Placental gene expression patterns of epidermal growth factor in intrauterine growth restriction

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

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Placental gene expression patterns of epidermal growth factor in intrauterine growth restriction Attila Rab a, Imre Szentpe´teri b, La´szlo´ Kornya c, Bala´zs Bo¨rzso¨nyi d, Csaba Demendi d, Jo´zsef Ga´bor Joo´ e,* a

Telki Hospital, Budapest, Hungary Praxis fu¨r Gyna¨kologie und Geburtshilfe und allgemeine Medizin, Wehingen, Baden-Wu¨rttemberg, Germany St. Stephen’s Hospital, Budapest, Hungary d Second Department of Obstetrics and Gynecology, Semmelweis University, Budapest, Hungary e First Department of Obstetrics and Gynecology, Semmelweis University, Budapest, Hungary b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 December 2012 Received in revised form 1 April 2013 Accepted 27 May 2013

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 a pregnancy (Ln2 : 1.54; p < 0.04). Within the IUGR group no fetal gender-dependent difference was a seen in EGF gene expression (Ln2 : 0.44; p < 0.06). Similarly, no significant difference in EGF expression a was noted in cases with more vs. less severe forms of IUGR (Ln2 : 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. ß 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: Gene expression Placenta Epidermal growth factor Intrauterine growth restriction Body mass index Maternal age

1. Introduction Intrauterine growth restriction (IUGR) is defined as fetal birthweight 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, erythroblastic 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: [email protected] (J.G. Joo´). 0301-2115/$ – see front matter ß 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 third percentiles 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 most common 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 multiple growth factors rise. Among these, insulin-like growth factor 1 and 2 (IGF-1, IGF-2) appear to be especially important in the pathogenesis of both IUGR and premature delivery [4,5]. Epidermal growth factor (EGF) has been found to play a role in stimulating placental growth [6]. Structurally, human EGF is a polypeptide consisting of 53 amino acids. Its precursor is substantially larger, consisting of a 1207 amino 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

have mitogenic activity [9]. Moreover, EGF also seems to play a role in placental growth and in the regulation of physiological changes of placental function during intrauterine fetal development [9–13]. The physiologic activity of EGF is mediated through the EGF receptor (EGFR) also known as erythroblastic leukemia viral oncogene homolog (ErbB-1). After binding to its receptor, EGF acts in the initiation of cell division. During human gestation this mechanism appears to be important primarily in promoting placental growth [14–16]. Other members of the EGF protein family may activate ErbB receptors 1–4 [17]. While ErbB receptors 2–4 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 in both IUGR and other medical conditions associated with increased risk during pregnancy, such as smoking. Quantitative changes in EGFR distribution may be associated either with alterations in EGF secretion or with changes in placental expression of the EGF gene [20,21]. Our primary objective in this study was to identify and characterize alterations in placental EGF gene expression patterns in IUGR pregnancies compared to normal pregnancies. We believed that clarifying these alterations would contribute to a better understanding of the role played by EGF in placental growth. Our secondary aim was to identify gender-related alterations of EGF gene expression in IUGR. We also investigated the relationship between the degree of growth restriction in IUGR (fetal birthweight 0–5 percentile vs. 5–10 percentile) and placental EGF expression. 2. Materials and methods We obtained 101 placental samples for characterization of EGF expression from all patients treated for IUGR in our clinic at the Second Department of Gynecology and Obstetrics, Semmelweis University, Budapest, in the study period between January 1, 2010 and January 1, 2011, as well as 140 placental samples from cases of normal pregnancy used as controls during the same time period. Maternal age, gestational weight gain and BMI increase during pregnancy were also evaluated. IUGR was diagnosed per standard criteria as fetal birthweight below the tenth percentile for fetal sex and gestational age. (In certain cases the fifth or 3rd percentile are also used to diagnose IUGR, but in the majority of cases the tenth percentile is the marked borderline.) We have taken into consideration those cases of IUGR in which the growth restriction was diagnosed prenatally by ultrasound, and the measurement of the birthweight confirmed the diagnosis postnatally. The IUGR group was subdivided into two groups by the degree of growth restriction as below: less severe growth restriction defined as birth weight of 5–10 percentiles vs. more severe growth restriction (0–5 percentile). Abdominal circumference (AC) determined through ultrasonography was also considered when establishing the clinical diagnosis of IUGR. Abdominal circumference values in cases with a clinical diagnosis of IUGR were compared to cases with similar gestational age in the normal pregnancy group. Only those placentas were included in the study where IUGR was likely to be due to placental dysfunction after the exclusion of intrauterine infections, chromosomal abnormalities, fetal malformations, developmental disorders, maternal malnutrition,

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multiple pregnancy and structural abnormalities in the placenta [22,25]. We also excluded cases of IUGR caused by maternal preeclampsia, because it would have strongly influenced the correct evaluation of the etiological role of EGF in the background of intrauterine growth restriction. Delivery was either vaginal or by cesarean section based on clinical decision. In the final analysis of data, no distinction was made with respect to the type of delivery. Placental tissue samples were taken in a uniform manner with approximate dimensions of 2 cm  2 cm  2 cm (8 cm3), which were then kept at 70 8C for genetic expression testing. The sampling of each placenta was random, so all areas of each placenta had an equal chance of being sampled. (In 10 cases of IUGR and 10 control cases the sampling of the placental tissue was performed from four different points of the placental tissue, and the gene expression values did not differ.) Maternal demographics and relevant clinical data during pregnancy or the postnatal period were collected including maternal 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 carbohydrate metabolism, neonatal weight and Apgar score. Consent was obtained in each case from the mother (signatures on file). Whole placental RNA content was isolated with Quick RNA microprep kit (Zymo Research). RNA concentration was determined using NanoDrop spectophotometer (NanoDrop). Reverse transcription was performed in 20 ml target volume using 5 mg whole RNS, 75 pmol random hexamer primer, 10 mM dNTP (Invitrogen), 20 U M-MuLV Reverse Transcriptase enzyme (MBI Fermentas) and 1-es buffer (MBI Fermentas). The reaction mix was incubated for 2 h at 42 8C. Subsequently, the enzyme was inactivated at 70 8C for 15 min. The reverse transcriptase reaction solution was diluted threefold with nuclease-free water. For the real-time PCR assay, 1 ml diluted cDNS (approximately15 ng RNA-equivalent) and 1 SYBR Green Master Mixet (Applied Biosystems) were used. Primers were designed using Primer Express Software (Applied Biosystems). Primer sequences are detailed in Table 1. Real-time PCR was performed in 20 ml target volume using 1 ml cDNA, 1 pmol, genespecific Forward and Reverse primer and 1 x SYBR Green PCR Master mix. All real-time PCR were performed using the MX3000 Real-time PCR (Stratagen) system with the following settings: 40 cycles at 95 8C, denaturing process for 15 s, annealing at 60 8C, chain elongation and detection for 60 s. For each gene, relative expression was normalized using the human b-actin gene as standard. 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 was performed using the Welch–Satterthwaite correction. Values of gene expression testing were interpreted in the following manner: (1) overexpression = Ln value >1, p < 0.05; (2) underexpression = 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 SPSS software. Logistic regression was used for dichotomous outcomes

Table 1 Primers and sequences in real-time PCR. Gene name and code

Forward primer

Reverse primer

EGF (NM_001963) b-Actin (M10277)

50 -AATACCGTTAAGATACAGTGTAGGCACTTTA-30 50 -GGCACCCAGCACAATGAAG-3

50 -ATCACAACTCATTTTGGCAAAATC-30 50 -GCCGATCCACACGGAGTACT-30

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

Table 5 Maternal age distribution in the IUGR and normal pregnancy groups. Age of mother (years)

Normal pregnancy

3. Results

IUGR pregnancy

A total of 101 placental samples were obtained for determination of EGF expression in the IUGR group vs. 140 in the normal pregnancy group (Table 2). The EGF gene was underexpressed in a IUGR compared to the control group (Ln2 : 1.54; p < 0.04). Within the IUGR group no fetal gender-dependent difference a was seen in placental EGF expression (Ln2 : 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) and less severe (5–10 percentile fetal birthweight) IUGR subgroups a (Ln2 : 0.08; p = 0.05) (Table 4). No significant difference was found in the placental EGF expression between the group of newborns delivered by cesarean a section versus those delivered vaginally (Ln2 cesaraean section: 0.53; a Ln2 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 the control group it was 73 males and 67 females with male to female ratio of 1.09, a significant difference (p < 0.05). There was no significant difference between median values of maternal age in the IUGR (30.82  4.34 years) vs. the normal pregnancy 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 to other age groups (Table 5). The median value of the gestational age at delivery was 34.95  2.61 weeks in the group of IUGR, and 38.41  1.39 weeks in the control group. In the group of patients with IUGR in 67/101 cases (66.3%) the newborn was delivered by cesarean section, while in the remaining

Total

17–24

25–31

32–34

35–44

Total

n % n %

24 48.0% 20 40.0%

49 66.2% 31 41.9%

42 68.8% 19 31.2%

25 44.6% 31 55.4%

140 58.1% 101 41.9%

n

50

74

61

56

241

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

Maternal age (years) Male:female ratio Gestational age at delivery (week) Way of delivery Cesarean section Per vias naturals Weight gain of the patient during the pregnancy (kg) BMI increase of the patient during the pregnancy

IUGR group

Control group

30.82  4.34 0.58 34.95  2.61

31.45  3.12 1.09 38.41  1.39

66.3% (67/101) 33.7% (57/140) 10.9

33.7% (34/101) 59.3% (83/140) 14.8

4.1

5.3

34/101 cases (33.7%) delivery was vaginal. Among the control cases the distribution of the mode of delivery was different (cesarean section: 57/140; 40.7%; vaginal delivery: 83/140; 59.3%): the difference proved to be significant (p < 0.05) (Table 6). A statistically significant difference between the groups was found both in gestational weight gain (p < 0.05), and gestational BMI increase (p < 0.05). Mean gestational weight gain was 10.9 kg in the IUGR group compared to 14.8 kg in the normal pregnancy group. Mean gestational BMI increase was 4.1 in the IUGR group vs. 5.3 in the normal pregnancy group. 4. Comments

Table 2 Gene expression patterns of placental EGF in IUGR vs. normal pregnancy (normal pregnancy used as control). a

Gene name

a value  SE (a)

Ln 2

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 bactin. Table 3 Comparison of placental EGF gene expression in IUGR pregnancies with male vs. female fetal gender (female fetal gender used as control). a

Gene name

a value  SE (a)

Ln 2

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 4 Comparison 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). a

Gene name

a value  SE (a)

Ln 2

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.

Intrauterine growth restriction has long remained one of the major challenges in obstetric practice, leading to increased neonatal morbidity and mortality. Although the pathology giving rise to the development of IUGR is rather complex, placental dysfunction can be identified as a major factor in the majority of cases. Since placental dysfunction leading to IUGR is relatively common and its pathomechanism is still largely unsettled, this group constitutes an ideal focus for studies aiming to clarify the pathological processes involved in the development of IUGR. It is generally accepted that the physiological processes of implantation, intrauterine fetal and placental growth are a result of a complex interplay of molecular and cellular level regulatory influences [23]. A number of regulatory substances including hormones, cytokines and growth factors are involved in this complex regulatory system. Among growth factors, EGF appears to be prominent in the implantation process, as well as the development of the placenta during gestation. Besides EGF, endometrium-derived VEGF and LIF also play a role in implantation [24]. Growth factors secreted by cytotrophoblasts facilitate adhesion and proliferation of placental cells. Among these growth factors, EGF seems to be crucial [24]. Previous reports also suggest that EGF stimulates placental secretion of several hormones [13,16]. In our study population, IUGR developed as a result of placental dysfunction. Although the precise mechanisms leading to placental dysfunction could not be identified, it is generally assumed that 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

pregnancies) with respect to gestational age contributes to the slower intrauterine development of the fetus in such cases [25]. If this is indeed the case, then the underexpression of placental EGF found in our study could be interpreted as a potentially important mechanism behind slower placental growth. This would suggest that underexpression of placental EGF is one of the genetic level mechanisms leading to the development of IUGR. Previous studies in rabbits reported a stimulatory effect of EGF on intrauterine fetal development. EGF administered into the amniotic fluid appeared to accelerate the speed of development of the rabbit embryo [26,27]. We could not find a fetal gender-dependent difference in the expression of EGF in IUGR pregnancies. This suggests that fetal gender does not influence the degree of underexpression of placental EGF in IUGR. This is in contrast to the IGF-2 gene, which is overexpressed in the placenta in IUGR pregnancies with male fetal gender [4]. The fact that the degree of growth restriction in IUGR did not correlate with the degree of underexpression of placental EGF suggests that factors other than EGF must be at play in more severe forms of IUGR. In our study population, the most important clinical characteristics associated with IUGR were female fetal gender and a maternal age exceeding 35 years. Advanced maternal age has been associated with an increased risk of several other gestational disorders, including premature delivery and gestational diabetes [28]. The median gestational age at delivery in cases of intrauterine growth restriction proved to be less than 35 weeks, which is due to the phenomenon that in cases of IUGR the danger of fetal asphyxia as well as of intrauterine death increases. This may make the immediate preventive termination of pregnancy necessary, independently from the gestational age. It is also noteworthy that IUGR pregnancies in our population appeared to be marked by reduced gestational weight gain and reduced gestational increase in BMI [4]. In summary, our present study suggests that placental EGF gene activity is lower in IUGR pregnancies compared to normal pregnancy. We speculate that decreased expression of the EGF gene will eventually lead to slower placental development with a small for gestational age placenta, which fails to keep up with physiological fetal demands. This decreased gene activity of placental EGF does not seem to be fetal gender dependent. The degree of growth restriction in IUGR does not seem to correlate with placental EGF expression. Regarding clinical characteristics, IUGR appears to be more common with maternal age exceeding 35 years. Furthermore, decreased gestational weight gain and reduced gestational increase in BMI are also associated with increased risk of the development of intrauterine growth restriction.

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Conflict of interest [23]

The authors report no conflict of interest.

[24]

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

[25]

[26]

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