Histomorphometric and Ultrastructural Study of Gestational Changes in Mouse Liver Using Haematoxylin and Eosin, Periodic acid Schiff stain

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Histomorphometric and Ultrastructural Study of Gestational Changes in Mouse Liver Using Haematoxylin and Eosin, Periodic acid Schiff stain

https://doi.org/10.21203/rs.3.rs-4050397/v1

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Background: Studying the makeup of a mouses liver, such, as its lobes, lobules and how hepatocytes are organized is important, for researching changes that occur during pregnancy.

Aim of Study: the purpose of this study is to assess the structural, morphological, and general histological differences in liver tissue during pregnancy, including weight changes in the liver and total body weight, morphometric analysis and statistical assessment of hepatocytes, and glycogen concentration in the liver.

Methodology: The study involved a comparative investigation between pregnant and non-pregnant mice on liver weight, total body weight, hepatocyte size, and glycogen concentration with utilized hematoxylin, eosin, and Periodic Acid Schiff stain for histological examination and applied statistical tools for data analysis.

Results: Pregnant mice showed a significant increase (p=0.0001), in both liver and overall body weight and examination of the cell measurements indicated that the liver cells, in mice were larger indicating alterations. Glycogen concentration significantly increased in the livers of pregnant mice (p=0.0001), indicating altered metabolic demands.

Conclusion: Pregnancy induces noticeable changes in liver weight, hepatocyte size, and glycogen storage, reflecting the liver's adaptive response to the increased metabolic needs during gestation.

glycogen

hepatocyte

hematoxylin and eosin (H&E)

Periodic Acid Schiff

Pregnancy

Pregnancy refers to the duration during which a woman harbours a foetus inside her uterus. Typically, the foetus undergoes growing inside the uterus, commencing when the egg is fertilised by sperm and then implanted in the uterine lining. Following this, it undergoes development into the placenta and embryo, and then progresses into a foetus (Coates et al., 2020).

Furthermore, human pregnancy typically lasts about 40 weeks, or just over 9 months, measured from the last menstrual period to childbirth. This duration is divided into three trimesters (Lakshmi et al., 2016). In contrast, the duration of pregnancy in mice is around 20-22 days. The duration of gestation in laboratory mice is typically uniform within a certain strain, although it might vary across different strains. The gestational period in mice is categorised into the first, second, and third weeks, during which several physiological alterations take place in pregnant mice to accommodate embryonic growth (Suckow et al., 2023).

Additionally, the organs associated with the digestive tract include major salivary glands, pancreas, liver, and gallbladder. Notably, the liver is the largest gland in the human body, as well as in vertebrates and other animals. It is reddish to brown in color and has four lobes of irregular size and shape in both humans and mice (Janqueira & Carneiro, 2005).

The average weight of a human liver is from 1.44 to 1.66 kg (3.2 to 3.7 lb), and it is situated in the right upper quadrant of the abdominal cavity. The organ in question is situated inferior to the diaphragm, positioned to the right of the stomach, and positioned above the gallbladder (Kumar et al., 2017; Tortora & Derrickson, 2018). Conversely, the liver of a mouse is composed of four primary lobes that exhibit a varied pattern of lobation. The liver's fibrous capsule extends connective tissue septa into the liver tissue, resulting in the division of the liver lobes into indistinct lobules. The central vein is located in the centre, while the portal triad is situated at the corners (Riggle et al., 2018). Glisson's capsule, a thin connective tissue, envelops the liver and thickens near the hilum area (Last, 1956).

Hepatocytes, which are the primary cells found in the main parenchymal tissue of the liver, make up the majority of the liver's cell type, accounting for around 80% of its total mass. The observed cells have a polygonal morphology, characterised by a rounded nucleus and a profusion of cellular organelles that are involved in metabolic and secretory processes (Alberts et al., 2018). The liver cells, in the mouse liver tissue are arranged in cord formations. The small blood vessels, lined with endothelial cells are positioned amidst these cord structures (Riggle et al., 2018).

Many research studies have used mice as a model to explore the structure and functions of the liver suggesting similarities, in the livers of humans, mice and rats (Yan et al., 2017). The hepatic organ receives blood from two primary sources, namely the hepatic portal vein and the hepatic artery. The majority of blood is then eliminated by the hepatic veins (Nozaki et al., 2020). One of the primary direct impacts of insulin on hepatocytes is the promotion of net hepatic glycogen synthesis, which plays a pivotal role in the inhibition of hepatic glucose generation (Guarino et al., 2020).

During the gestational period, the female undergoes physiological changes that facilitate the growth and development of the foetus. The concentrations of oestrogens and progesterone exhibit a gradual rise, affecting hepatic metabolic, synthesis, and excretory functions (Paschkis & Rakoff, 2013).

Moreover, in the treatment of liver problems during pregnancy, the use of serum liver function tests is crucial, including a range of particular assays (Iluz-Freundlich et al., 2020).

The measurement of serum bile acid concentrations can be particularly useful for managing cholestasis during pregnancy, although its availability is limited (Juusela et al., 2020).

Software plays a pivotal role in the morphometric study, allowing for various processing and analytical procedures. Image J, for instance, is user-friendly software capable of performing a multitude of imaging manipulations (López et al., 2022).

Histomorphometric measurements provide a quantitative and qualitative means of analyzing the shape, size, and quantity of stains, crucial for understanding biological morphology and its implications (Gabelaia et al., 2018). One of the programs used for morphometric analysis is Aperio Algorithms Image Scope, which offers advanced features for assessing digital slides quantitatively (Gabelaia et al., 2018).

The aims of this study include assessing structural, morphological, and general histological changes in liver tissue during pregnancy; analyzing weight changes in liver weight and total body weight; morphometrically analyzing and statistically assessing hepatocyte changes; and assessing glycogen concentration in the liver.

The project was approved by the Scientific Research and Ethical Committee of the College of Medicine, University of Al-Iraqia. Baghdad. Iraq.

A total of 30 female Mice with body weights ranging between 20-30 g and ages of 8-10 weeks and the animals were housed in the Center of Cancer and Medical Genetics Research (CCMG) and were kept under conventional conditions in an acclimatized room with free access to water (fresh tap water) and food (standard pellet diet). They were maintained under a 12h light: 12h darkness cycle and at a constant temperature (22 ± 2°C).

The mice were divided into two groups:

Group A: This control group consisted of 15 mature, non-pregnant female mice.

Group B: This group included 15 pregnant female mice between 15-19 days of gestation.

Each female mouse was placed in a cage with one healthy male mouse, and each morning the cages were checked for the presence of a vaginal plug. The day after the formation of a vaginal plug was designated as the first day of gestation. The total body weight of the pregnant mice was measured at two different times: first, at the time of the appearance of the vaginal plug, and second, in late pregnancy after the removal of embryos.

Dissection of the mice was carried out in the laboratory room. The animals from each group were dissected separately under a dissecting microscope to allow for better visualization of the liver.

Sampling and tissue preparation:

Following the CCMGR animal housing guidelines, the animals were anesthetized with ether, and the abdominal wall was dissected to remove the liver under anesthesia, after which the animals were euthanized through deep anesthesia. The liver samples were then prepared for histological examination, which included fixation in 10% formalin for 24 hours (although typically 48-72 hours is recommended), followed by washing with 70% ethyl alcohol to remove the fixative. Dehydration was achieved by transferring the sections through progressively higher concentrations of ethyl alcohol: 70% for 24 hours, 90% for 2-6 hours, and absolute alcohol for 1-2 hours. Clearing involved two 15-minute xylene baths to remove alcohol and enhance tissue transparency. Embedding was done in molten paraffin wax at 56-58°C in labeled copper metal molds, followed by solidification and storage at 4°C. Sectioning was performed at 4-7 μm thickness for staining. Sections were floated in a 48-50°C water bath before being mounted on slides. Each slide was marked with necessary information using a pencil, ensuring that every stage, from fixation to sectioning, adhered to strict protocols for accurate histological analysis and photography (Bancroft et al., 2013).

Histological staining (Haematoxylin and Eosin):

The general histological staining with Haematoxylin and Eosin (H&E), as detailed by Bancroft et al.(Bancroft et al., 2013), involves a simplified yet effective method for highlighting cellular and tissue structures. Initially, staining solutions are prepared: Harris-modified haematoxylin, containing alum and mercuric oxide for nuclear staining, and a 1% Eosin-yellowish solution for cytoplasmic and connective tissue staining. The staining procedure involves dewaxing sections in xylene, rehydrating through graded alcohols, staining with haematoxylin followed by eosin, and then dehydrating, clearing, and mounting the slides. Additionally, the Periodic Acid-Schiff (PAS) technique, used for carbohydrates, follows a protocol of dewaxing, rehydrating, treating with periodic acid, Schiff reagent, and counterstaining with haematoxylin. Each step, from the preparation of solutions to the final examination under a microscope, is meticulously performed to ensure precise and clear histological analysis.

Microscopical Examination and Morphometric analysis

The examination of tissue sections was conducted using a Genex microscope, which includes a built-in digital camera of undisclosed megapixels, offering various magnification powers of 4X, 10X, 20X, and 40X. For the photography aspect of the study, a strategy was employed where five random fields per slide—comprising the four corners and the center of the tissue section—were selected for imaging. These fields were captured using the microscope's digital camera, and images were saved in JPEG format.

Morphometric analysis was facilitated by ImageJ software, a Java-based image processing program developed by the National Institutes of Health, USA. The software is versatile, supporting a wide array of image formats and capable of performing detailed analyses, including area, pixel value statistics, distance, and angle measurements. It also allows for spatial calibration, enabling measurements in real-world units like micrometers. The procedure for morphometric analysis entailed downloading ImageJ, opening it, and then calibrating it using a calibration slide image to set the scale in micrometers. Subsequently, images captured from the microscope were opened in ImageJ for analysis, ensuring consistency in camera and microscope settings, magnification power, and camera zoom with the calibration slide.

Statistical analysis

In this investigation, Data description, analysis, and presentation were performed using GraphPad Prism version 9.5.0 software (GraphPad Software, Inc. USA) and Microsoft Excel 2013 were employed. The present study's data was carefully examined to evaluate the difference between groups using normality tests to determine if it was parametric or non-parametric. As a result, appropriate statistical test was employed T. test. The level of significance in this study as Not significant P>0.05, Significant P<0.05.

Observations of the liver's anatomical criteria in both groups revealed that the liver is situated in the right upper quadrant of the abdominal cavity, resting just below the diaphragm, to the right of the stomach, and overlying the gallbladder. A slight enlargement of the liver was noticed in the pregnant group. There were no anatomical differences between the livers of the pregnant and non-pregnant (control) groups in terms of location, shape, diffusion, and consistency. However, differences in external morphology were observed through naked eye examination, which showed clear color differences between the two groups. In the control group, the liver was dark brown, while in the pregnant group, it was light brown in color, as demonstrated in Figure 1.

The statistical analysis of liver weight indicated a significant increase in the weight of the liver in the pregnant group compared to the control group, with a p-value of 0.0001. The mean ± standard error (SE) for the liver weight was 2.14 ± 0.08 for the pregnant group and 1.47 ± 0.03 for the control group, as detailed in Table 1.

Table 1: Show the difference in the weight of liver in control and pregnant groups.

The Groups	Mean ± SE of Liver weight (g)	p-value
Control	1.47± 0.03	0.0001
Pregnant	2.14± 0.08	0.0001
p< 0. 05 a statistically significant.

The total body weight (TBW) analysis revealed a significant increase when comparing the TBW of pregnant group mice in their late pregnancy (after the removal of embryos) to their TBW before pregnancy. The mean TBW before pregnancy was 26.97 ± 0.15 grams, which increased to 32.66 ± 0.64 grams in late pregnancy (with embryos removed). The statistical analysis of these means indicated a significant increase in TBW, marked with a p-value of 0.001, as detailed in Table 2.

Table 2: Show the difference in body weight of mice between the pregnant group before and after pregnancy.

Pregnant (Time)	Mean ± SE Body weight (g)	P-value
Control	26.97± 0.15	0.0001
Late Pregnancy (with removed embryos)	32.66± 0.64
Significant at P value < 0. 05.

Sections of mouse liver stained with hematoxylin and eosin (H&E) display cellular labeling patterns consistent with those reported for other mammalian species. Portal areas, showcasing elements of the hepatic triad (branches of the portal vein and hepatic artery, a bile duct, lymphatic vessels, and minimal connective tissue), confirm the lobular structure of the mouse liver. Hepatocytes are organized in plates or cords radiating from central venules towards portal areas, with sinusoidal capillaries separating them. Liver sinusoids exhibit a regular distribution and are lined by small, flat endothelial cells. Additionally, Kupffer cells, macrophages associated with the endothelial lining, are observed as large, darkly stained cells often containing granules. Hepatocytes feature multiple narrow spaces around them, representing bile canaliculi. As shown in Figure 2.

Examination of liver sections from both pregnant and control groups revealed that the liver is encased in a thin connective tissue capsule (Glisson's capsule), composed of fine, dense irregular fibroelastic connective tissue, with no observed differences in capsule composition, arrangement, or thickness between groups. The liver parenchyma consists of hepatic lobules with a rough hexagonal arrangement, made up of hepatocyte plates radiating from a central vein. However, identifying hepatic lobules was challenging due to the limited and uneven arrangement of portal areas, leading to unclear boundaries within the liver parenchyma. Portal areas, showing no differences between the groups, contained a branch of the portal vein with a thin wall and regular lumen, a branch of the hepatic artery with a thick wall, and a bile duct lined by cuboidal epithelium. The overall number of portal areas was limited, with no recorded differences in the distribution and constituents of portal areas between the control and pregnant groupsn (Figure 3 and Figure 4).

Hepatocytes in both pregnant and control groups were arranged in plates radiating from the central vein. Notably, larger and often binucleated hepatocytes were more frequent in the pregnant group. Morphometric analysis using Image J on images from various slide sections indicated smaller cell sizes and higher cell numbers in the non-pregnant group, while larger cell sizes and fewer cells were observed in the pregnant group. This suggests adaptive changes in hepatocyte size during pregnancy as recorded in Figure (5) and Figure (6). Group A (non-pregnant) had an average hepatocyte area of 9,845.07±1,764.857 µm², compared to 20,346.03±2,000.942 µm² in Group B (pregnant), indicating a significant increase in hepatocyte size in the pregnant group (P=0.001). As illustrated in Table 3.

Table 3: Size of hepatocytes in study groups

	groups	Mean	±SD	±SE	t-test	p-value
hepatocytes	pregnant	20346.03	2000.942	365.320	21.557	0.0001
hepatocytes	non pregnant	9845.07	1764.857	322.217	21.557	0.0001
SD: Std. Deviation, SE: Std. Error Mean. Not significant P>0.05, Significant P<0.05

In hepatocytes, the cytoplasm exhibited notable differences between the control and pregnant groups. Control hepatocytes displayed a highly vacuolated cytoplasm with large pale areas around the nucleus, likely indicating glycogen accumulation. In contrast, the pregnant group showed an increased presence of glycogen vacuoles. Hepatocytes, characterized by their polygonal shape and arrangement in plates or cords within liver lobules, have central nucleus and abundant cytoplasm rich in organelles like the endoplasmic reticulum and mitochondria, indicative of their metabolic activity. Glycogen within these cells appears as granules, which can be stained and visualized under a microscope.

The PAS (Periodic Acid-Schiff) stain, used to identify glycogen arrangement and aggregation, revealed significant differences between the groups. Control group liver showed sparse, small eosinophilic glycogen aggregates, whereas the pregnant group exhibited larger, more numerous eosinophilic glycogen aggregations distributed throughout the liver. As shown in Figure 7 and Figure 8, respectively.

Quantitative assessment of PAS stain expression, indicating glycogen concentration, was performed using the Aperio program. The non-pregnant group had a mean expression value of 0.0527±0.01741 pixels/micron², while the pregnant group showed a significantly higher mean value of 0.0987±0.02738 pixels/micron², demonstrating a marked increase in glycogen concentration in the pregnant group (P=0.001). As recorded in Table 4.

Table 4: the mean value of Glycogen concentration in study groups

	groups	Mean	±SD	±SE	t-test	p-value
Glycogen	pregnant	0.0987	0.02738	0.00500	7.765	0.0001
Glycogen	non pregnant	0.0527	0.01741	0.00318	7.765	0.0001
SD: Std. Deviation, SE: Std. Error Mean. Not significant P>0.05, Significant P<0.05

The liver's crucial role extends beyond nutrient management; it neutralizes and eliminates numerous harmful substances. This multifaceted functionality has motivated research into liver cell composition and organization under various conditions, including pregnancy—a subject not yet fully explored.

With mice increasingly used in liver research, their resemblance to human liver structure and function underpins many studies. Given mice's growing importance in liver research, establishing a baseline for their liver's normal cellular composition is essential. This foundation will support future investigations into liver function and pathology (Baratta et al., 2009; Naugler et al., 2015).

This study utilized mice to examine cellular and tissue changes in the liver through various histological techniques (H&E, PAS). The findings reveal that the mouse liver shares fundamental structural similarities with livers of other mammals.

Anatomically, the liver is located in the right upper quadrant of the abdomen, presenting as a reddish-brown, wedge-shaped organ with four lobes of varying sizes and shapes. This observation aligns with previous research, highlighting the liver as the largest gland and a significant organ in the body, characterized by its wedge shape and divided into right and left lobes based on external morphology (Mitra & Metcalf, 2012).

The study also explored the impact of pregnancy on maternal body weight and its subsequent effects on the liver. Statistical analysis indicated significant changes in body weight during pregnancy, correlating with increases in liver weight in the pregnant group. These findings are consistent with earlier studies (Milona et al., 2010), which reported a normal weight increase in the liver of pregnant females, attributed to changes in maternal mass and the heightened metabolic demands of the fetoplacental unit.

In a study by done by Grimbert et al. (Grimbert et al., 1993) a marked increase body weight was established statistically with a parallel increase in liver weight and this was explained by fact that pregnancy tend to slightly decrease of amount of mitochondrial protein recovery from the liver showed a marked amount increased (51± 2g) in pregnant than the control which was (27±1g). These studies have a recorded in morphological frequent appearance of double or triple nuclei in pregnancy than in those seen in non-pregnant group.

The liver's cellular organization and its lobular structure are defined by the arrangement of hepatocytes around the central vein, supported by portal areas containing the hepatic triad. These lobules, hexagonal in shape, feature a central vein with radially arranged hepatocytes and intervening liver sinusoids, lined by endothelial and Kupffer cells (Nagarajan, 2019).

The liver's cellularity encompasses both parenchymal (primarily hepatocytes) and non-parenchymal cells (including Kupffer, stellate, and lymphocytes), distinguishable through histological and microscopic techniques (Bowman & Russell, 2006).

Pregnancy induces enhanced hepatic metabolism to meet the increased energy demand and detoxify fetal metabolites. Hepatocytes, often enlarged and multinucleated, show a capacity for increased metabolic activity, contributing to liver enlargement during pregnancy (Milona et al., 2010). This gestational hepatomegaly, alongside metabolic changes affecting enzyme activities, glycolysis, and gluconeogenesis, remains not fully understood. Complications like acute fatty liver and gestational diabetes indicate significant metabolic shifts, with systemic glucose production rising by 16% to support the fetus (Bancroft et al., 2013).

Liver endothelial cells, crucial for organ function, feature fenestrations forming sieve plates, facilitating the movement of small molecules, highlighting the liver's specialized vascular architecture (Pavelka & Roth, 2015).

The maintenance of normal glucose tolerance is facilitated by lactogenic activity, which involves the augmentation of pancreatic islet mass by β-cell hypertrophy and hyperplasia. This process also leads to an enhancement in insulin synthesis and glucose-stimulated insulin secretion, while simultaneously reducing the glucose-stimulation threshold. The endocrine pancreas undergoes modification during gestation in response to two primary stimuli, namely human placental lactogen and prolactin, which are known as lactogens. Ramos-Roman's work examines the impact of lactogens on maintaining glucose balance during pregnancy. It suggests a method via which the hormonal regulation of breastfeeding, primarily by prolactin, may influence the biology of adipocytes, glucose and lipid metabolism, and protect postpartum women from developing type 2 diabetes (Ramos-Roman, 2011)

The present study has observed liver enlargement on the 12th day of pregnancy, indicating that lactogens originating from the placenta may initiate this process. This is because during this time, there is significant development of the placenta, leading to the release of lactogens from the placenta. Additionally, there is a significant increase in maternal hepatic growth hormone and prolactin receptor expression during pregnancy (Cramer et al., 1992). Furthermore, the correlation between liver size and foetal number provides evidence that foetal or placental signals have a role in gestational hepatomegaly. Gaining insight into the signals that trigger liver growth, hepatocyte hypertrophy, and cell-cycle arrest at S/G2 in mice models during pregnancy might provide valuable information for developing safer methods to enhance liver function in women at times of heightened metabolic requirements.

Actually, rodents experience gestational hepatomegaly as a means to accommodate the heightened metabolic requirements placed on the mother liver during the course of pregnancy. The processes and signals behind pregnancy-induced liver expansion remain unknown, despite its significance as a physiological function. In this study, we demonstrate that liver growth in pregnant women occurs before the increase in maternal body weight. This growth is directly related to the number of foetuses and is caused by the enlargement of liver cells, which is connected with the development of the cell cycle, polyploidy, and changes in the expression of cell-cycle regulators such as p53, Cyclin-D1, and p27. Due to the elevated levels of circulating reproductive hormones and bile acids in pregnant women, these substances have the potential to stimulate liver development in rats, making them potential candidates for the signalling pathway responsible for gestational liver enlargement in rodents. The administration of reproductive hormones at pregnancy levels did not provide a significant effect on liver development. However, it was shown that mouse pregnancy was correlated with elevated levels of serum bile acids. The need of the bile acid sensor Fxr for the successful recuperation after partial hepatectomy has been established. In this study, we provide evidence that mice lacking Fxr−/− experience gestational liver expansion via the process of adaptive hepatocyte hyperplasia. This study represents the first discovery of a specific element necessary for the proper functioning of gestational hepatomegaly. Additionally, it suggests that Fxr plays a role in a physiologically normal process that regulates the cell cycle of hepatocytes. According to Milona et al. (Milona et al., 2010) who conclude that understanding pregnancy-induced hepatocyte hypertrophy in mice could suggest mechanisms for safely increasing functional liver capacity in women during increased metabolic demand.

The result of present study consistent with Al-Gayyim and Al-Habib (Al-Gayyim & Al-Habib, 2020) who found maternal liver growth during pregnancy is driven by hepatocyte hyperplasia and hypertrophy. Several waves of hepatocyte proliferation, manifested by increases in the numbers of Ki67-positive hepatocytes and mitotic figures and in maternal hepatic DNA content, were evident as pregnancy progresses. The most prominent waves of hepatocyte proliferation took place on gestation days 8, 13, and 15. Hepatocyte hypertrophy was concomitant with maternal liver mass expansion, especially during the second half of pregnancy. It is noteworthy that, by examining non-pregnant and gestation day 18 livers.

In addition, Gielchinsky et al. (2010) conducted a study that provided evidence for the regeneration of the elderly maternal liver by hepatocyte hypertrophy after partial hepatectomy during pregnancy (Gielchinsky et al., 2010). The results suggest that pregnancy has a significant impact on the recovery of the maternal liver after a substantial loss of mass. Significantly, more recent studies have emphasised the involvement of placental hormones, including prolactin and placental lactogens, in facilitating the hormonal response of the maternal pancreas to pregnancy (Kim et al., 2010).

Whether these hormones take part in regulating maternal liver growth during pregnancy is under investigation.

During pregnancy, there are physiological adaptations that occur in the maternal compartment. This study aimed to investigate the impact of pregnancy on the maternal liver in CD-1 mice. Significant alterations were seen in the dimensions of the maternal liver over the course of pregnancy. The weight of the livers increased twofold from the nonpregnant condition to the 18th day of pregnancy. The hepatomegaly seen during pregnancy was a natural occurrence characterised by liver expansion, as shown by an increase in DNA content and the identification of hepatocyte hyperplasia and hypertrophy. Hepatic growth started after implantation and reached its highest point after parturition. An investigation was conducted to examine the expression and/or activity of crucial genes that are responsible for regulating liver regeneration, which is a process of liver expansion that compensates for the loss of liver mass (Dai et al., 2011).

During pregnancy, noticeable physiological changes occur, including an increase in total body weight, largely attributed to adiposity, and a significant change in liver weight, driven by heightened metabolic enzyme activity to meet the demands of the fetoplacental unit. Concurrently, there's an elevation in liver glycogen levels in pregnant rats, indicative of altered storage and distribution to accommodate the increased metabolic requirements. Furthermore, the prevalence of multinucleated, enlarged hepatocytes during this period underscores the liver's adaptive response to the escalated metabolic needs, illustrating the profound systemic adaptations inherent to pregnancy.

ACKNOWLEDGMENTS

This article did not receive any specific grant from funding agencies in the public, commercial, or not‐for‐profit sectors.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

ETHICS STATEMENT

The project was approved by the Scientific Research and Ethical Committee of the College of Medicine, University of Al-Iraqia, and performed in accordance with the Swiss Animal Protection Law in the Center of Cancer and Medical Genetics Research (CCMGR), in which animals were housed and an experimental study was performed.

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No competing interests reported.

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Histomorphometric and Ultrastructural Study of Gestational Changes in Mouse Liver Using Haematoxylin and Eosin, Periodic acid Schiff stain

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