Molecular Human Reproduction, Vol. 7, No. 3, 287-292,
March 2001
© 2001 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
Expression of insulin-like growth factor-I and placental growth hormone mRNA in placentae: a comparison between normal and intrauterine growth retardation pregnancies*
1 Cell Biology Department, Research Society, BJ Wadia Hospital for Children and Institute of Child Health, Acharya Donde Marg, Parel, Mumbai 400 012 and 2 Department of Obstetrics and Gynaecology, King Edward Memorial Hospital, Acharya Donde Marg, Parel, Mumbai 400 012, India
Abstract
Intrauterine growth restriction (IUGR) is generally defined as the pathological restriction of fetal growth resulting in a fetus with birth weight below the 10th percentile for gestational age. Almost 75% of IUGR cases develop during third trimester. Studies on animals (rodents and sheep) as well as humans suggest that insulin-like growth factor-I (IGF-I), under the influence of placental growth hormone (PGH) plays crucial roles in fetal growth regulation during this period. Limited data are available with regard to IGF-I and PGH in placentae of normal and IUGR births. Therefore, in the present study, IGF-I and PGH mRNA expression has been studied in term placentae of normal (n = 10) and IUGR (n = 15) births by in-situ hybridization procedure. Their expression was also studied in first (n = 5) and second (n = 5) trimester placentae obtained from elective termination of normal pregnancies. Both IGF-I and PGH expression were found to be higher in the first and second trimester placentae compared to term placentae in normal pregnancies. However, IUGR term placentae showed increased expression of both IGF-I and PGH mRNA in comparison with normal placentae. Various mechanisms leading to the increased transcription of IGF-I and PGH mRNA in IUGR placenta are discussed. This increased transcription perhaps occurs in response to the reduction in the fetal growth.
IGF-I/in-situ hybridization/IUGR/PGH/placenta
Introduction
The term intrauterine growth restriction (IUGR) is generally assigned to the infants born with birth weights below the 10th percentile for gestational age in that community/population, as a result of a pathological restriction of fetal growth (Pollack and Divon, 1992
; Abu-Amero et al., 1998; Wollmann, 1998). These babies are at high risk of developing metabolic and/or cardiovascular diseases and other complications in post-natal life (Barker, 1995). Major factors leading to IUGR include placental/ fetal factors like chromosomal abnormalities, fetal congenital malformations, placental pathology, intrauterine infections and maternal factors such as pre-eclampsia, maternal cardiovascular disease, or other maternal complications. However, the aetiology of the majority of IUGR cases remains unexplained (Vander Veen and Fox, 1983; Gluckman and Harding, 1997; Abu-Amero et al., 1998; Wollmann, 1998).
Insulin-like growth factors I and II (IGF-I and IGF-II) have been implicated to play important roles in fetal growth regulation (Giudice et al., 1995; Klauwer et al., 1997), although their relative importance still remains controversial. Gene knock-out experiments done on mice and other studies conducted on animals (rodents and sheep) as well as humans suggest that in the third trimester, the key feature of fetal growth is the supply of nutrients from the mother to the fetus through placenta. During this period, IGF-I (in maternal, placental and fetal compartments) becomes the dominant regulator of fetal growth, since it not only influences partitioning of nutrients between placenta and fetus but also promotes fetal somatogenesis and fetal nutrient uptake (Baker et al., 1993; Gluckman, 1995; Gluckman and Harding, 1997).
Several investigators have studied the concentrations of IGF in cord blood and maternal circulation in IUGR versus normal pregnancies. IGF-I concentrations were found to be significantly lower in cord blood of IUGR infants compared to normal (Guidice et al., 1995; Klauwer et al., 1997; S.Sheik et al., unpublished observations). IGF-I concentrations in maternal circulation were also found to be significantly lower in pregnancies complicated by IUGR (Mirless et al., 1993; Holms et al., 1998; unpublished observations). Placental IGF, in both normal and IUGR pregnancies, has recently been studied for the first time (Abu-Amero et al., 1998). They reported an increased expression of IGF-II mRNA in IUGR term placentae compared to normal placentae, but found no significant difference in IGF-I mRNA expression between the two groups.
The majority of IUGR cases (almost 75%) generally develop during the second half of the pregnancy (Wollman et al., 1998), when IGF-I may be playing a major role in fetal growth regulation. Alterations in its concentrations and/or its activity in maternal, fetal and placental compartments may be one of the mechanisms involved in the pathogenesis of idiopathic IUGR cases.
In the present study, IGF-I mRNA expression was investigated in situ in normal versus IUGR placentae. Both maternal and placental IGF-I concentrations appear to be regulated by placental growth hormone (PGH), which replaces pituitary GH in maternal blood throughout pregnancy (Alsat et al., 1997; Evain-Brion, 1999). Therefore, we have also studied PGH mRNA expression in both groups.
Materials and methods
The present study was a prospective, observational study undertaken after approval by the Ethics Committee of King Edward Memorial Hospital, Mumbai and all subjects gave informed consent.
Collection of placental samples
Normal cases
First (6–10 weeks) and second (16–18 weeks) trimester placentae (five each) were collected after elective termination of normal pregnancies. Term placentae (n = 10) were collected from normal pregnancies immediately after delivery. All the women in the study had ultrasound confirmation of gestational age before 18 weeks of gestation.
IUGR cases
For the IUGR group, 26 women with clinical suspicion of IUGR were enrolled. These women were admitted to the antenatal care ward to confirm IUGR by fetal biometry and to investigate a possible cause for the same. All these women were followed with serial ultrasound examinations. Fifteen out of 26 women, who fulfilled the inclusion criteria for idiopathic IUGR, were included in the study. Their placenta samples (n = 15) were collected immediately after delivery. The remaining 11/26 cases were excluded from the study because either the mothers developed pregnancy-induced hypertension (n = 2) or babies were born with birth weight > the 10th percentile (n = 9) as compared to normal Indian new-born standards (Mohan et al., 1990).
Inclusion criteria for IUGR cases in the present study
Singleton pregnancy
The details of the IUGR cases included in the present study are summarized in Table I. The inclusion criteria for IUGR cases were: gestational age confirmed by ultrasound (first trimester by measurement of crown-rump length or early second trimester by measurement of biparietal diameter); IUGR suspected by a lag of 
3 weeks in fetal biometry on serial ultrasound examinations after 20 weeks; birth weight below 10th percentile for gestation age (although all babies actually had birth weight 
3rd percentile for gestational age, Table I); no history of pre-eclampsia or any other maternal complications, e.g. nutritional deficiency, anaemia, infection or diabetes associated with pregnancy; no evidence of ultrasound markers for fetal chromosomal defects and fetal congenital malformations; no evidence of intrauterine infections such as fever, TORCH positivity, vaginal discharge etc.; IUGR was confirmed clinically in neonates after birth on the basis of reduced birth weight and ponderal index in accordance with gestational age (Table I).
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Sample processing
Term placentae, obtained immediately after delivery, were grossly examined for infarcts and other abnormalities and weighed (Table I
). Placental tissues were collected from the site near the cord insertion (an area just deep to the basal plate in the central part of the placenta), fixed in 10% neutral buffered formalin and processed by the conventional method for preparing paraffin sections. Great care was taken to avoid degradation of mRNA in the tissue sections, e.g. use of gloves throughout processing, and wherever necessary solutions/reagents were made in DEPC (diethyl pyrocarbonate)-treated water and autoclaved. All glassware was autoclaved and baked prior to use.
In-situ localization of IGF-I and PGH m-RNA in placenta sections
Paraffin sections 5 µm thick were cut, deparaffinized, dehydrated, washed in DEPC-treated water and refixed in 2% paraformaldehyde for 10 min prior to being subjected to standard in-situ hybridization procedure (Boehringer Mannheim Technical Manual, 1992).
The oligoprobe selected for IGF-I mRNA localization in the present study is complementary to a conserved region in both IGF-IA and IGF-IB mRNA and encodes a part of exon 3 of the IGF-I gene. Probe sequence was taken from published literature with slight modification (Han et al., 1987). The probe for PGH mRNA is complementary to the region coding for amino acids 106 to 115 (Scippo et al., 1993). IGF-I probe: 5' CTC CGG AAG CAG CAC TCA TCC ACG ATA CCT 3'. PGH probe: 5' TCG GAC AGC AAC GTC TAT GAC CAC CTA AAG 3'. These oligoprobes were tail-labelled with Digoxigenin Oligonucleotide Tailing Kit (Boehringer Manneheim).
Briefly, after washing in phosphate-buffered saline (PBS, 0.1 mol/l), the slides were incubated in sodium saline citrate (2xSSC, 10 min). Pre-hybridization was carried out at room temperature for 1 h in a pre-hybridization cocktail (5xDenhardt's solution, 4xSSC, 50% formamide, 0.25% yeast tRNA, 0.5% herring sperm DNA, 10% Dextran sulphate). This was followed by overnight hybridization at 42°C with digoxigenin-labelled oligoprobes dissolved in the pre-hybridization cocktail at a concentration of 0.5 pmol/µl.
After stringent post-hybridization washes with varying concentrations of SSC and incubation in Tris buffer (0.1 mol/l, pH 7.4) for 10 min, the slides were blocked with normal sheep serum (2% in 0.1 mol/l Tris buffer containing 0.3 % Triton-X for 2 h at room temperature). Sections were then incubated overnight in alkaline phosphatase-conjugated anti-digoxigenin antibody (Boerhinger and Mannheim, diluted 1:500 in Tris buffer containing 2% normal sheep serum and 0.3% Triton-X). Next day slides were washed twice in Tris buffer for 5 min each and then incubated in Tris buffer (0.1 mol/l, pH 9.5, 25 min). Colour reaction was carried out by incubating the slides in a mixture of nitro-blue tetrazolium (NBT) and 5-bromo-4-chloro-3- indolyl phosphate (BCIP) for 20 min at room temperature (2.15 µl NBT, 1.75 µl BCIP in 500 µl of 0.1 mol/l Tris buffer, pH 9.5 containing 0.2% levamisole). After a final wash in distilled water, slides were mounted in an aqua mount. They were viewed and representative areas were photographed under an Olympus BX-60 microscope.
For negative controls, tissue sections were subjected to an identical procedure using digoxigenin-labelled sense oligoprobes.
Results
Clinical data of the study subjects is summarized in Table I.
IGF-I and PGH mRNA expression
The distribution pattern and relative abundance of IGF-I and PGH mRNA in normal placenta sections (at various gestational ages) and IUGR placenta (at term) are depicted in Figures 1–3 and summarized in Table II.
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Normal placenta
During early first trimester (6 weeks), when villi were undifferentiated, IGF-I and PGH mRNA expression was extremely abundant in most of the cells of developing placenta. By late first trimester (9–10 weeks), IGF-I mRNA was expressed primarily on syncytiotrophoblast, cytotrophoblasts, and some stromal cells of the well-developed villi (Figure 1A
), while PGH mRNA expression was predominantly concentrated in syncytiotrophoblast (data not shown).
The pattern of IGF-I and PGH mRNA expression did not change appreciably between first and second trimester. In second trimester (16–18 weeks), both IGF-I (Figure 1B) and PGH (not shown) mRNA expression was selectively concentrated in syncytiotrophoblast; however, some cytotrophoblasts and stromal cells of villi also expressed IGF-I mRNA.
Mature placental villi at term (with increased blood vessels, thinned syncytium layer and decreased cytotrophoblasts), showed a marked reduction in both IGF-I and PGH mRNA expression (Figures 1C, E and 2A). The chorion at term also showed weak expression of IGF-I (Figure 1D) and PGH mRNA (data not shown) in stromal cells. Interestingly co-expression of both IGF-I and PGH mRNA was observed in all samples studied at all time points.
IUGR placenta
All the IUGR placental sections (n = 15) obtained at term, exhibited an increased expression of both IGF-I (Figure 1F-H and Table II) and PGH (Figure 2B and Table II) mRNA in all the cellular elements of placenta (syncytiotrophoblast, cytotrophoblasts and stromal cells of the chorion layer) as compared with normal placentae.
No staining was observed in sections that were processed using digoxigenin-labelled sense probes (Figure 3).
Discussion
In normal pregnant women, the expression of PGH and IGF-I mRNA was observed to be greatest in the first and second trimester placentae and much lower in term placentae. The pattern of IGF-I mRNA localization (especially in syncytiotrophoblasts) noted in the present study was in agreement with earlier reports (Mills et al., 1986; Wang et al., 1988) and indicates the importance of the autocrine and paracrine role of IGF-I in early gestation as placental growth factor for the growth and function of placenta itself as well as for the embryo, since this stage is associated with embryonic induction, organogenesis, and rapid cell proliferation.
Furthermore, the similar pattern of expression for PGH and IGF-I mRNA across gestation, as noted in the present study, indicates that PGH, besides regulating the maternal circulatory IGF-I concentration (Alsat et al., 1997; Evain-Brion, 1999) perhaps also regulates local production of IGF-I in placenta. The positive correlation between PGH and IGF-I mRNA expression in placentae demonstrated in the present study is supported by earlier reports where IGF-I values in maternal plasma correlated with corresponding PGH values regardless of complications and gestational age (Caufriz et al., 1993; Mirless et al., 1993).
The expression of IGF-I and PGH mRNA was found to be increased in term placentae of IUGR infants compared with normal in the present study, and these observations were also confirmed by total RNA dot blot analyses (data not shown). The increased expression of IGF-I and PGH mRNA in IUGR placentae could be due to prematurity of IUGR cases. However, this is not likely since, in all IUGR cases, gestational age was confirmed by both ultrasound and last menstrual period. All IUGR cases included in the present study were born full term (37.8 ± 1.3 weeks) and were idiopathic (Table I).
The increased expression of IGF-I and PGH mRNA in IUGR term placentae noted in the present study is in contrast to earlier reports. Chowen et al. (1996) demonstrated a significantly reduced number of cells expressing PGH mRNA in IUGR term placentae compared to normal (Chowen et al., 1996). Additionally, no significant difference in placental IGF-I mRNA expression was observed by Abu-Amero et al. (1998) between IUGR and normal cases by reverse transcription-polymerase chain reaction (Abu-Amero et al., 1998). However, Fatayerji et al. (1996) reported an over-expression of IGF-I mRNA in placenta, similar to that noted in present study, in alcohol-fed rats who delivered IUGR infants (Fatayerji et al., 1996).
Inconsistency in the pattern of observations noted among various studies could be due to the difference in selection criteria of IUGR. Earlier reports have included cases with birth weights < the 10th/5th percentile and growth restriction in utero whereas this study focused on IUGR cases where, in addition to the reduced birth weight (< the 3rd percentile, Table I), all other known maternal or fetal factors associated with defective fetal growth were also excluded.
The other possible explanation for the discrepancy between the present study and the earlier report (Abu-Amero et al., 1998) could be the site of IGF-I gene analysis. It is known that the IGF-I gene in humans and rats is governed by two promoters adjacent to the 5' end of exons 1 and 2 respectively, and the gene also has several polyadenylation sites within exon 6. The mRNA derived from promoters 1 and 2 are spliced and processed differently. The alternative splicing affects exons 1, 2 and 5 and may cause excision of part of the untranslated regions within these exons. Such alternative splicing and alternative polyadenylation results in many kinds of IGF-I mRNA transcripts ranging from <1 kb to >7.5 kb. Therefore, in order to analyse the total amount of IGF-I mRNA in any tissue, it is essential that the site or region selected for analysis should be a common region in all the transcripts (Lowe et al., 1987; Kajimoto and Umayahara, 1998). Abu-Amero et al. (1998) have analysed the region between exons 1 and 2 and possibly have missed some of the transcripts in their analysis. The probe used in the present study encodes a part of exon 3 of IGF-I gene, which is common to all the transcripts.
Increased transcription of IGF-I and PGH mRNA in IUGR term placentae could have several explanations. Earlier studies have mentioned that IGF binding protein-1 (IGFBP-1) has high affinity for IGF-I and, if present in high amounts (both locally or in circulation) can sequester IGF-I and hence may not only decrease the bioavailability/transport of IGF-I peptides to tissues, but also inhibit IGF-I-induced DNA and protein synthesis (Ritvos et al., 1989; Giudice et al., 1995; Klauwer et al., 1997; Osgerby et al., 1999). The major source of IGFBP-1 during pregnancy is the decidua (Martina et al., 1997), but decidual function has received little attention from researchers in any pregnancy complication. It is possible that decidual dysfunction in IUGR may lead to increased production of decidually derived IGFBP-1 which may be sequestering IGF-I and thereby resulting in over-expression of IGF-I mRNA and hence PGH mRNA. In humans, elevated concentrations of IGFBP-I have been shown in cord blood and maternal circulation in pregnancies complicated by IUGR (Giudice et al., 1995; Fowler et al., 1999; S.Sheik et al., unpublished observations). Similar observations were noted in an earlier report on alcohol-fed rats (Fatayerji et al., 1996). The reason for the increase in IGFBP-I production is currently not known.
Another possibility could be that IGF-I peptide is secreted and degraded more rapidly in IUGR cases than in normal placentae or that the IGF-I mRNA itself is unstable or partly non-functional because of some post-transcriptional and/or translational modifications. Such translational and post-transcriptional mechanisms have been reported in protein restricted rats (Thissen et al., 1991), in human Wilms' tumour and pheochromocytoma (Hasselbacher et al., 1987). In all these studies, over-expression of IGF mRNA was found not to be associated with a corresponding increase in protein.
In conclusion, IGF-I and PGH mRNA expression in normal pregnancy was found to be higher in first and second trimester compared to term placentae; however, in IUGR term placentae, an increased transcription of both IGF-I and PGH mRNA was noted. Regardless of the mechanisms involved, this over-expression of IGF-I and PGH mRNA in placentae of IUGR infants appears to be a response to growth restriction of the fetus.
Notes
* Some of the data were presented at the VII International conference of Reproductive Immunology, New Delhi, India, October, 1998. 
3 To whom correspondence should be addressed. E-mail: deepabhartiya{at}yahoo.com
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Submitted on July 11, 2000; accepted on December 28, 2000.
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