Morphological Characteristics of Microenvironment in the Human Thymus During Fetal Development

doi:10.21203/rs.3.rs-5194610/v1

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Morphological Characteristics of Microenvironment in the Human Thymus During Fetal Development

https://doi.org/10.21203/rs.3.rs-5194610/v1

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Background

The thymus is a key organ for the development of T cells. T cell precursors first migrate from the bone marrow to the thymus. During maturation, these precursors require interactions with various types of cells that form the thymic microenvironment, such as epithelial, mesenchymal, and other immune cells not belonging to the T lineage. The aim of this study was to examine the changes in the number and diameter of Hassall's corpuscles, as well as the density and distribution of epithelial cells (p63+) and macrophages (CD68+).

Methods

Twenty-five fetal thymus samples were examined, divided into five groups according to gestational age. The samples were processed using standard histological methods and immunohistochemical staining.

Results

The study showed that the number and diameter of Hassall's corpuscles gradually increased during fetal development, with a significant increase from the 14th to the 38th gestational week. The average diameter of Hassall's corpuscles was largest in the age group of 34–38 weeks. The density of p63 + epithelial cells decreased in correlation with gestational age, while the density of CD68 + macrophages significantly increased, particularly in the thymic medulla, towards the end of the fetal period.

Conclusions

An increase in the number and size of Hassall's corpuscles during fetal development was recorded, while the density of epithelial cells decreased and the density of macrophages increased.

Thymus

Hassall's corpuscles

epithelial cells

macrophages

The thymus is a primary lymphoid organ, and research into its embryology represents a true mystery of biology and medicine, particularly in pediatric immunology [1]. In childhood, the thymus is a relatively large structure that is gradually replaced by fatty retrosternal tissue during the aging process. By the age of around 20, 80% of the thymus undergoes fatty involution, which is typically complete by the age of 60 [2]. The lymphoid component within the cortex and medulla decreases by approximately 3% annually until middle age (35–45 years) and 1% annually thereafter [3].

The thymus consists of two compartments: the true epithelial space and the perivascular space. The epithelial space of the thymus is divided into the cortex and medulla, where T cells mature and achieve self-tolerance. The cortex is the site of early thymocyte development, while the medulla is the site of subsequent development. In the medulla, thymocytes undergo negative selection to eliminate autoreactivity. The perivascular space consists of adipocytes, peripheral lymphocytes, and stroma [4]. Histologically, the cortex is characterised by the presence of densely packed thymocytes, making it appear significantly darker than the medulla when observed on histological sections. The most striking feature of the medulla is the presence of thymic corpuscles—Hassall's corpuscles—which are composed of keratinised epithelial cells. It is assumed that the number and diameter of Hassall's corpuscles per unit area increase during fetal development [5]. Additionally, lymphocytes are present in the medulla, but their density is lower, making the medulla appear lighter than the cortex in histological sections.

The thymic microenvironment at birth consists of various cell types. In addition to T lymphocytes at different stages of maturation, there are other cell types, including epithelial cells, fibroblasts, macrophages, dendritic cells, endothelial cells, cytokines, chemokines, as well as smaller populations of plasma cells, basophils, and eosinophils [6]. During their journey to become mature T cells, progenitors require interaction with many different types of cells within the thymic microenvironment. There are two key selection steps necessary for producing mature T cells: positive and negative selection. Both of these processes must be carried out efficiently to produce functional T cells [7]. The selection process begins when lymphocyte precursors derived from the bone marrow reach the outer cortical region of the thymus and begin to mature into functional T lymphocytes, which will ultimately leave the thymus and populate peripheral lymphoid organs [8].

Although the basic characteristics of thymus development and function are known, detailed studies on the changes in the number and diameter of Hassall's corpuscles during fetal development, as well as the distribution of macrophages and epithelial cells, can provide deeper insight into the processes of T-lymphocyte maturation. The aim of this study is to quantitatively and qualitatively analyse the morphological characteristics of the thymus during fetal development, focussing on the changes in the number and diameter of Hassall's corpuscles, as well as the density and distribution of p63 + cells (epithelial cells) and CD68 + cells (macrophages). Ultimately, the purpose of this research is to gain a better understanding of the role of these cells and the dynamics involved in the maturation of the thymus.

The study is retrospective and was conducted at the Center for Pathology and Histology of the University Clinical Center of Vojvodina (UKCV). Samples were collected from October 1, 2019, to February 1, 2020, with some being retrieved from the center’s archives, while the remaining samples were obtained during fetal autopsies at the Center for Pathology and Histology of UKCV.

The study included 25 fetal thymus samples collected during standard fetal autopsies. Samples were taken from fetuses with gestational ages between the 14th and 38th weeks, with gestational age precisely determined based on available clinical documentation, including gynecological reports and macroscopic assessment of the fetus. Macroscopic characteristics considered included fetal weight and length, crown-rump length, femur length, and foot length, which enabled an accurate assessment of age. The samples were divided into 5 groups according to gestational age to allow comparative analysis of thymic morphological changes at different stages of fetal development. The first group included fetuses aged 14–18 gestational weeks, the second group 19–23 weeks, the third 24–28 weeks, the fourth 29–33 weeks, and the fifth group included fetuses aged 34–38 weeks. Each group contained five samples.

The samples obtained from fetal autopsies were fixed in 4% formalin to preserve tissue structure and then processed through a dehydration process using increasing concentrations of ethanol to gradually remove water from the tissue. After dehydration, the samples were cleared in xylene to enable paraffin infiltration, and then embedded in paraffin blocks. The paraffin blocks were sectioned using a Sakura Tissue-TekR microtome into thin histological sections 5 micrometers thick, which is the optimal thickness for detailed microscopic examination. The sections were placed in a water bath to ensure proper spreading on slides and then transferred to a thermostat for one hour to dry and adhere to the slides. After drying, the sections were stained using the standard hematoxylin-eosin (HE) method, which allows for the differentiation of tissue components. Hematoxylin stains nuclei blue, while eosin stains cytoplasm and other structures pink, providing a clear depiction of the morphological features of the thymus for further analysis.

From the paraffin blocks, cylindrical samples of fetal thymus tissues 5 mm in diameter were taken using a microarray device. These samples were placed in new paraffin molds and then sectioned into thin histological slices using a microtome and placed on slides. The sections were then stained using immunohistochemical methods to visualise specific cell populations, including CD68 + macrophages and p63 + epithelial cells. The aim of this staining was to determine the density and distribution of these target cells in different topographical regions of the thymus (cortex and medulla) during various stages of fetal development. Antibodies used in the study: Monoclonal Mouse p63, manufacturer: "Dako" (target cells: epithelial cells), Monoclonal Mouse CD68, manufacturer: "Thermoscientific" (target cells: macrophages).

Immunohistochemical staining was conducted according to the manufacturer's instructions. Positive and negative controls were applied to ensure quality. Positive control confirmed specific staining of target cells, while the negative control verified the absence of nonspecific antibody binding, ensuring high precision and reliability of the staining results.

Qualitative analysis of histological sections stained with routine HE methods and immunohistochemical antibodies was conducted using a DMLB100T light microscope. The aim of the analysis was to assess the morphological structure of the thymus and the presence of p63 + epithelial cells and CD68 + macrophages, in relation to fetal gestational age. Qualitative analysis enabled a visual evaluation of the changes in distribution and density of target cells during different stages of fetal development.

Quantitative analysis of all histological sections, stained with both routine HE methods and immunohistochemical antibodies, was performed using the VisionTek TM Sakura device. The samples were photographed and morphometrically analyzed using VisionTekLive2.6 software, allowing precise measurement and quantification of the target structures.

In HE-stained sections, the number of Hassall’s corpuscles was counted, and their diameter was measured. The area of the thymic medulla was measured using the software's "Ruler-Free from area" option, and the density of Hassall’s corpuscles per square millimeter was calculated by dividing the total number of corpuscles by the total medullary area. These values were used to calculate the average values for each group defined by fetal gestational age, including the average sizes of Hassall’s corpuscles for each group.

In immunohistochemically stained sections, CD68 + and p63 + cells in the thymic cortex and medulla were counted. The number of these cells was counted over an area of 0.2 mm², and cell density on this defined surface was calculated. For each sample, average cell counts were calculated, and these data were aggregated for each gestational age group. This quantitative analysis provided detailed insight into the changes in density and distribution of target cells in different topographical regions of the thymus during fetal development.

All collected data were analysed using Microsoft Excel and SPSS (IBM SPSS Statistics, version 27.0.2). Mean values (arithmetic means) and standard deviations were calculated for all quantitative variables, including the number and diameter of Hassall’s corpuscles, as well as the density of p63 + and CD68 + cells in different topographical regions of the thymus (cortex and medulla).

Pearson's correlation coefficient was used to assess the relationship between gestational age and cell density, as well as the diameter of Hassall’s corpuscles, to determine the strength and direction of the linear relationship. Linear regression analysis was conducted to estimate the change in cell density and the diameter of Hassall’s corpuscles depending on gestational age. The coefficient of determination (R²) was used to evaluate the percentage of variation explained by the regression model. One-way analysis of variance (ANOVA) was used to compare the mean diameters of Hassall’s corpuscles between different gestational groups. The ANOVA results, along with F-values and corresponding p-values, allowed conclusions regarding statistically significant differences between the groups.

Statistical significance was set at p < 0.05, and values below this threshold were considered statistically significant differences between the groups. Results were presented in the form of graphs and tables, with statistically significant differences marked.

All steps of the study were conducted in accordance with the Declaration of Helsinki [9], and the study protocol was approved by the UKCV Ethics Committee in Novi Sad (decision from December 31, 2019, decision number: 00-1212). All samples were processed anonymously, respecting the privacy and confidentiality of information about the fetuses and their families. The material used in the study was collected exclusively from the archives of the Center for Pathology and Histology and from autopsies conducted in accordance with legal and ethical guidelines. All tissue analysis procedures were performed under laboratory conditions with strict adherence to ethical and professional standards. No aspect of the study compromised the physical or psychological well-being of the families, and all data were used solely for scientific purposes, without identifying personal information.

Figure 1. shows the increase in the average number of Hassall's corpuscles with gestational age, divided into groups ranging from 14 to 38 weeks. The average number of corpuscles gradually increases as the pregnancy progresses, while the vertical lines display the variability within each group. It is evident that the number of Hassall's corpuscles is the lowest in the 14–18 week group, while the highest average number is observed in the 34–38 week group. Pearson's correlation coefficient between the diameter of Hassall's corpuscles and gestational age is 0.58, indicating a moderate positive correlation (p = 0.007). This result shows that as gestational age increases, the diameter of Hassall's corpuscles also increases, further supporting the findings presented in the graph, where the growth of the average number of Hassall's corpuscles is clearly visible as the pregnancy advances.

The results of linear regression (Fig. 2.) show that there is a statistically significant positive relationship between gestational age and the number of Hassall's corpuscles. The regression coefficient is 0.3355, indicating that the number of Hassall's corpuscles increases by 0.3355 units with each additional week of gestational age (p = 0.007). The coefficient of determination (R²) is 0.336, meaning that approximately 33.6% of the variation in the number of Hassall's corpuscles can be explained by gestational age. These results confirm the findings that the number of Hassall's corpuscles significantly increases as pregnancy progresses.

Figure 3. shows the average dimensions of Hassall's corpuscles in micrometers (µm) for different gestational age groups (14–38 weeks). A gradual increase in the size of the corpuscles is evident as gestational age progresses, with average values increasing from the 14–18 to the 34–38 week group. The vertical lines represent the variability within each group, while the overall trend shows that the size of Hassall's corpuscles increases with gestational age. The results of the one-way ANOVA analysis yielded an F-value of 2.58 and a p-value of 0.001.

The results of linear regression shown in Fig. 4. indicate that there is a statistically significant positive relationship between gestational age and the average diameter of Hassall's corpuscles. The analysis revealed that the average diameter increases by approximately 0.996 µm with each additional week of gestational age, supported by high statistical significance (p < 0.001). The coefficient of determination (R²) is 0.550, meaning that about 55% of the variation in the average diameter of Hassall's corpuscles can be explained by gestational age. While the actual data show some variability, the overall trend suggests an increase in corpuscle diameter as pregnancy progresses.

Figure 5. shows the density of p63 + cells in the cortex (blue bars) and medulla (red bars) across different gestational age groups. The density of p63 + cells in the medulla is higher in the early gestational weeks (14–18) and gradually decreases in the older gestational groups (34–38 weeks). Similarly, the density of p63 + cells in the cortex also decreases as gestational age progresses, but the decline is less pronounced compared to the medulla.

Pearson's correlation results show a moderately strong negative correlation between gestational age and the density of p63 + cells in both regions – the cortex and medulla of the thymus. For the density of p63 + cells in the cortex, Pearson's coefficient is -0.53, indicating that cell density decreases with increasing gestational age (p = 0.042). Similarly, for the density of p63 + cells in the medulla, Pearson's coefficient is -0.51, also indicating a negative correlation (p = 0.051). These results suggest that the density of p63 + cells decreases in both regions as gestational age advances, with this effect being more pronounced in the medulla.

Figure 6. shows the results of the regression analysis of the density of p63 + cells in the cortex (blue dots and line) and medulla (red dots and line) in relation to gestational age. A negative trend is observed for both regions, where cell density decreases as gestational age increases. For the density of p63 + cells in the cortex, the regression analysis shows that the R-squared value is 0.969, meaning that approximately 97% of the variation in the density of p63 + cells can be explained by gestational age. The coefficient is -3.707, indicating a decrease in the density of p63 + cells in the cortex by approximately 3.71 units for each additional week of gestational age (p = 0.002). For the density of p63 + cells in the medulla, the R-squared value is 0.929, meaning that about 93% of the variation in cell density can be explained by gestational age. A coefficient of -5.98 indicates that the density of cells in the medulla decreases by approximately 5.98 units per week of gestational age (p = 0.008).

The histological image (Fig. 7) shows a thymus stained using the immunohistochemical technique, with prominent blue-brown colours indicating the presence of specific cell markers, namely p63 + cells. The thymus tissue is organised into lobules, separated by connective septa. Dark-stained cells, represented by p63 + cells, are predominantly located within the blue regions. Subcapsularly, in the immediate vicinity of the thymus capsule, a higher density of p63 + cells are observed compared to other parts of the tissue, where these cells are diffusely distributed.

In the medulla of the thymus, a uniform distribution of p63 + cells is observed (Fig. 8) across all gestational periods. p63 + cells are diffusely distributed throughout the tissue, without significant concentration along the corticomedullary boundary, indicating a relatively even distribution of these cells in the thymic medulla.

Figure 8. Histological Section of the Thymus Medulla, 18 Weeks Gestation, p63 + Immunohistochemistry, 20x.

The results show that the number of CD68 + cells increases with gestational age in both regions of the thymus – in the cortex and the medulla (Fig. 8). Pearson's correlation results indicate a very strong positive association between gestational age and the number of CD68 + cells in both the cortex and medulla of the thymus. For the density of CD68 + cells in the cortex, Pearson's correlation coefficient is 0.993, indicating a very strong positive correlation (p = 0.007). Similarly, for the density of CD68 + cells in the medulla, Pearson's correlation coefficient is 0.99 (p > 0.05).

The regression analysis for the density of CD68 + cells in the thymus shows a significant positive association between gestational age and cell density in both regions, the cortex and medulla (Fig. 9). For the density of CD68 + cells in the cortex, the R-squared value is 0.986, meaning that about 98.6% of the variation can be explained by gestational age, while the regression coefficient of 0.398 indicates that the density of CD68 + cells in the cortex increases by 0.398 units per week of gestational age. For the density of CD68 + cells in the medulla, the R-squared value is even higher at 0.999, meaning that nearly 100% of the variation can be explained by gestational age. The regression coefficient for the medulla is 2.638, indicating a larger increase in cell density in the medulla of 2.638 units per week of gestational age.

The histological image of the thymus cortex, stained using the immunohistochemical technique marking CD68 + cells with brown colour, is shown (Fig. 10). The thymus cortex is organised into lobules, which are separated by light connective septa. CD68 + cells are present in the cortex, diffusely distributed, without forming clusters. Their density appears uniform, with clearly distinguishable individual cells within the blue background, which represents the nuclei of other cells.

The histological image of the thymus medulla, stained using the immunohistochemical technique for CD68 + cells, marked in brown, is shown (Fig. 11). The image shows the uniform distribution of CD68 + cells throughout the thymus medulla. CD68 + cells are diffusely distributed without visible clusters or increased density in specific regions. Their presence is evenly spread, with clearly distinguishable individual cells located within the blue background, which represents the nuclei of other cells.

The thymic microenvironment plays a key role in T cell development [10, 11]. This specialised environment ensures the proper differentiation and selection of T lymphocytes, thus securing their functionality in the immune system. This is vital for developing a strong adaptive immune response against pathogens and tumors without causing autoimmune disease [11].

The thymus is highly sensitive to various environmental factors, particularly radiation and chemical agents. For example, hematopoietic stem cell transplantation (HSCT) can acutely damage the thymus due to the application of chemotherapy, radiotherapy, and antibody therapies [12]. This can be further exacerbated by infections that an immunodeficient patient may acquire, and in the case of allogeneic HSCT, thymic graft-versus-host disease (GVHD) [13, 14]. However, after the acute damage subsides, the thymus has the capacity for intrinsic regeneration, though this process is hindered by the natural involution of the thymus.

Thymic epithelial cells (TECs) are crucial for T cell development, but the cellular mechanisms that maintain TECs in adults are still poorly understood. Most of the supporting tissue of the thymus consists of epithelial cells that form a unique cytoreticulum [15]. This cytoreticulum is inhabited by blast lymphocytes derived from the bone marrow. When these cells (T lymphocyte precursors) reach the thymus, their immunological education begins. Thymic epithelial cells can be classified into different populations that vary in distribution, structure, function, and their ability to synthesise thymic hormones such as thymulin, thymopoietin, thymosin, and thymic humoral factor [16]. These cells play a key role in regulating the maturation of lymphoid progenitors and immune responses. Morphologically, thymic epithelial cells can be divided into two main subtypes—cortical (cTEC) and medullary (mTEC) [17]—both of which are essential for the development of the T cell repertoire. Immunohistochemically, thymic epithelial cells can be classified into four subtypes: subcapsular, cortical, medullary, and Hassall’s corpuscles, each with a specific role in the organisation of the thymus and its immune function [18]. Strong evidence suggests that during fetal development and in the perinatal thymus, there is a bipotent progenitor that can generate both cTECs (cortical thymic epithelial cells) and mTECs (medullary thymic epithelial cells) [19].

In this study, the presence of epithelial cells was demonstrated by staining with Monoclonal Mouse p63. This antigen is predominantly expressed in thymic epithelial cells, especially in cortical epithelial cells (cTEC) [20]. The postnatal thymic cortex contains several distinct microenvironments, including the subcapsular region, central cortex, and perimedullary cortex [21]. Staining with monoclonal antibodies, microscopic morphology, and location within the thymic cortex revealed at least four different subtypes of cortical thymic epithelial cells (cTEC) [22]. However, the functional distinction between these four cTEC subtypes remains unclear. A global gene expression analysis of the subcapsular region, central cortex, and perimedullary cortex showed variability in gene expression profiles across these regions [21, 23]. These cells play a crucial role in T lymphocyte maturation, where p63 contributes to their development and maintenance. One isoform of p63, ΔNp63, is particularly important for the proliferation and differentiation of thymic epithelial cells, which are key to the proper development and function of the thymus [24]. These cells are essential for the positive selection of T lymphocytes during their maturation in the thymus. Specifically, ΔNp63 aids in the proliferation and differentiation of epithelial progenitor cells in the thymus, which is vital for maintaining the structural integrity and function of this organ [20, 25].

The role of ΔNp63 has been investigated in the context of its regulation of signaling pathways such as FgfR2 and Jag2, which are critical for proper thymic epithelial formation [25]. Disruptions in this regulation can lead to reduced epithelial cell proliferation, negatively impacting T cell development and resulting in decreased thymic functionality, especially in mice with experimental models that exclude ΔNp63 [26]. Additionally, studies have shown that ΔNp63 influences the long-term maintenance of thymic epithelial cells, enabling the creation of an appropriate microenvironment for T lymphocytes, which is crucial for a normal immune response [21, 26]. Furthermore, cTECs express various self-antigens that are presented via major histocompatibility complex (MHC) molecules. These antigens also play a significant role in the positive selection of T cells, a process that determines the diversity of the T cell repertoire [27].

Lymphoid progenitor cells enter the postnatal thymus via blood vessels, typically localised around the corticomedullary junction. Upon entering the thymus, lymphoid progenitor cells, or immature CD4-CD8 double-negative thymocytes, migrate to the subcapsular region of the thymic cortex, where they begin to proliferate and differentiate into T-lineage cells [21]. This is why p63 + cells are most abundant in this region, as confirmed in our study through qualitative data analysis.

Although it is known that cTEC cells belong to endodermal epithelial cells and are neither lymphoid nor hematopoietic, cTEC cells are a crucial component of the adaptive immune system because they express molecules, including b5t and MHC class II, whose functions are primarily significant in the adaptive immune system and whose expression is detectable only in vertebrates with adaptive immunity [21, 28].

While cTECs are responsible for positive selection during T lymphocyte maturation, medullary epithelial cells (mTECs) are responsible for negative selection during T lymphocyte development. Thymic epithelial cells, particularly mTECs, play a key role in the three-dimensional organisation of the thymus structure [29]. This three-dimensional network allows for proper T lymphocyte selection, which is crucial for maintaining central immune tolerance and preventing autoimmune diseases. mTECs express a wide range of tissue-specific antigens (TRAs) that facilitate the negative selection of autoreactive T lymphocytes. mTECs express a different set of self-antigens that enable the elimination (negative selection) of autoreactive T cells that bind to these antigens with high affinity, thereby establishing central tolerance [27, 30]. Additionally, the self-antigens expressed by mTECs regulate the production of regulatory T cells (Tregs), which suppress immune responses in peripheral tissues, a process known as peripheral tolerance [27, 31].

In this study, the analysis of epithelial cell density revealed that their density decreases during fetal development. A greater number of epithelial cells are present in the thymic medulla and in the subcapsular region. Research by Wang and colleagues shows that during fetal thymic development, the number of epithelial cells significantly increases as the organ matures and forms the structure necessary for T lymphocyte development [20]. In the early stages of development, cortical epithelial cells (cTECs) dominate, while medullary epithelial cells (mTECs) appear later and become key for negative lymphocyte selection [20]. However, our study assessed epithelial cell density, which showed that epithelial cell density decreases during fetal development. This decrease can be explained by the faster growth of thymic tissue compared to the rate of epithelial cell division. Our results suggest that the rapid growth of thymic tissue outpaces the proliferation rate of epithelial cells, resulting in lower epithelial cell density in the later stages of fetal development.

Hassall's corpuscles (HC) (also known as thymic corpuscles) are specific structures found in the thymic medulla and are formed by type VI epithelial cells, which are arranged concentrically [32]. HCs are unique, antigen-specific, functionally active, multicellular components of the non-lymphocytic cellular microenvironment of the thymic medulla and are involved in the physiological activities of both prenatal and adult thymus, crucial for thymus function and immune regulation [33]. Although the function of Hassall's corpuscles is not completely understood, it is known that they produce cytokines, which contribute to the maturation of dendritic cells and the induction of regulatory T cells, important for maintaining immune tolerance [34]. Recent research has revealed that these corpuscles may play a role in the pathogenesis of various autoimmune diseases, such as type 1 diabetes, rheumatoid arthritis, and multiple sclerosis [34, 35]. It has also been found that they synthesise chemokines that affect other cells in the thymic medulla, but the relationships between Hassall's corpuscles and other thymic cells (such as dendritic and neuroendocrine cells) have not yet been fully explored [34]. Some studies suggest that Hassall's corpuscles participate in the negative selection of lymphocytes, the removal of autoreactive T lymphocytes, and the production and storage of antibodies and antigens [5].

During thymic ontogeny, HCs appear when lymphopoiesis is already established, and the cortex, medulla, and corticomedullary junction become capable of conducting positive and negative selection of T lymphocytes, which undergo progressive maturation [33]. HCs are structurally organised from reticular epithelial (RE) cells with lamellar keratinisation, which usually undergo hypertrophy before becoming part of the outer layer of corpuscle cells [36]. Observations by Bodey et al. showed that the greatest developmental progress and primary organisation of HCs occur between the 45th and 54th days of gestation in dogs and between the 6th and 10th lunar months in humans [33].

In this study, histological analysis of a sample from the 14th gestational week (GW) revealed only one Hassall's corpuscle. This finding aligns with previous studies that showed no HCs appeared in the 12th GW, with the first corpuscle observed between the 12th and 14th GW, and after this period, the number and diameter of the corpuscles increased during fetal development [37]. Our research also indicates that the average areas of Hassall's corpuscles significantly increase with fetal age, as confirmed by a 2022 study, in which the largest difference was observed between the 16–19 week age group and the 20–23 week group [32]. Another study found that the number and size of Hassall's corpuscles increased between the 17th and 24th weeks [38]. In our study, the largest increase in HC area was recorded between the 19th and 28th gestational weeks. Additionally, it was found that the number of HCs gradually increases during fetal development, with the most intense increase occurring between the 29th and 38th GWs. These results suggest the continuous development and growth of Hassall's corpuscles as the fetus approaches the later stages of gestation.

Nearly all thymocytes (95–97%) are removed through apoptosis during T cell development in the thymus [39]. Macrophages play the role of "cleaners," removing many thymocytes that undergo apoptosis due to failure in positive selection or as a result of negative selection [40]. These cells are important for the efficient phagocytosis of dead thymocytes, as well as for supporting the immune microenvironment in T cell development [6, 41]. Additionally, thymic macrophages play a significant role in tissue repair, especially after injuries such as radiation, which is applied in some oncology patients. They secrete cytokines, growth factors, and other molecules that promote epithelial regeneration and maintain thymic structure after injury [6, 42].

For many years, thymic macrophages were not well- characterised or understood due to technical limitations in analysing these cells and conducting functional studies. There are only a few well-known macrophage markers identified on thymic macrophages (ED1 and ED2 in rats, CD68, F4/80, and CD11b in mice), making it difficult to study the origin of these macrophages and identify their heterogeneity in the thymus [43, 44]. Macrophages in the fetal thymus originate from various hematopoietic progenitors. Some derive from the yolk sac and fetal liver, while others come from bone marrow progenitors. Studies show that different populations of thymic macrophages develop at different stages of the fetus, with each having a specific function in the organisation and development of the thymus [45]. Thymic macrophages predominantly come from two populations: Timd4 + cells, of embryonic origin, located in the thymic cortex, and Cx3cr1 + cells, derived from adult hematopoietic progenitors, located in the medulla [45]. Since macrophages are recruited from different sources, including hematopoietic progenitors from the fetal liver and yolk sac, their number significantly increases during fetal development [45]. As the thymus becomes more active during fetal development, its volume increases, as does the number of apoptotic thymocytes, leading to an increase in macrophage density. Our study shows similar results. During the fetal period, macrophage density continuously rises until the end of fetal development, with a higher macrophage density observed in the thymic medulla. These findings confirm the role of macrophages in eliminating apoptotic cells and supporting thymic immune function during development.

The limitations of this study include a relatively small sample size, which may limit the generalisability of the results to a broader population. The study is retrospective in nature, and it depends on the quality and availability of previously collected samples and documentation, which may introduce certain biases. The samples were collected exclusively during standard fetal autopsies, so the quality of the samples may vary. Finally, the study did not consider possible additional factors such as fetal sex or health status, which could have influenced the results.

Further research on the thymus is needed as its microenvironment plays a crucial role in generating functional cellular immunity. Disruptions in this environment can impair T lymphocyte education and weaken the adaptive immune response, increasing the risk of autoimmune diseases. While thymic involution is often associated with aging, new data suggest the influence of sex hormones, obesity, infections, and oxidative stress [46]. Studies have also shown that thymectomy in adults can lead to an increased risk of cancer and reduced immunity, highlighting the importance of preserving the thymus [47, 48].

During fetal development, the thymus undergoes significant morphological changes. The number and diameter of Hassall's corpuscles gradually increase, while the density of epithelial cells (p63+) decreases. In contrast, the density of macrophages (CD68+) increases in correlation with gestational age. These results provide important insight into the development of the thymus, which plays a key role in the maturation of T lymphocytes and immune development.

UKCV - University Clinical Center of Vojvodina

HE - hematoxylin-eosin

ANOVA - One-way analysis of variance

HSCT - hematopoietic stem cell transplantation

GVHD - graft-versus-host disease

TECs - Thymic epithelial cells

cTECs - cortical thymic epithelial cells

mTECs - medullary thymic epithelial cells

MHC - major histocompatibility complex

TRAs - tissue-specific antigens

Tregs - gulatory T cells

HC - Hassall's corpuscles

RE - reticular epithelial cells

GW - gestational week

Ethics approval and consent to participate

All steps of the study were conducted in accordance with the Declaration of Helsinki (9), and the study protocol was approved by the UKCV Ethics Committee in Novi Sad (decision from December 31, 2019, decision number: 00-1212). All samples were processed anonymously, respecting the privacy and confidentiality of information about the fetuses and their families. The material used in the study was collected exclusively from the archives of the Center for Pathology and Histology and from autopsies conducted in accordance with legal and ethical guidelines. All tissue analysis procedures were performed under laboratory conditions with strict adherence to ethical and professional standards. No aspect of the study compromised the physical or psychological well-being of the families, and all data were used solely for scientific purposes, without identifying personal information.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the Provincial Secretariat for Higher Education and Scientific Research project No. 142- 451-3531/2023-01.

Authors' contributions

N.M., A.F.L. and J.A. contributed to the conception and design of the study. S.B, A.R., N.D. and N.M. contributed to collecting the data. A.R, N.M., Z.G. and N.D. participated in the statistical analysis and interpretation of data. N.M., N.D., S.B., A.F.L. and J.A. contributed to drafting and revising the manuscript. All authors have read and agreed to the published version of the manuscript.

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No competing interests reported.

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Morphological Characteristics of Microenvironment in the Human Thymus During Fetal Development

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