Histological, Immunofluorescence, and Biochemical Evaluation of the First 24-Hour Effects of Betamethasone on Fetal Lung Maturation: An Experimental Rat Study

Histological, Immunofluorescence, and Biochemical Evaluation of the First 24-Hour Effects of Betamethasone on Fetal Lung Maturation: An Experimental Rat Study | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 4 September 2025 V1 Latest version Share on Histological, Immunofluorescence, and Biochemical Evaluation of the First 24-Hour Effects of Betamethasone on Fetal Lung Maturation: An Experimental Rat Study Authors : Burak Cakmak 0000-0001-8371-6183 [email protected] , Merve Acikel Elmas , Samed Ozer , Sezin Çevik , Serap Arbak , and Alev Atis Aydin Authors Info & Affiliations https://doi.org/10.22541/au.175698572.28287467/v1 126 views 97 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Objective: To examine the short-term (first 24 hours) effects of antenatal betamethasone on fetal lung maturation using an experimental rat model, including evaluation of histology, surfactant protein A1 (SP-A1) levels, and surfactant localization. Study Design: Nine pregnant Sprague Dawley rats were randomly assigned to either a single-dose, double-dose, or control group. Cesarean sections were performed at 6, 12, and 24 hours after the intervention. Lung tissues were analyzed histologically with hematoxylin–eosin staining. Surfactant localization was evaluated by immunofluorescence, and SP-A1 levels in amniotic fluid were determined using ELISA. Statistical analyses included ANOVA and Kruskal–Wallis tests. Results: There were no significant histological differences observed between groups at any time point. Surfactant protein expression detected by immunofluorescence was minimal and showed no meaningful variation between groups. SP-A1 levels did not differ significantly at 6, 12, or 24 hours (all p > 0.05). A non-significant trend toward higher SP-A1 levels was noted in the single-dose group at 6 hours. No differences were found between single- and double-dose regimens. Conclusion: Betamethasone did not significantly speed up fetal lung development within the first 24 hours in this animal model. These results indicate that the positive effects of antenatal corticosteroids may not appear immediately after administration, emphasizing the need for careful use in pregnancies at risk of delivery within 24 hours. INTRODUCTION Preterm birth, defined as delivery before 37 completed weeks of gestation, remains a leading cause of neonatal morbidity and mortality worldwide 1 . Pulmonary immaturity, particularly respiratory distress syndrome (RDS), is one of the most significant contributors to adverse neonatal outcomes. 2 To mitigate these risks, antenatal corticosteroids (ACS) have become a cornerstone in the management of pregnancies at risk of preterm labor. Among ACS options, betamethasone is widely used due to its potent glucocorticoid activity, long half-life, and ability to cross the placenta in its active form. Its primary benefit lies in enhancing fetal lung maturation by inducing surfactant production and promoting structural changes that improve pulmonary compliance. 3 However, despite its proven efficacy, recent literature has raised growing concerns about the potential overuse of betamethasone, particularly in cases where preterm delivery does not occur within the optimal therapeutic window. Emerging data suggest that unnecessary or mistimed ACS exposure may carry long-term neurodevelopmental, metabolic, cardiovascular, and reproductive consequences, particularly for term infants who are inadvertently exposed. 4 Studies have linked ACS to increased risks of mental and behavioral disorders, 5 alterations in glucose metabolism, cardiovascular abnormalities, 6 and impaired reproductive health in animal models 7 and human cohorts. These concerns have prompted calls for more judicious use of corticosteroids, particularly in scenarios where the timing or necessity of preterm delivery is uncertain. Betamethasone’s mechanism of action involves binding to intracellular glucocorticoid receptors, resulting in the transcription of genes that drive cellular differentiation and surfactant synthesis in type II alveolar cells. 8 Pulmonary surfactant is a complex lipoprotein mixture composed of phospholipids and four primary surfactant-associated proteins (SP-A, SP-B, SP-C, and SP-D). 9 Among these, surfactant protein A1 (SP-A1) plays a crucial role in surfactant metabolism, alveolar stability, and innate immune defense. SP-A1 expression increases significantly in late gestation and is a sensitive marker of fetal lung maturity. Although the benefits of ACS generally begin after 24–48 hours and peak within seven days, many pregnancies at risk for preterm birth deliver within 24 hours of steroid administration. 10 There is limited evidence evaluating the short-term biological efficacy of betamethasone within this early window. Understanding whether a single or double dose of betamethasone can meaningfully enhance lung maturation within 24 hours is essential for refining ACS protocols and minimizing unnecessary exposure. This study aimed to evaluate the short-term (first 24 hours) effects of antenatal betamethasone administration on fetal lung development using a pregnant rat model. We assessed histological changes in lung architecture, SP-A1 levels in amniotic fluid, and surfactant localization via immunofluorescence following single- and double-dose corticosteroid regimens. By analyzing multiple time points post-administration (6, 12, and 24 hours), our goal was to determine whether early betamethasone exposure offers measurable benefits and to inform more targeted and responsible clinical use. MATERIALS AND METHODS Rationale for Animal Model Rodent models are widely used in experimental research investigating fetal lung maturation during pregnancy. In particular, Sprague Dawley rats are preferred due to their well-defined pregnancy physiology, clearly delineated fetal developmental stages, and consistent reproductive performance. Rats have an average gestation period of approximately 21 days, with the most rapid phase of fetal lung development occurring between gestational days 17 and 21, which corresponds to the last trimester of human pregnancy. This short but critical time frame enables precise assessment of prenatal interventions over narrow intervals. Based on these scientific, ethical, and practical considerations, the Sprague Dawley rat was selected as the model organism in this study. Laboratory Animals Animals were supplied by the Acıbadem Mehmet Ali Aydınlar University, Experimental Animals Application and Research Center (ACU-DEHAM). The sample size was calculated using G*Power Version 3.1.6, assuming a medium effect size (f = 1.0), a significance level of α = 0.05, and 80% power, resulting in a total of nine pregnant rats (250–300 g). Animals were housed in individually ventilated cages under a 12-h light/12-h dark cycle (lights on at 07:00, off at 19:00), with a temperature of 21–23°C and a relative humidity of 60–65%. They were provided with autoclaved bedding and nesting material for environmental enrichment. Standard laboratory chow (Altromin rat/mouse maintenance diet, Lage, Germany) and water were provided ad libitum. Ethical approval was obtained from the Acıbadem Mehmet Ali Aydınlar University Animal Experiments Local Ethics Committee (ACU-HADYEK; Decision No. 2023/46, dated 23.06.2023). All experimental procedures were conducted by the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals and the ARRIVE reporting guidelines. Pregnancy Determination Nine female Sprague Dawley rats (250–300 g) were mated with male rats between 09:00 and 11:00 for five consecutive days (one estrous cycle). Vaginal plugs were checked twice daily to determine pregnancy. Vaginal smears were performed on animals without visible plugs; dams with sperm detected on cytological examination were considered pregnant. Pregnant rats were housed individually under the controlled conditions described above. Experimental Design and Groups To evaluate the effects of betamethasone on fetal lung maturation, pregnant rats were randomly allocated into three groups (n = 3 per group): single-dose betamethasone (SD), double-dose betamethasone (DD), and a control group. Each group was further divided into three subgroups according to sacrifice time (6 h, 12 h, and 24 h after injection; Figure 1). Betamethasone (Celestone Chronodose, 6 mg/mL) was diluted by adding 9 mL of 0.9% Sodium Chloride to 1 mL of stock to obtain a 10 mL solution containing 6 mg of betamethasone. From this solution, 1 mL was withdrawn into an insulin syringe to administer 0.10 mg/kg -1 betamethasone acetate. Single-dose group (SD): Cesarean sections were performed at six h (15:00 on GD 16), 12 h (21:00 on GD 16), or 24 h (09:00 on GD 16) post-injection. Double-dose group (DD): Dams received two injections 12 h apart. Cesarean sections were performed at six h (15:00 on GD 16), 12 h (21:00 on GD 16), or 24 h (09:00 on GD 16) after the second injection. Control group: Dams received no injections; cesarean sections were performed at 09:00, 15:00, and 21:00 on GD 16. The number of pups per litter varied, and insufficient amniotic fluid was obtained from some fetuses, resulting in different sample sizes for surfactant protein A1 (SP-A1) analyses across groups and time points. Tissue Collection At the specified time points, pregnant rats were anesthetized with 2–3% isoflurane in oxygen at a flow rate of 1.0 L/min using a calibrated vaporizer. The absence of a withdrawal reflex confirmed the depth of anesthesia. Euthanasia was performed after cesarean section by cervical dislocation. Following laparotomy, uterine horns were exposed, and fetuses were exteriorized under sterile conditions. Amniotic fluid was aspirated into insulin syringes for biochemical analysis. Fetuses were then euthanized by rapid cooling on dry ice, and fetal lung and placenta tissues were dissected, fixed, or snap-frozen as required. Histological Examination Lung tissue was fixed in 10% neutral buffered formalin and processed through ascending ethanol (70%, 90%, 96%, 100%) and xylene before paraffin embedding. Paraffin-embedded tissues were sectioned at 5 µm thickness on a rotary microtome (Thermo-Shandon™ Finesse™ ME+). Sections were stained with hematoxylin and eosin (H&E) to evaluate lung architecture. Representative areas were imaged under a light microscope (Zeiss A1 Axio Scope, Germany) at 20× and 40× magnification. Surfactant Immunofluorescence Staining To detect surfactant protein A (SP-A), 5 µm lung sections fixed with 4% paraformaldehyde were deparaffinized in xylene, rehydrated through graded ethanol, and subjected to antigen retrieval in citrate buffer (pH 6.0) at 100 Watts for 20 min in a microwave oven. Nonspecific binding was blocked with 5% goat serum for one hour. Sections were incubated overnight at 4°C with rabbit anti-surfactant protein A antibody (Abcam, USA; Cat. No. ab51891; 1:200), followed by three washes in PBS and incubation with DyLight™ 488-conjugated goat anti-rabbit secondary antibody (Invitrogen, USA; Cat. No. 35552; 1:500) for one h at room temperature. Nuclei were counterstained with DAPI-containing mounting medium (Abcam, UK; Cat. No. ab104139). Slides were examined and imaged using a confocal microscope (Zeiss LSM 700, Germany). Investigators were blinded to group allocation during image acquisition and analysis. Biochemical Analysis SP-A1 levels in amniotic fluid were measured using a rat SP-A1 ELISA kit (BT-Lab, China) with a sensitivity of 0.05 ng/mL and intra- and inter-assay coefficients of variation of <10% and antibodies were incubated with amniotic fluid samples. After sequential incubation with biotinylated detection antibody, streptavidin-HRP, and chromogenic substrate, absorbance was measured at 450 nm using a BIO-TEK ELx800 microplate reader. Washing steps were performed on a BIO-TEK ELx50 automated washer. Statistical Analysis Data were analyzed using GraphPad Prism Version 9.0 (GraphPad Software, San Diego, CA, USA). Normality was tested with the Shapiro–Wilk test, and homogeneity of variance was assessed with Levene’s test. Parametric data were compared using one-way ANOVA with Tukey’s post-hoc test, while non-parametric data were analyzed using Kruskal–Wallis with Dunn’s post-hoc test. A p-value < 0.05 was considered statistically significant. Histological Findings The morphological evaluation of fetal lung tissue sections from different experimental groups is presented in Figure 2. Across all groups, immature alveolar structures displayed similar histological characteristics. Expansion of the air spaces (incipient alveolar spaces), characteristic of this embryonic stage, was observed, with active participation of the surrounding mesenchymal tissue in the development of these spaces. In the control group (Figure 2A), the developmental structures appeared to be in the early stages of formation, with no apparent signs of acceleration or maturation associated with pharmacological intervention. Following single-dose betamethasone administration, histological examinations at 6 hours (Figure 2B), 12 hours (Figure 2C), and 24 hours (Figure 2D) revealed no significant morphological differences compared to the control group. The immature alveolar morphology remained preserved throughout these time points. Similarly, in the double-dose betamethasone groups, lung tissue assessments at 6 hours (Figure 2E), 12 hours (Figure 2F), and 24 hours (Figure 2G) showed no meaningful alterations in alveolar architecture. The structural features of immature alveoli were comparable to those of the control and single-dose groups. Collectively, these findings suggest that betamethasone does not exert a notable maturational effect on fetal lung histology within the first 24 hours following administration. All histological assessments were performed on sections stained with hematoxylin and eosin (H&E), with images captured at 20× magnification and corresponding inset images at 40× (Figure 2). Immunofluorescence Findings Figure 3 presents the results of immunofluorescence staining performed on fetal lung tissue sections from all experimental groups. Surfactant protein-positive cells appear as bright green fluorescent signals, while cellular nuclei are counterstained with DAPI, emitting blue fluorescence. Areas where both stains overlap clearly indicate cells expressing surfactant protein. Surfactant-positive cells were observed in limited numbers across all groups and were predominantly localized within the alveolar regions. Cells exhibiting positive staining are marked with white arrows in the figure. All images were acquired at 20× magnification and represent merged fluorescence channels. These findings indicate that there was no notable increase in the distribution or intensity of surfactant protein expression among the experimental groups . Thus, betamethasone administration did not significantly enhance surfactant production at the immunofluorescence level within the first 24 hours following treatment. SP-A1 (Pulmonary Surfactant-Associated Protein A1) Levels In this study, fetal surfactant-associated protein A1 (SP-A1) levels were compared across the single-dose (SD), double-dose (DD), and control groups at 6, 12, and 24 hours following antenatal corticosteroid (betamethasone) administration. For each time point, the mean, standard deviation (SD), and median values were calculated, and statistical analyses were conducted using the independent samples t-test. Table 1 summarizes the comparisons between SD and DD groups versus the control group at each time point. No statistically significant differences in SP-A1 levels were detected at any time point (p > 0.05). Although the SD group at 6 hours showed a higher mean SP-A1 concentration compared to the control, this difference was not statistically significant (p = 0.567). This trend is visually illustrated in Figure 4, where the SD group at 6 hours displays a broader distribution and higher mean value; however, the “n.s.” (non-significant) label on the boxplot confirms the lack of statistical significance. Table 2 presents the direct comparison between the SD and DD groups at 6, 12, and 24 hours. These analyses also revealed no statistically significant differences in SP-A1 levels between the two dosing regimens at any time point. In summary, no significant increase in fetal surfactant protein A1 levels was observed within the first 24 hours following betamethasone administration , and dose variation did not confer a measurable early-phase benefit . These findings suggest that the early effect of betamethasone on surfactant production may be limited during the first 24 hours of exposure. DISCUSSION This study investigated the early effects of antenatal corticosteroid administration—specifically betamethasone—on fetal lung maturation within the first 24 hours using an experimental animal model. A notable strength of this work lies in its design, which involved evaluating three distinct time points (6, 12, and 24 hours) following both single- and double-dose administrations of betamethasone. As such, this study is one of the few in the literature that aims to characterize the time-dependent nature of corticosteroid efficacy in the immediate post-administration period. Our findings demonstrate that neither surfactant protein A1 (SP-A1) levels in amniotic fluid nor histological assessments of fetal lung tissue exhibited significant changes within the first 24 hours of corticosteroid exposure. These results suggest that betamethasone does not exert a measurable maturational effect during the earliest phase post-administration. Considering the potential adverse effects associated with corticosteroids, this raises an important clinical consideration: in cases where preterm birth is anticipated within 24 hours, the risk–benefit profile of antenatal corticosteroid (ACS) use may be more limited than previously assumed. A 2015 World Health Organization report noted that ACS is most effective within 7 days of administration, with benefits typically beginning to emerge after 24–48 hours. 11 These timelines align with our results, which emphasize that clinically meaningful effects may not be observable within the first 24 hours. Recent large-scale population-based studies have also highlighted potential long-term safety concerns associated with ACS. A Finnish prospective cohort study by Räikkönen et al. involving 670,097 singleton children born between 2006 and 2017 found a significant association between ACS exposure and an increased risk of childhood mental and behavioral disorders. The cumulative incidence of such disorders was 12.01% in exposed children versus 6.45% in unexposed peers (absolute risk difference: 5.56%; HR: 1.33; 95% CI: 1.26–1.41). In term-born infants specifically, ACS exposure increased the risk from 6.31% to 8.89% (HR: 1.47; 95% CI: 1.36–1.69). 5 Moreover, a discordant sibling-pair analysis within the same study revealed that ACS-exposed siblings had a 6.56% incidence of psychiatric disorders, compared to 4.17% in their non-exposed counterparts (HR: 1.38; 95% CI: 1.21–1.58), suggesting that these effects may be independent of shared genetic and environmental factors. In contrast, among preterm infants, although the incidence of disorders remained higher in the ACS group (14.59% vs. 10.71%), the relative risk was not statistically significant (HR: 1.00; 95% CI: 0.92–1.09), possibly indicating a balance between the short-term neonatal benefits (e.g., reduced RDS or IVH) and long-term risks. Tijsseling et al. (2012) conducted a histopathological study of hippocampal tissues in preterm neonates and found a reduction in neuronal density and increased gliosis in infants exposed to antenatal glucocorticoids, suggesting possible structural alterations in brain regions critical for learning and memory. 12 Kelly et al. (2012) found associations between prenatal glucocorticoid exposure and increased aortic stiffness as well as impaired glucose metabolism in adolescence, highlighting possible long-term cardiovascular and metabolic sequelae. 6 In an animal study, Ni et al. (2018) reported that prenatal betamethasone exposure led to increased placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) expression, reduced birth weights, and long-term metabolic impairments in adult offspring, including hyperglycemia, glucose intolerance, reduced insulin secretion, and dyslipidemia. 13 A recent national cohort study by Ho et al. (2025) analyzed the relationship between the timing of ACS exposure and the risk of childhood mental health disorders. The study found that ACS administration before 34 weeks’ gestation was associated with a significantly increased risk of ADHD and developmental delays in early preterm infants, but not in those born at term. 14 Furthermore, two experimental studies by Borges et al. (2017) evaluated the long-term effects of antenatal betamethasone exposure on reproductive health in male and female rats. In males, exposure led to reduced body weight, sperm motility, and count, with increased abnormal sperm ratios, testicular changes, and hormonal alterations. In females, diminished ovarian reserve and altered follicle profiles were noted, implying reduced fertility potential in adulthood. 7,15 Taken together, these findings suggest that ACS should be reserved for cases with a high likelihood of imminent preterm delivery, and caution should be exercised in term pregnancies or borderline indications. The long-term neurodevelopmental, cognitive, metabolic, and reproductive consequences of unnecessary ACS exposure—particularly in infants who ultimately deliver at term—warrant careful risk-benefit evaluation. Our results support this approach by reinforcing that ACS administration may not offer significant benefit within the first 24 hours, especially in borderline cases where early delivery is uncertain. Limitations This study has several limitations. First, the effects of betamethasone were assessed only within the first 24 hours following administration. Given that prior literature indicates optimal corticosteroid efficacy typically begins after 24–48 hours and lasts for up to 7 days, our time frame may not have captured the drug’s peak effect. Second, as an animal-based study, the results cannot be directly extrapolated to human physiology. Differences in placental structure, fetal development rate, and hormonal regulation across species must be considered when interpreting these findings. Third, sample size was deliberately limited, as is common in animal studies, which may have reduced the statistical power of some comparisons. Future studies should include larger cohorts to validate these findings. In addition, no subgroup analysis was conducted based on fetal sex, despite known sex-related differences in physiological responses to corticosteroids. Despite these limitations, our study provides original and valuable insights by characterizing the early time course of ACS effects and highlighting the importance of timing in clinical decision-making. Future research should be designed to evaluate both longer post-treatment windows (e.g., beyond 24 hours) and long-term systemic outcomes. Consideration of additional variables, such as fetal sex, placental enzyme activity, and epigenetic modulation , will be essential for developing more comprehensive models. CONCLUSION This study examined the early effects of the antenatal corticosteroid betamethasone on fetal lung development. Specifically, it aimed to evaluate the impact of betamethasone within the first 24 hours of administration and to assess whether dosing differences (single vs. double dose) influenced its effectiveness. In this experimental model, histological and immunofluorescence analyses were performed on fetal lung tissue, and surfactant protein A1 (SP-A1) levels in amniotic fluid were measured following single- and double-dose betamethasone administration. Our findings indicate that neither dosing regimen resulted in significant changes in fetal lung histology nor did they lead to a statistically meaningful increase in surfactant production within the first 24 hours. Although the single-dose group showed a higher mean SP-A1 level at 6 hours, this difference did not reach statistical significance. Furthermore, no significant differences were observed between the two dosing groups in terms of SP-A1 levels. These results suggest that betamethasone has limited efficacy in promoting fetal lung maturation during the first 24 hours following administration . The findings imply that in pregnancies at high risk of preterm delivery, where birth is expected to occur within 24 hours, betamethasone administration may not exert a substantial maturational effect on the fetal lungs . This highlights the need for individualized clinical decision-making, weighing the potential benefits and risks before administering antenatal corticosteroids. Previous literature has raised concerns regarding the long-term neurodevelopmental, cognitive, and metabolic consequences of antenatal corticosteroid exposure, particularly when administered unnecessarily. In this context, re-evaluating the use of betamethasone in patients with imminent but uncertain delivery within 24 hours is warranted . In conclusion, the early effectiveness of betamethasone appears limited within the first 24 hours, and its administration during this timeframe should be carefully considered , especially in light of its potential long-term effects. Future research with larger cohorts and extended follow-up periods is needed to understand better the long-term impact of antenatal corticosteroids and their relationship with various physiological, developmental, and molecular parameters. ACKNOWLEDGMENTS The authors thank the staff of the Acibadem Mehmet Ali Aydinlar University Experimental Animals Application and Research Center (ACU-DEHAM) for their technical assistance. FUNDING This study received no external funding. CONFLICT OF INTEREST The authors declare no conflicts of interest. AUTHOR CONTRIBUTIONS B.C. conceived and designed the study and wrote the manuscript. M.A.E. and S.A. conducted the histological and immunofluorescence analyses. S.O. assisted with the surgical procedures on animals and sample collection. A.A.A. supervised the research process and contributed to manuscript editing and critical review. ETHICAL APPROVAL Animal experimentation was approved by the Acibadem Mehmet Ali Aydinlar University Animal Experiments Ethics Committee (ACU-HADYEK; No. 2023/46, dated 23.06.2023). REFERENCES 1. Manuck TA, Rice MM, Bailit JL, Grobman WA, Reddy UM, Wapner RJ, et al; Eunice Kennedy Shriver National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network. Preterm neonatal morbidity and mortality by gestational age: a contemporary cohort. Am J Obstet Gynecol. 2016;215(1):103.e1-103.e14. doi:10.1016/j.ajog.2016.01.004 2. Ma YJ, Sun Y, Zhang CH. Adverse perinatal outcomes associated with respiratory distress syndrome in preterm infants: a retrospective analysis. Ital J Pediatr. 2025;51(1):235. doi:10.1186/s13052-025-02061-0 3. Schmidt AF, Kannan PS, Bridges JP, et al. Dosing and formulation of antenatal corticosteroids for fetal lung maturation and gene expression in rhesus macaques. Sci Rep. 2019;9:9039. doi:10.1038/s41598-019-45171-6 4. Chung HW, Yu CH, Huang CY, Liang FW. Antenatal corticosteroid exposure and neonatal outcomes in term infants. Early Hum Dev. 2025;208:106325. doi:10.1016/j.earlhumdev.2025.106325 5. Räikkönen K, Gissler M, Kajantie E. Associations between maternal antenatal corticosteroid treatment and mental and behavioral disorders in children. JAMA. 2020;323(19):1924-1933. doi:10.1001/jama.2020.3937 6. Kelly BA, Lewandowski AJ, Worton SA, Davis EF, Lazdam M, Francis J, et al. Antenatal glucocorticoid exposure and long-term alterations in aortic function and glucose metabolism. Pediatrics. 2012;129(5):e1282-e1290. doi:10.1542/peds.2011-3175 7. Borges CD, Dias AF, Silva PV, Rosa JL, Guerra MT, Silva RF, et al. Long-term adverse effects on reproductive function in male rats exposed prenatally to the glucocorticoid betamethasone. Toxicology. 2017;376:15-22. doi:10.1016/j.tox.2016.04.005 8. Anti NAO, Gheorghe CP, Deming DD, Adeoye OO, Zhang L, Mata-Greenwood E. Perinatal glucocorticoid sensitivity in the preterm newborn: molecular mechanisms, endogenous determinants, and clinical implications. Front Endocrinol (Lausanne). 2025;16:1587891. doi:10.3389/fendo.2025.1587891 9. Sati L, Seval-Celik Y, Demir R. Lung surfactant proteins in the early human placenta. Histochem Cell Biol. 2010;133(1):85-93. doi: 10.1007/s00418-009-0642-9. 10. Berger R, Stelzl P, Maul H. Administration of antenatal corticosteroids: optimal timing. Geburtshilfe Frauenheilkd. 2024;84(1):48-58. doi:10.1055/a-2202-5363 11. World Health Organization. WHO Recommendations on Interventions to Improve Preterm Birth Outcomes. Geneva: World Health Organization; 2015. 12. Tijsseling D, Wijnberger LDE, Derks JB, van Velthoven CTJ, de Vries WB, van Bel F, et al. Effects of antenatal glucocorticoid therapy on hippocampal histology of preterm infants. PLoS One. 2012;7(3):e33369. doi:10.1371/journal.pone.0033369 13. Ni L, Pan Y, Tang C, Xiong W, Wu X, Zou C. Antenatal exposure to betamethasone induces placental 11β-hydroxysteroid dehydrogenase type 2 expression and the adult metabolic disorders in mice. PLoS One. 2018;13(9):e0203802. doi:10.1371/journal.pone.0203802 14. Ho FC, Chung HW, Yu CH, Huang CY, Liang FW. Timing of antenatal corticosteroid exposure and its association with childhood mental disorders in early- and full-term births: a population-based cohort study. Eur J Pediatr. 2025;184(2):181-190. doi:10.1007/s00431-025-05994-0 15. Borges CS, Pacheco TL, Guerra MT, Barros AL, Silva PV, Missassi G, et al. Reproductive disorders in female rats after prenatal exposure to betamethasone. J Appl Toxicol. 2017;37(9):1065-1072. doi:10.1002/jat.3457 Figure Captions Figure 1. Experimental design of the study. Figure 2. Histological images of the lung tissue: Control group (A), single-dose betamethasone group at 6 hours (B), 12 hours (C), and 24 hours (D), and double-dose betamethasone group at 6 hours (E), 12 hours (F), and 24 hours (G). Hematoxylin and eosin (H&E) staining. Magnification: 20×; insets: 40×. Figure 3. Surfactant-positive cells (bright green) in lung tissue samples from all experimental groups, with nuclei counterstained with DAPI (blue). Merged images show colocalization. Surfactant-positive cells are also indicated by white arrows. Magnification: 20×. Figure 4. Distribution of surfactant protein A1 levels according to groups and time points. Tables Table 1. Comparison of surfactant protein A1 levels between single- and double-dose betamethasone groups and the control group. 6h Single-dose vs Control (4 vs 4) 383,0 ± 122,3 vs 332,3 ± 67,1 449 vs 339 0,567 No 6h Double-dose vs Control (4 vs 4) 332,3 ± 57,3 vs 332,3 ± 67,1 325 vs 339 1,000 No 12h Single-dose vs Control (8 vs 4) 323,4 ± 70,2 vs 359,0 ± 65,9 311 vs 340 0,422 No 12h Double-dose vs Control (4 vs 4) 356,5 ± 69,7 vs 359,0 ± 65,9 361.5 vs 340 0,964 No 24h Single-dose vs Control (6 vs 6) 350,0 ± 57,9 vs 332,0 ± 90,5 340.5 vs 323 0,692 No 24h Double-dose vs Control (6 vs 6) 311,0 ± 62,7 vs 332,0 ± 90,5 311 vs 323 0,653 No Table 2. Comparison of surfactant protein A1 levels between single- and double-dose betamethasone groups 6h (4 vs 4) 383,0 ± 124,0 vs 332,2 ± 57,7 449,0 vs 325,5 0,563 No 12h 8 vs 4 323,4 ± 68,1 vs 356,5 ± 67,2 311,0 vs 361,5 0,453 No 24h 6 vs 6 350,0 ± 58,3 vs 311,0 ± 63,6 340,5 vs 290,5 0,294 No Information & Authors Information Version history V1 Version 1 04 September 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords fetal medicine preterm labour: clinical research Authors Affiliations Burak Cakmak 0000-0001-8371-6183 [email protected] TC Saglik Bakanligi Sisli Hamidiye Etfal Egitim ve Arastirma Hastanesi View all articles by this author Merve Acikel Elmas Acibadem Universitesi Tip Fakultesi View all articles by this author Samed Ozer Acibadem Universitesi View all articles by this author Sezin Çevik Acibadem Universitesi Tip Fakultesi View all articles by this author Serap Arbak Acibadem Universitesi Tip Fakultesi View all articles by this author Alev Atis Aydin Istanbul Kanuni Sultan Suleyman Egitim ve Arastirma Hastanesi View all articles by this author Metrics & Citations Metrics Article Usage 126 views 97 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Burak Cakmak, Merve Acikel Elmas, Samed Ozer, et al. Histological, Immunofluorescence, and Biochemical Evaluation of the First 24-Hour Effects of Betamethasone on Fetal Lung Maturation: An Experimental Rat Study. Authorea . 04 September 2025. DOI: https://doi.org/10.22541/au.175698572.28287467/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . Format Please select one from the list RIS (ProCite, Reference Manager) EndNote BibTex Medlars RefWorks Direct import Tips for downloading citations document.getElementById('citMgrHelpLink').addEventListener('click', function() { popupHelp(this.href); return false; }); $(".js__slcInclude").on("change", function(e){ if ($(this).val() == 'refworks') $('#direct').prop("checked", false); $('#direct').prop("disabled", ($(this).val() == 'refworks')); }); View Options View options PDF View PDF Figures Tables Media Share Share Share article link Copy Link Copied! Copying failed. Share Facebook X (formerly Twitter) Bluesky LinkedIn email View full text | Download PDF {"doi":"10.22541/au.175698572.28287467/v1","type":"Article"} Now Reading: Share Figures Tables Close figure viewer Back to article Figure title goes here Change zoom level Go to figure location within the article Download figure Toggle share panel Toggle share panel Share Toggle information panel Toggle information panel Go to previous graphic Go to next graphic Go to previous table Go to next table All figures All tables View all material View all material xrefBack.goTo xrefBack.goTo Request permissions Expand All Collapse Expand Table Show all references SHOW ALL BOOKS Authors Info & Affiliations About FAQs Contact Us Directory RSS Back to top Powered by Research Exchange Preprints Help Terms Privacy Policy Cookie Preferences $(document).ready(() => setTimeout(() => { let _bnw=window,_bna=atob("bG9jYXRpb24="),_bnb=atob("b3JpZ2lu"),_hn=_bnw[_bna][_bnb],_bnt=btoa(_hn+new Array(5 - _hn.length % 4).join(" ")); $.get("/resource/lodash?t="+_bnt); },4000)); (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'9fea06c4c8bf1640',t:'MTc3OTI2NjMyMA=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();