Placenta
Volume 31, Issue 3 , Pages 178-185, March 2010

Differences in gene expression dependent on sampling site in placental tissue of fetuses with intrauterine growth restriction

  • A.A. Tzschoppe

      Affiliations

    • Department of Pediatrics and Adolescent Medicine, University of Erlangen-Nuremberg, Loschgestr. 15, D-91054 Erlangen, Germany
    • ,
  • E. Struwe

      Affiliations

    • Department of Pediatrics and Adolescent Medicine, University of Erlangen-Nuremberg, Loschgestr. 15, D-91054 Erlangen, Germany
    • ,
  • H.G. Dörr

      Affiliations

    • Department of Pediatrics and Adolescent Medicine, University of Erlangen-Nuremberg, Loschgestr. 15, D-91054 Erlangen, Germany
    • ,
  • T.W. Goecke

      Affiliations

    • Department of Obstetrics & Gynaecology, University of Erlangen-Nuremberg, Germany
    • ,
  • M.W. Beckmann

      Affiliations

    • Department of Obstetrics & Gynaecology, University of Erlangen-Nuremberg, Germany
    • ,
  • R.L. Schild

      Affiliations

    • Department of Obstetrics & Gynaecology, Diakonische Dienste Hannover, Hannover, Germany
    • ,
  • J. Dötsch

      Affiliations

    • Department of Pediatrics and Adolescent Medicine, University of Erlangen-Nuremberg, Loschgestr. 15, D-91054 Erlangen, Germany
    • Corresponding Author InformationCorresponding author. Tel.: +49 9131 85 33117; fax: +49 9131 85 33706.

Accepted 3 December 2009. published online 04 January 2010.

Article Outline

Abstract 

Objective

The human placenta as part of the feto-placental unit may influence fetal endocrine systems and may therefore represent a very important link between intrauterine growth restriction (IUGR) and metabolic disorders in later life. We aimed to analyze the effect of sample origin on gene expression of placental factors potentially involved in fetal programming in IUGR versus appropriate for gestational age growth (AGA) to standardize sample collection procedure for a multicenter approach.

Design

Placental gene expression of insulin-like growth factor-binding protein (IGFBP)-1, prolactin, corticotropin releasing hormone (CRH) and leptin was measured and compared between proximal, intermediate and peripheral region of the placenta in 22 IUGR (proven by anomalous placental Doppler velocimetry) and 19 AGA neonates.

Results

Whereas no difference in gene expression was seen in the proximal portion, in the intermediate placental region mRNA expression of IGFBP-1 (p = 0.01), prolactin (p = 0.04), CRH (p = 0.01) and leptin (p = 0.04) was increased in IUGR samples compared to controls. At the placental periphery, gene expression of these placental transcripts showed a higher expression level in IUGR placentas without statistical significance, except for leptin (p = 0.03).

Conclusion

Placental sampling site seems to be relevant for detecting differences in gene expression between IUGR and AGA neonates.

Keywords: Corticotropin releasing hormone, Insulin-like growth factor-binding protein-1, Intrauterine, Growth restriction, Leptin, Prolactin

 

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1. Introduction 

Intrauterine growth restriction (IUGR) is not only associated with an increased risk for perinatal morbidity and mortality, but can also lead to the development of the metabolic syndrome later in life including obesity, diabetes type 2, hypertension, and coronary artery disease [1], [2]. Among the various causes of IUGR, placental dysfunction associated with poor placental perfusion and hypoxia is one factor for ‘idiopathic’ IUGR [3]. Metabolic alterations in the fetal milieu change the early programming process [4], which may influence the regulation of endocrine function later in life. In this respect the central role of the placenta is well recognized and may therefore represent a very important link between IUGR and metabolic disorders in later life.

Elevated placental gene expression and cord blood concentrations associated with IUGR have been described for different placental endocrine regulators, e.g. insulin-like growth factor-binding protein-1 (IGFBP-1) [5], [6], [7], corticotropin releasing hormone (CRH) [8], [9], [10], leptin [11], [12] and prolactin [13].

When comparing gene expression of specific placental transcripts, it is important to consider the sampling site and control for placental inhomogeneity, as placental architecture and blood flow are not uniform across the chorioallantoic human placental disk [14], [15]. It has been shown that the expression of hypoxia-related transcripts is dependent on the sampling site and reflects the pattern of maternal arterial blood flow in the human placenta [16]. Therefore, differences in the sampling site may contribute to variability in gene expression across the placental disk. IUGR placentas represent diseased tissue and show characteristic hypoxic/ischemic changes, including increased syncytial knots, infarction, or hypercapillarization [17], [18], which might result in more dramatic changes in gene expression levels.

We established a prospective multicenter study (FIPS-study) to identify placental genes with predictive value for the development of obesity and metabolic disorders after intrauterine growth restriction based on the hypothesis of an alteration in fetal programming. As we define IUGR by anomalous placental Doppler velocimetry, all of the IUGR pregnancies in this study experience placental insufficiency. In annual follow-up examinations, clinical and biochemical characteristics of the enrolled IUGR and AGA infants especially with regard to childhood obesity, growth failure and hypertension are monitored until the age of 6 years. The ultimate goal is to prevent childhood morbidity in neonates after IUGR.

In view of this multicenter approach, we tried to establish a feasible concept of sample collection for all participants. To provide a basis for reliable investigations, we compared placental gene expression of factors potentially involved in fetal programming between IUGR and AGA newborns at different sampling sites.

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2. Methods 

2.1. Patients 

Placental tissue was collected from 22 mothers with IUGR neonates (10 males, 12 females) and 19 healthy women with AGA newborns (10 males, 9 females) born prematurely or at term. Inclusion and exclusion criteria are presented in Table 1. All children participated in the FIPS-study and had regular follow-up examinations after birth, therefore strict exclusion criteria comprising any condition affecting postnatal growth were applied according to the study protocol. The clinical characteristics of the 2 groups at birth are shown in Table 2. For the purpose of the study, percentiles of Voigt et al. for German newborns were used [19]. Birth weight Z-score was calculated using the following formula: birth weight Z-score = (neonates' birth weight – mean birth weight)/standard deviation. Gestational age was determined from the date of the last menstrual period of the mother and was confirmed by ultrasound measurements before the 14th week of gestation. Prematurity was defined as a gestational age <37 weeks.

Table 1. Inclusion and exclusion criteria for the study.
Inclusion criteria for IUGR

•Gestational age ≥30+0 and <41+0 weeks

•Birth weight <10th percentile [19]

•Ultrasound examination within the first trimester of gestation to confirm gestational age and to evaluate fetal morphology

•Proven placental insufficiency [31]:

elevated pulsatility index (PI) in at least one uterine artery (>1,2) with or without early diastolic notches

and elevated pulsatility index (PI) in the umbilical arteries (>2 SD) or absent/reverse diastolic flow

•Informed consent of parent

Inclusion criteria for AGA

•Gestational age ≥30+0 and <41+0 weeks

•Birth weight >25th and <75th percentile [19]

•All pregnancies were dated correctly by ultrasound during the first trimester of gestation

•Normal uterine and umbilical artery Doppler velocimetry during pregnancy [32]

•Informed consent of parent

Exclusion criteria

•Multiple (twins/triplets) pregnancies

•Chromosomal aberration/syndrome

•Congenital infection/TORCH

•Primary sepsis evidenced by clinical symptoms or biochemical signs (immature-to-total neutrophil ratio (IT ratio)>0,25, CRP>10mg/l or positive blood cultures)

•Preeclampsia

•Fetal alcohol syndrome or maternal drug ingestion

•Other congenital or acquired disease leading to postnatal growth restriction (severe cardiac malformation, bronchopulmonary dysplasia, intracranial hemorrhage >II°, hydrocephaly, short bowel syndrome or other severe malformation)

•Large for gestational age (LGA)-newborns>75. percentile [19]

Table 2. Birth characteristics of IUGR (n=22) and AGA (n=19) neonates.
IUGRAGA
Maternal age (years)28.1±1.3(16–38)31.0±1.3(20–38), NS
Gestational age (weeks)34.5±0.6(30–39)36.0±0.6(30–39), NS
Birth weight1556±118(770–2700)2722±138(1420–3570)†
Birth weight Z-score−2.3±0.2(−3.9–1.0)−0.4±0.1(−1.1–0.2)†
Male/female10/1210/9, NS
Spontaneous delivery/Section‡1/219/10*
Maternal smoking‡22, NS
Placental weight (g)367±28(190–660)470±32(320–780)*

Values are shown as mean±SEM (range). Significant differences between the IUGR and AGA group are marked (unpaired t-test, ‡Fishers's exact test).

NS, not significant.

*p<0.05.

†p<0.0001.

As a subgroup, we furthermore analyzed placental tissue of 10 SGA neonates (5 males, 5 females). SGA was defined by a birth weight Z-score <10th percentile and normal uterine and umbilical artery Doppler velocimetry during pregnancy. Exclusion criteria were the same as for IUGR and AGA newborns (Table 1). Birth weight Z-score ranged from a minimum of −2.5 to a maximum of −1.4 with a mean of −2.0. Mean gestational age was 39.0 weeks (range 36–41). Caesarean section was performed in 4 cases, 6 neonates were delivered spontaneously. Mean placental weight was 442g (range 315–690g).

The study was reviewed and approved by the ethics committee of the University of Erlangen–Nuremberg. It was explained to each parent who signed a written consent.

2.2. Placental tissue acquisition 

Fresh samples of human placentas derived from 22 IUGR to 19 AGA infants were obtained within 30 min after placental delivery in collaboration with the Department of Obstetrics and Gynecology at the University of Erlangen-Nuremberg. Using a sterile scalpel, we excised three quadrangular segments (approximately 2 × 2 cm) along the placental thickness from basal towards chorionic surface, which were localized at specific portions of the placenta. To establish a feasible standardized concept of sample collection even for cases with lateral insertion of the cord or very irregularly shaped placenta, we defined sample location by the percentage of distance between umbilical cord insertion site (0%) and placental margin (100%). Thus, probes taken from an area of up to 10% were called proximal, the sample location at 30–50% was designated as intermediate and at 60–80% as peripheral.

After a rinse of the samples with normal saline, the amniotic membranes and the maternal decidua were removed, then the samples were snap frozen in liquid nitrogen and stored at −80 °C until further processing.

2.3. RNA extraction and reverse transcription 

Total cellular RNA was extracted from the placental tissue by TRIzol® reagent (TRIzol®, Invitrogen GmbH, Karlsruhe, Germany). RNA concentrations were determined spectrophotometrically. One μg of RNA was reversely transcribed in a volume of 20 μl at 37 °C for 60 min to synthesize cDNA (chemicals from Boehringer Mannheim, Germany).

2.4. TaqMan real-time PCR 

TaqMan real-time PCR (Perkin–Elmer, Foster City, CA) was used to quantify the expression of genes in placenta of IUGR and AGA newborns. The mRNA expression was normalized to 2 different housekeeping genes, hypoxanthine guanine phosphoribosyl transferase (HPRT) and β2-microglobulin (β2-MG), that have been shown not to respond to hypoxia and that can be detected pseudogene free. The method is established and validated in our group and has been applied successfully for the quantification of gene expression in placental biopsies [20], [21].

Commercial reagents (TaqMan PCR reagent kit, Perkin–Elmer) and conditions were applied according to the manufacturer's protocol. 2.5 μl of complementary DNA (reverse transcription mixture) and oligonucleotides at a final concentration of 300 nmol/L (HPRT, IGFBP-1 forward) or 600 nM (β2-MG, prolactin, leptin, IGFBP-1 reverse, CRH forward) or 900 nM (CRH reverse) of primers and 200 nmol/L of TaqMan hybridization probe were analyzed in a 25 μl-volume. All of the primers and probes were purchased from Eurogentec (Belgium) and Sigma (Germany). The thermocycler parameters were 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C and 60 °C for 1 min. Serial dilutions of 1 of the samples served as reference providing relative quantification of the unknown samples.

The following primers and TaqMan probes were used. HPRT: forward 5′-CCGGCTCCGTTATGGC-3′, reverse 5′-GGTCATAACCTGGTTCATCATCA-3′, TaqMan probe 5′(FAM)-CGCAGCCCTGGCGTCGTGATTA-(TAMRA)3′; β2-MG: forward 5′-TGACTTTGTCACAGCCCAAGATA-3′, reverse 5′-CCAAATGCGGCATCTTC-3′, TaqMan probe 5′(FAM)-TGATGCTGCTTACATGTCTCGATCCCA-(TAMRA)3′; IGFBP-1: forward 5′-CTCTCCATGTCACCAACATCAAA-3′, reverse 5′-GTGCCTTGGCTAAACTCTCTACGA-3′, TaqMan probe 5′(FAM)-AATGGAAGGAGCCCTGCCGAATAGAACTC-(TAMRA)3′; Leptin: forward 5′-ACAATTGTCACCAGGATCAATGAC-3′, reverse 5′-TCCAAACCGGTGACTTTCTGT-3′, TaqMan probe 5′(FAM)-TTTCACACACGCAGTCAGTCTCCTCCA-(TAMRA)3′; CRH: forward 5′-CAG CCA GTG TCA GGA CCT CAC CAC G-3′, reverse 5′-CGG CAG CCG CAT GTT AG-3′, TaqMan probe 5′(FAM)-AAA GGA GAC AAT TTG GCT CTG C-(TAMRA)3′; Prolactin: forward 5′-AAGCTGTAGAGATTGAGGAGCAAAC-3′, reverse 5′-CCAGACAGGGTAGATCTCATTTTCTT-3′, TaqMan probe 5′(FAM)-TTTCAGGATGAACCTGGCTGACTATCAGCTC-(TAMRA)3′.

2.5. Statistical analysis 

Results were expressed as mean ± Standard Error of the Mean (SEM) and median, minimum, maximum, where appropriate. Statistical analyses were performed using GraphPad Prism® Version 4.0c for Windows, GraphPad Software, San Diego, CA (www.graphpad.com). After testing for Gaussian distribution, the data were analyzed using the unpaired parametric (Student's t-test) and non-parametric tests (Mann–Whitney test). The correlation analysis was performed using Pearson's or Spearman's linear regression analyses as required. Fisher's exact test was used for the analysis of categorical data. The limit of significance was set at a p value of <0.05.

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3. Results 

Gene expression of specific placental factors was compared between IUGR and AGA infants. Hereby, samples were taken from the proximal, intermediate and peripheral placental portion. These three samples were analyzed in parallel to control for placental inhomogeneity with respect to the potential gradient of oxygenation from placental central region to lateral margin.

Considering the proximal placental portion, there was no difference in the relative mRNA expression of IGFBP-1 (p = 0.09), prolactin (p = 24), CRH (p = 0.90) and leptin (p = 0.50) between IUGR and AGA placentas. The direct comparison of gene expression between males and females did not reveal any significant differences for IGFBP-1 (IUGR: p = 0.77, AGA: p = 0.44), prolactin (IUGR: p = 0.69, AGA: p = 0.55), CRH (IUGR: p = 0.19, AGA: p = 0.11), or for leptin mRNA expression (IUGR: p = 0.43, AGA: p = 0.17). In the AGA group, where 9 from 19 children were born by spontaneous delivery, there was no difference in IGFBP-1 (p = 0.44), prolactin (p = 0.57), CRH (p = 0.78) and leptin gene expression (p = 0.28) between spontaneous and caesarean section delivery.

In the placental intermediate region, the relative expression of IGFBP-1, prolactin, CRH and leptin was significantly higher in IUGR compared to control infants (Fig. 1). Comparing gene expression between male and female IUGR patients, there was no difference neither for IGFBP-1 (p = 0.74), nor for prolactin (p = 0.87), CRH (p = 0.64) or leptin mRNA expression (p = 0.81), indicating that gender did not influence these findings. In addition, placental gene expression of IGFBP-1 (p = 0.39), prolactin (p = 0.16), CRH (p = 0.06) and leptin (p = 0.21) did not differ significantly between male and female AGA neonates. In the AGA group, no difference was found in IGFBP-1 (p=0.36), prolactin (p=0.46), CRH (p=0.90) and leptin gene expression (p=0.36) between spontaneous and caesarean section delivery.

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  • Fig. 1 

    Relative gene expression in samples taken from different areas of IUGR and AGA placentas. Only in placental intermediate samples, there is a significant difference between IUGR (n=22) and AGA (n=19) infants in IGFBP-1 (p=0.01, Mann–Whitney test), prolactin (p=0.04, Mann–Whitney test), CRH (p=0.01, unpaired t-test) and leptin (p=0.04, Mann–Whitney test) mRNA expression Except for leptin (p=0.03, unpaired t-test), no significant difference was found for IGFBP-1 (p=0.56, Mann–Whitney test), prolactin (p=0.62, Mann–Whitney test) and CRH (p=0.10, Mann–Whitney test) mRNA expression, when placental peripheral samples were analyzed. Considering the proximal placental portion, there is no difference in the relative mRNA expression of IGFBP-1 (p=0.09, unpaired t-test), prolactin (p=24, unpaired t-test), CRH (p=0.90, unpaired t-test) and leptin (p=0.50, Mann–Whitney test) between IUGR and AGA placentas. Comparing the relative expression between different sampling sites, there is a non-significant trend towards higher gene expression levels at the placental periphery compared to the intermediate portion of IUGR placentas (IGFBP-1: p=0.92, Mann–Whitney test; prolactin: p=0.99, Mann–Whitney test; CRH: p=0.21, Mann–Whitney test; leptin: p=0.13, unpaired t-test).In the proximal placental part, gene expression is significantly different for IGFBP-1 (highest expression, compared to the intermediate (p=0.001, Mann–Whitney test) and peripheral area (p=0.005, Mann–Whitney test)) and for leptin (lowest expression, compared to the intermediate (p=0.03, unpaired t-test) and peripheral area (p=0.03, unpaired t-test)). In AGA placentas; mRNA expression of IGFBP-1 (p=0.47, Mann–Whitney test), prolactin (p=0.41, Mann–Whitney test), CRH (p=0.80, unpaired t-test), and leptin (p=0.75, Mann–Whitney test) is increased in samples taken from the peripheral area compared to the intermediate portion of the placenta without reaching statistical significance. Gene expression of IGFBP-1 is highest in the proximal placental area (versus intermediate area: p<0.0001, Mann–Whitney test). Leptin mRNA is significantly lower in the proximal compared to the intermediate (p=0.003, Mann–Whitney test) and peripheral (p=0.01, Mann–Whitney test) placental site, like in IUGR placentas. Gene expression is related to the housekeeping gene hypoxanthine guanine phosphoribosyl transferase (HPRT). The lines show the mean±SEM. *significant.

Analysis of gene expression of these placental factors in samples derived from the placental periphery revealed only a significant increment in leptin gene expression in IUGR infants. The relative mRNA expression of IGFBP-1, CRH and prolactin was not significantly different between the IUGR and AGA group, but showed a trend towards an increased gene expression in IUGR children (Fig. 2). A direct comparison of gene expression between male and female IUGR patients did not demonstrate any significant differences for IGFBP-1 (p=0.25), prolactin (p=0.25), CRH (p=0.60) or for leptin mRNA expression (p=0.25). Also, in the AGA group there was no difference in gene expression of IGFBP-1 (p=0.60), prolactin (p=0.58), CRH (p=0.61) or leptin (p=0.64) between male and female neonates. Considering delivery mode, we found no significant differences in IGFBP-1 (p=0.62), prolactin (p=0.42), CRH (p=0.25) and leptin gene expression (p=0.62) at the peripheral placental site within the group of AGA infants.

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  • Fig. 2 

    Absolute expression of housekeeping genes and genes of interest in samples taken from different areas of IUGR and AGA placentas. In addition to IGFBP-1, prolactin, CRH and leptin, the two housekeeping genes HPRT and β2-MG are regulated in their expression dependent on sampling site. The lines show the mean±SEM. *significant.

When comparing the relative expression of these four placental transcripts between the three sampling sites of the placental tissue in the IUGR group, we found a slightly higher, but statistically not significant expression level at the placental periphery compared to the intermediate portion. In the proximal placental part, gene expression was significantly different for IGFBP-1 (highest expression) and for leptin (lowest expression, Fig. 1). Analysis of gene expression in AGA placentas also demonstrated an increased mRNA expression of IGFBP-1, prolactin and CRH in samples taken from the peripheral area of the placenta. This difference was not statistically significant. Gene expression of IGFBP-1 was significantly higher and leptin mRNA was significantly lower in the proximal compared to the intermediate and peripheral placental site, like in IUGR placentas (Fig. 1).

Considering the delivery characteristics of our patients, gestational age did not differ significantly between IUGR and AGA neonates (Table 2). When analyzing gene expression data within each subgroup, we found no differences in mRNA expression of IGFBP-1, prolactin, CRH and leptin between premature babies and neonates born at term in intermediate as well as in peripheral samples of IUGR placental tissue (intermediate part: IGFBP-1 (p=0.56), prolactin (p=0.33), CRH (p=0.50), leptin (p=0.15); placental periphery: IGFBP-1 (p=0.16), prolactin (p=0.70), CRH (p=0.55), leptin (p=0.66)). Considering the proximal part of IUGR placentas, however, we observed a significantly higher gene expression of IGFBP-1 (p<0.01) and prolactin (p<0.001) in preterm compared to term placentas. Placental mRNA expression of CRH (p=0.25) and leptin (p=0.26) did not differ between IUGR and AGA neonates.

In AGA placentas there was no significant difference in gene expression of these four placental transcripts between preterm and term delivery in the placental proximal, intermediate and peripheral region (proximal part: IGFBP-1 (p=0.44), prolactin (p=0.56), CRH (p=0.77), leptin (p=0.39); intermediate part: IGFBP-1 (p=0.30), prolactin (p=1.00), CRH (p=0.07), leptin (p=0.66); placental periphery: IGFBP-1 (p=0.31), prolactin (p=0.17), CRH (p=0.20), leptin (p=0.22)).

In addition to the relative gene expression of the four placental transcripts, we separately analyzed the expression pattern of the housekeeping genes and changes in the pure expression data of IGFBP-1, prolactin, CRH and leptin between the different placental sites and between IUGR and AGA placentas. Beside the latter genes of interest, HPRT and β2-MG were regulated in their expression as well dependent on sampling site, even within a single placenta. The expression of these housekeeping genes was highest in the proximal portion and differed for β2-MG also between intermediate and peripheral placental site (Fig. 2). As a consequence, changes seen in the relative expression of the genes of interest were due to combined alterations in gene expression.

Considering the pure expression data of HPRT, β2-MG, IGFBP-1, prolactin, CRH and leptin, no correlation was found between gestational age and mRNA expression of the two housekeeping genes as well as the genes of interest in the placental intermediate and peripheral region. However, in the proximal portion of IUGR placentas, gene expression of IGFBP-1 and prolactin correlated positively to gestational age (Table 3).

Table 3. Correlation between gene expression of placental IGFBP-1, prolactin, CRH, leptin, and gestational age in the proximal, intermediate and peripheral portion of IUGR and AGA placentas.
Correlation to gestational ageIUGR (n=22)AGA (n=19)
Proximal placental portion
HPRTr=−0.20, nsp=0.54r=−0.20, nsp=0.49
β2-MGr=0.00, nsp=0.99r=−0.28, nsp=0.32
IGFBP-1r=0.72, *p<0.01r=0.24, nsp=0.42
Prolactinr=0.75, *p<0.01r=0.32, nsp=0.26
CRHr=−0.43, nsp=0.16r=0.34, nsp=0.23
Leptinr=−0.47, nsp=0.13r=−0.06, nsp=0.84

Intermediate placental portion
HPRTr=0.09, nsp=0.71r=0.05, nsp=0.83
β2-MGr=0.01, nsp=0.98r=−0.07, nsp=0.79
IGFBP-1r=−0.24, nsp=0.28r=−0.06, nsp=0.79
Prolactinr=−0.19, nsp=0.40r=0.14, nsp=0.58
CRHr=0.02, nsp=0.94r=0.35, nsp=0.15
Leptinr=−0.34, nsp=0.14r=0.25, nsp=0.33

Peripheral placental portion
HPRTr=−0.10, nsp=0.69r=−0.04, nsp=0.86
β2-MGr=0.13, nsp=0.60r=−0.02, nsp=0.92
IGFBP-1r=−0.10, nsp=0.70r=0.04, nsp=0.88
Prolactinr=−0.02, nsp=0.95r=0.31, nsp=0.20
CRHr=0.15, nsp=0.56r=0.17, nsp=0.49
Leptinr=−0.03, nsp=0.92r=0.01, nsp=0.95

HPRT, hypoxanthine guanine phosphoribosyl transferase.

β2-MG, β2-microglobulin.

IGFBP-1, insulin-like growth factor-binding protein-1.

CRH, corticotropin releasing hormone.

NS, not significant.

*p<0.01.

To prove the concept of differential expression and placental inhomogeneity independent on placental size, we exemplarily analyzed a subgroup of 10 SGA placentas. Placental weight did not differ significantly between IUGR and SGA placentas. When comparing the expression pattern of the housekeeping genes and changes in the pure expression data of prolactin and CRH between IUGR and SGA placentas, we found regional differences in gene expression. Especially in the intermediate placental portion, significant differences could be observed with higher expression levels in SGA placentas (Fig. 3). Moreover, like in IUGR placentas, mRNA expression differed significantly between different sampling sites in SGA placentas as well (Fig. 3).

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  • Fig. 3 

    Comparison between IUGR and SGA placentas: Absolute expression of housekeeping genes and of Prolactin and of CRH in samples of different placental areas. When comparing the pure expression data between IUGR (n=22) and a subgroup of SGA (n=10) placentas, there are significant differences in gene expression dependent on sampling site, even within a single placenta. The lines show the mean±SEM. *significant.

All data for placental gene expression are presented as relative gene expression normalized for hypoxanthine guanine phosphoribosyl transferase. The use of the alternative housekeeping gene β2-Microglobulin did not change the main results (data not shown).

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4. Discussion 

The aim of our study was to analyze whether the sampling site of placental tissue might influence the gene expression of specific placental factors potentially involved in fetal programming. This was done by analyzing three samples taken from different parts of the placenta in parallel and by comparing gene expression patterns between IUGR and AGA neonates. The basic idea behind this study was to establish a feasible concept of sample collection for all participants of a multicenter study.

Considering the results from samples taken from the intermediate part of the placenta, our data support earlier studies on placental IGFBP-1, CRH, leptin and prolactin expression in pregnancies complicated by IUGR. However, the same did not apply if samples had been taken from the proximal or peripheral portion of the placentas.

Increased circulating IGFBP-1 is a consistent observation in human IUGR fetuses and newborns, and in animal models of IUGR [5], [6], [7]. Gene expression of IGFBP-1 is upregulated in IUGR placentas [22]. IGFBP-1 is a potent inhibitor of IGF-I and IGF-II interactions with the IGF-1 receptor, thereby limiting the mitogenic actions of the IGFs during periods of hypoxia, undernutrition or placental insufficiency. Our data are in line with these findings, but a significantly increased placental IGFBP-1 gene expression in placental tissues of IUGR neonates could only be found when placental intermediate probes were analyzed.

In IUGR placental tissue, CRH was shown to be upregulated using microarray analysis [22], [23]. Measuring placental CRH mRNA expression, we were able to detect significantly higher CRH expression levels only in IUGR samples taken from the placental intermediate region. However, our results would have been different if samples from placental proximal or peripheral sites were used for analysis. In the human placenta, CRH seems to modulate vasodilation [24], prostaglandin production and ACTH secretion. Maternal plasma CRH concentrations are significantly elevated in pregnancies associated with abnormal placental function such as preeclampsia and IUGR [8], [9]. Similarly, mean umbilical cord plasma CRH levels in growth-restricted fetuses are higher in IUGR than in AGA neonates matched for gestational age, presence or absence of labour, and mode of delivery [10].

The ob-gene product leptin was initially identified as an adipocyte-derived hormone that decreases food intake and body weight via its receptor in the hypothalamus [25], [26]. During pregnancy, leptin is involved in the regulation of fetal growth, placental angiogenesis and immunomodulation, as well as mobilization of maternal fat [11], [27]. In studies where IUGR was confirmed by prenatal Doppler ultrasound examination, microarray technique identifying genes with possible relevance in the pathogenesis of IUGR reveals a significant increment of leptin gene expression in IUGR placentas [12], [23]. In this context, we could show that leptin is highly upregulated in IUGR placental tissue on mRNA level, too, but only if analysis was conducted in samples obtained from the placental intermediate or peripheral area.

Elevated placental gene expression in IUGR could also be confirmed for prolactin in our patients, when measurements were performed in placental intermediate samples. Prolactin concentrations are increased in cord blood of IUGR neonates with a ponderal index <2 [28].

In our study, however, when measuring gene expression of these placental factors in samples derived from the placental proximal or peripheral part, the statistically significant difference between IUGR and AGA is lost. With the notable exception of leptin, only a non-significant trend towards increased mRNA expression of IGFBP-1, CRH and prolactin in IUGR tissue taken from placental periphery can be shown, a fact that may be attributable to the relative small number of subjects studied.

In previous studies, gene expression in non-IUGR placentas was analyzed at different sites within the placental disk and sampling location was demonstrated to be an important consideration [16], [29], [30]. It was shown that the gene expression of hypoxia-related placental transcripts depended on sampling site and reflected the pattern of maternal arterial blood flow in the human placenta [16], [31]. Thus, gene expression seems to be altered dependent on placental oxygenation. However, in these studies, only term human placental tissues from uncomplicated pregnancies were analyzed.

IUGR placentas represent diseased tissue and show characteristic hypoxic/ischemic changes, including increased syncytial knots, infarction, or hypercapillarization [17], [18]. Placental insufficiency associated with a progressive deterioration in placental function leads to a decrease in transplacental transfer of oxygen and nutrients to the fetus. The resulting fetal hypoxemia suppresses fetal growth as an attempt to reduce metabolic demands by the growing fetus.

In this context, we speculate that changes in gene expression levels could be more dramatic in diseased placental tissue. This might especially be the case when considering specific placental factors potentially involved in fetal programming. We therefore analyzed three samples in parallel taken from different parts of the placentas of IUGR and AGA infants and controlled for placental inhomogeneity. In AGA placentas, mRNA expression of CRH and prolactin in samples taken from the peripheral part of the placenta was increased, although the difference was not statistically significant. The highest expression of IGFBP-1 mRNA was found in the proximal placental area. Leptin gene expression was decreased in the proximal placental area compared to the intermediate portion. This could be explained by an hypoxia-induced placental leptin mRNA-upregulation [32], [33] following the gradient of oxygenation from the center to the periphery of the placenta.

Considering IUGR placental tissue, we found a higher expression level of IGFBP-1, leptin, CRH and prolactin at the placental peripheral portion, which was not statistically significant. Like in AGA placentas, in the proximal portion of IUGR placentas, IGFBP-1 mRNA expression was increased, whereas leptin gene expression was decreased compared to the intermediate placental area. In spite of an abnormal placental environment in IUGR, in healthy placentas the same difference in gene expression of placental factors potentially involved in fetal programming seems to persist between the three different placental portions. These findings are in line with data from the literature, where regional differences, which are found in placentas derived from uncomplicated pregnancies, are reported to be also characteristic of underperfused villi [34]. However, detection sensitivity is considerably reduced.

In addition to the relative gene expression, the pure expression data of the housekeeping genes and the genes of interest were analyzed separately. Thus, changes seen in the relative expression of the genes of interest resulted from combined alterations in gene expression, as HPRT and β2-MG were shown to be regulated in their expression as well dependent on placental sampling site. The expression of these housekeeping genes was highest in the proximal portion and differed for β2-MG also between intermediate and peripheral placental site. The exact reasons and underlying mechanisms cannot be clarified in the study. We speculate that HPRT as an important enzyme in the purine salvage pathway might play a role especially in the proximal placental portion, where free purine bases arrive via the umbilical cord. β2-MG is considered an excellent candidate for use as internal control in RT-PCR due to its expression stability [35], [36]. Recently, β2-MG was shown to be a potential factor for the expansion of mesenchymal stem cells [37], which might explain higher expression levels in a placental area close to the umbilical cord insertion. However, although the underlying molecular mechanisms cannot be elucidated in our study design, our findings underline the importance of placental sampling site with regard to regional variation of gene expression.

To prove the concept of differential expression and placental inhomogeneity independent on placental size, a subgroup of 10 SGA placentas was exemplarily analyzed. Like in IUGR placentas, mRNA expression differed significantly between different sampling sites in SGA placentas. Moreover, when comparing regional differences in the expression pattern between IUGR and SGA placentas, we interestingly found higher expression levels of the housekeeping genes and the genes of interest especially in the intermediate placental area.

As SGA placentas are small in size, but otherwise are normal, resulting gene expression differences between SGA (normal tissue) and IUGR placentas (diseased tissue) would be relevant. Unfortunately, we were not able to match our SGA subgroup with the IUGR patient cohort, as, in contrast to IUGR, prematurity and primary caesarean section only randomly occur in SGA pregnancies. Further studies are needed to elucidate potential differences in gene expression patterns between IUGR and SGA placentas.

With regard to delivery characteristics of our patients, there is a marked difference in the rate of spontaneous and caesarean section delivery between the IUGR and AGA group, introducing a potential confounding factor. However, we were unable to find any significant differences in IGFBP-1, leptin, CRH and prolactin gene expression between infants born by Caesarean section and infants born by vaginal delivery in the AGA group, where the number of patients for each subgroup was almost equal. Also, the effect of gender on gene expression of these four placental transcripts analyzed as another potential confounding factor did not affect our results. Of note, there was no difference in placental gene expression when prematurely born infants and infants born at term were compared within each subgroup. Furthermore, no correlation was found between gestational age and gene expression in the intermediate placental portion.

When gene expression between IUGR and AGA in the intermediate placental region was analyzed, our results were in line with previously reported results. Importantly, we compared gene expression of specific placental factors at different sampling sites separately within each group. This approach made it likely that the potentially confounding factors mentioned introduced no bias. Of course, further investigations in larger cohorts and with adjustment to the mode of delivery will be necessary to confirm our results.

In conclusion, placental sampling site seems to be relevant and should be exactly defined when analyzing gene expression of factors potentially involved in human fetal programming. This important aspect will be the basis for future studies, as sampling from only one site of the placenta might not be sufficient to detect differences in gene expression between IUGR and AGA neonates.

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Acknowledgements 

The authors wish to thank Pfizer Pharma GmbH, Germany and Novo Nordisk for financial support (Educational grant) as well as Ms. Bitterer, Ms. Schmied and Ms. Allabauer for measurements of placental gene expression and their excellent technical assistance.

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PII: S0143-4004(09)00392-0

doi:10.1016/j.placenta.2009.12.002

Placenta
Volume 31, Issue 3 , Pages 178-185, March 2010

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