Fetal growth restriction (FGR) or intrauterine growth restriction (IUGR) is the significant reduction of fetal growth rate, resulting in birth weight in the lowest 10th percentile for the gestation age.1 IUGR occurs in 5% to 7% of all pregnancies, and approximately 5% to 10% of all pregnancies complicated by IUGR result in stillbirth or neonatal death.2 IUGR pregnancies have many antepartum and intrapartum complications, such as antepartum steroid treatment, premature delivery, and cesarean delivery.3 Other investigations have indicated that IUGR offsprings have enhanced susceptibility to the low bone mineral content at birth and an increased risk of osteoporosis in later life.4,5 Fetal growth is controlled by numerous genetic, environmental, and nutritional factors. It is widely accepted that diet restriction very likely induced IUGR because of undernutrition.6,7 And study reported that prenatal food restriction induces IUGR and poor-quality articular cartilage in female rat offspring.8 However, there appears to be a significant knowledge gap regarding its mechanism of bone dysplasia in IUGR offsprings.
The abnormal growth and development of human bones are related to the imbalance of calcium and phosphate regulation and the disorder of vitamin D metabolism.5 It has been established that fibroblast growth factor 23 (FGF23) plays a central role in regulating phosphate homeostasis in adults.9 Known that FGF23 belongs to the FGF family member, is produced by osteocytes and targets distant organs in an endocrine fashion.10 And it could be detected in the circulation, and associated with renal phosphate homeostasis and vitamin D metabolism.11 FGF23 also needs the transmembrane coreceptor Klotho to increase the binding affinity of FGF23 to FGF receptors.12 Shao et al. observed the increased Klotho expression in placentas from gestations with fetal macrosomia compared with a normal birth weight group.13 More specifically, in cases of IUGR the low volume of the placenta has been associated with reduced extrauterine skeletal size and increased fracture risk.14 These findings suggested that FGF23 levels in maternal and fetal might be associated with IUGR.
Bone gla protein (BGP), or “osteocalcin”, is the most abundant noncollagenous protein deposited in the bone matrix during bone formation.15 BGP is a special marker of the bone turnover marker, especially the function of newly formed osteoblasts and bone mineralization.16 Thus, we indirectly evaluated the relationship between FGF23 and bone development by detecting the expression of FGF23 in maternal and fetal during different pregnancy and its correlation with BGP expression.
Animals and experimental groups
Adult Sprague Dawley (6–8 weeks) rats (license number: SCXK (Sichuan), 2008-24) weighing 230–270 g (males, n = 20) and 210–245 g (females, n = 50) were purchased from the Chengdu Dashuo Biotechnology Co., Ltd (Sichuan, China). The Committee on the Ethics of Animal Experiments of Sichuan University approved all protocols related to the animal experiments in this study (permit number: 2016-010), and the study was carried out in accordance with the National Institute of Health guidelines for the Care and Use of Laboratory Animals. Adult Sprague Dawley rats were housed in humidity- and temperature-controlled rooms at a 12:12-h light-dark cycle and mated overnight. The presence of a vaginal mucous plug the morning after mating marked gestation day 0 (GD0).
Pregnant rats were housed in individual steel cages, randomly divided into two groups and fed either 50% of their daily food requirement, undernutrition group (UN group, n = 25) or rat chow ad libitum (AD group, n = 25). Both groups had ad libitum access to drinking water. In this study, the pregnant rats were anesthetized with 2% isoflurane and sacrificed on different stage, progestation, GD10, GD15, GD20, and postpartum, respectively (n = 5 per group). The maternal blood samples were collected via the carotid artery, centrifuged, and stored at −80°C until subsequent assays. And placenta and fetal tissue samples (including skull, spine, and thighbone) were also collected and stored at −80°C or fixed in a 10% formaldehyde solution for further analysis. The head-hip diameter was measured as the fetal length. Postpartum pups in each group were weighed, after which the average weight () and standard deviation (SD) of the AD group were calculated. IUGR was defined as weight less than () − 2SD. The survival rats were euthanized by inhaling 5% isoflurane for 30–60 s after the study.
Enzyme-linked immunosorbent assay
FGF23 levels in the serum and tissue homogenates were determined using a commercial FGF23 Kit (Cusabio Company, Wuhan, China) by following the manufacturer's recommendations to determine the dam serum, pup tissue, and placental FGF23 profiles. The BGP expression in fetal rats was also determined by a commercial kit (Cusabio Company). All experiments were run in duplicate, and their mean values are reported.
Real-time quantitative polymerase chain reaction (qPCR) analysis
Total RNA was extracted using TRIzol reagent (Tiangen Biotech Co., Beijing, China), and its integrity was verified via agarose gel electrophoresis. Reverse transcription reactions were performed using ReverTra Ace MMLV reverse transcriptase RNaseH (Thermo Fisher Scientific Inc., Shanghai, China). Real-time qPCR (RT-qPCR) analyses to determine the number of complementary DNA (cDNA) molecules in the reverse-transcribed samples were performed using Mastercycler ep gradient S (Eppendorf, Germany). PCR was performed using 8.7 μL of 2× Maxima SYBR Green qPCR Master Mix (Fermentas Inc., Canada), 0.15 μL of each 5′ and 3′ primer, 1 μL of the cDNA samples, and 10 μL of H2O in a final volume of 20 μL. After the samples were denatured at 95°C for 10 minutes, amplification was performed in three steps as follows: denaturation at 95°C for 15 seconds, annealing at 60°C for 1 minute, and extension at 72°C for 30 seconds. Moreover, SYBR Green fluorescence was detected at the end of the extension, and this value reflected the amount of double-stranded DNA in the reaction mixture. The amplification cycle was carried out 40 times. A melting curve was obtained at the end of each run to discriminate specific from nonspecific cDNA products. The data were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels in the samples. FGF23 primers (forward: 5′-CCCATCAGACTATCTACAGTGCCC-3′ and reverse: 5′-GCTTCGGTGACAGGTAGACGTC-3′) and GAPDH primers (forward: 5′-GCAAGTTCAATGGCACAG-3′ and reverse: 5′-AAGTTCTTCCTGGCCGGTAT-3′) were used in RT-qPCR analysis to assess the expression of the FGF23 gene.
Placental samples were fixed in formalin and embedded in paraffin. Sections (4 μm) were deparaffinized, rehydrated, treated with 4% hydrogen peroxide for 10 minutes in the dark at room temperature, and autoclaved at 95°C for 10 minutes in 0.05 mol/L Tris-EDTA (pH = 9.0) before incubation with rabbit anti-rat FGF23 polyclonal antibody (1:100 dilution, Bioss, China) at 37°C for 2 hours. The sections were incubated with secondary antibody (goat anti-rabbit 1:1 000; Beijing Zhong Shan Biotechnology, Beijing, China) for 45 minutes at 37°C after being washed three times in phosphate buffer (PBS). The sections were counterstained with hematoxylin and mounted on a cover glass. The negative control samples were incubated with PBS as an alternative for the anti-rat FGF23 antibody.
Cell proliferation assay
A human osteoblast-like cell line (MG-63) was provided by the State Key Laboratory of Human Disease Biotherapy, West China Hospital, Sichuan University. The water soluble tetrazolium (WST-1) (Beyotime, China) assay is based on the cleavage of the tetrazolium salt WST-1 in viable cells only, leading to the formation of a soluble formazan salt that can then be measured photometrically. Cells were seeded in a 96-well plate at a density of 1 × 104 cells/well. Then, the cells were cultured in a CO2 incubator at 37°C in conditioned medium with recombinant human FGF23 (rhFGF23) at different concentrations and for different time periods after 12 hours of starvation. After termination of the cell culture experiment, the cells were washed twice with PBS, and 50 μL WST-1 in 500 μL cell culture medium was added to the cells. After 2 hours, the absorbance was measured at 450 nm using an Infinite 200 PRO microplate reader (Tecan Group, Ltd., Switzerland).
Statistical analyses were performed using SPSS 19.0 (SPSS, Inc., Chicago, IL, USA). All experiments were performed in triplicate. The data were analyzed using an independent two-tailed t test and are represented as the mean ± SD. In addition, one-way analysis of variance was used when more than two groups were compared. Spearman rank-order correlation coefficients (continuous variables) and the Mann–Whitney U test (categorical variables) were performed to determine the relationship between the results and clinical parameters. A P < 0.05 according to a two-tailed distribution indicated statistical significance.
In the postpartum period, the AD group gave birth to a total of 46 offsprings, whereas the UN group gave birth to a total of 41 offspring with no stillbirths. Excluded the overweight group (three pups weighing more than 7.08 g (7.90 − 2 × 0.41)), and the average weight of the AD group (7.90 ± 0.41) g was significantly greater than that of the UN group (6.38 ± 0.24) g (P < 0.05, Fig. 1A). In addition, the lengths of the newborn rat in the UN group (5.5 ± 0.3) cm were shorter than AD groups (6.4 ± 0.2) cm (P < 0.05, Figs. 1B, C). No difference was observed in the litter size or the male-to-female ratio between the two groups.
Maternal serum FGF23 level
In the AD group, the maternal serum FGF23 level gradually increased from the progestational stage to the progression of pregnancy until it peaked at GD15, and that at GD20 was significantly lower than that at other stages of pregnancy (P < 0.05, Fig. 2). At the same stages through gestation, the UN group had a lower serum FGF23 level than that in the AD group, particularly at the late stages of pregnancy (P < 0.05, Fig. 2).
Tissue homogenate FGF23 and BGP levels in fetal rats
The FGF23 level slightly decreased from GD10 to GD15 in the fetal rat tissues of both the AD and UN groups and reached a statistically significant maximum value at GD20. All UN fetal rats exhibited lower FGF23 expression than the contemporaneous AD group, and this difference was significant in the fetal rats at GD20 (P < 0.05, Fig. 3A). BGP expression followed a similar trend as FGF23 in fetal rats, but no significant difference was observed between BGP expression in the AD and UN groups (Fig. 3B). The levels of FGF23 and BGP were positively correlated according to correlation analysis in AD group (Fig. 3C) and UN group (Fig. 3D) (r = 0.923, P < 0.05; r = 0.925, P < 0.05, respectively).
Placental FGF23 mRNA and protein expression
Placental FGF23 in both groups were localized to both the decidual and labyrinth zones (Figs. 4A–D), and placental FGF23 level was decreased at GD15 and increased at GD20 significantly (P < 0.05, Figs. 4E–F, H and I). Both the FGF23 protein and mRNA expression levels in the placentas of rats in the UN group were significantly lower than those in the AD group at GD20 (P < 0.05, Figs. 4G, J).
Viability of osteoblast-like cells
We performed a WST-1 assay to investigate the effect of rhFGF23 on the viability of osteoblast-like MG-63 cells. As the OD values could reflect the cell viability, rhFGF23 (0–10 μg/mL) could enhance MG-63 cell viability in a dose- and time-dependent manner (P < 0.05, Fig. 5).
It is clear that poor nutrient provision induced by diet restriction to the fetus is a critical component in the pathogenesis of IUGR. Fetal development is largely dependent on the availability of nutrients to the fetus via maternal circulation, and as placental transport.17 In this study, diet restriction during pregnancy decreased the weight and length of newborn rats. BGP, as an osteoblast-specific marker, is an osteoblast–osteoclast coupling factor.18 The complex mechanism of deposition, liberation, and activation requires the concerted action between osteoblasts and osteoclasts, thus rendering BGP activity finely controllable at different points.19 And levels of the bone turnover marker osteocalcin reflect bone development.20 In the current study, we found that the content of BGP of fetal bone was decreased in UN group without significance. It suggested that prenatal undernutrition partially hindered the ossification and induced bone dysplasia.
FGF23 is a phospholipid hormone that down-regulates the membrane abundance of endoepithelial sodium phosphate co-transporter in proximal renal tubules.21,22 High levels of FGF23 lead to renal phosphate wasting and impaired bone mineralization in patients with autosomal dominant hypophosphatemic rickets, and tumor-induced osteomalacia.23 Series of evidence has suggested the important role of FGF23 in regulating vitamin D and phosphate metabolism and maintaining bone development.9 In our study, the results showed that all UN fetal rats exhibited lower FGF23 expression than the contemporaneous AD group, and this difference was significant in both late pregnancy fetal rats (GD20), and the levels of FGF23 and BGP in both groups were positively correlated according to correlation analysis. In vitro, rhFGF23 enhanced MG-63 cell viability in a dose- and time-dependent manner. It indicated that the decreased BGP level was determined by the low FGF23 expression, thus the bone development was associated with FGF23 in IUGR offsprings by undernutrition.
The previous study reported that the levels of FGF23 in umbilical cord blood were significantly lower than those in neonates 4 days after birth, mothers, and adult volunteers with normal term pregnancies.24 The placental weight and birth weight are predictors of total bone mineral content in neonates.25,26 It indicated that the FGF23 levels are varied during pregnancy and associated with birth weight. In the current study, we revealed the temporal evolution of FGF23 levels in gestation. Our findings suggested that maternal plasma FGF23 levels peaked in GD15 and then decreased gradually in both normal and underfed pregnant rats. However, the peak level of FGF23 was reached in fetal rat and placenta tissues in GD20, these might be associated with the compensation of maternal placenta. Moreover, the expression of FGF23 was decreased compared to controls, and studies have confirmed that iron deficiency may be associated with changes in FGF23 levels.27 It has been reported that the increased levels of circulating FGF23 in mice with pathological conditions such as Hyp mice exerted direct effects on the placenta and affected fetal vitamin D metabolism.28 Hence, we speculated that the change of FGF23 level in the IUGR offsprings was mainly caused by the mother through the placenta.
Undeniably, our study has some limitations. To some extent, we demonstrated that FGF23 promotes the proliferation of human osteoblasts in vitro, understanding the relationship between maternal and fetal FGF23 expression and IUGR with bone metabolism during pregnancy. However, the specific mechanism still needs further research to explore.
In conclusion, prenatal undernutrition could decrease the FGF23 expression in fetal rats caused by the mother through the placenta, and induced IUGR and hindered the ossification. The FGF23 levels are peaked in GD15 mother but peaked in GD20 placenta and fetuses, these requires further investigation. This study explored the pathological mechanism underlying bone dysplasia in IUGR offsprings is essential to develop therapeutic interventions for IUGR.
This work was supported by the National Natural Science Foundation of China (No. 81571465, No. 81871175).
Shun-Ping Gui: data curation, investigation, and writing original draft; Heng Zou: data curation, investigation, and software operation; Yi Bai: data curation and investigation; Min Liu: writing and editing manuscript; Tao Wang: data curation, investigation, and project administration (Supporting); Rong Zhou: funding acquisition, project administration, and writing editing. All authors have read and approved the final version of the manuscript. As well as, they have agreed to be personally accountable for the authors’ own contributions.
Conflicts of Interest
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