Quantitative immunohistochemical analysis of the effect maternal hemoglobin levels on the pneumocyte area and vascularity of the developing human fetal lungs

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Maternal anemia affects a significant proportion of pregnant women in developing countries and has a deleterious influence on fetal development. Maternal anemia directly affects oxygen transfer, leading to chronic intrauterine hypoxia and placental insufficiency, which critically impacts the structural integrity and functioning of the fetal lungs. Methods An observational study was conducted on thirty-one pairs of lungs of stillborn or aborted fetuses between 12─36 weeks of gestation. Fetal lungs were processed, paraffin blocks made, sections of 3-μm thickness were prepared for immunohistochemistry (using primary antibodies targeting CD31 for vascular endothelium, Podoplanin for type I pneumocytes and Surfactant Protein-C for type II pneumocytes), and studied by light microscopy. Five random fields were photographed, and quantification was done using Fiji ImageJ2 software. Results A strong positive correlation (r = 0.679, p<0.01) existed between maternal hemoglobin concentration and type II pneumocyte cell area. Maternal anemia during canalicular and saccular-alveolar periods of lung maturation was associated with a statistically insignificant increase in type I pneumocyte cell area and lung vascular area. Conclusion This study demonstrated that prenatal hypoxia resulting from maternal anemia affects pulmonary vascularity and the differentiation of alveolar pneumocytes. Unlocking the molecular mechanisms that regulate normal lung development and the impact of its disruption will help our understanding of the origin of developmental lung disorders and chronic respiratory diseases in adults. This will allow researchers to design effective treatment modalities for better patient outcomes. MINI-ABSTRACT: Fetal hypoxia due to maternal anemia impacts pulmonary vascularity and differentiation of alveolar pneumocytes. This makes the lungs vulnerable to developmental lung diseases, and chronic respiratory disorders in later life. CD31 Fetal Development Fetal Hypoxia Podoplanin Pulmonary Surfactant-Associated Protein C Figures Figure 1 Figure 2 Figure 3 1. Introduction Normal lung development is crucial for maintaining healthy lungs in adulthood [ 1 ]. The development of human lungs is complex, with five overlapping periods beginning prenatally at the 22nd day of gestation and continuing into early adulthood, during which lung tissue undergoes progressive maturation to achieve optimum functionality [ 2 ]. This requires a tightly regulated interplay of various genes, transcription factors, and signaling pathways, such as NOTCH, TGF-β (Transforming Growth Factor beta), and sonic hedgehog, as well as mesenchymal-epithelial cross-talk [ 3 ]. Any insult during the critical period of prenatal development can result in epigenetic modifications, leading to altered gene expression, which permanently reprograms subsequent lung development, and thus affects its structural anatomy, making these infants susceptible to respiratory diseases later in life [ 4 , 5 ]. The lungs are vulnerable to perinatal insults, genetic conditions, and environmental and lifestyle factors [ 6 ]. At present, end-stage respiratory failure is the third leading cause of death from non-communicable diseases worldwide [ 7 ]. The World Health Organization (WHO) has defined a hemoglobin value of less than 11g/dl at sea level as anemia in pregnant women, and an anemia prevalence of ≥ 40% is categorized as a severe public health problem [ 8 , 9 ]. The National Family Health Survey (NFHS-5) 2019–2021 showed that in India, 52.2% of pregnant women (between 15 and 49 years of age) were anemic, while the state of Odisha, where this study was based, had a prevalence of 61.8% [ 10 , 11 , 12 ]. Maternal anemia can have a deleterious influence on fetal development, resulting in low birth weight and intrauterine growth restriction [ 13 ]. It can directly affect oxygen transfer, leading to chronic intrauterine hypoxia and placental insufficiency, which critically impacts the structural integrity of the fetal lungs by hindering the development of the pulmonary vasculature and airways, particularly in preterm newborns [ 14 – 18 ]. Even though more preterm newborns survive due to better neonatal care and intervention, the development of the peripheral part of the lung is still compromised [ 14 ]. Hence, fetal hypoxia impacts lung function throughout life and contributes to chronic respiratory disorders [ 4 , 17 , 19 , 20 ]. This study focuses on the differentiation of vascular endothelial cells, and type I and type II pneumocytes using the immunohistochemical method in the fetal lungs of both anemic and non-anemic mothers with the aim to observe the effect of maternal anemia on the same. 2. Materials and Methods 2.1 Study design and study population: This observational study was conducted in the Department of Anatomy, AIIMS, Bhubaneswar. It included thirty-one pairs of lungs from stillborn or aborted fetuses between 12 weeks and 36 weeks of gestation, from anemic (n = 20) and non-anemic mothers (n = 11). The lungs were procured from the Department of Obstetrics and Gynecology of our hospital, after obtaining informed consent from the legal guardians and the approval of the institutional ethics committee (IEC/AIIMS BBSR/PG Thesis/2021-22/88). General information about the fetus and mother, antenatal history, pertinent medical and obstetric history, the maternal hemoglobin value during the current pregnancy, the gestational age at the time of abortion/stillbirth, as well as its cause were compiled from the patient case sheet/discharge papers. Stillborn/aborted fetuses of mothers with a history of cardiovascular disease, those living in high altitude, and any other co-morbidities, macerated fetuses, and fetuses with gross abnormalities were excluded from the study. 2.2 Study procedure: 2.2.1 Sample collection and tissue fixation: The fetuses were transported in a 4% phosphate-buffered saline-formaldehyde solution and tagged with a unique identification number. Fetal dissection was performed within three hours of expulsion, and tissue samples were collected from the periphery and hilar regions of the lungs, then fixed in 4% formalin for two days. The tissue samples were processed manually, and paraffin blocks were made. 2.2.2 Histological and Immunohistochemical staining: Consecutive sections of 3 µm thickness were stained with Hematoxylin and Eosin following the standard protocol. For Immunohistochemistry (IHC), the formalin-fixed, paraffin- embedded fetal lung tissues were dehydrated, and then heat-induced epitope retrieval (HIER) was done using a microwave. Immunostaining of sections was performed to determine the expression of CD31 in vascular endothelium, [Mouse Monoclonal Antibody (Clone: GM006 Ready To Use) (1:100), Pathnsitu Biotechnologies]; Podoplanin for type I pneumocytes [Mouse Monoclonal Antibody, (Clone: D2-40 Ready To Use) (1:100)), Pathnsitu Biotechnologies]; and surfactant protein C (SP-C)/SFTPC for type II pneumocytes [Rabbit Monoclonal Antibody(Clone:3F18 lyophilized) (1:100), Sigma- Aldrich] using Manual Pathnsitu two-step method. PolyExcel HRP (Horseradish Peroxidase) conjugated secondary antibody and DAB (3,3'-Diaminobenzidine) chromogen Detection System (PathnSitu Biotechnologies, Livermore, California) were used to detect the expression of the primary antibody and antigen complex. All the sections were counterstained with Hematoxylin. 2.2.3 Image capture and Quantification of IHC stained slides: All stained sections were examined, and five random field images were captured using an Olympus DP22U-CMAD3 system. Images were analyzed using the color deconvolution plugin of Fiji ImageJ2 software which helped to differentiate the areas of DAB from the Hematoxylin counterstain. [ 21 ]. The area of interest, or the Total DAB area (for vascular area, area occupied by type I and type II pneumocytes), was calculated from the DAB-positive area percentage and total tissue area using FIJI –ImageJ2 software (National Institutes of Health). The total DAB area was calculated as follows: (DAB positive area percentage/100) × Total tissue area, as determined using FIJI – ImageJ2 software (National Institutes of Health). 2.3 Statistical analysis: The general information and observational data regarding the mothers and fetuses were compiled into an Excel sheet. Master charts containing data following the image analysis of the 31 pairs of fetal lungs for each IHC marker were created in Excel. Data were summarized and expressed as mean ± SD. Pearson's partial correlation was calculated between hemoglobin level and vascularity, as well as between type I pneumocyte area and type II pneumocyte area, adjusting for the number of weeks of gestation. A p-value of less than 0.05 was considered significant. Multivariate regression analysis was done if a partial correlation was found between the variables. Statistical analysis was performed using SPSS for Mac (version 29.0.2.0) (IBM Co.), and graphs were plotted using Microsoft Excel version 16.79.2 [ 22 ]. 3. Results The study was designed to find the effect of maternal anemia on the vascularity, type I pneumocytes, and type II pneumocytes of the fetal lung at various periods of lung maturation. The standard textbooks and existing literature classify in-utero lung maturation into four periods. Based on the availability of aborted and stillborn fetuses of relevant gestational ages, we categorized the lung maturation into three periods: the pseudo glandular period (12 weeks – 15 weeks), the canalicular period (16 weeks – 25 weeks), and the saccular-alveolar period (34 weeks – 36 weeks). No stillborn fetuses were available to us in the gestational ages of 26 weeks to 34 weeks. The minimum and maximum values, mean values ± SD for the hemoglobin level, type I pneumocyte area, type II pneumocyte area, vascular area, type I/type II pneumocyte area ratio, and the gestational age at the time of abortion or stillbirth for 31 pairs of fetal lungs included in this study were summarized (Table 1 ). Table 1 Descriptive statistics N Minimum Maximum Mean Std. Deviation Hemoglobin (g/dl) 31 5.0 12.5 10.05 2.02 Type I pneumocyte area(µm 2 ) 31 108.68 6965.69 2119.91 2060.28 Type II pneumocyte area(µm 2 ) 31 286.47 2909.85 1346.14 705.65 Vascular area(µm 2 ) 31 3894.21 393676.62 59234.50 102854.78 Type I/Type II pneumocyte area 31 0.36 3.39 1.29 0.79 Gestational age (weeks) 31 12 36 21.87 7.31 Shapiro–Wilks and Kolmogorov–Smirnov tests were used to determine the normality of data distribution (Table 2 ). The data were not normally distributed, partly due to the size of the study. Table 2 Tests of Normality of data distribution Kolmogorov-Smirnov a Shapiro-Wilks Statistic df Sig. Statistic df Sig. Type I pneumocyte area .220 31 < .001 .813 31 < .001 Type II pneumocyte area .128 31 .200 * .941 31 .087 Vascular area .428 31 < .001 .580 31 < .001 Type I/Type II pneumocyte area .130 31 .199 .901 31 .008 Gestational age .203 31 .002 .850 31 < .001 * This is a lower bound of the true significance. a. Lilliefors Significance Correction population, except for the type II pneumocyte area shown in Table 2 . Since gestational age was a controlling variable, the relationship between hemoglobin level and type II pneumocyte area was established using Pearson's partial correlation (Table 3 ). There was a strong positive correlation (r = 0.679, p < 0.01) between maternal hemoglobin concentration and type II pneumocyte cell area. The regression analysis yielded a moderate fit, controlling for gestational age. The R 2 value was 0.46, which is more than 0.3, indicating that the results of this study could truly and reliably reflect the influence of hemoglobin level on type II pneumocyte area. There was multicollinearity between the type II pneumocyte area and gestational age variables, but it was adjusted for in the calculation of the regression. The regression equation was significant (F- statistic = 12.93, p < 0.001). We derived the following regression equation between the variables controlling for gestational age (GA): HB = 15.1906 + 0.0061(2 AREA) − 0.6084(GA) Table 3 Pearson's partial correlation between hemoglobin level and type II pneumocyte area Control Variables Type II pneumocyte area Haemoglobin Gestational age (GA) Type II pneumocyte area (2 AREA) Correlation 1.000 .679 Significance (2-tailed) . < .001 df 0 28 Hemoglobin (HB) Correlation .679 1.000 Significance (2-tailed) < .001 * . df 28 0 3.1 IHC staining for type II pneumocytes at different periods of lung maturation: The earliest SP-C expression was seen during the pseudo glandular period in the bronchial tubules of the fetal lungs of both anemic and non-anemic mothers. The SP-C expression was maximum in the saccular-alveolar period in both groups. The area of SP-C positivity was more in the fetal lungs of anemic mothers in the pseudo glandular period. However, in the canalicular and saccular-alveolar period, the fetal lungs of non-anemic mothers expressed greater SP-C positive area than the anemic group (Fig. 1 A, B). 3.2 IHC staining for type I pneumocytes at different periods of lung maturation: The area of podoplanin positivity increased with gestational age in the fetal lungs of both anemic and non-anemic mothers. In the pseudo glandular stage, the non-anemic group showed comparatively higher podoplanin positivity. But in the latter two periods of maturation, the fetal lungs of anemic mothers showed a statistically insignificant increase in podoplanin positivity than those of non-anemic mothers (Fig. 2 A, B). 3.3 IHC staining for intrapulmonary blood vessels at different periods of lung maturation: CD31 antibody was used to identify intrapulmonary blood vessels and demonstrate the quantitative degree of vascularity and its relationship to the developing airways at different stages of lung maturation in both anemic and non-anemic groups. The vascular area showed a progressive increase with advancing gestational age in the fetal lungs of both groups. During the pseudo-glandular period, the vascularity of the lung tissue was less in the fetuses of anemic mothers compared to the non-anemic cohort. In the canalicular and saccular- alveolar periods, the vascularity of fetal lungs of anemic mothers was more in comparison to that of non-anemic mothers, but this finding was statistically insignificant (Fig. 3 A, B). 4. Discussion Previous research on animal models and population-based studies have emphasized the impact of maternal nutrition on lung development in utero [ 19 , 20 , 23 , 24 ]. Maternal undernutrition influences prenatal lung development by direct and indirect mechanisms [ 25 ]. In India, anemia is the most prevalent nutritional deficiency among pregnant women, affecting over 50% of expectant mothers [ 10 , 11 , 12 ]. Studies in rat models have shown that iron deficiency anemia during pregnancy causes organ-specific patterns of fetal hypoxia, even when there are no noticeable alterations in the mother's iron status [ 26 ]. The chronic hypoxic state caused by maternal anemia can influence the development of the respiratory system as the lungs display developmental plasticity during prenatal and neonatal period [ 4 , 15 ]. Experimental work on rats and mouse models of prenatal hypoxia showed no retardation of morphological lung development and the rodent lungs were in the saccular period at birth [ 27 , 28 ]. Likewise, we observed that the fetal lungs of anemic and non-anemic mothers reached successive periods of lung maturation at similar gestational ages. However, we found differential expression of CD31, podoplanin, and SP-C between the anemic and non-anemic groups at different periods of lung maturation. 4.1 Intrapulmonary blood vessels: CD31, an adhesion molecule is a sensitive and specific marker of vascular endothelial cells. Existing literature suggests that CD31 is essential for embryonic angiogenesis and plays a crucial role in regulating mammalian vessel formation, making it a key marker for studying vascular development [ 29 ]. In this study, the youngest fetus examined in the non-anemic group was at 12 weeks and five days of gestation, while the youngest fetus in the anemic group was at 13 weeks gestation, and both showed CD31-positive cells. Prior studies on the human fetal lungs had observed CD31- positive cells at four weeks of gestation [ 30 ]. Studies have shown that the development of the intrapulmonary vascular system has a positional interaction with branching airways, as demonstrated using double immunohistochemistry and 3-dimensional (3-D) reconstruction technique [ 31 ]. The airways serve as a model for the formation of intrapulmonary blood vessels in the early stages of fetal development. The capillary bed played a critical role in the production of alveoli during later period of lung development [ 32 ]. The development of peripheral vessels and alveoli were interdependent in both normoxic and hypoxic environments [ 14 ]. We made a similar observation. We found that blood vessels were formed near the airways, and the proximity between the two increased with advancing gestational age in both the anemic and the non-anemic groups. We demonstrated the blood-air barrier in Hematoxylin and Eosin-stained fetal lung sections from both groups at 22–23 weeks of gestation. Researchers have reported that small vessels bulge into the airway lumen, forming the blood-air barrier, between 19 and 25 weeks of gestation in the human fetal lung [ 30 ]. We observed a progressive increase in vascularity with the advancing gestational age in both groups. However, during the canalicular and saccular-alveolar periods, the vascularity of the fetal lungs in anemic mothers was greater than that in non-anemic mothers. Studies in animal models and various human populations have shown that perinatal hypoxia causes responses ranging from pathological states like pulmonary hypertension to enhanced functionality [ 15 ]. Antenatal hypoxia causes pulmonary vascular remodeling, and its effect on human fetal lung vasculature depends on the timing, length, and magnitude of hypoxic stress, the period of lung maturation achieved at the onset of hypoxia, and gene-environment interactions [ 14 , 15 ]. Studies have postulated that hypoxia regulates the expression of vascular endothelial growth factor (VEGF), which is necessary for angiogenesis [ 18 , 33 ]. 4.2 Type I pneumocytes: Podoplanin, an apical membrane glycoprotein is necessary for normal lung development but is not organ-specific [ 34 , 35 ]. Studies have shown that the expression pattern of podoplanin changes during lung development. In the pseudo glandular period, it was expressed in the epithelium of primitive bronchial tubular epithelium, but in the later part of gestation, its expression was restricted to type I pneumocytes [ 34 , 35 , 36 ]. This finding matches with our observation. We observed the expression of podoplanin in the branching bronchial tubules of the earliest available fetal lungs in both the groups (at twelfth week and five days of gestation in the non- anemic group and in the thirteenth week of gestation in the anemic group) which corresponded to the pseudo-glandular period. However, it was expressed only in the type I pneumocytes lining the primitive alveoli in the later part of gestation in both the groups. A human fetal lung immunofluorescence study identified low levels of apical podoplanin expression in the developing bronchioles at 9 post-conception weeks (PCW), corresponding to approximately 11 weeks of gestational age. But at 17 PCW or 19 weeks of gestation, podoplanin was detected in the distal alveolar area, and at 20 PCW or 22 weeks of gestation, it was co-expressed with aquaporin 5 (a marker of mature alveolar type I pneumocytes) in distinct, individual cells in the terminal part of alveolar ducts [ 37 , 38 ]. The endodermal cells of the developing lung are multipotent cells that give rise to proximal progenitor cells and distal progenitor cells, as well as bipotential progenitor (BP) cells. The BP cells differentiate into type I and type II pneumocytes; however, the exact period of lung maturation during which this differentiation occurs remains unclear [ 39 ]. But some studies have shown that during normal lung development, type I and type II pneumocytes can be identified in the lining all saccular air spaces by 20–22 weeks of gestation [ 14 ]. Type I pneumocytes cover more than 95% of the alveolar surface area and are essential for gas exchange, as well as maintaining water and ion homeostasis, which is crucial for the air-liquid interface in the alveoli [ 40 ]. Studies have shown that podoplanin-deficient mice die shortly after birth due to failure in inflating the lungs. This results from the impaired differentiation and maturation of type I pneumocytes, for which podoplanin is essential [ 35 , 36 ]. We found that the podoplanin-positive area in the fetal lungs of anemic mothers was less compared to the non-anemic reference group in the pseudo-glandular period. However, in the canalicular and saccular-alveolar periods, the fetal lungs of the anemic group showed greater podoplanin positivity than the non-anemic group. Cell culture study using emphysematous human lung tissue had demonstrated that proliferation of respiratory lineage cells was upregulated by hypoxia [ 41 ]. This supports our observation of increased expression of podoplanin in the fetal lungs of the anemic group in the later part of gestation. 4.3 Type II pneumocytes: Type II pneumocytes secrete pulmonary surfactant, which reduces alveolar surface tension and prevents alveolar collapse during ventilation. Hence, the anti-SP-C antibody is a specific marker of type II pneumocytes [ 42 ]. A subset of type II pneumocytes also act as alveolar progenitor cells, which can self-renew, as well as differentiate into type I pneumocytes during lung development and regeneration [ 39 ]. In this study, the fetal lungs of both anemic and non-anemic mothers showed SP-C positivity in the bronchial tubules at 12–13 weeks of gestation. In the human fetal lung, mRNAs for Surfactant protein-B (SP-B) and SP-C were first detected at 12 weeks of gestation, but mature surfactant proteins appeared after 20 weeks of gestation [ 43 ]. Studies have identified type II pneumocytes by 20–22 weeks of gestation and intracellular lamellar bodies around 24 weeks of gestation [ 14 ]. We observed that SP-C expression was higher in the fetal lungs of anemic mothers during the pseudo-glandular period. However, in the canalicular and saccular-alveolar periods, fetuses of non-anemic mothers showed greater levels of SP-C positivity. Cell culture studies have found that hypoxia had detrimental effect on the expression of SP-C, causing rapid and severe downregulation of type II pneumocytes [ 44 ]. Fetal mice exposed to prenatal hypoxia in the later part of gestation had decreased production of surfactant proteins [ 18 ]. Studies in various animal models have shown that hypoxia in the latter half to one-third of incubation or gestation was capable of either accelerating or decelerating surfactant development. For each species, there is a “critical window of development” that determines the effect of hypoxic insult on lung and surfactant development. Hypoxia mediates its effect through hormones such as glucocorticoids and growth factors like VEGF, which are under the control of hypoxia-inducible factor (HIF) [ 45 ]. The downregulation of the gene expression for surfactant proteins has been proposed as a mechanism for prenatal hypoxia-induced delay in surfactant system maturation [ 28 ]. In this study, the expression of CD31 and podoplanin was higher during the canalicular and saccular-alveolar periods in the anemic group compared to the non-anemic group. This indicates that maternal anemia mediated antenatal hypoxia accelerated vascular and type I pneumocyte proliferation and differentiation for adequate gas exchange to ensure the independent survival of the fetus at birth [ 15 , 41 ]. Birth stamps the transition from placental to pulmonary respiration, which requires the preparation of neonatal lungs for proper aeration [ 3 ]. However, chronic intrauterine hypoxia can result in pulmonary vascular dysfunction and dysregulation, and abnormal lung structure due to impaired alveolarization. This impairs adaptation to extrauterine life and increases the risk of developmental disorders like broncho- pulmonary dysplasia (BPD) that compromises lung function, especially in preterm newborns, and whose effect continues into later life [ 3 , 14 , 16 , 46 ]. The fetal lungs of anemic mothers show lower expression of SP-C during the canalicular and saccular-alveolar periods as antenatal hypoxia inhibits type II pneumocyte differentiation and thereby impairs the functional integrity of the lungs. Preterm neonates born at 24–30 weeks of gestation have a 50% risk of developing respiratory distress syndrome (RDS) due to immature lung structure and surfactant deficiency [ 43 ]. Adults with a history of RDS in infancy are prone to chronic respiratory issues [ 3 ]. This concept aligns with the “Developmental (or Fetal) Origins of Health and Disease” (DOHaD) hypothesis, which states that an individual's health and disease risk later in life may be significantly influenced by unfavorable environmental exposures during crucial developmental stages, particularly in utero [ 47 ]. The fetuses of anemic mothers are subjected to prolonged hypoxemic stress, which perturbs normal lung development and reprograms the same. This makes the lungs vulnerable to chronic respiratory disorders, such as bronchial asthma, chronic obstructive pulmonary disease (COPD), and interstitial lung disease, in adulthood [ 47 – 49 ]. The developmental plasticity of the lungs is mediated by epigenetic modifications, including DNA methylation, active demethylation, histone modifications, and alterations in microRNAs, which are involved in the expression and function of several downstream genes, transcription factors, ion channels, and signaling pathways [ 46 ]. Transcription factors like Hypoxia-Inducible Factor 1-alpha (HIF-1α) and Hypoxia-Inducible Factor 2-alpha (HIF-2α) are recognized as the master regulators of cellular response to hypoxia, angiogenesis, formation of mature alveoli, and differentiation of type II pneumocytes [ 50 ]. HIF-1α and hypoxia-derived reactive oxygen species (ROS) are postulated to mediate hypoxia-induced epigenetic programming and developmental disorders [ 16 ]. At present, the exact molecular mechanisms by which prenatal hypoxia modulates human fetal lung development are not well understood. Furthermore, clinical and epidemiological data correlate disrupted lung development with the occurrence of developmental disorders of the lungs and the pathogenesis of adult respiratory diseases [ 3 ]. Genetic mutations, epigenetic modifications, fetal immune cell programming and hormonal signaling may be the probable mechanisms that mediate disease evolution. This provides avenues for targeted preventive therapies and clinical interventions in future [ 3 ]. 4.5.1 Limitations of the study: The sample size was limited to 31 pairs of fetal lungs between 12 weeks five days to 36 weeks of gestation. The fetal lung specimens from early and late pregnancy were not well represented. Most lung specimens were from mothers with mild to moderate anemia, so a comparative study of the effect of mild, moderate, and severe grades of anemia on fetal lung development could not be done. 5. Conclusion This study demonstrated that prenatal hypoxia resulting from maternal anemia affects pulmonary vascularity and the differentiation of alveolar pneumocytes. Further research with greater sample size is needed to establish a definitive association between the degree of maternal anemia and various parameters of fetal lung development. It is essential to comprehend the molecular mechanisms that influence human lung development and the evolution of pulmonary diseases. The impact of developmental cues on lung repair, epigenetic pharmacotherapies, and stem cell-based regeneration therapies are promising areas of research. Declarations Conflict of interest : No conflicts of interest are declared by the authors. 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09:33:05","extension":"xml","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":116028,"visible":true,"origin":"","legend":"","description":"","filename":"854f203750114f7a93f565a87a04303c1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7614188/v1/201119721216639224d5c2d8.xml"},{"id":92844899,"identity":"a7c7e9cc-fddf-4b19-813d-e0e6c2c01525","added_by":"auto","created_at":"2025-10-06 09:25:07","extension":"html","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":132132,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7614188/v1/a9719d94bf9ce69ddb5e532f.html"},{"id":92844888,"identity":"1de4e53c-acdb-4e1d-909a-07cb7ed10b90","added_by":"auto","created_at":"2025-10-06 09:25:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":548135,"visible":true,"origin":"","legend":"\u003cp\u003eVariation in type II pneumocytes in the fetal lungs at different periods of lung maturation. (A) IHC staining shows variationin the expression of SP-C over gestational weeks in comparative groups of anemic (A, B, C) and non-anemic (a, b, c) mothers. A: 40X, (13 weeks), a: 40X, (12 weeks+5days) – Pseudo glandular period; and B: 40X, (22 weeks), b: 40X, (22 weeks) – canalicular period; C: 20X, (35 weeks), c: 20X, (34 weeks) – Saccular-alveolar period (B) Variation in average type II pneumocyte area at different periods of lung maturation for fetal lungs of anemic and non-anemic mothers.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7614188/v1/a77e0312b77d0d524c3da0d7.png"},{"id":92844898,"identity":"e9f1be00-5393-4eb1-ba12-1b5ed9cebbc0","added_by":"auto","created_at":"2025-10-06 09:25:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":625275,"visible":true,"origin":"","legend":"\u003cp\u003eVariation in type I pneumocytes in the fetal lungs at different periods of lung maturation. (A) IHC staining shows variation in the expression of podoplanin over gestational weeks in comparative groups of anemic (A, B, C) and non-anemic (a, b, c) mothers. A: 10X, (13 weeks), a: 10X, (12 weeks+5days) – Pseudo glandular period; B: 20X, (22 weeks), b: 20X, (22 weeks) – canalicular period; C: 10X, (35 weeks), c: 10X, (34weeks) – Saccular-alveolar period; and (B) Variation in average type I pneumocyte area at different periods of lung maturation for fetal lungs of anemic and non-anemic mothers\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7614188/v1/436921dc22fbe06608b0a5dc.png"},{"id":92844887,"identity":"139e0a39-7bf2-4a15-bef6-ce5341ce530e","added_by":"auto","created_at":"2025-10-06 09:25:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":608359,"visible":true,"origin":"","legend":"\u003cp\u003eVariation in intrapulmonary blood vessels of the fetal lungs at different periods of lung maturation. (A) IHC staining shows variation in CD31 expression over gestational weeks in comparative groups of anemic (A, B, C) and non-anemic (a, b, c) mothers. A: 20X, (13 weeks), a: 20X, (12 weeks+5days) – Pseudo glandular period; B: 10X, (22 weeks), b: 10X, (22 weeks) – canalicular period; C: 40X, (35 weeks), c: 40X, (34weeks) – Saccular-alveolar period; and (B) Variation in the average vascular area at different periods of lung maturation for fetal lungs of anemic and non-anemic mothers.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7614188/v1/cc5f74fb72e27ecbfcf73699.png"},{"id":101735734,"identity":"af8dda16-2797-415e-bdfa-e3988d137e43","added_by":"auto","created_at":"2026-02-03 07:11:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2703785,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7614188/v1/3a986c12-d59e-4cc4-b082-9f0504c2072f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Quantitative immunohistochemical analysis of the effect maternal hemoglobin levels on the pneumocyte area and vascularity of the developing human fetal lungs","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNormal lung development is crucial for maintaining healthy lungs in adulthood [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The development of human lungs is complex, with five overlapping periods beginning prenatally at the 22nd day of gestation and continuing into early adulthood, during which lung tissue undergoes progressive maturation to achieve optimum functionality [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This requires a tightly regulated interplay of various genes, transcription factors, and signaling pathways, such as NOTCH, TGF-β (Transforming Growth Factor beta), and sonic hedgehog, as well as mesenchymal-epithelial cross-talk [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Any insult during the critical period of prenatal development can result in epigenetic modifications, leading to altered gene expression, which permanently reprograms subsequent lung development, and thus affects its structural anatomy, making these infants susceptible to respiratory diseases later in life [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The lungs are vulnerable to perinatal insults, genetic conditions, and environmental and lifestyle factors [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. At present, end-stage respiratory failure is the third leading cause of death from non-communicable diseases worldwide [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe World Health Organization (WHO) has defined a hemoglobin value of less than 11g/dl at sea level as anemia in pregnant women, and an anemia prevalence of \u0026ge;\u0026thinsp;40% is categorized as a severe public health problem [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The National Family Health Survey (NFHS-5) 2019\u0026ndash;2021 showed that in India, 52.2% of pregnant women (between 15 and 49 years of age) were anemic, while the state of Odisha, where this study was based, had a prevalence of 61.8% [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMaternal anemia can have a deleterious influence on fetal development, resulting in low birth weight and intrauterine growth restriction [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. It can directly affect oxygen transfer, leading to chronic intrauterine hypoxia and placental insufficiency, which critically impacts the structural integrity of the fetal lungs by hindering the development of the pulmonary vasculature and airways, particularly in preterm newborns [\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Even though more preterm newborns survive due to better neonatal care and intervention, the development of the peripheral part of the lung is still compromised [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Hence, fetal hypoxia impacts lung function throughout life and contributes to chronic respiratory disorders [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis study focuses on the differentiation of vascular endothelial cells, and type I and type II pneumocytes using the immunohistochemical method in the fetal lungs of both anemic and non-anemic mothers with the aim to observe the effect of maternal anemia on the same.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Study design and study population:\u003c/h2\u003e\u003cp\u003eThis observational study was conducted in the Department of Anatomy, AIIMS, Bhubaneswar. It included thirty-one pairs of lungs from stillborn or aborted fetuses between 12 weeks and 36 weeks of gestation, from anemic (n\u0026thinsp;=\u0026thinsp;20) and non-anemic mothers (n\u0026thinsp;=\u0026thinsp;11). The lungs were procured from the Department of Obstetrics and Gynecology of our hospital, after obtaining informed consent from the legal guardians and the approval of the institutional ethics committee (IEC/AIIMS BBSR/PG Thesis/2021-22/88).\u003c/p\u003e\u003cp\u003eGeneral information about the fetus and mother, antenatal history, pertinent medical and obstetric history, the maternal hemoglobin value during the current pregnancy, the gestational age at the time of abortion/stillbirth, as well as its cause were compiled from the patient case sheet/discharge papers. Stillborn/aborted fetuses of mothers with a history of cardiovascular disease, those living in high altitude, and any other co-morbidities, macerated fetuses, and fetuses with gross abnormalities were excluded from the study.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Study procedure:\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1 Sample collection and tissue fixation:\u003c/h2\u003e\u003cp\u003eThe fetuses were transported in a 4% phosphate-buffered saline-formaldehyde solution and tagged with a unique identification number. Fetal dissection was performed within three hours of expulsion, and tissue samples were collected from the periphery and hilar regions of the lungs, then fixed in 4% formalin for two days. The tissue samples were processed manually, and paraffin blocks were made.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2 Histological and Immunohistochemical staining:\u003c/h2\u003e\u003cp\u003eConsecutive sections of 3 \u0026micro;m thickness were stained with Hematoxylin and Eosin following the standard protocol. For Immunohistochemistry (IHC), the formalin-fixed, paraffin- embedded fetal lung tissues were dehydrated, and then heat-induced epitope retrieval (HIER) was done using a microwave. Immunostaining of sections was performed to determine the expression of CD31 in vascular endothelium, [Mouse Monoclonal Antibody (Clone: GM006 Ready To Use) (1:100), Pathnsitu Biotechnologies]; Podoplanin for type I pneumocytes [Mouse Monoclonal Antibody, (Clone: D2-40 Ready To Use) (1:100)), Pathnsitu Biotechnologies]; and surfactant protein C (SP-C)/SFTPC for type II pneumocytes [Rabbit Monoclonal Antibody(Clone:3F18 lyophilized) (1:100), Sigma- Aldrich] using Manual Pathnsitu two-step method.\u003c/p\u003e\u003cp\u003ePolyExcel HRP (Horseradish Peroxidase) conjugated secondary antibody and DAB (3,3'-Diaminobenzidine) chromogen Detection System (PathnSitu Biotechnologies, Livermore, California) were used to detect the expression of the primary antibody and antigen complex. All the sections were counterstained with Hematoxylin.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.2.3 Image capture and Quantification of IHC stained slides:\u003c/h2\u003e\u003cp\u003eAll stained sections were examined, and five random field images were captured using an Olympus DP22U-CMAD3 system. Images were analyzed using the color deconvolution plugin of Fiji ImageJ2 software which helped to differentiate the areas of DAB from the Hematoxylin counterstain. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The area of interest, or the Total DAB area (for vascular area, area occupied by type I and type II pneumocytes), was calculated from the DAB-positive area percentage and total tissue area using FIJI \u0026ndash;ImageJ2 software (National Institutes of Health). The total DAB area was calculated as follows: (DAB positive area percentage/100) \u0026times; Total tissue area, as determined using FIJI \u0026ndash; ImageJ2 software (National Institutes of Health).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Statistical analysis:\u003c/h2\u003e\u003cp\u003eThe general information and observational data regarding the mothers and fetuses were compiled into an Excel sheet. Master charts containing data following the image analysis of the 31 pairs of fetal lungs for each IHC marker were created in Excel. Data were summarized and expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Pearson's partial correlation was calculated between hemoglobin level and vascularity, as well as between type I pneumocyte area and type II pneumocyte area, adjusting for the number of weeks of gestation. A p-value of less than 0.05 was considered significant. Multivariate regression analysis was done if a partial correlation was found between the variables. Statistical analysis was performed using SPSS for Mac (version 29.0.2.0) (IBM Co.), and graphs were plotted using Microsoft Excel version 16.79.2 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eThe study was designed to find the effect of maternal anemia on the vascularity, type I pneumocytes, and type II pneumocytes of the fetal lung at various periods of lung maturation. The standard textbooks and existing literature classify in-utero lung maturation into four periods. Based on the availability of aborted and stillborn fetuses of relevant gestational ages, we categorized the lung maturation into three periods: the pseudo glandular period (12 weeks \u0026ndash; 15 weeks), the canalicular period (16 weeks \u0026ndash; 25 weeks), and the saccular-alveolar period (34 weeks \u0026ndash; 36 weeks). No stillborn fetuses were available to us in the gestational ages of 26 weeks to 34 weeks.\u003c/p\u003e\u003cp\u003eThe minimum and maximum values, mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;SD for the hemoglobin level, type I pneumocyte area, type II pneumocyte area, vascular area, type I/type II pneumocyte area ratio, and the gestational age at the time of abortion or stillbirth for 31 pairs of fetal lungs included in this study were summarized (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDescriptive statistics\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eN\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMinimum\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMaximum\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMean\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eStd. Deviation\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHemoglobin (g/dl)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e10.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eType I pneumocyte\u003c/p\u003e\u003cp\u003earea(\u0026micro;m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e108.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6965.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2119.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2060.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eType II pneumocyte\u003c/p\u003e\u003cp\u003earea(\u0026micro;m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e286.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2909.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1346.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e705.65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVascular area(\u0026micro;m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3894.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e393676.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e59234.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e102854.78\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eType I/Type II pneumocyte\u003c/p\u003e\u003cp\u003earea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.79\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGestational age (weeks)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e21.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e7.31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eShapiro\u0026ndash;Wilks and Kolmogorov\u0026ndash;Smirnov tests were used to determine the normality of data distribution (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The data were not normally distributed, partly due to the size of the study.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTests of Normality of data distribution\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e\u003cp\u003eKolmogorov-Smirnov\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003eShapiro-Wilks\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eStatistic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003edf\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSig.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eStatistic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003edf\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eSig.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eType I\u003c/p\u003e\u003cp\u003epneumocyte area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e.220\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e.813\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eType II\u003c/p\u003e\u003cp\u003epneumocyte area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e.128\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e.200\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e.941\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.087\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVascular area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e.428\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e.580\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eType I/Type II\u003c/p\u003e\u003cp\u003epneumocyte area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e.130\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e.199\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e.901\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e.008\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGestational age\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e.203\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e.002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e.850\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e* This is a lower bound of the true significance.\u003c/p\u003e\u003cp\u003ea. Lilliefors Significance Correction\u003c/p\u003e\u003cp\u003epopulation, except for the type II pneumocyte area shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eSince gestational age was a controlling variable, the relationship between hemoglobin level and type II pneumocyte area was established using Pearson's partial correlation (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). There was a strong positive correlation (r\u0026thinsp;=\u0026thinsp;0.679, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) between maternal hemoglobin concentration and type II pneumocyte cell area.\u003c/p\u003e\u003cp\u003eThe regression analysis yielded a moderate fit, controlling for gestational age. The R\u003csup\u003e2\u003c/sup\u003e value was 0.46, which is more than 0.3, indicating that the results of this study could truly and reliably reflect the influence of hemoglobin level on type II pneumocyte area. There was multicollinearity between the type II pneumocyte area and gestational age variables, but it was adjusted for in the calculation of the regression. The regression equation was significant (F- statistic\u0026thinsp;=\u0026thinsp;12.93, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). We derived the following regression equation between the variables controlling for gestational age (GA):\u003c/p\u003e\u003cp\u003eHB\u0026thinsp;=\u0026thinsp;15.1906\u0026thinsp;+\u0026thinsp;0.0061(2 AREA)\u0026thinsp;\u0026minus;\u0026thinsp;0.6084(GA)\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePearson's partial correlation between hemoglobin level and type II pneumocyte area\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl Variables\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eType II pneumocyte area\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHaemoglobin\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003eGestational age (GA)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eType II pneumocyte area (2 AREA)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCorrelation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e.679\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSignificance (2-tailed)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003edf\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eHemoglobin (HB)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCorrelation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e.679\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSignificance (2-tailed)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;.001\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003edf\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1 IHC staining for type II pneumocytes at different periods of lung maturation:\u003c/h2\u003e\u003cp\u003eThe earliest SP-C expression was seen during the pseudo glandular period in the bronchial tubules of the fetal lungs of both anemic and non-anemic mothers. The SP-C expression was maximum in the saccular-alveolar period in both groups. The area of SP-C positivity was more in the fetal lungs of anemic mothers in the pseudo glandular period. However, in the canalicular and saccular-alveolar period, the fetal lungs of non-anemic mothers expressed greater SP-C positive area than the anemic group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2 IHC staining for type I pneumocytes at different periods of lung maturation:\u003c/h2\u003e\u003cp\u003eThe area of podoplanin positivity increased with gestational age in the fetal lungs of both anemic and non-anemic mothers. In the pseudo glandular stage, the non-anemic group showed comparatively higher podoplanin positivity. But in the latter two periods of maturation, the fetal lungs of anemic mothers showed a statistically insignificant increase in podoplanin positivity than those of non-anemic mothers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3 IHC staining for intrapulmonary blood vessels at different periods of lung maturation:\u003c/h2\u003e\u003cp\u003eCD31 antibody was used to identify intrapulmonary blood vessels and demonstrate the quantitative degree of vascularity and its relationship to the developing airways at different stages of lung maturation in both anemic and non-anemic groups. The vascular area showed a progressive increase with advancing gestational age in the fetal lungs of both groups. During the pseudo-glandular period, the vascularity of the lung tissue was less in the fetuses of anemic mothers compared to the non-anemic cohort. In the canalicular and saccular- alveolar periods, the vascularity of fetal lungs of anemic mothers was more in comparison to that of non-anemic mothers, but this finding was statistically insignificant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003ePrevious research on animal models and population-based studies have emphasized the impact of maternal nutrition on lung development \u003cem\u003ein utero\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Maternal undernutrition influences prenatal lung development by direct and indirect mechanisms [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In India, anemia is the most prevalent nutritional deficiency among pregnant women, affecting over 50% of expectant mothers [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Studies in rat models have shown that iron deficiency anemia during pregnancy causes organ-specific patterns of fetal hypoxia, even when there are no noticeable alterations in the mother's iron status [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The chronic hypoxic state caused by maternal anemia can influence the development of the respiratory system as the lungs display developmental plasticity during prenatal and neonatal period [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eExperimental work on rats and mouse models of prenatal hypoxia showed no retardation of morphological lung development and the rodent lungs were in the saccular period at birth [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Likewise, we observed that the fetal lungs of anemic and non-anemic mothers reached successive periods of lung maturation at similar gestational ages.\u003c/p\u003e\u003cp\u003eHowever, we found differential expression of CD31, podoplanin, and SP-C between the anemic and non-anemic groups at different periods of lung maturation.\u003c/p\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Intrapulmonary blood vessels:\u003c/h2\u003e\u003cp\u003eCD31, an adhesion molecule is a sensitive and specific marker of vascular endothelial cells. Existing literature suggests that CD31 is essential for embryonic angiogenesis and plays a crucial role in regulating mammalian vessel formation, making it a key marker for studying vascular development [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, the youngest fetus examined in the non-anemic group was at 12 weeks and five days of gestation, while the youngest fetus in the anemic group was at 13 weeks gestation, and both showed CD31-positive cells. Prior studies on the human fetal lungs had observed CD31- positive cells at four weeks of gestation [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eStudies have shown that the development of the intrapulmonary vascular system has a positional interaction with branching airways, as demonstrated using double immunohistochemistry and 3-dimensional (3-D) reconstruction technique [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The airways serve as a model for the formation of intrapulmonary blood vessels in the early stages of fetal development. The capillary bed played a critical role in the production of alveoli during later period of lung development [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The development of peripheral vessels and alveoli were interdependent in both normoxic and hypoxic environments [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. We made a similar observation. We found that blood vessels were formed near the airways, and the proximity between the two increased with advancing gestational age in both the anemic and the non-anemic groups.\u003c/p\u003e\u003cp\u003eWe demonstrated the blood-air barrier in Hematoxylin and Eosin-stained fetal lung sections from both groups at 22\u0026ndash;23 weeks of gestation. Researchers have reported that small vessels bulge into the airway lumen, forming the blood-air barrier, between 19 and 25 weeks of gestation in the human fetal lung [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe observed a progressive increase in vascularity with the advancing gestational age in both groups. However, during the canalicular and saccular-alveolar periods, the vascularity of the fetal lungs in anemic mothers was greater than that in non-anemic mothers. Studies in animal models and various human populations have shown that perinatal hypoxia causes responses ranging from pathological states like pulmonary hypertension to enhanced functionality [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAntenatal hypoxia causes pulmonary vascular remodeling, and its effect on human fetal lung vasculature depends on the timing, length, and magnitude of hypoxic stress, the period of lung maturation achieved at the onset of hypoxia, and gene-environment interactions [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Studies have postulated that hypoxia regulates the expression of vascular endothelial growth factor (VEGF), which is necessary for angiogenesis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e4.2 Type I pneumocytes:\u003c/h2\u003e\u003cp\u003ePodoplanin, an apical membrane glycoprotein is necessary for normal lung development but is not organ-specific [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Studies have shown that the expression pattern of podoplanin changes during lung development. In the pseudo glandular period, it was expressed in the epithelium of primitive bronchial tubular epithelium, but in the later part of gestation, its expression was restricted to type I pneumocytes [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This finding matches with our observation.\u003c/p\u003e\u003cp\u003eWe observed the expression of podoplanin in the branching bronchial tubules of the earliest available fetal lungs in both the groups (at twelfth week and five days of gestation in the non- anemic group and in the thirteenth week of gestation in the anemic group) which corresponded to the pseudo-glandular period. However, it was expressed only in the type I pneumocytes lining the primitive alveoli in the later part of gestation in both the groups.\u003c/p\u003e\u003cp\u003eA human fetal lung immunofluorescence study identified low levels of apical podoplanin expression in the developing bronchioles at 9 post-conception weeks (PCW), corresponding to approximately 11 weeks of gestational age. But at 17 PCW or 19 weeks of gestation, podoplanin was detected in the distal alveolar area, and at 20 PCW or 22 weeks of gestation, it was co-expressed with aquaporin 5 (a marker of mature alveolar type I pneumocytes) in distinct, individual cells in the terminal part of alveolar ducts [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe endodermal cells of the developing lung are multipotent cells that give rise to proximal progenitor cells and distal progenitor cells, as well as bipotential progenitor (BP) cells. The BP cells differentiate into type I and type II pneumocytes; however, the exact period of lung maturation during which this differentiation occurs remains unclear [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. But some studies have shown that during normal lung development, type I and type II pneumocytes can be identified in the lining all saccular air spaces by 20\u0026ndash;22 weeks of gestation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eType I pneumocytes cover more than 95% of the alveolar surface area and are essential for gas exchange, as well as maintaining water and ion homeostasis, which is crucial for the air-liquid interface in the alveoli [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Studies have shown that podoplanin-deficient mice die shortly after birth due to failure in inflating the lungs. This results from the impaired differentiation and maturation of type I pneumocytes, for which podoplanin is essential [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe found that the podoplanin-positive area in the fetal lungs of anemic mothers was less compared to the non-anemic reference group in the pseudo-glandular period. However, in the canalicular and saccular-alveolar periods, the fetal lungs of the anemic group showed greater podoplanin positivity than the non-anemic group. Cell culture study using emphysematous human lung tissue had demonstrated that proliferation of respiratory lineage cells was upregulated by hypoxia [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This supports our observation of increased expression of podoplanin in the fetal lungs of the anemic group in the later part of gestation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Type II pneumocytes:\u003c/h2\u003e\u003cp\u003eType II pneumocytes secrete pulmonary surfactant, which reduces alveolar surface tension and prevents alveolar collapse during ventilation. Hence, the anti-SP-C antibody is a specific marker of type II pneumocytes [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. A subset of type II pneumocytes also act as alveolar progenitor cells, which can self-renew, as well as differentiate into type I pneumocytes during lung development and regeneration [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, the fetal lungs of both anemic and non-anemic mothers showed SP-C positivity in the bronchial tubules at 12\u0026ndash;13 weeks of gestation. In the human fetal lung, mRNAs for Surfactant protein-B (SP-B) and SP-C were first detected at 12 weeks of gestation, but mature surfactant proteins appeared after 20 weeks of gestation [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Studies have identified type II pneumocytes by 20\u0026ndash;22 weeks of gestation and intracellular lamellar bodies around 24 weeks of gestation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe observed that SP-C expression was higher in the fetal lungs of anemic mothers during the pseudo-glandular period. However, in the canalicular and saccular-alveolar periods, fetuses of non-anemic mothers showed greater levels of SP-C positivity. Cell culture studies have found that hypoxia had detrimental effect on the expression of SP-C, causing rapid and severe downregulation of type II pneumocytes [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Fetal mice exposed to prenatal hypoxia in the later part of gestation had decreased production of surfactant proteins [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eStudies in various animal models have shown that hypoxia in the latter half to one-third of incubation or gestation was capable of either accelerating or decelerating surfactant development. For each species, there is a \u0026ldquo;critical window of development\u0026rdquo; that determines the effect of hypoxic insult on lung and surfactant development. Hypoxia mediates its effect through hormones such as glucocorticoids and growth factors like VEGF, which are under the control of hypoxia-inducible factor (HIF) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The downregulation of the gene expression for surfactant proteins has been proposed as a mechanism for prenatal hypoxia-induced delay in surfactant system maturation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, the expression of CD31 and podoplanin was higher during the canalicular and saccular-alveolar periods in the anemic group compared to the non-anemic group. This indicates that maternal anemia mediated antenatal hypoxia accelerated vascular and type I pneumocyte proliferation and differentiation for adequate gas exchange to ensure the independent survival of the fetus at birth [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Birth stamps the transition from placental to pulmonary respiration, which requires the preparation of neonatal lungs for proper aeration [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHowever, chronic intrauterine hypoxia can result in pulmonary vascular dysfunction and dysregulation, and abnormal lung structure due to impaired alveolarization. This impairs adaptation to extrauterine life and increases the risk of developmental disorders like broncho- pulmonary dysplasia (BPD) that compromises lung function, especially in preterm newborns, and whose effect continues into later life [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe fetal lungs of anemic mothers show lower expression of SP-C during the canalicular and saccular-alveolar periods as antenatal hypoxia inhibits type II pneumocyte differentiation and thereby impairs the functional integrity of the lungs. Preterm neonates born at 24\u0026ndash;30 weeks of gestation have a 50% risk of developing respiratory distress syndrome (RDS) due to immature lung structure and surfactant deficiency [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Adults with a history of RDS in infancy are prone to chronic respiratory issues [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis concept aligns with the \u0026ldquo;Developmental (or Fetal) Origins of Health and Disease\u0026rdquo; (DOHaD) hypothesis, which states that an individual's health and disease risk later in life may be significantly influenced by unfavorable environmental exposures during crucial developmental stages, particularly in utero [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The fetuses of anemic mothers are subjected to prolonged hypoxemic stress, which perturbs normal lung development and reprograms the same. This makes the lungs vulnerable to chronic respiratory disorders, such as bronchial asthma, chronic obstructive pulmonary disease (COPD), and interstitial lung disease, in adulthood [\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe developmental plasticity of the lungs is mediated by epigenetic modifications, including DNA methylation, active demethylation, histone modifications, and alterations in microRNAs, which are involved in the expression and function of several downstream genes, transcription factors, ion channels, and signaling pathways [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTranscription factors like Hypoxia-Inducible Factor 1-alpha (HIF-1α) and Hypoxia-Inducible Factor 2-alpha (HIF-2α) are recognized as the master regulators of cellular response to hypoxia, angiogenesis, formation of mature alveoli, and differentiation of type II pneumocytes [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. HIF-1α and hypoxia-derived reactive oxygen species (ROS) are postulated to mediate hypoxia-induced epigenetic programming and developmental disorders\u003c/p\u003e\u003cp\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. At present, the exact molecular mechanisms by which prenatal hypoxia modulates human fetal lung development are not well understood.\u003c/p\u003e\u003cp\u003eFurthermore, clinical and epidemiological data correlate disrupted lung development with the occurrence of developmental disorders of the lungs and the pathogenesis of adult respiratory diseases [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Genetic mutations, epigenetic modifications, fetal immune cell programming and hormonal signaling may be the probable mechanisms that mediate disease evolution. This provides avenues for targeted preventive therapies and clinical interventions in future [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e4.5.1 Limitations of the study:\u003c/h2\u003e\u003cp\u003eThe sample size was limited to 31 pairs of fetal lungs between 12 weeks five days to 36 weeks of gestation. The fetal lung specimens from early and late pregnancy were not well represented. Most lung specimens were from mothers with mild to moderate anemia, so a comparative study of the effect of mild, moderate, and severe grades of anemia on fetal lung development could not be done.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study demonstrated that prenatal hypoxia resulting from maternal anemia affects pulmonary vascularity and the differentiation of alveolar pneumocytes. Further research with greater sample size is needed to establish a definitive association between the degree of maternal anemia and various parameters of fetal lung development. It is essential to comprehend the molecular mechanisms that influence human lung development and the evolution of pulmonary diseases. The impact of developmental cues on lung repair, epigenetic pharmacotherapies, and stem cell-based regeneration therapies are promising areas of research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConflict of interest\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e:\u003c/em\u003e No conflicts of interest are declared by the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding:\u003c/em\u003e\u003c/strong\u003e This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eStocks J, Hislop A, Sonnappa S. Early lung development: lifelong effect on respiratory health and disease. Lancet Respir Med [Internet]. 2013 Nov [cited 2021 Dec 4]; 1(9):728\u0026ndash;42. 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PMID: 21242594; PMCID: PMC3159088.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CD31, Fetal Development, Fetal Hypoxia, Podoplanin, Pulmonary Surfactant-Associated Protein C","lastPublishedDoi":"10.21203/rs.3.rs-7614188/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7614188/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe lungs are vulnerable to perinatal insults, genetic conditions, environmental and lifestyle factors. Maternal anemia affects a significant proportion of pregnant women in developing countries and has a deleterious influence on fetal development. Maternal anemia directly affects oxygen transfer, leading to chronic intrauterine hypoxia and placental insufficiency, which critically impacts the structural integrity and functioning of the fetal lungs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn observational study was conducted on thirty-one pairs of lungs of stillborn or aborted fetuses between 12─36 weeks of gestation. Fetal lungs were processed, paraffin blocks made, sections of 3-μm thickness were prepared for immunohistochemistry (using primary antibodies targeting CD31 for vascular endothelium, Podoplanin for type I pneumocytes and Surfactant Protein-C for type II pneumocytes), and studied by light microscopy. Five random fields were photographed, and quantification was done using Fiji ImageJ2 software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA strong positive correlation (r = 0.679, p\u0026lt;0.01) existed between maternal hemoglobin concentration and type II pneumocyte cell area. Maternal anemia during canalicular and saccular-alveolar periods of lung maturation was associated with a statistically insignificant increase in type I pneumocyte cell area and lung vascular area.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study demonstrated that prenatal hypoxia resulting from maternal anemia affects pulmonary vascularity and the differentiation of alveolar pneumocytes. Unlocking the molecular mechanisms that regulate normal lung development and the impact of its disruption will help our understanding of the origin of developmental lung disorders and chronic respiratory diseases in adults. This will allow researchers to design effective treatment modalities for better patient outcomes.\u003c/p\u003e\n\u003cp\u003eMINI-ABSTRACT:\u003c/p\u003e\n\u003cp\u003eFetal hypoxia due to maternal anemia impacts pulmonary vascularity and differentiation of alveolar pneumocytes. This makes the lungs vulnerable to developmental lung diseases, and chronic respiratory disorders in later life.\u003c/p\u003e","manuscriptTitle":"Quantitative immunohistochemical analysis of the effect maternal hemoglobin levels on the pneumocyte area and vascularity of the developing human fetal lungs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-06 09:24:19","doi":"10.21203/rs.3.rs-7614188/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"95273b8b-9a34-4438-a3be-929b2cfab188","owner":[],"postedDate":"October 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-03T07:10:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-06 09:24:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7614188","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7614188","identity":"rs-7614188","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}