European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 96–99

Placental gene expression patterns of epidermal growth factor inintrauterine growth restriction

Attila Rab a, Imre Szentpeteri b, Laszlo Kornya c, Balazs Borzsonyi d, Csaba Demendi d,Jozsef Gabor Joo e,*a Telki Hospital, Budapest, Hungaryb Praxis fur Gynakologie und Geburtshilfe und allgemeine Medizin, Wehingen, Baden-Wurttemberg, Germanyc St. Stephen’s Hospital, Budapest, Hungaryd Second Department of Obstetrics and Gynecology, Semmelweis University, Budapest, Hungarye First Department of Obstetrics and Gynecology, Semmelweis University, Budapest, Hungary

A R T I C L E I N F O

Article history:

Received 15 December 2012

Received in revised form 1 April 2013

Accepted 27 May 2013

Keywords:

Gene expression

Placenta

Epidermal growth factor

Intrauterine growth restriction

Body mass index

Maternal age

A B S T R A C T

Objective: In this study, we compared human placental gene expression patterns of epidermal growth

factor (EGF) in pregnancies with intrauterine growth restriction (IUGR) vs. normal pregnancies as

control.

Study design: Gene expression of EGF was determined from human placental samples collected from all

pregnancies presenting with IUGR at our institution during the study period January 1, 2010–January 1,

2011. Multiple clinical variables were also assessed including maternal age, gestational weight gain,

increase of BMI during pregnancy and fetal gender.

Results: A total of 241 samples were obtained (101 in the IUGR pregnancy group, 140 in the normal

pregnancy group). EGF was found to be underexpressed in the IUGR group compared to normal

pregnancy (Ln2a

: �1.54; p < 0.04). Within the IUGR group no fetal gender-dependent difference was

seen in EGF gene expression (Ln2a: 0.44; p < 0.06). Similarly, no significant difference in EGF expression

was noted in cases with more vs. less severe forms of IUGR (Ln2a: �0.08; p = 0.05). IUGR pregnancies

were significantly more common in the maternal age group 35–44 years compared to other age groups.

Gestational weight gain and gestational BMI increase were significantly lower in IUGR pregnancies

compared to controls.

Conclusions: Placental expression of EGF was found to be reduced in IUGR pregnancies vs. normal

pregnancies. This may partly explain the smaller placental size and placental dysfunction commonly

seen with IUGR. An increased incidence of IUGR was observed with maternal age exceeding 35 years. The

probability of IUGR correlated with lower gestational weight gain and lower BMI increase during

pregnancy.

� 2013 Elsevier Ireland Ltd. All rights reserved.

Contents lists available at SciVerse ScienceDirect

European Journal of Obstetrics & Gynecology andReproductive Biology

jou r nal h o mep ag e: w ww .e lsev ier . co m / loc ate /e jo g rb

1. Introduction

Intrauterine growth restriction (IUGR) is defined as fetalbirthweight below the tenth percentile for sex and gestational

Abbreviations: IUGR, intrauterine growth restriction; IGF-1, insulin-like growth

factor 1; IGF-2, insulin-like growth factor 2; VEGF-A, vascular endothelial growth

factor A; TGF-b, transforming growth factor beta; EGF, epidermal growth factor;

TGF-a, transforming growth factor alfa; EGFR, EGF-receptor; ErbB-1-4, erythro-

blastic leukemia viral oncogene homolog 1–4; AC, abdominal circumference; BMI,

body mass index; LIF, leukemia inhibiting factor.

* Corresponding author at: 1st Department of OB/GYN, Semmelweis University,

Hungary, Baross utca 27, 1088 Budapest, Hungary. Tel.: +36 1 459 15 00;

fax: +36 1 317 61 74.

E-mail address: joogabor@hotmail.com (J.G. Joo).

0301-2115/$ – see front matter � 2013 Elsevier Ireland Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.ejogrb.2013.05.020

age [1] (though it must be remarked that the fifth and thirdpercentiles as a borderline of IUGR are also used in obstetrics).IUGR may result from placental dysfunction, fetal malformation,intrauterine infection or maternal factors. Although the mostcommon etiology for IUGR is thought to be placental dysfunction,its pathology at molecular level remains largely unknown [2,3].

During human gestation, maternal serum levels of multiplegrowth factors rise. Among these, insulin-like growth factor 1 and2 (IGF-1, IGF-2) appear to be especially important in thepathogenesis of both IUGR and premature delivery [4,5]. Epidermalgrowth factor (EGF) has been found to play a role in stimulatingplacental growth [6].

Structurally, human EGF is a polypeptide consisting of 53 aminoacids. Its precursor is substantially larger, consisting of a 1207amino acid polypeptide chain [7,8]. In all tissues, EGF appears to

A. Rab et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 96–99 97

have mitogenic activity [9]. Moreover, EGF also seems to play a rolein placental growth and in the regulation of physiological changesof placental function during intrauterine fetal development [9–13].

The physiologic activity of EGF is mediated through the EGFreceptor (EGFR) also known as erythroblastic leukemia viraloncogene homolog (ErbB-1). After binding to its receptor, EGF actsin the initiation of cell division. During human gestation thismechanism appears to be important primarily in promotingplacental growth [14–16]. Other members of the EGF proteinfamily may activate ErbB receptors 1–4 [17]. While ErbB receptors2–4 can be identified in both villous and extravillous trophoblasts,EGFR (ErbB-1) only occurs in villous trophoblasts [18,19].

Changes in EGFR receptor distribution have been observed inboth IUGR and other medical conditions associated with increasedrisk during pregnancy, such as smoking. Quantitative changes inEGFR distribution may be associated either with alterations in EGFsecretion or with changes in placental expression of the EGF gene[20,21].

Our primary objective in this study was to identify andcharacterize alterations in placental EGF gene expression patternsin IUGR pregnancies compared to normal pregnancies. Webelieved that clarifying these alterations would contribute to abetter understanding of the role played by EGF in placental growth.Our secondary aim was to identify gender-related alterations ofEGF gene expression in IUGR. We also investigated the relationshipbetween the degree of growth restriction in IUGR (fetal birth-weight 0–5 percentile vs. 5–10 percentile) and placental EGFexpression.

2. Materials and methods

We obtained 101 placental samples for characterization of EGFexpression from all patients treated for IUGR in our clinic at theSecond Department of Gynecology and Obstetrics, SemmelweisUniversity, Budapest, in the study period between January 1, 2010and January 1, 2011, as well as 140 placental samples from cases ofnormal pregnancy used as controls during the same time period.Maternal age, gestational weight gain and BMI increase duringpregnancy were also evaluated. IUGR was diagnosed per standardcriteria as fetal birthweight below the tenth percentile for fetal sexand gestational age. (In certain cases the fifth or 3rd percentile arealso used to diagnose IUGR, but in the majority of cases the tenthpercentile is the marked borderline.) We have taken intoconsideration those cases of IUGR in which the growth restrictionwas diagnosed prenatally by ultrasound, and the measurement ofthe birthweight confirmed the diagnosis postnatally. The IUGRgroup was subdivided into two groups by the degree of growthrestriction as below: less severe growth restriction defined as birthweight of 5–10 percentiles vs. more severe growth restriction (0–5percentile). Abdominal circumference (AC) determined throughultrasonography was also considered when establishing theclinical diagnosis of IUGR. Abdominal circumference values incases with a clinical diagnosis of IUGR were compared to caseswith similar gestational age in the normal pregnancy group.

Only those placentas were included in the study where IUGRwas likely to be due to placental dysfunction after the exclusion ofintrauterine infections, chromosomal abnormalities, fetal mal-formations, developmental disorders, maternal malnutrition,

Table 1Primers and sequences in real-time PCR.

Gene name and code Forward primer

EGF (NM_001963) 50-AATACCGTTAAGATACAGTGTAG

b-Actin (M10277) 50-GGCACCCAGCACAATGAAG-3

multiple pregnancy and structural abnormalities in the placenta[22,25]. We also excluded cases of IUGR caused by maternalpreeclampsia, because it would have strongly influenced thecorrect evaluation of the etiological role of EGF in the backgroundof intrauterine growth restriction.

Delivery was either vaginal or by cesarean section based onclinical decision. In the final analysis of data, no distinction wasmade with respect to the type of delivery.

Placental tissue samples were taken in a uniform manner withapproximate dimensions of 2 cm � 2 cm � 2 cm (8 cm3), whichwere then kept at �70 8C for genetic expression testing. Thesampling of each placenta was random, so all areas of eachplacenta had an equal chance of being sampled. (In 10 cases ofIUGR and 10 control cases the sampling of the placental tissue wasperformed from four different points of the placental tissue, andthe gene expression values did not differ.)

Maternal demographics and relevant clinical data duringpregnancy or the postnatal period were collected includingmaternal and paternal age, obstetric history, genetic history,general medical history, maternal birthweight, gestational age,fetal gender, weight gain and BMI increase during pregnancy,pregnancy-related pathology including disorders of carbohydratemetabolism, neonatal weight and Apgar score. Consent wasobtained in each case from the mother (signatures on file).

Whole placental RNA content was isolated with Quick RNAmicroprep kit (Zymo Research). RNA concentration was deter-mined using NanoDrop spectophotometer (NanoDrop). Reversetranscription was performed in 20 ml target volume using 5 mgwhole RNS, 75 pmol random hexamer primer, 10 mM dNTP(Invitrogen), 20 U M-MuLV Reverse Transcriptase enzyme (MBIFermentas) and 1�-es buffer (MBI Fermentas). The reaction mixwas incubated for 2 h at 42 8C. Subsequently, the enzyme wasinactivated at 70 8C for 15 min.

The reverse transcriptase reaction solution was diluted three-fold with nuclease-free water. For the real-time PCR assay, 1 mldiluted cDNS (approximately15 ng RNA-equivalent) and 1� SYBRGreen Master Mixet (Applied Biosystems) were used. Primers weredesigned using Primer Express Software (Applied Biosystems).Primer sequences are detailed in Table 1. Real-time PCR wasperformed in 20 ml target volume using 1 ml cDNA, 1 pmol, gene-specific Forward and Reverse primer and 1 x SYBR Green PCRMaster mix. All real-time PCR were performed using the MX3000Real-time PCR (Stratagen) system with the following settings: 40cycles at 95 8C, denaturing process for 15 s, annealing at 60 8C,chain elongation and detection for 60 s. For each gene, relativeexpression was normalized using the human b-actin gene asstandard.

For gene expression studies of the EGF gene in the IUGR vs.normal pregnancy groups two-sample t-test was used with 95%confidence interval. Determination of degree of freedom wasperformed using the Welch–Satterthwaite correction. Values ofgene expression testing were interpreted in the following manner:(1) overexpression = Ln value >1, p < 0.05; (2) underexpres-sion = Ln value <�1, p < 0.05; (3) normal expression = Ln value<1, >�1, p < 0.05. GraphPad Prism 3.0 (GraphPad Software Inc.)software was used in all statistical analytic procedures.

Demographics and clinical data were analyzed with SPSSsoftware. Logistic regression was used for dichotomous outcomes

Reverse primer

GCACTTTA-30 50-ATCACAACTCATTTTGGCAAAATC-30

50-GCCGATCCACACGGAGTACT-30

Table 5Maternal age distribution in the IUGR and normal pregnancy groups.

Age of mother (years)

17–24 25–31 32–34 35–44 Total

Normal pregnancy n 24 49 42 25 140

% 48.0% 66.2% 68.8% 44.6% 58.1%

IUGR pregnancy n 20 31 19 31 101

% 40.0% 41.9% 31.2% 55.4% 41.9%

Total n 50 74 61 56 241

Table 6The main clinical characteristics of the IUGR group and the control group.

IUGR group Control group

Maternal age (years) 30.82 � 4.34 31.45 � 3.12

Male:female ratio 0.58 1.09

Gestational age at delivery (week) 34.95 � 2.61 38.41 � 1.39

Way of delivery

Cesarean section 66.3% (67/101) 33.7% (34/101)

Per vias naturals 33.7% (57/140) 59.3% (83/140)

Weight gain of the patient

during the pregnancy (kg)

10.9 14.8

BMI increase of the patient

during the pregnancy

4.1 5.3

A. Rab et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 96–9998

with multiple independent variables. For continuous outcomes,analysis of variance (ANOVA) and linear regression were used asappropriate. p value of <0.05 was accepted for statisticalsignificance.

3. Results

A total of 101 placental samples were obtained for determina-tion of EGF expression in the IUGR group vs. 140 in the normalpregnancy group (Table 2). The EGF gene was underexpressed inIUGR compared to the control group (Ln2

a: �1.54; p < 0.04).

Within the IUGR group no fetal gender-dependent differencewas seen in placental EGF expression (Ln2

a: 0.44; p < 0.06)

(Table 3).There was no significant difference in placental EGF expression

between the more severe (0–5 percentile fetal birthweight) andless severe (5–10 percentile fetal birthweight) IUGR subgroups(Ln2

a: �0.08; p = 0.05) (Table 4).

No significant difference was found in the placental EGFexpression between the group of newborns delivered by cesareansection versus those delivered vaginally (Ln2

acesaraean section: 0.53;

Ln2a

vaginal delivery: 0.62; p > 0.05)Fetal gender distribution in the IUGR group was as follows: 37

males, 64 females, with a male to female ratio of 0.58; in thecontrol group it was 73 males and 67 females with male to femaleratio of 1.09, a significant difference (p < 0.05).

There was no significant difference between median values ofmaternal age in the IUGR (30.82 � 4.34 years) vs. the normalpregnancy group (31.45 � 3.12 years; p > 0.05).

When stratifying maternal age in the age period of 35–44 years,a significantly higher incidence of IUGR was found compared toother age groups (Table 5).

The median value of the gestational age at delivery was34.95 � 2.61 weeks in the group of IUGR, and 38.41 � 1.39 weeks inthe control group.

In the group of patients with IUGR in 67/101 cases (66.3%) thenewborn was delivered by cesarean section, while in the remaining

Table 2Gene expression patterns of placental EGF in IUGR vs. normal pregnancy (normal

pregnancy used as control).

Gene name a value � SE (a) Ln 2a

p Change in gene

expression

EGF �1.54 � 0.86 �1.06 0.06 Underexpression

nnormal pregnancy = 140; nIUGR = 101; a = DCtnormal pregnancy� DCtIUGR; control gene b-

actin.

Table 3Comparison of placental EGF gene expression in IUGR pregnancies with male vs.

female fetal gender (female fetal gender used as control).

Gene name a value � SE (a) Ln 2a

p Change in gene

expression

EGF 0.64 � 0.79 0.44 0.03 No change

nfemale = 64; nmale = 37; a = DCtfemale� DCtmale; control gene b-actin.

Table 4Comparison of placental EGF expression in IUGR pregnancies with more severe (0–5

percentile birthweight; B) vs. less severe (5–10 percentile birthweight; A) growth

restriction (less severe growth restriction used as control).

Gene name a value � SE (a) Ln 2a

p Change in gene

expression

EGF �0.12 � 0.65 �0.08 0.05 No change

a = DCtA� DCtB; DCtA = CEGF� Ctcontrol gene (5–10 percent of IUGR placental

samples); DCtB = CtEGF� Ctcontrol gene (0–5 percent of IUGR placental samples);

(nA = 61, nB = 40), Control gene: b-actin.

34/101 cases (33.7%) delivery was vaginal. Among the control casesthe distribution of the mode of delivery was different (cesareansection: 57/140; 40.7%; vaginal delivery: 83/140; 59.3%): thedifference proved to be significant (p < 0.05) (Table 6).

A statistically significant difference between the groups wasfound both in gestational weight gain (p < 0.05), and gestationalBMI increase (p < 0.05). Mean gestational weight gain was 10.9 kgin the IUGR group compared to 14.8 kg in the normal pregnancygroup. Mean gestational BMI increase was 4.1 in the IUGR group vs.5.3 in the normal pregnancy group.

4. Comments

Intrauterine growth restriction has long remained one of themajor challenges in obstetric practice, leading to increasedneonatal morbidity and mortality. Although the pathology givingrise to the development of IUGR is rather complex, placentaldysfunction can be identified as a major factor in the majority ofcases. Since placental dysfunction leading to IUGR is relativelycommon and its pathomechanism is still largely unsettled, thisgroup constitutes an ideal focus for studies aiming to clarify thepathological processes involved in the development of IUGR.

It is generally accepted that the physiological processes ofimplantation, intrauterine fetal and placental growth are a result ofa complex interplay of molecular and cellular level regulatoryinfluences [23]. A number of regulatory substances includinghormones, cytokines and growth factors are involved in thiscomplex regulatory system. Among growth factors, EGF appears tobe prominent in the implantation process, as well as thedevelopment of the placenta during gestation. Besides EGF,endometrium-derived VEGF and LIF also play a role in implanta-tion [24]. Growth factors secreted by cytotrophoblasts facilitateadhesion and proliferation of placental cells. Among these growthfactors, EGF seems to be crucial [24]. Previous reports also suggestthat EGF stimulates placental secretion of several hormones[13,16].

In our study population, IUGR developed as a result of placentaldysfunction. Although the precise mechanisms leading to placentaldysfunction could not be identified, it is generally assumedthat reduced placental size (which is very common in IUGR

A. Rab et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 170 (2013) 96–99 99

pregnancies) with respect to gestational age contributes to theslower intrauterine development of the fetus in such cases [25]. Ifthis is indeed the case, then the underexpression of placental EGFfound in our study could be interpreted as a potentially importantmechanism behind slower placental growth. This would suggestthat underexpression of placental EGF is one of the genetic levelmechanisms leading to the development of IUGR.

Previous studies in rabbits reported a stimulatory effect of EGFon intrauterine fetal development. EGF administered into theamniotic fluid appeared to accelerate the speed of development ofthe rabbit embryo [26,27].

We could not find a fetal gender-dependent difference in theexpression of EGF in IUGR pregnancies. This suggests that fetalgender does not influence the degree of underexpression ofplacental EGF in IUGR. This is in contrast to the IGF-2 gene, which isoverexpressed in the placenta in IUGR pregnancies with male fetalgender [4].

The fact that the degree of growth restriction in IUGR did notcorrelate with the degree of underexpression of placental EGFsuggests that factors other than EGF must be at play in more severeforms of IUGR.

In our study population, the most important clinical character-istics associated with IUGR were female fetal gender and amaternal age exceeding 35 years. Advanced maternal age has beenassociated with an increased risk of several other gestationaldisorders, including premature delivery and gestational diabetes[28]. The median gestational age at delivery in cases of intrauterinegrowth restriction proved to be less than 35 weeks, which is due tothe phenomenon that in cases of IUGR the danger of fetal asphyxiaas well as of intrauterine death increases. This may make theimmediate preventive termination of pregnancy necessary,independently from the gestational age. It is also noteworthythat IUGR pregnancies in our population appeared to be marked byreduced gestational weight gain and reduced gestational increasein BMI [4].

In summary, our present study suggests that placental EGF geneactivity is lower in IUGR pregnancies compared to normalpregnancy. We speculate that decreased expression of the EGFgene will eventually lead to slower placental development with asmall for gestational age placenta, which fails to keep up withphysiological fetal demands. This decreased gene activity ofplacental EGF does not seem to be fetal gender dependent. Thedegree of growth restriction in IUGR does not seem to correlatewith placental EGF expression. Regarding clinical characteristics,IUGR appears to be more common with maternal age exceeding 35years. Furthermore, decreased gestational weight gain and reducedgestational increase in BMI are also associated with increased riskof the development of intrauterine growth restriction.

Conflict of interest

The authors report no conflict of interest.

Acknowledgements

I would like to acknowledge the significant contribution of mycolleagues at the Semmelweis University in performing the study.

References

[1] Wollmann HA. Intrauterine growth restriction: definition and etiology. HormRes 1998;49(Suppl. 2):1–6.

[2] Sheikh S, Satoskar P, Bhartiya D. Expression of insulin-like growth factor-I andplacental growth hormone mRNA in placentae: a comparison between normal

and intrauterine growth retardation pregnancies. Mol Hum Reprod2001;7:287–92.

[3] Gluckman PD, Harding JE. The physiology and pathophysiology of intrauterinegrowth retardation. Horm Res 1997;48(Suppl. 1):11–6.

[4] Borzsonyi B, Demendi C, Nagy Z, et al. Gene expression patterns of insulin-likegrowth factor 1, insulin-like growth factor 2 and insulin-like growth factorbinding protein 3 in human placenta from pregnancies with intrauterinegrowth restriction. J Perinat Med 2011;39:701–7.

[5] Demendi C, Borzsonyi B, Nagy ZB, et al. Gene expression patterns of insulin-like growth factor 1, 2 (IGF-1, IGF-2) and insulin-like growth factor bindingprotein 3 (IGFBP-3) in human placenta from preterm deliveries: influence ofadditional factors. Eur J Obstet Gynecol Reprod Biol 2012;160:40–4.

[6] Hofmann GE, Drews M, Scott RT, et al. Epidermal growth factor and its receptorin human implantation trophoblast: immunohistochemical evidence for au-tocrine–paracrine function. J Clin Endocrinol Metab 1992;74:981–8.

[7] Cohen S. Isolation of a mouse submaxillary gland protein accelerating incisoreruption and eyelid opening in the newborn animal. J Biol Chem1962;237:1555–62.

[8] Scott J, Urgea M, Quiroga M, et al. Structure of a mouse submaxillary messen-ger RNA encoding epidermal growth factor and seven related proteins. Science1983;221:236–40.

[9] Casalini P, Iorio MV, Galmozzi E, et al. Role of HER receptors family indevelopment and differentiation. J Clin Physiol 2004;200:343–50.

[10] Morrish DW, Bhardwaj D, Dabbagh LK, et al. Epidermal growth factor inducesdifferentiation and secretion of human chorionic gonadotropin and placentallactogen in human placenta. J Clin Endocrin Metab 1987;65:1282–90.

[11] Maruo T, Matsuo H, Oishi M, et al. Induction of differentiated trophoblastfucntion by epidermal growth factor. Relation of immunohistochemicallydetected cellular epidermal growth factor receptor levels. J Clin EndocrinolMetab 1987;64:744–50.

[12] Maruo T, Matsuo H, Murata K. Gestational age-dependent dual action ofepidermal growth factor on human placenta early in gestation. J Clin Endo-crinol Metab 1992;75:1362–7.

[13] Barnea ER, Feldman D, Kaplan M, et al. The dual effect of epidermal growthfactor upon chorionic gonadotropin secretion by the first trimester placenta invitro. J Clin Endocrin Metab 1990;71:923–8.

[14] Normanno N, De Luca A, Bianco C, et al. Epidermal growth factor receptor(EGFR) signaling in cancer. Gene 2006;366:2–16.

[15] Prenzel N, Fischer OM, Streit S, et al. The epidermal growth factor receptorfamily as a central element for cellular signal transduction and diversification.Endocr Relat Cancer 2001;8:11–31.

[16] Fondacci C, Alsat E, Gabriel R, et al. Alterations of human placental epidermalgrowth factor receptor in intrauterine growth restriction. J Clin Invest1994;93:1149–55.

[17] Harris LK, Chung E, Coffey RJ. Adhesion molecules in human trophoblast – areview II. Extravillous trophoblast. Placenta 2009;30:299–304.

[18] Tuncer ZS, Vegh GL, Fulop V, et al. Expression of epidermal growth factorreceptor-related family products in gestational trophoblastic diseases andnormal placenta and its relationship with development of postmolar tumor.Gynecol Oncol 2000;77:389–93.

[19] Tanimura K, Nakago S, Murakoshi H, et al. Changes in the expression andcytological localization of beta-cellulin and its receptors (ErbB-1 and ErbB-4)in the trophoblasts in human placenta over the course of pregnancy. Eur JEndocrinol 2004;151:93–101.

[20] Wang SL, Lucier GW, Everson RB, et al. Smoking-related alterations in epider-mal growth factor and insulin receptors in human placenta. Mol Pharmacol1988;34:265–71.

[21] Fujita Y, Kurachi H, Morishige K, et al. Decrease in epidermal growth factorreceptor and its messenger ribonucleic acid levels in intrauterine growth-retarded and diabetes mellitus-complicated pregnancies. J Clin EndocrinMetab 1991;72:1340–5.

[22] (a) Lee JJ. Birth weight for gestational age patterns by sex, plurality and parityin Korean populations. Korean J Pediatr 2007;50:732–9;(b) Singh M, Chaudry P, Asselin E, et al. Bridging endometrial receptivity andimplantation: network of hormones, cytokines and growth factors. J Endo-crinol 2011;210:5–14.

[23] Knofler M. Critical growth factors and signalling pathways controlling humantrophoblast invasion. Int J Dev Biol 2010;54:269–80.

[24] Calvo MT, Romo A, Gutierrez JJ, et al. Study of genetic expression of intrauter-ine growth factors IGF-I and EGFR in placental tissue from pregnancies withintrauterine growth retardation. J Pediatr Endocrin Metab 2004;17(Suppl.3):445–50.

[25] Cellini C, Xu J, Buchmiller-Crair T. Effect of epidermal growth factor on smallintestinal sodium/glucose cotransporter expression in a rabbit model ofintrauterine growth retardation. J Pediatr Surg 2005;40:1892–7.

[26] Buchmiller T, Shaw K, Choupourian H, et al. Effect of transamnionic adminis-tration of epidermal growth factor on fetal rabbit small intestinal nutrienttransport and dissacharidase development. J Pediatr Surg 1993;28:1239–44.

[27] Cunningham FG, Leveno KJ. Childbearing among older women – the message iscautiously optimistic. N Engl J Med 1995;333:1002–4.

[28] Hytten FE, Thomson AM. Maternal physiological adjustments. In: Assali NS,editor. Biology of Gestation. Vol. I. The maternal organism. New York: Aca-demic Press; 1968.