered term served as controls (controlgroup 2) for the comparison of placental expression ofFSTL3. Because multiple gestations and the labormay affect placental gene expression, all subjects weresingleton and underwent elective cesarean section. Theindications for cesarean section were pre-eclampsia forthe pre-eclamptic women and maternal requirementfor the control women. The control women had no signof gestational complications or fetal distress and gavebirth to healthy neonates of appropriate size for theirgestational age.

Sample collection

Fasting blood samples were taken from the patientswith pre-eclampsia at the time of admission before anymedication and from the control women who had nottaken any medication except for vitamins and minerals.All the blood samples were centrifuged at 3000 gfor 15 min to clarify serum. Serum was collected andstored at -80°C until assay. Placenta samples wereimmediately collected after the placenta was delivered.Placental cotyledons were dissected from an area inthe middle of the placenta, washed thoroughly with

Follistatin-like 3 in pre-eclampsia

© 2012 The Authors 989Journal of Obstetrics and Gynaecology Research © 2012 Japan Society of Obstetrics and Gynecology

ice-cold normal saline after the amniotic membranes,deciduas and connective tissues were removed, snap-frozen with liquid-nitrogen after excessive liquid wasabsorbed with paper toweling and then stored at -80°Cuntil assay. The total time taken for sample collectionwas less than 10 min.

Determination of serum FSTL3 and myostatin

Serum levels of myostatin and FSTL3 were determinedby enzyme-linked immunosorbent assay. The reagentswere purchased from Groundwork BiotechnologyDiagnosticate (San Diego, CA, USA) and R&D systems(Minneapolis, MN, USA), respectively. The assays wereconducted in duplicate according to the manufac-turer’s protocols.

Assessment of gene expression withreal-time PCR

Total RNA was extracted with TRIzol (Invitrogen,Carlsbad, CA, USA) and reversely transcribed tocomplementary DNA (cDNA) using the PrimeScriptreverse transcription reagent kit (TakaRa, Otsu, Japan)according to the manufacturer’s protocol. The two-stepmethod real-time polymerized chain reaction (PCR)was performed using SYBR Premix EX Taq (TakaRa) onABI PRISM 7900HT Fast Real-Time PCR System(Applied Biosystems, Foster City, CA, USA). The reac-tion was conducted in a system of 25 mL containingSYBR Green, forward and reverse primers and cDNAtemplate. The housekeeping gene b-actin was used asan internal control. Primers for b-actin, myostatin andFSTL3 were designed using Primer Express 3.0 soft-ware (Applied Biosystems). The sequences of primerswhose amplicons span over introns were as follows:

• b-actin: 5′-ACCCACACTGTGCCCATCTACGA-3′(F)

5′-GCCGTGGTGGTGAAGCTGTAGCC-3′(R)

• myostatin: 5′-TGGTCATGATCTTGCTGTAACCTT-3′(F)

5′-TGTCTGTTACCTTGACCTCTAAAA-3′(R)

• FSTL3: 5′-GGAAGGACTGAGGAAGGGAGGC-3′(F)

5′-CGGCAATAGGTCTTAGGGTAGGG-3′(R)

The cycling conditions were as follows: 95°C for 30 sfollowed by 40 cycles of 60°C for 30 s and 95°C for 5 s.The melting curves were generated and experimentswere conducted in duplicate. Data were analyzed by

the comparative threshold cycle (CT) method and the2-DCT method was calculated using the followingformula: relative expression = 2-DCT, where DCT = CT(target gene - CT internal control).20,21

Analysis of FSLT3 and myostatin protein inplacental tissue

Placental tissues of approximately 400 mg were mecha-nically homogenized with the Kinematica homogenizer(Lucerne, Switzerland) on ice in 1 mL of a cold lysisbuffer containing 50 mM tris-HCl (pH 8.0), 150 mMNaCl, 0.02% sodium azide, 1% NP-40, 0.5% sodiumdeoxycholate and 1 mM phenylmethanesulfonyl fluo-ride, and centrifuged at 15 000 g for 10 min at 4°C.

After boiling in loading buffer, samples containing100 mg protein were loaded on 10% sodium dodecylsulfate-polyacrylamide gel, electrophoresed and trans-ferred onto nitrocellulose membranes (GE Healthcare,Amersham, UK). The membranes were blocked for 1 hat room temperature in blocking buffer containing 5%nonfat dry milk in TBST (50 mM tris-HCL, 100 mMNaCl, 0.1% Tween-20, pH 7.4), then incubated over-night at 4°C with appropriate primary antibodies:mouse monoclonal immunoglobulin G (IgG) anti-myostatin, diluted at 1:4000, purchased from Abcam(Cambridge Science Park, Cambridge, UK); mousemonoclonal IgG anti-FSTL3: diluted at 1:250, pur-chased from R&D systems; mouse monoclonal IgGanti-b-actin: diluted at 1:2000, obtained from Sigma(St Louis, MO, USA). After being washed three timeswith TBST, the membranes were incubated for 2 h atroom temperature with horseradish peroxidase-conjugated polyclonal rabbit anti-mouse IgG second-ary antibody from Sigma (1:10 000 for myostatin;1:7500 for FSTL3; 1:4000 for b-actin) and then washedas previous. The blotting signals were visualized withchemiluminescence ECL Plus (GE Healthcare) andimaged with Image Quant LAS 4000 mini bio-molecular imager (GE Healthcare Bio-Sciences AB,Uppsala, Sweden). The expression of the target proteinwas standardized against internal control b-actin withImage Quant TL Analysis software (GE HealthcareBio-Sciences AB).

Data analysis

All the data were normally distributed and presentedas mean � SD, and comparison of the two groupswas evaluated using Student’s t-test. The c2-testwas used for the analysis of number of cases withproteinuria. The statistics package SPSS (Statistical

J. Guo et al.


Analysis System, Chicago, IL, USA) was used for dataanalysis. Values of P < 0.05 were considered to bestatistically significant.

Results

Table 1 summarizes the clinical characteristics of thepre-eclamptic women and the control groups. Therewere no significant differences in maternal age betweenthe pre-eclamptic women (28.4 � 5.6 years) and controlgroup 1 (28.7 � 3.2 years, P = 0.56) or control group 2(28.6 � 3.6 years, P = 0.66). Gestational age at the time ofblood sampling was not significantly different betweenthe women with pre-eclampsia (35.2 � 3.0 weeks) andcontrol group 1 (36.8 � 1.3 weeks, P = 0.06), but the ges-tational age at the time of placenta sampling was signifi-cantly different between the women with pre-eclampsiaand control group 2 (38.7 � 2.2 weeks, P < 0.001). Dia-stolic and systolic blood pressures were significantlyhigher in the pre-eclamptic women than in controlgroup 1 (P < 0.001 for both) or control group 2 (P < 0.001for both). Proteinuria was detected in all women withpre-eclampsia, but was not detectable in any womenwith a normal pregnancy (P < 0.001).

Maternal serum concentrations of myostatinand FSTL3

Myostatin and FSTL3 were detected in all serumsamples. Maternal serum levels of myostatin andFSTL3 were significantly increased in women with pre-eclampsia compared with the controls (P < 0.001 forboth, Fig. 1). There were no significant differences inthe maternal serum levels of FSTL3 (P = 0.095) or myo-statin (P = 0.681) between mild and severe pre-eclamptic women.

Placental expression of myostatin and FSTL3

Myostatin and FSTL3 expressions at mRNA andprotein levels were detected in all placental samples

and significantly elevated in cases of pre-eclampsiacompared with the controls. The expressions of mRNAfor FSTL3 and myostatin were also significantlyincreased in the pre-eclamptic pregnancies comparedwith the controls (P < 0.001 and P = 0.028, respectively;Fig. 2). There was no significant difference in placen-tal mRNA levels of FSTL3 (P = 0.986) or myostatin(P = 0.493) between mild and severe pre-eclampticpregnancies.

Western blotting analysis detected the FSTL3 proteinproduct (27 kDa), and 52 kDa and 26 kDa products cor-responding to the myostatin precursor and maturedimer, respectively. Immunoreactive bands of FSTL3and myostatin precursor and mature dimer were sig-nificantly enhanced in pre-eclamptic pregnancies com-pared with the controls (P = 0.001 for the myostatinprecursor, P < 0.001 for the myostatin mature dimerand P < 0.001 for FSTL3, Fig. 3). There was no signifi-cant difference in the placenta protein levels of myo-statin (P = 0.277 for the precursor, P = 0.762 for themature dimer) or FSTL3 (P = 0.479) between mild andsevere pre-eclamptic pregnancies.

Discussion

In the current study, we found that maternal serumlevel and placental expression of FSTL3 were signifi-cantly increased in women with pre-eclampsia com-pared with the controls: more importantly, we reportedfor the first time the significant elevation of myostatin,a ligand of FSTL3 and a member of the TGF-b super-family, in the circulation and placentas of pre-eclampticwomen. Our data added new evidence that TGF-bsuperfamily members and their binding proteins mightbe involved in pre-eclampsia.

Multiple lines of evidence revealed placental expres-sion and maternal circulating levels of activins andrelated proteins (mainly including activin, inhibin,follistatin and FSTL3) were altered, implying that

Table 1 Clinical characteristics

Pre-eclampsia(n = 40)

Controlgroup 1(n = 40)

P-value Controlgroup 2(n = 40)

P-value

Maternal age (years) 28.4 � 5.6 28.7 � 3.2 P = 0.56 28.6 � 3.6 P = 0.66Gestational age at blood sampling (weeks) 35.2 � 3.0 36.8 � 1.3 P = 0.06 — —Gestational age at placenta sampling (weeks) 36.2 � 1.5 — — 38.7 � 2.2 P < 0.001Systolic blood pressure (mmHg) 156 � 20 111 � 11 P < 0.001 110 � 10 P < 0.001Diastolic blood pressure (mmHg) 103 � 15 70 � 8 P < 0.001 75 � 5 P < 0.001Proteinuria (�1+) (n) 40 0 P < 0.001 0 P < 0.001



derangement of the activin pathway might be involvedin pre-eclampsia. Petraglia et al. first described theelevation of maternal serum activin A levels in pre-eclamptic women,22 and subsequently, Muttukrishnaet al.9 and others7,10,11,14,23–27 have consistently reportedincreases in the maternal serum level and placentalexpression of activin A and inhibin A in pre-eclampsia-complicated pregnancies; however, two publicationsdescribed different findings of follistatin, the bindingprotein for activins and inhibins. D’Antona et al. foundthat the maternal serum follistatin level was not signifi-cantly different between pre-eclamptic and normoten-sive pregnancies,28 while Keelan et al. observed thatfollistatin concentrations in maternal serum were mod-estly (less than two-fold), but significantly elevated inpre-eclampsia compared with normotensive pregnan-cies.15 Maternal serum levels of activin A and inhibin Aincrease prior to the onset of pre-eclampsia and areassociated with disease severity.24,29 Furthermore,Spencer et al. reported that maternal serum inhibin Aand activin A levels were increased in the first trimesterof pregnancies developing pre-eclampsia.30

Pryor-Koishi et al. determined the maternal serumconcentration and placental expression of FSTL3 inpre-eclamptic and normotensive pregnancies, andfound that FSTL3 was significantly enhanced in thecirculation and placenta of pre-eclamptic women com-pared with the controls,18 which confirmed the findingsof the current investigation. More importantly, wereported for the first time that maternal serum andplacental myostatin was significantly elevated in pre-eclampsia. The findings of the current investigationprovide new evidence that the derangement of theactivin pathway is involved in pre-eclampsia. It wasreported that the maternal concentration of circulatinginhibin A began to increase rapidly at 9–10 weeks’ ges-tation, fell after 12 weeks’ gestation31,32 and graduallyrose again in the third trimester.31–34 The alteration ofcirculating activin A in the pregnancy was similar.Maternal activin A levels increased after the establish-ment of pregnancy, fell from 8 to 16 weeks’ gestation31,35

and rose progressively in the third trimester with amaximum at term.31,33,35 Schneider-Kolsky et al. foundan increase of 7.8-fold in the maternal serum activin A

Figure 1 Comparison of maternalserum follistatin-like 3 (FSTL3)and myostatin between thepre-eclamptic women and thecontrols. Maternal serum levelsof FSTL3 and myostatin weresignificantly higher in womenwith pre-eclampsia than in thecontrols (P < 0.001) (a,b). Therewas no significant differencesin serum FSTL3 (P = 0.095) ormyostatin (P = 0.681) betweenwomen with mild and severepre-eclampsia (c,d).

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and a three-fold increase in serum follistatin in a termpregnancy compared with the first trimester preg-nancy.36 Similarly, circulating levels of both myostatinand FSTL3 were significantly increased with gestation(unpublished data from our laboratory). In the currentinvestigation, we found that myostatin and FSTL3levels in both serum and placenta were significantlyelevated in pre-eclampsia, implying that the placentamight be one of the sources of increased serum myo-statin and FSTL3 in pre-eclampsia.

Placental hypoxia is one of the characteristics of pre-eclampsia and is likely to be the cause of enhancedFSTL3 in maternal circulation and placenta. Biron-Shental et al. found that primary placental trophoblastsisolated from term placentas exhibited a 4–6-foldincrease in the expression of FSTL3 as early as 4 h afterexposure to hypoxia (FiO2 < 1%) and that hypoxiaincreased the secretion of FSTL3 to the medium;37

however, no publication is available describing theeffect by hypoxia on the expression of myostatin.

The significance of the increased expression of myo-statin and FSTL3 in pre-eclampsia needs further obser-vation. There were controversial reports on the effect

of myostatin on placental glucose uptake.38–41 Generally,myostatin, as a member of the TGF-b superfamily,regulates the proliferation and differentiation of cells,16

and FSTL3, as a binding protein for TGF-b membersof the superfamily, is involved in the proliferation anddifferentiation of cells.17 Due to the specialty of cellularcomponents at the feto–maternal interface, attentionshould be paid to the effects of myostatin and itsbinding proteins on the maintenance of normalpregnancy and the regulating mechanisms at thefeto–maternal interface.

We recognize that the inability to match the casesand controls for gestational age when placental geneexpression was to be compared is one of the limitationsof the current investigation. This is an intrinsic problemfaced by researchers in this field because pre-eclampticwomen, by the nature of the pathology, are usuallydelivered before term. The design of a cross-sectionalstudy led to the inability to draw a clear conclusionregarding the causal relationship of altered expressionof myostatin and FSTL3 in pre-eclampsia. A longitudi-nal observation on circulating myostatin and FSTL3 inboth pre-eclamptic women and normally pregnant

Figure 2 Comparison of mRNAexpression of follistatin-like 3(FSTL3) and myostatin in the pla-centas from women with pre-eclampsia and those who hadhad a normal pregnancy. Therelative expression of FSTL3(P < 0.001) and myostatin mRNAwas significantly increased inthe pre-eclamptic women com-pared with control (P = 0.028)(a,b). There were no significantdifferences in placental FSTL3(P = 0.986) and myostatin (P =0.493) mRNA levels betweenmild and severe pre-eclampsia(c,d).

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Figure 3 Comparison of follistatin-like 3 (FSTL3; 27 kDa), myostatin precursor (52 kDa) and mature dimer (26 kDa) in theplacentas from pre-eclamptic and normal pregnancies. Placental FSTL3 (P < 0.001) and myostatin (P = 0.001 for theprecursor and P < 0.001 for the mature dimer) were significantly elevated in those from pre-eclamptic pregnancies thanfrom the controls (a,b). There was no significant difference in the placental FSTL3 (P = 0.479) and myostatin (P = 0.277 forthe precursor and P = 0.762 for the mature dimer) between mild and severe pre-eclamptic pregnancies (c,d).

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women would enhance our insights into the role ofmyostatin and binding proteins in the pathogenesis ofpregnancy.

Acknowledgments

This work was financially supported by the NaturalScience Foundation of Zhejiang Province (Y2100416),Natural Science Foundation of China (81170572)and National Basic Research Program of China(2012CB944903).

Disclosure

None declared.

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Alterations of maternal serum and placental follistatin-like 3 and myostatin in pre-eclampsia