From July 2023 to June 2024, pregnant women and their newborns admitted to the Nanning Maternal and Child Health Hospital of Guangxi were recruited. The inclusion criteria were as follows: (1) Gestational age between 34 + 0 and 41 + 6 weeks (singleton pregnancy confirmed by early ultrasound); (2) Birth weight ≦90th percentile for gender and gestational age(using reference standards and growth curves for neonatal birth weight at various gestational ages and sexes by Zhu Li, Zhang Rong); (3) Availability of complete prenatal growth monitoring data (≥3 ultrasound records); (4) Signed informed consent from both pregnant women and their families. The exclusion criteria were: (1) Fetal conditions: major congenital anomalies (ICD-10 codes Q00-Q99), chromosomal abnormalities, stillbirth or termination of pregnancy cases; (2) Maternal conditions: critical obstetric conditions (amniotic fluid embolism, uterine rupture, sepsis, et al), multiple pregnancies, gestational age <34 weeks, infectious diseases(HIV,HBV,syphilis, et al); (3) Incomplete prenatal examination information and materials; (4) Discrepancy >2 weeks between LMP and early ultrasound dating; (5) Participant withdrawal at any stage Participants were divided into two groups: IUGR group and AGA group. This study was approved by the Ethics Committee of the Nanning Maternal and Child Health Hospital, and written informed consent was obtained from all participants or their families prior to enrollment.

Reference standards and growth curves for neonatal birth weight at various gestational ages and sexes were reported by Zhu Li, Zhang Rong, and colleagues in 2015 (16). Intrauterine growth restriction (IUGR) was defined according to the following criteria: (1) Birth weight below the 10th percentile for the corresponding sex and gestational age (BW < 10th percentile); (2) First-trimester ultrasound assessment to confirm gestational age and evaluate fetal morphology;(3) Unlike SGA, IUGR focuses on the fact that a fetus fails to reach its genetic potential due to pathological factors such as placental insufficiency, infection, maternal disease, essentially pathological growth disorder, SGA encompasses pathological IUGR as well as physiological low birthweight infants. The diagnostic criteria for appropriate-for-gestational-age (AGA) neonates were as follows: (1) Birth weight within the 10th to 90th percentiles for the corresponding sex and gestational age; (2) Confirmation of gestational age through early pregnancy ultrasound examination.

The placental tissue was frozen, sectioned into 6 µm sections and stained with HE staining kit (Solarbio, #G1120). Specifically, the 6 µm thick sections of the placenta were air-dried at room temperature for 10–15 min and then stained with hematoxylin solution for 5 min, washed with distilled water to remove excess stain, dipped in differentiating solution for 3–5 s, and then rinsed with water twice for 3–5 min each time. The sections were then stained with eosin solution for 30 s to 2 min, excess stain was removed, and the sections were rapidly dehydrated. The slides were sequentially immersed in ethanol gradients of 75%, 85%, 95%, and 100% for 2 to 3 s each. The slides were immersed in 100% ethanol for 1 min and subsequently cleared with xylene twice, each for 1 min, and mounted using neutral balsam mounting medium (Shenggong Bioengineering Shanghai Co. LTD, #F418FD0271). A total of 5 biological samples were analyzed per group,The placental tissue was then assessed by scoring the H&E-stained sections under 200 ×  magnification as reported previously.

The samples were soaked in a 3% hydrogen peroxide solution at room temperature for 15 min to eliminate endogenous catalase. The tissue sections were then immersed in phosphate-buffered saline (PBS) three times, with each soak lasting 3 min. The samples were treated with 5% normal goat serum for 1 h at room temperature to block nonspecific antigens. The excess serum was removed without washing. The sections were incubated with a blocking solution at a 1:500 dilution for 90 min at room temperature. The samples were subsequently r rinsed with PBS three times, for 5 min each. Next, the slides were incubated with an HRP-conjugated secondary antibody at a 1:70 dilution for 60 min. The samples were subsequently collected and washed with PBS three times, each for 5 min. DAB was then applied to the slides until the chromogenic reaction was visible, resulting in a brown color under microscopic observation, typically within 1–10 min. The slides were washed with distilled water twice, each for a duration of 5 min. Following this, the sections were stained with hematoxylin for 5 min. If excess dye was observed, the slides were rinsed with 1% hydrochloric acid ethanol for about 5 s, followed by a rinse in tap water until the stain turned blue. The sections were dehydrated with a gradient series of alcohols at 70%, 80%, 90%, 95%, and 100% concentration for 3 min each. The slides were then cleared twice with xylene for 5 min. Neutral balsam was used to seal the sections, and photographs were taken. The antibodies used in this study included a primary antibody against Klotho (Rabbit anti-Human/Mouse/Rat, Proteintech, Cat# 28100-1-AP) and an HRP-conjugated secondary antibody [Goat anti-Rabbit IgG (H + L), ZSGB-Bio, Cat# SP-9001], both of which were used for immunohistochemical analysis. IHC-stained sections were imaged using a light microscope at ×200 magnification. A total of 10 biological samples were analyzed per group. Quantification of IHC staining was performed using ImageJ software. Ten randomly selected fields per section at ×200 magnification were analyzed to calculate mean optical density (MOD), which served as the indicator of expression level.

The tissue specimens were air-dried at room temperature, then immersed in anhydrous methanol (DAMAO, #67-56-1) at −20°C for 10 min. Following this, they were rinsed three times with PBS, each wash lasting 5 min, dried completely, and outlined with a hydrophobic barrier pen to prevent overflow. The slides were blocked with PBS containing 1% BSA at room temperature for 40 min to block nonspecific binding. They were then incubated with primary antibodies overnight. After incubation, the samples were rinsed three times with PBS. Subsequently, they were incubated with secondary antibodies at room temperature for 1 h and washed three times with PBS in the dark. Finally, the placenta samples were mounted with coverslips using Anti-Fade Mounting Medium (Beyotime, #P0126-25 ml).Immunofluorescence images were acquired using a fluorescence microscope at ×200 magnification. Quantification of positive staining was done using ImageJ, calculating fluorescence intensity across 5 randomly selected fields.

RNA was extracted from the maternal placenta, and the concentration was assessed using a Nanodrop 2000 (Thermo, USA). cDNA synthesis was performed using reverse transcription with the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) from TaKaRa. The reverse transcription conditions included 37 ℃ for 15 min, 85 °C for 5 s, and completion of the reactions on ice. qPCR was performed using the PerfectStart® Green qPCR SuperMix Kit with the qTOWER 3G Real-Time PCR System (Analytik Jena AG, Germany) according to the manufacturer's instructions. The primers used for Klotho were designed as follows: Klotho-F: 5′-ATGCCGAGCAAGACTCACTGA-3′ and Klotho-R: 5′-ACGCAAAGTAGCCACAAAGGT-3′. For GAPDH, the primers included GAPDH-F: 5′-AGGTCGGTGTGAACGGATTTG-3′ and GAPDH-R: 5′-TGTAGACCATGTAGTTGAGGTCA-3′. All of these primers were obtained from Sangon Biotech Ltd., located in Shanghai, China. We have specified that 200–1,000 ng of total RNA was used per RT-qPCR reaction, depending on sample concentration. Three biological replicates were analyzed per group (n = 3). Performed using SYBR Green. Relative gene expression was calculated using the 2^-ΔΔCt method, normalized to GAPDH. Each reaction was run in technical duplicate or triplicate.

Maternal venous and umbilical cord blood were collected and centrifuged at 3,500 rpm for 10–15 min. The collected supernatant was transferred to a new centrifuge tube and stored at −20 °C or −80 °C until further use. Human serum and umbilical cord blood concentrations of GH, IGF-1, and IGFBP-1 were measured using ELISA kits specific to human targets: GH (Abbexa, Cat# abx250478), IGF-1 (Abcam, Cat# ab100545), and IGFBP-1 (Thermo Fisher Scientific, Cat# EHIGFBP1), All procedures were performed strictly according to the manufacturers' instructions. Following the kit instructions, serial dilutions of Klotho standards, typically from high to low concentrations, are prepared in 6–8 different concentrations. For example, 0 pg/ml, 50 pg/ml, 100 pg/ml, 200 pg/ml, 400 pg/ml, 800 pg/ml, and 1,600 pg/ml. Each concentration is tested in duplicate or triplicate. Different concentrations of standard solutions were added to the corresponding wells of the microplate, with 100 μl typically added to each well. The prepared samples were added to the wells of the microplate, with 100 μl added to each well. Similarly, each sample was tested in duplicate or triplicate. After sample loading, the microplate was sealed with a plate sealing membrane and incubated in an incubator maintained at 37 °C for 1–2 h to allow the Klotho antigen in the sample to bind to the antibody coated on the microplate. At the end of incubation, the microplate was removed, and the liquid in the wells was discarded, followed by washing with a wash buffer. Generally, the wells were washed 3–5 times, with 300–400 μl of wash buffer added to each well each time. After soaking for 1–2 min, the liquid in the wells was blotted dry. 100 μl of biotin-conjugated anti-Klotho detection antibody was added to each well, and the microplate was sealed with a plate sealing membrane and incubated in an incubator maintained at 37 °C for 1 h. The microplate was washed 3–5 times with wash buffer to remove unbound assay antibodies. 100 μl of enzyme-labeled avidin was added to each well, and plates were sealed and incubated at 37°C for 30–60 min. The washing operation was repeated to ensure adequate removal of unbound enzyme-labeled avidin. 100μl substrate solution was added to each well, gently mixed, and then the microplate was placed into a constant temperature incubator at 37 °C for 15–30 min in the dark for color development, and the liquid color change in the well was observed. When the appropriate degree of color development was reached, 50 μl of termination solution was added to each well to terminate the enzymatic reaction, at which point the color no longer changed. The microplate was placed into the microplate reader and the appropriate wavelength (usually 450 nm) was selected to read the absorbance value (OD) of each well.

The 6 µm-thick slide sample was carefully removed and air-dried at room temperature for 10–15 min. Subsequently, the cells were stained with freshly prepared Weigert iron hematoxylin for 5–10 min, followed by differentiation using an acidic ethanol solution. The samples were then rinsed thoroughly with distilled water, treated with Masson's bluing solution to restore blue coloration, and washed again with distilled water. Ponceau fuchsin staining solution was applied for 5–10 min. During this process, a weak acid working solution (prepared in a ratio of distilled water to weak acid solution = 2:1) was used to wash the samples for 1 min. The samples were further washed with phosphomolybdic acid solution for 1–2 min, followed by another wash with the weak acid working solution for 1 min. Aniline blue staining solution was then applied for 1–2 min, followed by an additional wash with the weak acid working solution for 1 min. Finally, the slides were rapidly dehydrated with 95% ethanol, dehydrated in three cycles with absolute ethanol (each lasting 5–10 s), cleared with xylene in three cycles (each lasting 1–2 min), and mounted with neutral resin. Five biological replicates were analyzed per group (n = 5). Masson's trichrome-stained sections were observed and photographed using an optical microscope at ×200 magnification.

All data were plotted and subjected to statistical analysis using GraphPad Prism version 10.1.2. An unpaired t-test with Welch's correction was employed to compare the means between two independent samples. For comparisons involving multiple groups, one-way ANOVA was used. Measurement data are presented as mean ± standard deviation (mean ± SD). Statistical significance was defined as two-tailed p < 0.05.

Fifty-two subjects who met the inclusion criteria were recruited from Nanning Maternal and Child Health Hospital. The subjects' basic demographic characteristics are shown in Table 1. The subjects were divided into two groups: AGA groups (n = 30) and IUGR groups (n = 22). The mean ± standard deviation of the age of the pregnant women in the AGA group was 30.53 ± 5.29 years (range 20–40 years), and that in the IUGR group was 31.04 ± 4.57 years (range 22–38 years). Statistical analysis was performed using an unpaired t-test for continuous variables and a chi-square test for categorical variables, it revealed that the gestational age at delivery and birth weight in the IUGR group were significantly lower than those in the AGA group (****p < 0.0001) (Table 1).

GH1 and IGF-1 in placental samples were measured by immunofluorescence. The fluorescence intensity was quantified with ImageJ software to assess changes in protein expression associated with intrauterine growth restriction (IUGR). Immunofluorescence staining revealed IGF-1 (green) and nuclear (DAPI) fluorescence (Figure 6), with the mean fluorescence intensity of IGF-1 protein being significantly increased in the placentas of the IUGR group (n = 5) compared to the AGA group (n = 5) (***p < 0.001) (Figure 7). Similarly, GH1 (green) and nuclear (DAPI) fluorescence staining (Figures 2, 3) demonstrated that the mean fluorescence intensity of GH1 protein was significantly reduced in the placentas of the IUGR group (n = 5) compared to the AGA group (n = 5) (***p < 0.001) (Figure 8). Fluorescence intensity was quantified by measuring the mean fluorescence intensity (MFI) using ImageJ software, with background fluorescence subtracted to ensure accurate quantification. An unpaired t-test with Welch's correction was employed to compare the meanings between two independent samples. For comparisons involving multiple groups.

Serum levels of GH, IGF-1, and IGFBP-1 were determined using ELISA. The results showed that, compared with the AGA group venous blood GH levels were significantly increased in the IUGR group (p < 0.05), while IGF-1 levels were significantly decreased (p < 0.01). No significant difference was observed for IGFBP-1 levels. In the IUGR group, GH levels of umbilical cord blood were significantly reduced (p < 0.01), IGF-1 levels were markedly decreased (p < 0.001), and IGFBP-1 levels were significantly elevated (p < 0.01). These findings are summarized in Figures 9. An unpaired t-test with Welch's correction was employed to compare the meanings between two independent samples. For comparisons involving multiple groups.