Expression of the IGF‑1Ea isoform in human placentas from third trimester normal and idiopathic intrauterine growth restriction singleton pregnancies: Correlations with clinical and histopathological parameters
Intrauterine growth restriction (IUGR) is the second most common obstetric complication after preterm labor, affecting 5–10% of all pregnancies . Patients with growth restricted fetuses often develop preeclampsia  and fetuses with IUGR have a 5–10 times higher risk of dying in uterο . Additionally, fetuses affected by IUGR have a higher risk for perinatal morbidity, impaired neurodevelopment and long-term complications such as diabetes and cardiovascular and renal disease . IUGR occurs under the influence of fetal, maternal and placental factors that prevent the fetus attaining the optimal growth and placental insufficiently is the main cause for the development of IUGR . The molecular mechanisms mediating impaired fetal growth are unclear and therapeutic interventions in overcoming placental insufficiency and fetal growth restriction (FGR) remain unsatisfactory . The human placenta is a fetal organ of embryological origin that develops from the trophoectoderm, which is the outer layer of the blastocyst and the extraembryonic mesoderm shortly after blastocyst implantation. The placenta serves a key role in ensuring optimal fetal growth and development during pregnancy . Appropriate trophoblast differentiation and placental structure, growth and function are key for the maintenance of pregnancy and normal fetal growth, development and survival . Abnormal trophoblast proliferation and differentiation are associated with severe pregnancy complications, including recurrent miscarriage, preeclampsia and FGR . In early placentation, trophoblast precursors differentiate into highly invasive trophoblast cells, known as extravillous trophoblasts . Extravillous trophoblast formation occurs when placental villi attach to the uterine decidua constituting the anchoring villi, which are also termed ‘cell columns’. Extravillous trophoblast cells proliferate, migrate and invade the uterine decidua, endometrial glands, spiral arteries and inner third of the myometrium . One of the key roles of extravillous trophoblasts is remodeling of the maternal spiral arteries. To achieve this, extravillous trophoblast cells interact with maternal immune cells, disrupt the tunica media, induce elastolysis in the arterial wall, replace the endothelial cells and line the spiral arteries . This spiral artery remodeling provides a steady perfusion of intervillous spaces with maternal blood, unhindered by the influence of vasoactive agents . Poor spiral artery remodeling in the uterus is associated with shallow extravillous trophoblast cell invasion and is a key pathological feature of the placenta in preeclampsia, IUGR, placental abruption and spontaneous preterm premature rupture of membranes . Extravillous trophoblast cell proliferation, migration and invasion are positively or negatively regulated by numerous molecules produced by the fetomaternal interface to maintain a normal utero-placental homeostasis . These molecules include autocrine factors produced by the trophoblast such as growth factors [including insulin-like growth factor (IGF)-1, IGF-2, heparin binding epidermal growth factor-like, vascular epidermal, placental and hepatocyte growth factor], growth factor-binding proteins [including IGF binding proteins (IGFBPs)], proteoglycans (including decorin and biglycan), sialoproteins (including osteopontin), cytokines (including IL-6 and IL-15), chemokines [including C-X3-C motif chemokine ligand 1, C-C motif chemokine ligand (CCL)4 and CCL14], lipid derivatives (including prostaglandin E 2 ), matrix metalloproteinases, plasminogen activator inhibitors (PAI)-1 and PAI-2 or paracrine factors produced by the decidua, such as decidual leukocytes and immune cells . In the third trimester of pregnancy the terminal villous structure regulates the maternal nutrient transport and respiratory gas exchange to fetal circulation to aid fetal growth and development . Placental chorionic tertiary villi are bathed in maternal blood, which fills the trophoblastic lacunae. Nutrients, oxygen and waste products are transported across the two cell layers of the tertiary villi between the fetal and maternal blood, which are the syncytiotrophoblast and fetal endothelial cells . The syncytiotrophoblast, which is the outer layer of chorionic villi, cannot regenerate but is formed by continuous fusion of the underlying proliferating villous cytotrophoblasts into a continuous multinuclear cell layer . Following fusion of cytotrophoblasts, nuclei demonstrate signs of degeneration . In the third trimester of pregnancy, some nuclei in the syncytiotrophoblast gather together into clusters termed syncytial trophoblastic knots (STKs) . Notably, there is an increased number of STKs in pregnancies complicated by IUGR and preeclampsia . In addition, IUGR is associated with maternal vascular malperfusion (MVM) of the placental bed and decreased villous branching and terminal villi volume and exchange surface area between fetal and maternal circulation . Furthermore, in patients with IUGR, impaired cytotrophoblast cell-cell fusion is associated with decreased area of the syncytiotrophoblast within the villous and increased syncytiotrophoblast apoptosis, interrupting placental functions such as nutrient and oxygen transport to the fetus and the production and release of placental hormones . In the placenta, cellular turnover and renewal are regulated to maintain a bilayer of chorionic villus structure . The IGF system promotes trophoblast turnover by promoting both proliferation and differentiation of the cytotrophoblast to the syncytiotrophoblast for normal trophoblast function and fetal growth and development . Insulin, IGF-1 and IGF-2 serve a key role in fetal growth and development . IGF-1 and IGF-2 are highly homogenous single chain polypeptides with molecular weight of ~7 kDa that are structurally homologous to proinsulin . Alternative splicing of exons of the IGF-1 gene produces multiple heterogeneous IGF-1 mRNA transcripts, such as the mRNA isoforms IGF-1Ea, IGF-1Eb and IGF-1Ec. The translation of these mRNA isoforms produces various IGF-1 peptides . IGF-1 isoforms are associated with various gynecological conditions and pathologies, such as endometrial carcinoma, endometriosis and leiomyomas . However, there is a lack of knowledge regarding the effects of the IGF-1 isoforms, particularly IGF-1Ea peptide, on the growth of the human placenta and the remodeling of maternal uterine arteries by the extravillous trophoblast. In addition, expression profile of IGF-1Ea peptide within the endothelium of villous and endometrial blood vessels on the maternal side of the maternal-fetal interface remains unknown, as well as the autocrine effects of IGF-1Ea peptide in human placenta. Therefore, the aim of the present study was to investigate the role of IGF-1Ea expression in the placenta from pregnancies complicated by IUGR and uneventful pregnancies. Additionally, placental IGF-1Ea expression in IUGR pregnancies compared with appropriate-for-gestational-age (AGA) pregnancies were examined in connection with clinical and histopathological parameters to improve understanding of the pathophysiology of IUGR pregnancy.   Patient population A total of 62 placentas (15 AGA and 47 IUGR) were obtained from patients with singleton AGA pregnancies or pregnancies complicated by IUGR, delivered by either vaginal labor or cesarean section at the General Maternity Hospital of Athens ‘Elena Venizelou’ (Athens, Greece). The age range of patients was 16–46 years-old and the gestational age was 26–41 weeks. Informed written consent to participate was obtained from all patients and the study was approved by the Scientific Committee of the General Maternity Hospital of Athens ‘Elena Venizelou’, Athens, Greece (approval no. 2nd Scientific Committee Meeting/8th agenda/23-1-2018) and the Research and Bioethics Committee of the Medical School of the National and Kapodistrian University of Athens, Athens, Greece (approval no. 1718016683). The inclusion criteria for the IUGR group included pregnant patients with a fetal weight <5th percentile for gestational age and either of the following, diagnosed via antenatal ultrasound: i) Abnormal umbilical artery Doppler waveform with absent or reverse end-diastolic flow velocity during pregnancy; ii) oligohydramnios, defined as a deepest fluid pocket of ≤2 cm or an amniotic fluid index ≤5 cm via antenatal ultrasound performed prior to delivery or iii) asymmetric growth of the fetus with increased head to abdominal circumference ratio >1.2. IUGR pregnancies associated with pre-eclampsia were also included. The diagnosis of preeclampsia was based on hypertension during pregnancy (a systolic blood pressure ≥140 mmHg and/or a diastolic blood pressure of ≥90 mmHg) in a previously normotensive patient and proteinuria (≥300 mg protein in a 24-h urine collection). The control patients had AGA pregnancies without complications and delivered healthy neonates with birth weights from the 5 to 90th percentile. All control placentas were selected from full-term pregnancies (>37 weeks of gestation) and were grossly normal. The exclusion criteria for both AGA and IUGR pregnancies were abnormal oral glucose tolerance screening test results between 24 and 28 weeks of pregnancy, use of nutritional supplements during pregnancy, maternal hormonal treatment, smoking or use of recreational drugs, pre-existing maternal hypertension, liver, cardiovascular or kidney disease, endocrinological disorders, multiple pregnancies, chorioamnionitis, placental abruption, prolonged rupture of membranes, chromosomal abnormality, fetal anatomical defects and intrauterine viral infection. The appropriate gestational age was estimated using the date of the last menstrual period and confirmed by the crown-rump length of the fetus in the first trimester scan. The baseline maternal and fetal demographic characteristics [including maternal age and body mass index (BMI), gestational age, neonatal and placental weight and fetal sex] were recorded. Tissue sampling The sample collection was performed from February 2018 to August 2021. All placentas were obtained <15 min after labor and were weighed after the removal of the umbilical cord and fetal membranes. For mRNA isolation, fresh tissue samples were obtained from 28 placentas, excised from the medial areas, ~5 cm apart from the insertion of the umbilical cord and excluding the peripheral margins. These samples included villous parenchyma from the decidua basalis to the fetal surface, avoiding areas with gross calcification, infarcts, marked fibrin deposition and intervillous thrombi. The samples were cut into small pieces, washed in 0.9% PBS to remove blood contaminants and then snap-frozen and stored at −80°C until RNA extraction. The rest of the placenta was fixed in 10% buffered formalin at room temperature for one week, for histopathological and immunohistochemical examination. Histopathology Formalin-fixed paraffin-embedded sections were obtained using an automated tissue processor (Donatello, Diapath) and then stained with ready to use Hematoxylin H (Biognost) for 5 min and counterstained with Eosin Y 1% alcoholic (Biognost) for 10 sec, at room temperature. The gross and histological description of the placental lesions followed the Amsterdam criteria . The placentas were evaluated for the following major histological patterns: Changes consistent with i) maternal vascular malperfusion of the placental bed (MVM); ii) fetal vascular malperfusion (FVM); iii) massive perivillous fibrin/fibrinoid deposition (MPFD); iv) acute or chronic inflammatory lesions, including chronic villitis of unknown etiology (VUE) and v) delayed villous maturation (DVM). Other miscellaneous lesions were also recorded. RNA isolation and cDNA synthesis The frozen placental samples were from 13 normal and 15 IUGR third trimester pregnancies. Total RNA was extracted from frozen tissue samples using TRItidy (TRitidy G™ reagent; PanReac AppliChem GmbH) according to the manufacturer's instructions. The frozen placental tissues were cut into segments (12×8 mm) and homogenized in 0.5 ml TRItidy G. Following addition of 500 µl isopropanol, the resulting mixture was centrifuged (13,226 × g for 15 min at room temperature. The pellet containing the total RNA was washed once in 75% ethanol and dissolved in 20 µl diethylpyrocarbonate-treated water. Reverse transcription (RT) was performed using the ProtoscriptR II First Strand cDNA synthesis kit according to the manufacturer's instructions (New England BioLabs; cat. no. NE, E6560L). Quantitative PCR (qPCR) Oligonucleotide sequences were as follows: IGF-1Ea Forward, 5′-GTGGAGACAGGGGCTTTTATTTC-3′ and IGF-1Ea Reverse, 5′-CTTGTTTCCTGCACTCCCTCTACT-3′ generating a 251 bp product. This set of primers was designed to lie within different exons of the IGF-1 gene to detect and amplify only the IGF-1Ea transcript; 5′ sense primer is on exon 4, while the antisense primer is on the exon 6. 18S ribosomal RNA was used as the housekeeping gene (sense, 5′-GGCCCTGTAATTGGAATGAGTC-3′ and antisense, 5′-CCAAGATCCAACTACGAGCTT-3′). qPCR was performed using a Thermal Cycler (Bio-Rad iCycler Thermal Cycler IQ5 Multicolor Real-Time PCR Detection System; Bio-Rad Laboratories, Inc.). The reaction was conducted using 12.5 µl iQTM SYBR Green Supermix (Bio-Rad Laboratories, Inc.), 50 ng cDNA and 0.4 µM each primer, adjusted to a 20 µl total volume with ddH 2 O. A no template control was performed in each plate to verify the absence of extraneous nucleic acid contamination. The thermocycling conditions were as follows: Initial denaturation at 95°C for 4 min, followed by 45 cycles of 12 sec at 95°C, 30 sec at 61°C and 30 sec at 72°C and final extension at 72°C for 5 min. 2- ΔΔCt formula was used to calculate the fold-differences between the IUGR and normal pregnancy samples using 18S ribosomal RNA as the internal control . All the samples were analyzed in duplicate. Immunohistochemistry A total of 15 AGA and 47 IUGR formalin-fixed-paraffin-embedded placental samples were used for immunohistochemical analysis using EnVision FLEX+, Mouse, High pH (Link) cat. no. K8002, Dako, Agilent Technologies. The 4 µm-thick microtome sections were dried at 37°C overnight, de-waxed in xylene and rehydrated in serial dilutions of ethanol. Antigen retrieval was performed by heating at 97°C in a PT module immersed in DAKO high ph solution (pH 9) for 20 min and cooling at room temperature. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide (provided in the kit) for 5 min in a dark place at room temperature, followed by two washes with distilled water and then DAKO Wash buffer. The sections were then incubated with polyclonal anti-Rabbit IGF-1Eα antiserum (gifted by Dr Elisabeth Barton, University of Pennsylvania, PA, USA) at a dilution of 1:700 in Dako Diluent and incubated for 24 h at 4°C. After rinsing in Wash buffer, the sections were incubated with EnVision FLEX+Rabbit (LINKER) cat. no. K8009; Dako; Agilent Technologies, Inc.) for 15 min at room temperature, followed by two rinses in Wash buffer, followed by incubating slides in polymer Envision for 30 min and rinsed twice in Wash buffer. Visualization of the immunocomplex was performed by incubating the sections in DAB for 10 min (Dako; Agilent Technologies, Inc.). The sections were stained in hematoxylin for 5 min at room temperature, washed in distilled water, dehydrated in serial dilutions of ethanol and xylene and mounted in dibutyl phthalate xylate. Adrenal carcinoma was used as a positive control for IGF-1Ea immunostaining (obtained from archival surgical pathology material at Aretaieion University Hospital; Medical School of the National and Kapodistrian University of Athens). Negative controls were incubated without the primary antibody. Immunohistochemical evaluation was performed by examining the tissue sections under a light microscope at 100× magnification for the initial screening. For each specimen, 5 optical fields at 100× magnification were randomly selected and semi-quantitative measurements were performed using a slide grid under ×200 magnification. Measurements were simultaneously assessed by two qualified pathologists and consensus results were recorded for each case. Immunostaining in the perivillous trophoblast Semi-quantitative scoring of IGF-1Ea immunolabeling in the perivillous syncytiotrophoblast was used; cases were classified into three groups according to the percentage (scores) of positively stained perivillous syncytial areas as follows: 0, 0–10% immunopositive syncytial areas (negative); 1, 11–50% immunopositive syncytial areas (moderate); 2, >51% immunopositive syncytial areas (high). The staining intensity in the perivillous syncytiotrophoblast was defined using a semiquantitative scale: 0, negative (no staining); 1, weak; 2, moderate and 3, strong. Iimmunostaining in the extravillous trophoblast Immunostaining in the extravillous trophoblastic cells was defined as negative (no expression) or positive (any positive cells identified). The staining intensity in the extravillous trophoblastic cells was graded as follows: 0, negative (no staining); 1 weak; 2, moderate and 3, strong. Immunostaining in the vascular endothelium Immunostaining in the endothelium of the maternal decidual vessels was classified as negative (no staining) or positive (staining observed in one maternal vessel included in the basal plate). Similarly, immunostaining in the endothelium of the villous fetal blood vessels was classified as negative (no staining) or positive (staining observed in one fetal blood vessel). Statistical analysis Data are presented as the mean of two independent experimental repeats ± SD. The associations between categorical variables were assessed using exact Pearson's χ 2 or Fisher's exact test. For continuous variables, the differences were assessed using the Mann-Whitney U test. A two-tailed P<0.05 was considered to indicate a statistically significant difference. Data were analyzed using SPSS version 28.0 (IBM Corp).   A total of 62 placentas (15 AGA and 47 IUGR) were obtained from patients with singleton AGA pregnancies or pregnancies complicated by IUGR, delivered by either vaginal labor or cesarean section at the General Maternity Hospital of Athens ‘Elena Venizelou’ (Athens, Greece). The age range of patients was 16–46 years-old and the gestational age was 26–41 weeks. Informed written consent to participate was obtained from all patients and the study was approved by the Scientific Committee of the General Maternity Hospital of Athens ‘Elena Venizelou’, Athens, Greece (approval no. 2nd Scientific Committee Meeting/8th agenda/23-1-2018) and the Research and Bioethics Committee of the Medical School of the National and Kapodistrian University of Athens, Athens, Greece (approval no. 1718016683). The inclusion criteria for the IUGR group included pregnant patients with a fetal weight <5th percentile for gestational age and either of the following, diagnosed via antenatal ultrasound: i) Abnormal umbilical artery Doppler waveform with absent or reverse end-diastolic flow velocity during pregnancy; ii) oligohydramnios, defined as a deepest fluid pocket of ≤2 cm or an amniotic fluid index ≤5 cm via antenatal ultrasound performed prior to delivery or iii) asymmetric growth of the fetus with increased head to abdominal circumference ratio >1.2. IUGR pregnancies associated with pre-eclampsia were also included. The diagnosis of preeclampsia was based on hypertension during pregnancy (a systolic blood pressure ≥140 mmHg and/or a diastolic blood pressure of ≥90 mmHg) in a previously normotensive patient and proteinuria (≥300 mg protein in a 24-h urine collection). The control patients had AGA pregnancies without complications and delivered healthy neonates with birth weights from the 5 to 90th percentile. All control placentas were selected from full-term pregnancies (>37 weeks of gestation) and were grossly normal. The exclusion criteria for both AGA and IUGR pregnancies were abnormal oral glucose tolerance screening test results between 24 and 28 weeks of pregnancy, use of nutritional supplements during pregnancy, maternal hormonal treatment, smoking or use of recreational drugs, pre-existing maternal hypertension, liver, cardiovascular or kidney disease, endocrinological disorders, multiple pregnancies, chorioamnionitis, placental abruption, prolonged rupture of membranes, chromosomal abnormality, fetal anatomical defects and intrauterine viral infection. The appropriate gestational age was estimated using the date of the last menstrual period and confirmed by the crown-rump length of the fetus in the first trimester scan. The baseline maternal and fetal demographic characteristics [including maternal age and body mass index (BMI), gestational age, neonatal and placental weight and fetal sex] were recorded.   The sample collection was performed from February 2018 to August 2021. All placentas were obtained <15 min after labor and were weighed after the removal of the umbilical cord and fetal membranes. For mRNA isolation, fresh tissue samples were obtained from 28 placentas, excised from the medial areas, ~5 cm apart from the insertion of the umbilical cord and excluding the peripheral margins. These samples included villous parenchyma from the decidua basalis to the fetal surface, avoiding areas with gross calcification, infarcts, marked fibrin deposition and intervillous thrombi. The samples were cut into small pieces, washed in 0.9% PBS to remove blood contaminants and then snap-frozen and stored at −80°C until RNA extraction. The rest of the placenta was fixed in 10% buffered formalin at room temperature for one week, for histopathological and immunohistochemical examination.   Formalin-fixed paraffin-embedded sections were obtained using an automated tissue processor (Donatello, Diapath) and then stained with ready to use Hematoxylin H (Biognost) for 5 min and counterstained with Eosin Y 1% alcoholic (Biognost) for 10 sec, at room temperature. The gross and histological description of the placental lesions followed the Amsterdam criteria . The placentas were evaluated for the following major histological patterns: Changes consistent with i) maternal vascular malperfusion of the placental bed (MVM); ii) fetal vascular malperfusion (FVM); iii) massive perivillous fibrin/fibrinoid deposition (MPFD); iv) acute or chronic inflammatory lesions, including chronic villitis of unknown etiology (VUE) and v) delayed villous maturation (DVM). Other miscellaneous lesions were also recorded.   The frozen placental samples were from 13 normal and 15 IUGR third trimester pregnancies. Total RNA was extracted from frozen tissue samples using TRItidy (TRitidy G™ reagent; PanReac AppliChem GmbH) according to the manufacturer's instructions. The frozen placental tissues were cut into segments (12×8 mm) and homogenized in 0.5 ml TRItidy G. Following addition of 500 µl isopropanol, the resulting mixture was centrifuged (13,226 × g for 15 min at room temperature. The pellet containing the total RNA was washed once in 75% ethanol and dissolved in 20 µl diethylpyrocarbonate-treated water. Reverse transcription (RT) was performed using the ProtoscriptR II First Strand cDNA synthesis kit according to the manufacturer's instructions (New England BioLabs; cat. no. NE, E6560L).   Oligonucleotide sequences were as follows: IGF-1Ea Forward, 5′-GTGGAGACAGGGGCTTTTATTTC-3′ and IGF-1Ea Reverse, 5′-CTTGTTTCCTGCACTCCCTCTACT-3′ generating a 251 bp product. This set of primers was designed to lie within different exons of the IGF-1 gene to detect and amplify only the IGF-1Ea transcript; 5′ sense primer is on exon 4, while the antisense primer is on the exon 6. 18S ribosomal RNA was used as the housekeeping gene (sense, 5′-GGCCCTGTAATTGGAATGAGTC-3′ and antisense, 5′-CCAAGATCCAACTACGAGCTT-3′). qPCR was performed using a Thermal Cycler (Bio-Rad iCycler Thermal Cycler IQ5 Multicolor Real-Time PCR Detection System; Bio-Rad Laboratories, Inc.). The reaction was conducted using 12.5 µl iQTM SYBR Green Supermix (Bio-Rad Laboratories, Inc.), 50 ng cDNA and 0.4 µM each primer, adjusted to a 20 µl total volume with ddH 2 O. A no template control was performed in each plate to verify the absence of extraneous nucleic acid contamination. The thermocycling conditions were as follows: Initial denaturation at 95°C for 4 min, followed by 45 cycles of 12 sec at 95°C, 30 sec at 61°C and 30 sec at 72°C and final extension at 72°C for 5 min. 2- ΔΔCt formula was used to calculate the fold-differences between the IUGR and normal pregnancy samples using 18S ribosomal RNA as the internal control . All the samples were analyzed in duplicate.   A total of 15 AGA and 47 IUGR formalin-fixed-paraffin-embedded placental samples were used for immunohistochemical analysis using EnVision FLEX+, Mouse, High pH (Link) cat. no. K8002, Dako, Agilent Technologies. The 4 µm-thick microtome sections were dried at 37°C overnight, de-waxed in xylene and rehydrated in serial dilutions of ethanol. Antigen retrieval was performed by heating at 97°C in a PT module immersed in DAKO high ph solution (pH 9) for 20 min and cooling at room temperature. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide (provided in the kit) for 5 min in a dark place at room temperature, followed by two washes with distilled water and then DAKO Wash buffer. The sections were then incubated with polyclonal anti-Rabbit IGF-1Eα antiserum (gifted by Dr Elisabeth Barton, University of Pennsylvania, PA, USA) at a dilution of 1:700 in Dako Diluent and incubated for 24 h at 4°C. After rinsing in Wash buffer, the sections were incubated with EnVision FLEX+Rabbit (LINKER) cat. no. K8009; Dako; Agilent Technologies, Inc.) for 15 min at room temperature, followed by two rinses in Wash buffer, followed by incubating slides in polymer Envision for 30 min and rinsed twice in Wash buffer. Visualization of the immunocomplex was performed by incubating the sections in DAB for 10 min (Dako; Agilent Technologies, Inc.). The sections were stained in hematoxylin for 5 min at room temperature, washed in distilled water, dehydrated in serial dilutions of ethanol and xylene and mounted in dibutyl phthalate xylate. Adrenal carcinoma was used as a positive control for IGF-1Ea immunostaining (obtained from archival surgical pathology material at Aretaieion University Hospital; Medical School of the National and Kapodistrian University of Athens). Negative controls were incubated without the primary antibody. Immunohistochemical evaluation was performed by examining the tissue sections under a light microscope at 100× magnification for the initial screening. For each specimen, 5 optical fields at 100× magnification were randomly selected and semi-quantitative measurements were performed using a slide grid under ×200 magnification. Measurements were simultaneously assessed by two qualified pathologists and consensus results were recorded for each case.   Semi-quantitative scoring of IGF-1Ea immunolabeling in the perivillous syncytiotrophoblast was used; cases were classified into three groups according to the percentage (scores) of positively stained perivillous syncytial areas as follows: 0, 0–10% immunopositive syncytial areas (negative); 1, 11–50% immunopositive syncytial areas (moderate); 2, >51% immunopositive syncytial areas (high). The staining intensity in the perivillous syncytiotrophoblast was defined using a semiquantitative scale: 0, negative (no staining); 1, weak; 2, moderate and 3, strong.   Immunostaining in the extravillous trophoblastic cells was defined as negative (no expression) or positive (any positive cells identified). The staining intensity in the extravillous trophoblastic cells was graded as follows: 0, negative (no staining); 1 weak; 2, moderate and 3, strong.   Immunostaining in the endothelium of the maternal decidual vessels was classified as negative (no staining) or positive (staining observed in one maternal vessel included in the basal plate). Similarly, immunostaining in the endothelium of the villous fetal blood vessels was classified as negative (no staining) or positive (staining observed in one fetal blood vessel).   Data are presented as the mean of two independent experimental repeats ± SD. The associations between categorical variables were assessed using exact Pearson's χ 2 or Fisher's exact test. For continuous variables, the differences were assessed using the Mann-Whitney U test. A two-tailed P<0.05 was considered to indicate a statistically significant difference. Data were analyzed using SPSS version 28.0 (IBM Corp).   Clinical and pathological findings A total of 62 pregnant patients were recruited. There were significant differences in the mean placental and neonatal birth weight and the gestational age at delivery (all P<0.001) with the IUGR group exhibiting lower values compared with AGA pregnancies . Notably, preterm birth, defined as gestational age at delivery of <37 weeks, was observed only in the IUGR group (100%; ). All cases in the AGA group and 21 cases in the IUGR group gave birth at a gestational age of ≥37 weeks of pregnancy . The fetal sex distribution was 15 male (34.9%) and 28 female (65.1%; n=43; unreported in 19 IUGR cases). In the IUGR group, fetal sex distribution was 53.3% male and 71.4% females, with a male to female ratio of 0.746, while in the AGA group there were 46.7% male and 28.60% female fetuses with a male to female ratio of 1.633, . IUGR pregnancies were highly represented in the group of placentas with changes of MVM (83.3%), VUE (83.3%) and DVM (71.4%), though without any statistically significant differences when compared with the AGA group . Notably placentas from IUGR pregnancies showed histological changes consistent with MVM in 74.5% of cases, VUE in 10.6% and DVM in 10.6%. No differences could be statistically confirmed between the IUGR and AGA groups for any of the histopathological characteristics examined in our sample. Placental IGF-1Ea mRNA expression The placental IGF-1Ea mRNA expression levels were determined by qPCR. The mean IGF-1Ea mRNA expression levels were similar between the IUGR and AGA groups, with no significant difference (P=0.991; ). Additionally, no significant associations were observed in the placental IGF-1Ea mRNA expression between the IUGR and AGA pregnancies in relation to the clinical parameters such as maternal age and BMI, gestational age at delivery, fetal sex, neonatal and placental weight at delivery or the histopathological parameters such as MVM, MPFD, FVM, VUE, DVM and other lesions . Furthermore, there was no significant difference in placental IGF-1Ea mRNA expression in IUGR vs. AGA pregnancies in the full-term cases (>37 weeks of gestation; ). Moreover, placental IGF-1Ea mRNA expression in IUGR vs. the AGA pregnancies was not significant in cases with fetal birth weight >2,500 g . Finally, placental IGF-1Ea mRNA expression levels were similar between the IUGR and AGA groups in cases with placental weight >400 g, with no significant difference . Immunostaining localization Positive expression of IGF-1Ea protein was observed as granular or homogeneous brown staining localized to the cytoplasm and the cytoplasmic membrane of the perivillous syncytiotrophoblast ( and ) and the extravillous trophoblast of the basal plate and diaphragmatic columns . Positive IGF-1Ea expression was also detected in the endothelium of the fetal vessels within the stem, intermediate and distal chorionic villi, as well as in the endothelium of the maternal decidual vessels . IGF-1Ea isoform expression in perivillous syncytiotrophoblast of human placentas Of the 47 IUGR pregnancies, six (12.8%) exhibited high expression, 15 cases (31.9%) exhibited moderate expression and 26 (55.3%) had negative IGF-1Ea expression in the perivillous syncytiotrophoblast. Of 15 AGA pregnancies, one case (6.75%) exhibited high expression, two (13.3%) exhibited moderate expression and 12 cases (80%) had negative expression in the perivillous syncytiotrophoblast. These immunohistochemical IGF-1Ea placental expression scores were not significantly different between the IUGR and AGA pregnancies . Additionally, immunohistochemical IGF-1Ea expression scores of the IUGR and AGA pregnancies were not significantly associated with the clinical parameters such as maternal age, neonatal (>2,500 g) and placental weight (>400 g), maternal BMI, fetal sex and the histopathological parameters including MVM, VUE and DVM . In the IUGR group, one case (2.1%) exhibited strong IGF-1Ea intensity, nine (19.1%) exhibited moderate intensity, while 37 cases (78.7%) exhibited weak intensity. In the AGA group, no cases exhibited strong intensity, four (26.7%) exhibited moderate intensity, while 11 (73.3%) exhibited weak expression. The difference in IGF-1Ea expression intensity was not significantly different between the two groups . Additionally, intensity of the IGF-1Ea immunopositivity was not significantly associated with maternal age and gestational age (>37 weeks), neonatal birth weight (>2,500 g), placental weight (>400 g), maternal BMI, fetal sex, MVM and DVM . and showIGF-1Ea immunopositive expression in the perivillous syncytiotrophoblast, respectively. IGF-1Ea isoform expression in extravillous trophoblastic cells of human placenta The positive immunohistochemical IGF-1Ea staining in the extravillous trophoblast was significantly different between AGA and IUGR placentas . In the AGA placentas, IGF-1Ea immunopositive cells were observed in 73.3% of cases, while in the IUGR placentas, the IGF-1Ea immunopositivity was much lower . Additionally, there was a significant difference in the IGF-1Ea immunopositive cells in the extravillous trophoblast according to maternal age <40 years. Notably, patients <40 years in the IUGR group had negative IGF-1Ea immunohistochemical staining in the extravillous trophoblast in most cases; for the AGA group, positive staining was observed in most cases. A significant difference in IGF-1Ea immunopositive cells in the extravillous trophoblast was found between the IUGR and AGA groups in relation to gestational age. Notably, patients with a gestational age of 37 weeks in the IUGR group had negative IGF-1Ea immunohistochemical staining in the extravillous trophoblast in most cases; for AGA group, immunopositive cells were observed in most cases. Moreover, there was a significant difference in the IGF-1Ea immunopositive cells in the extravillous trophoblast between the IUGR and AGA groups according to neonatal birth weight. Newborns weighing >2,500 g had an absence of IGF-1Ea immunopositive cells in the extravillous trophoblast in most of IUGR placentas; in the AGA group, positive staining was observed in 73.3% of placentas. Furthermore, there was a significant difference in the IGF-1Ea immunopositive cells in extravillous trophoblast between the IUGR and AGA groups in relation to placental weight. In total, more than half of IUGR cases with a placental weight >400 g had negative IGF-1Ea staining in the extravillous trophoblast; for AGA, most cases had positive staining. In addition, there was a significant difference in IGF-1Ea immunopositive cells in the extravillous trophoblast between IUGR and AGA groups according to maternal ΒΜI of <30 kg/m 2 . In total, 72.7% of IUGR cases with a maternal BMI of <30 kg/m 2 had negative IGF-1Ea staining in the extravillous trophoblast; for AGA, most cases had positive staining (75.0%). Furthermore, fetal female sex was associated expression of IGF-1Ea in the extravillous trophoblastic cells. Overall, most of IUGR cases of fetal female sex had negative IGF-1Ea staining in the extravillous trophoblast; in AGA, in most cases had positive staining. Additionally, there was a statistically significant difference in IGF-1Ea immunopositive cells in the extravillous trophoblast between the IUGR and AGA groups according to MVM of the placental bed. IGF-1Ea expression was positive in 29.4 and negative in 70.6% of IUGR cases with MVM, while IGF-1Ea expression was positive in 66.7 and negative in 33.3% of AGA cases with MVM . The intensity of IGF-1Ea expression in extravillous trophoblastic cells was not significantly different between AGA and IUGR placentas . Additionally, there was no association with IGF-1Ea staining intensity between the two groups according to maternal age and gestational age (>37 weeks), neonatal birth weight (>2,500 g), placental weight (>400 g), maternal ΒΜI, fetal male sex, MVM and DVM . However, fetal female sex was associated with intensity of IGF-1Ea expression in extravillous trophoblast of the AGA pregnancies compared with the IUGR . The immunohistochemical detection of cytoplasmic IGF-1Ea in the extravillous trophoblast from the third trimester human placentas is shown in . IGF-1Ea expression in endothelium of maternal decidual and the fetal blood vessels in the villi in human placenta The positive immunohistochemical IGF-1Ea staining in endothelium of the maternal decidual vessels from human placentas was not significantly different between the IUGR and AGA third trimester pregnancies. However, when the placental weight was >400 g, more IUGR placentas (81.2%) exhibited IGF-1Ea immunopositivity in uterine vessels from the decidua basalis compared with AGA pregnancies (33.3%; ). In addition, when maternal BMI was taken into consideration, more IUGR placentas exhibited IGF-1Ea immunopositivity in uterine vessels compared with the AGA pregnancies . The immunohistochemical positivity of IGF-1Ea in the endothelium of the fetal vessels within the stem-intermediate and distal-peripheral villi of the IUGR human placentas was significant compared with AGA placentas . As aforementioned, AGA cases were well-matched to the IUGR cases in terms of gestational age >37 weeks and neonatal >2,500 g and placental weight >400 g. Immunopositivity of the IGF-1Ea peptide in the endothelium of the fetal vessels in the stem-intermediate villi of IUGR placentas was significantly associated with gestational age, neonatal and placental weight, maternal ΒΜI and fetal sex compared with AGA placentas . In addition, the positivity of IGF-1Ea in endothelium of the fetal vessels within the distal villi of the IUGR human placentas was significantly associated with gestational age, neonatal and placental weight, maternal ΒΜI >30 kg/m 2 and fetal female sex compared with AGA placentas . When gestational age was ≥37 weeks, more IUGR placentas showed IGF-1Ea immunopositivity in endothelium of the fetal vessels within the stem-intermediate and distal villi compared with the AGA placenta . Similarly, when the neonatal birth weight was ≥2,500 g, more frequent IGF-1Ea immunopositivity was observed in endothelium of the fetal vessels within the stem-intermediate and distal villi of the IUGR placentas compared with AGA placenta. In addition, when the placental weight at birth was ≥400 g, more frequent IGF-1Ea immunohistochemical expression was observed in the endothelium of the fetal vessels within the stem-intermediate and distal villi of the IUGR placentas compared with the AGA placentas. Furthermore, there was a significant difference in the IGF-1Ea expression in endothelium of the fetal vessels within the stem-intermediate and distal villi of the IUGR placentas according to MVM of the placental bed compared with AGA placentas. There was more frequent IGF-Ea immunohistochemical expression in endothelium of fetal vessels within the stem-intermediate and distal villi of IUGR placentas, compared with the AGA placentas. Endothelial immunostaining is shown in .   A total of 62 pregnant patients were recruited. There were significant differences in the mean placental and neonatal birth weight and the gestational age at delivery (all P<0.001) with the IUGR group exhibiting lower values compared with AGA pregnancies . Notably, preterm birth, defined as gestational age at delivery of <37 weeks, was observed only in the IUGR group (100%; ). All cases in the AGA group and 21 cases in the IUGR group gave birth at a gestational age of ≥37 weeks of pregnancy . The fetal sex distribution was 15 male (34.9%) and 28 female (65.1%; n=43; unreported in 19 IUGR cases). In the IUGR group, fetal sex distribution was 53.3% male and 71.4% females, with a male to female ratio of 0.746, while in the AGA group there were 46.7% male and 28.60% female fetuses with a male to female ratio of 1.633, . IUGR pregnancies were highly represented in the group of placentas with changes of MVM (83.3%), VUE (83.3%) and DVM (71.4%), though without any statistically significant differences when compared with the AGA group . Notably placentas from IUGR pregnancies showed histological changes consistent with MVM in 74.5% of cases, VUE in 10.6% and DVM in 10.6%. No differences could be statistically confirmed between the IUGR and AGA groups for any of the histopathological characteristics examined in our sample.   The placental IGF-1Ea mRNA expression levels were determined by qPCR. The mean IGF-1Ea mRNA expression levels were similar between the IUGR and AGA groups, with no significant difference (P=0.991; ). Additionally, no significant associations were observed in the placental IGF-1Ea mRNA expression between the IUGR and AGA pregnancies in relation to the clinical parameters such as maternal age and BMI, gestational age at delivery, fetal sex, neonatal and placental weight at delivery or the histopathological parameters such as MVM, MPFD, FVM, VUE, DVM and other lesions . Furthermore, there was no significant difference in placental IGF-1Ea mRNA expression in IUGR vs. AGA pregnancies in the full-term cases (>37 weeks of gestation; ). Moreover, placental IGF-1Ea mRNA expression in IUGR vs. the AGA pregnancies was not significant in cases with fetal birth weight >2,500 g . Finally, placental IGF-1Ea mRNA expression levels were similar between the IUGR and AGA groups in cases with placental weight >400 g, with no significant difference .   Positive expression of IGF-1Ea protein was observed as granular or homogeneous brown staining localized to the cytoplasm and the cytoplasmic membrane of the perivillous syncytiotrophoblast ( and ) and the extravillous trophoblast of the basal plate and diaphragmatic columns . Positive IGF-1Ea expression was also detected in the endothelium of the fetal vessels within the stem, intermediate and distal chorionic villi, as well as in the endothelium of the maternal decidual vessels .   Of the 47 IUGR pregnancies, six (12.8%) exhibited high expression, 15 cases (31.9%) exhibited moderate expression and 26 (55.3%) had negative IGF-1Ea expression in the perivillous syncytiotrophoblast. Of 15 AGA pregnancies, one case (6.75%) exhibited high expression, two (13.3%) exhibited moderate expression and 12 cases (80%) had negative expression in the perivillous syncytiotrophoblast. These immunohistochemical IGF-1Ea placental expression scores were not significantly different between the IUGR and AGA pregnancies . Additionally, immunohistochemical IGF-1Ea expression scores of the IUGR and AGA pregnancies were not significantly associated with the clinical parameters such as maternal age, neonatal (>2,500 g) and placental weight (>400 g), maternal BMI, fetal sex and the histopathological parameters including MVM, VUE and DVM . In the IUGR group, one case (2.1%) exhibited strong IGF-1Ea intensity, nine (19.1%) exhibited moderate intensity, while 37 cases (78.7%) exhibited weak intensity. In the AGA group, no cases exhibited strong intensity, four (26.7%) exhibited moderate intensity, while 11 (73.3%) exhibited weak expression. The difference in IGF-1Ea expression intensity was not significantly different between the two groups . Additionally, intensity of the IGF-1Ea immunopositivity was not significantly associated with maternal age and gestational age (>37 weeks), neonatal birth weight (>2,500 g), placental weight (>400 g), maternal BMI, fetal sex, MVM and DVM . and showIGF-1Ea immunopositive expression in the perivillous syncytiotrophoblast, respectively.   The positive immunohistochemical IGF-1Ea staining in the extravillous trophoblast was significantly different between AGA and IUGR placentas . In the AGA placentas, IGF-1Ea immunopositive cells were observed in 73.3% of cases, while in the IUGR placentas, the IGF-1Ea immunopositivity was much lower . Additionally, there was a significant difference in the IGF-1Ea immunopositive cells in the extravillous trophoblast according to maternal age <40 years. Notably, patients <40 years in the IUGR group had negative IGF-1Ea immunohistochemical staining in the extravillous trophoblast in most cases; for the AGA group, positive staining was observed in most cases. A significant difference in IGF-1Ea immunopositive cells in the extravillous trophoblast was found between the IUGR and AGA groups in relation to gestational age. Notably, patients with a gestational age of 37 weeks in the IUGR group had negative IGF-1Ea immunohistochemical staining in the extravillous trophoblast in most cases; for AGA group, immunopositive cells were observed in most cases. Moreover, there was a significant difference in the IGF-1Ea immunopositive cells in the extravillous trophoblast between the IUGR and AGA groups according to neonatal birth weight. Newborns weighing >2,500 g had an absence of IGF-1Ea immunopositive cells in the extravillous trophoblast in most of IUGR placentas; in the AGA group, positive staining was observed in 73.3% of placentas. Furthermore, there was a significant difference in the IGF-1Ea immunopositive cells in extravillous trophoblast between the IUGR and AGA groups in relation to placental weight. In total, more than half of IUGR cases with a placental weight >400 g had negative IGF-1Ea staining in the extravillous trophoblast; for AGA, most cases had positive staining. In addition, there was a significant difference in IGF-1Ea immunopositive cells in the extravillous trophoblast between IUGR and AGA groups according to maternal ΒΜI of <30 kg/m 2 . In total, 72.7% of IUGR cases with a maternal BMI of <30 kg/m 2 had negative IGF-1Ea staining in the extravillous trophoblast; for AGA, most cases had positive staining (75.0%). Furthermore, fetal female sex was associated expression of IGF-1Ea in the extravillous trophoblastic cells. Overall, most of IUGR cases of fetal female sex had negative IGF-1Ea staining in the extravillous trophoblast; in AGA, in most cases had positive staining. Additionally, there was a statistically significant difference in IGF-1Ea immunopositive cells in the extravillous trophoblast between the IUGR and AGA groups according to MVM of the placental bed. IGF-1Ea expression was positive in 29.4 and negative in 70.6% of IUGR cases with MVM, while IGF-1Ea expression was positive in 66.7 and negative in 33.3% of AGA cases with MVM . The intensity of IGF-1Ea expression in extravillous trophoblastic cells was not significantly different between AGA and IUGR placentas . Additionally, there was no association with IGF-1Ea staining intensity between the two groups according to maternal age and gestational age (>37 weeks), neonatal birth weight (>2,500 g), placental weight (>400 g), maternal ΒΜI, fetal male sex, MVM and DVM . However, fetal female sex was associated with intensity of IGF-1Ea expression in extravillous trophoblast of the AGA pregnancies compared with the IUGR . The immunohistochemical detection of cytoplasmic IGF-1Ea in the extravillous trophoblast from the third trimester human placentas is shown in .   The positive immunohistochemical IGF-1Ea staining in endothelium of the maternal decidual vessels from human placentas was not significantly different between the IUGR and AGA third trimester pregnancies. However, when the placental weight was >400 g, more IUGR placentas (81.2%) exhibited IGF-1Ea immunopositivity in uterine vessels from the decidua basalis compared with AGA pregnancies (33.3%; ). In addition, when maternal BMI was taken into consideration, more IUGR placentas exhibited IGF-1Ea immunopositivity in uterine vessels compared with the AGA pregnancies . The immunohistochemical positivity of IGF-1Ea in the endothelium of the fetal vessels within the stem-intermediate and distal-peripheral villi of the IUGR human placentas was significant compared with AGA placentas . As aforementioned, AGA cases were well-matched to the IUGR cases in terms of gestational age >37 weeks and neonatal >2,500 g and placental weight >400 g. Immunopositivity of the IGF-1Ea peptide in the endothelium of the fetal vessels in the stem-intermediate villi of IUGR placentas was significantly associated with gestational age, neonatal and placental weight, maternal ΒΜI and fetal sex compared with AGA placentas . In addition, the positivity of IGF-1Ea in endothelium of the fetal vessels within the distal villi of the IUGR human placentas was significantly associated with gestational age, neonatal and placental weight, maternal ΒΜI >30 kg/m 2 and fetal female sex compared with AGA placentas . When gestational age was ≥37 weeks, more IUGR placentas showed IGF-1Ea immunopositivity in endothelium of the fetal vessels within the stem-intermediate and distal villi compared with the AGA placenta . Similarly, when the neonatal birth weight was ≥2,500 g, more frequent IGF-1Ea immunopositivity was observed in endothelium of the fetal vessels within the stem-intermediate and distal villi of the IUGR placentas compared with AGA placenta. In addition, when the placental weight at birth was ≥400 g, more frequent IGF-1Ea immunohistochemical expression was observed in the endothelium of the fetal vessels within the stem-intermediate and distal villi of the IUGR placentas compared with the AGA placentas. Furthermore, there was a significant difference in the IGF-1Ea expression in endothelium of the fetal vessels within the stem-intermediate and distal villi of the IUGR placentas according to MVM of the placental bed compared with AGA placentas. There was more frequent IGF-Ea immunohistochemical expression in endothelium of fetal vessels within the stem-intermediate and distal villi of IUGR placentas, compared with the AGA placentas. Endothelial immunostaining is shown in .   The IGF system is composed of three ligands (insulin, IGF-1 and IGF-2), tyrosine kinase receptors [IGF-1 receptor, (IGF-1R), insulin receptor, mannose 6-phosphate IGF-2R, and the IR/IGF-1R hybrid], IGFBP 1–6 and downstream target proteins including insulin receptor substrate-1, protein kinase B (Akt) and mTOR . IGF-1 is a cell growth factor with a molecular structure similar to insulin, which serves a key role in normal body growth, development and maintenance, as well as cell proliferation, differentiation, migration and survival . IGF-1 gene contains six exons, which undergo alternative splicing during transcription, giving rise to heterogeneous mRNAs, including the three mRNA isoforms IGF-1Ea, IGF-1Eb and IGF-1Ec . The post-translational cleavage of these pro-forms of IGF-1 results in common mature IGF-1 peptide and extension peptides, namely Ea, Eb and Ec, which have a common 16 amino acid sequence in the N-terminal region and different amino acid sequences in the C-terminal region . The most predominant peptide is IGF-1Ea, which has a 35 amino acid Ea-peptide . IGF-1Ea and IGF-1Eb peptides bind strongly to the extracellular matrix, preventing their release into the circulation and therefore exhibit local effects . IGF-1 is secreted by the liver under stimulation of the growth hormone (GH) . IGF-1 is produced by other organs such as skeletal muscle, kidney and brain . In addition, the three isoforms of IGF-1 are expressed in various types of human tissue and may bind to different receptors with different actions. IGF-1 actions are mediated mainly via its binding to the type 1 IGF receptor (IGF-1R), however IGF-1 signaling via insulin receptor (IR) and hybrid IGF-1/IR is also evident. Moreover, there is evidence that the IGF-1Ec isoform may regulate prostate cancer growth via Ec-peptide specific and IGF-1R/IR-independent signaling . During pregnancy, IGF-1 availability in the maternal circulation is primarily regulated by IGF-BPs such as IGFBP-1, synthesized by the decidua and the liver . Phosphorylation of human IGFBP-1 markedly increases its binding affinity for IGF-1 and effectively decreases IGF-1 availability and function . IGF-1 is found in human fetal serum from 15 to 23 week of gestation . Furthermore, birth and placental weights are positively associated with IGF-1 cord blood levels . In addition, maternal IGF-1 levels are positively associated with neonatal and placental weights suggesting that higher maternal IGF-1 levels at mid- and late-gestation indicate greater placental and fetal growth . In obese patients during pregnancy, the increased serum levels of IGF-1 and the low levels of IGFBP-1 increase placental weight and stimulate placental mTOR signaling, which positively regulates key placental functions, including glucose trasporter-1 and amino acid transport as well as mitochondrial biosynthesis, leading to fetal overgrowth . Conversely, IUGR pregnancies are associated with decreased maternal serum IGF-1 and increased IGFBP-1 levels . Additionally, higher IGFBP-1 levels in fetal serum have been found in IUGR pregnancies . Compared with placentas from normal pregnancies, expression of IGF-1 in IUGR full-term placentas is either increased , which may indicate a paracrine and/or endocrine function , or decreased , which may contribute to inhibition of the placental insulin/IGF-1 signaling pathway . Abu-Amero et al  observed no significant difference in placental IGF-1 mRNA expression in IUGR compared with AGA pregnancies. The IGF-1Ea isoform has similar endocrine effects as IGF-1 . To the best of our knowledge, the present study is the first to detect localization of IGF-1Ea peptide in placentas from IUGR and AGA pregnancies, including in syncytiotrophoblast, the extravillous cytotrophoblast and the endothelium of villous fetal vessels, as well as the maternal spiral arteries from the decidua basalis. The prevalence of VUE in IUGR placentas was 10.6%. There are highly variable reported rates of incidence of VUE in IUGR/FGR placentas, ranging from 1.6 to 86.0%, reflecting notable heterogeneity in sample size and study design . The prevalence observed in the present study (10.6%) falls within the lower limits of the reported rates and is in accordance with Nordenvall and Sandstedt , which reported VUE in 7.5% of 161 FGR cases. The present study indicated a negative IGF-1Ea expression in extravillous trophoblast in most IUGR placentas. This suggested selective presence of IGF-1Ea peptide in extravillous trophoblast may reflect the involvement of this isoform in normal placentation as extravillous trophoblast serves a critical role in spiral artery remodeling for normal maternal blood supply . This is further supported by the negative IGF-1Ea expression in extravillous trophoblast in IUGR placentas with MVM, which also included changes in decidual vasculopathy. In patients with IUGR, negative expression of IGF-1Ea in the extravillous trophoblast was also associated with a younger maternal age (<40 years), gestational age, neonatal birth weight and maternal BMI (<30 kg/m 2 ). These findings have an unclear mechanism and may reflect multiple and complex molecular pathways involved in the regulation of IGF1 biological activity . However, the association between reduced IGF-1Ea expression in extravillous trophoblast and the aforementioned clinical and pathological parameters of patients with IUGR may implicate IGF-1Ea isoform in key pathophysiological mechanisms underlying the development of IUGR. In the present study, negative expression of IGF-1Ea was associated with fetal female sex; however, there is no clear explanation for this. Decreased or negative IGF-1Ea expression in extravillous trophoblast in IUGR female fetuses may reflect an inhibitory role of fetal estrogen on IGF-1Ea synthesis, either directly via the IGF-1/GH axis or indirectly via regulation of IGFBP-1 . Moreover, fibroblast cultures show an interaction between IGF-1 and testosterone in controlling IGFBP production . Thus, exposure of IGF-1Ea protein to androgens or estrogens circulating in the fetal blood may lead to decreased synthesis (selective regulation of the synthesis) of certain IGF-1Ea binding proteins, resulting in upregulation of IGF-1Ea expression in endothelium of fetal vessels . Alternatively, other mechanisms and complex pathways may account for sex differences in the IGF-1/IUGR association. A potential role of IGF-1 DNA methylation rate in the development of IUGR was investigated in a previous study, and a fetal sex difference in IGF-1 DNA methylation rate was noted, which is of unclear significance . The present hypotheses concerning the role and interactions of the various IGF-1 binding proteins with sex hormones in regulating IGF-1Ea synthesis, as well as the implication of other mechanisms such as sex differences in DNA methylation and IGF-1 gene regulation, require further elucidation. Extravillous trophoblast serves a critical role in maternal spiral artery remodeling . Immunopositivity for IGF-1Ea peptide within the endothelium of the villous blood vessels in IUGR indicated a potential autocrine function of the IGF-1Ea peptide in the endothelial fetal vessels and suggested that the presence of the IGF-1Ea peptide may induce villous angiogenesis and hypercapillarization to compensate for hypoxic/ischemic and malperfused villi in IUGR placentas. Moreover, IGF-1Ea secretion in the endothelium of the villous blood vessels from IUGR placentas may contribute to enhanced vasodilation of fetal vasculature to enhance the compromised fetal-maternal exchange, demonstrating an autocrine function of IGF-1Ea in chorionic villi. In the present study, the immunopositivity of IGF-1Ea peptide in the endothelium of the villous fetal blood vessels was associated with MVM of the placental bed in IUGR. This further supports the hypothesis of a reactive autocrine IGF-1Ea secretion in malperfused villi. Cho et al  suggested that IGF-1 peptide promotes angiogenesis. In addition, IGF-1 stimulates the expression of angiogenesis-associated growth factors and promotes angiogenesis in endothelial cells via activation of the PI3K/Akt signaling pathway  or through VEGF induction . Through autocrine, paracrine and endocrine mechanisms, IGF-1 induces cellular activity such as DNA synthesis, differentiation, migration and glucose uptake . In addition, IGF-1 stimulation of adipose tissue-derived microvascular fragments enhances capacity for vascularization . IGF-1 promotes vasodilation by upregulating nitric oxide synthase activity in the endothelium and increases production of nitric oxide . In humans, low serum IGF-1 levels are associated with decreased endothelium-dependent vasodilation . By contrast, upregulation of IGF-1R decreases nitric oxide bioavailability and insulin sensitivity in the endothelium . The aforementioned mechanisms may be involved in regulating biological activity of the IGF-1Ea isoform. The association between IGF-1Ea expression in the endothelium of villous fetal vessels with maternal BMI, gestational age and neonatal and placental weight in IUGR pregnancies may suggest, as in the case of the extravillous trophoblast, that multiple and complex molecular pathways are involved in the regulation of IGF-1 biological activity. In addition, a significant difference in immunohistochemically detected IGF-1Ea expression was found in the endothelium of uterine spiral arteries of the decidua basalis from IUGR pregnancies compared with AGA pregnancies, which was associated with placental weight and maternal BMI. These findings suggested that IGF-1Ea protein may promote angiogenesis and vasodilation of maternal uterine vasculature to compensate for maternal malperfusion in IUGR placentas. This effect appears to be influenced by placental weight and maternal BMI. In the present study, differences in mRNA expression levels detected by RT-qPCR, were not consistent with the differences at the protein levels detected immunohistochemically. The enhanced endothelial IGF-1Ea expression in hypoxic villi from IUGR pregnancies shown by immunohistochemistry was not detected by RT-qPCR and immunohistochemically detected IGF-1Ea expression in the extravillous trophoblast was observed in the placentas from AGA pregnancies. This is likely due to the placenta being a histologically heterogeneous organ , showing notable variability in IGF-1Ea protein expression between its structures; this was depicted in this study by the variable immunolocalization of the protein within the optical fields where it was assessed, e.g. areas with variable protein expression in the villous vessels or in the perivillous syncytiotrophoblast, which could only be reflected in a semi-quantitative way of measurement within several optical fields; By contrast, total mRNA levels were obtained from focally resected frozen placental samples and were overall measured (including all histological structures, positive or negative) from only one full-thickness site of the placenta. This procedure and the variability of IGF-1Ea immune-detected expression in different histological placental structures may account for the discrepancy between mRNA and protein expression assessments in the IUGR and AGA groups. Due to the nature of the present study (IUGR vs. AGA), groups could not be matched for gestational age. In IUGR pregnancies birth often takes place before the 37th week of pregnancy, while normal pregnancies can be completed at later weeks (up to 41 weeks). Hence, IUGR births typically have lower gestational age compared with AGA, which is a limitation of the present study. Additionally, the number of IUGR cases completed after the 37th week of pregnancy was almost the same as that of AGA pregnancies (18 and 15, respectively). Moreover, significant associations were revealed, despite the small number of AGA cases. In addition, demographic variables, such as maternal age, BMI, fetal sex and numerous pathological characteristics were not significantly different, ensuring that the differences were mainly due to IUGR rather than other factors. Overall, the present study demonstrated that expression of the IGF-1Ea isoform in extravillous trophoblast was negatively associated with development of IUGR pregnancies and that this IGF-1 isoform may be involved in pathogenesis of placental malperfusion as well as regulation of villous angiogenesis, in the IUGR cases. To the best of our knowledge, the present study is the first to implicate IGF-1Ea expression in IUGR pregnancy development and progression. However, the potential regulatory role of the IGF-1Ea isoform in pathophysiology of IUGR requires further investigation to confirm whether this IGF-1 isoform could be used as a prognostic biomarker and/or a novel therapeutic target in IUGR pregnancy. However use of immunohistochemistry and RT-qPCR without complementary techniques weakens the robustness of the conclusions. Mechanistic studies that include the in vitro treatment of placental cells with synthetic IGF-1Ea peptide may determine IGF-1Ea induced cellular and molecular responses and the potential regulatory role of this IGF-1 isoform in pathophysiology of IUGR. In conclusion, the IGF-1Ea peptide is expressed in full- and preterm placentas and may serve a role in IUGR. Negative expression of the IGF-1Ea peptide in the extravillous trophoblast of IUGR placentas and its association with MVM suggest involvement in defective placentation and development of placental insufficiency. The expression of IGF-1Ea peptide in the endothelium of villous blood vessels may also suggest an autocrine role in regulating villous angiogenesis and vasodilation in malperfused villi of IUGR pregnancies. Similarly, secretion of the IGF-1Ea peptide in endothelium of the maternal decidual vessels may also reflect a reactive response to induce vasodilation in uterine spiral vessels at the maternal side of IUGR placentas, influenced by placental weight and the maternal BMI. Thus, IGF-1Ea peptide in human placentas may mediate trophoblastic invasion and normal placentation, contributing to development of normal pregnancies. The negative placental expression of IGF-1Ea peptide in the extravillous trophoblast and its positive expression within fetal and maternal vessels of IUGR pregnancies also appears to be influenced by clinical parameters such as maternal age (<40 years), gestational age, maternal BMI (<30 kg/m 2 ) and neonatal weight, likely reflecting the multiple and complex molecular pathways that have been implicated in the various biological functions of IGF-1.   