The placenta is composed of two distinct regions, the placentomal and interplacentomal areas, which develop and differentiate as pregnancy progresses. The interplacentomal area has been found to be simply located between the fetal membranes and the uterine epithelium. Placentomies, on the other hand, function as functional units of the placenta, forming the connection between the caruncular uterine and fetal cotyledons in later pregnancies³. The placenta, formed from the uterine mucosa and the offspring's chorion at the beginning of pregnancy, plays a role in the fetal development and protection. Healthy placentation not only provides nutrition, development, excretion, and respiration for the embryo, but also provides a healthy connection between the mother and the developing fetus by secreting various hormones throughout embryonic development⁴. Maternal endocrine glands are paracrine, autocrine and endocrine secretory organs that synthesize a wide variety of steroids and peptide hormones, primarily progesterone, estrogen, relaxin, hCG, and placental hormones, which support fetal development by providing hormonal support to the fetus via the fetoplacental unit and change maternal physiology in the process⁵.

Irisin is an adipokine and myokine that increases with pregnancy and regularly functions to maintain energy homeostasis throughout pregnancy^4,6. Irisin, first identified in 2012 as a myokine polypeptide released from muscle tissue in response to exercise. It results from the proteolytic degradation of FNDC5, regulates glucose and lipid metabolism in adipose tissue⁷. Recently, it has been demonstrated that irisin is not only involved in energy storage and secretion, but also plays a role in meeting energy needs related to reproductive function and fetal growth, and in balancing pregnancy-related metabolic changes⁸.

Serum irisin levels were found to be higher in pregnant women than in non-pregnant women during pregnancy^9,10. Furthermore, the expression of the fibronectin type III domain-containing protein 5 (FNDC5) protein was determined in the placenta of pregnant women⁹. Irisin has been shown to have cytoprotective properties in human preeclamptic placentas and in placental disease models (10, 11).Recently, it has been demonstrated that irisin may play a role in the placenta by regulating trophoblast differentiation through AMPK activation¹².

Studies on irisin hormone in twelve different species (including humans) have revealed the presence of irisin protein sequences⁴. It has been reported that irisin hormone is expressed in smaller amounts in the testes, liver, pancreas, brain, spleen, heart, and stomach, in addition to skeletal muscle and adipose tissue^13-15. Expression has also been observed in the ovaries, placental tissues, and umbilical cord serum of newborns⁹. In human placental tissue, it is localized in cytotrophoblasts and syncytiotrophoblast cells in the placental decidua⁹. A study specific to ruminants showed that irisin hormone is expressed in the placenta during pregnancy in Merino sheep and that serum levels are higher throughout pregnancy¹⁶. In recent years, studies have focused on the possible endocrine functions of adipose tissue and the adipokines it secretes on the reproductive system. Investigating the functions of adipokines in the reproductive system is essential for understanding energy homeostasis. Studies exist on farm animals as well, but they are insufficient.

In consideration of the aforementioned background, the objective of this study is to elucidate the histological alterations that occur in the placentomal and interplacentomal region in goats during the early, middle and late stages of pregnancy. Furthermore, the study seeks to ascertain the presence of irisin and its subsequent localization within the tissue using immunohistochemical methods.

Sample Collection and Gestational Age Determination: The study was conducted using fetal samples obtained from private slaughterhouses in Siirt province. After slaughter, an incision was made in the uterine horn, where the pregnancy is located, wide enough to allow for the removal of the fetus. After the fetal membranes and fluids were removed, the crown-rump length of each resulting fetus was measured. Gestational age was determined using the following formula: X = 2.1 (Y + 17) (X = gestational age (days), Y = crown-anus length^17,18. Singleton pregnancies were considered in the study. Eighteen pieces of uterine tissue from different periods of pregnancy including first trimester (0-66 days), second trimester (67-99 days) and third trimester (100-140 days) were taken together with the placenta and uteroplacental tissue was used¹⁹.

Tissue Processing and Hematoxylin-Eosin: Tissue samples were taken from the placentome and interplacentome regions in three groups formed in the study and were fixed in 10% neutral formalin solution for 24 hours. Tissues were dehydrated with alcohol series and cleared with xylene in accordance with standard histological tissue processing procedures. The tissues were then subjected to routine tissue processing in xylene (2 x xylene, 1.5 hours each step) for clarification. The paraffin-embedded tissues were then blocked. 5 ?m-thick sections were cut from the paraffin blocks using a rotary microtome (Thermo Scientific, USA), and the sections were placed on slides. The slides were dried in a 37°C oven and prepared for staining. The slides were stained with hematoxylin and eosin for histopathological analysis. After staining was completed, the slides were dehydrated by passing through a series of alcohols of increasing concentrations, made transparent with xylene, and coverslipped with entellan²⁰.

Irisin Immunohistochemical Staining: The immunoperoxidase method was used for the immunohistochemical analyses performed in the study²¹. First, 5 ?m-thick sections were cut from paraffin blocks of placentome and interplacentome region tissues. After deparaffinization with xylene, they were rehydrated by passing through a graded alcohol series. To inactivate endogenous enzymes, the tissues were held in 3% H2O2 for 10 minutes. Washing was carried out with phosphate buffered saline (PBS). Then, they were treated with Ultra V Block solution for 10 minutes and incubated with primary antibodies (Irisin, 1/100 dilution, 201r-0335, China) at +4°C overnight. For the negative control, phosphate-buffered saline (PBS) was applied in place of the primary antibody. The slides were incubated with a secondary antibody, followed by streptavidin-peroxidase, in a humid environment at 37 °C for 30 minutes. AEC (3-amino-9-ethylcarbazole substrate + AEC chromogen, LabVision Corporation, USA) was applied as a chromogen, and the sections were stained with Mayer's haematoxylin for nuclear staining. They were then mounted with CC/mount Aqueous Mounting Medium and examined under a light microscope. Histopathological examination and immunostaining evaluation were performed blindly by two independent researchers.

Immunohistochemical histoscores were performed according to the extent and intensity of immunoreactivity as previously described²². (Histoscore = extent (0.1: <25%, 0.4: 26%?50%, 0.6: 51%?75%, 0.9: 76%?100%) × intensity (0: absent, +0.5: very slight, +1 slight, +2: moderate, +3: severe). Irisin immunostaining in placentome and interplacentome tissue samples were examined microscopically at 10x, 20x, and 40x objective magnifications. For evaluation purposes, 4 random fields were selected from each section of placentome and interplacentome of each animal. A single value was determined for each animal by averaging the results obtained²³. The luminal epithelium, glandular epithelium, stromal tissue, and muscle cells of the interplacentomal region, and the maternal epithelium, fetal single and dual-nucleated trophoblast cells, and stromal cells of the placentomal region were evaluated.

Statistical Analysis: The data obtained were expressed as mean ± standard deviation, and statistical analyses were performed using the SPSS 22 program (IBM Corp., Armonk, NY, USA). When data showed a normal distribution, one-way ANOVA and Tukey's post hoc test were applied for comparisons of more than two groups. If the data did not meet the normality assumptions, the Kruskal-Wallis test was used, and pairwise comparisons were made using the Mann-Whitney U test. A p-value < 0.05 was calculated to determine whether the results were statistically significant. For the sample size analysis, the power of the test was determined to be 80%, and the type-1 error of 5% (G*Power statistics program, ver.3.1.9.4).

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Figure 1: Histological images of the goat placenta and interplacentomal region during early (0-66 days) (a-d), mid (67-100 days) (e-h), and late (100-150 days) (i-l) stages of gestation. Gestation. Fe: Fetal Epithelium; FS: Fetal Stroma; Bv: Blood Vessel; Me: Maternal Epithelium; Ms: Maternal Stroma; Le: Luminal Epithelium; S: Stroma; Gl: Gland Epithelium; M: Myometrium; Pv: primary chorionic villi; Sv: primary chorionic villi; Ts: primary chorionic villi. Scale bars: 500 µm (ı, j); 200 µm (a, b, e, f, k, l); 50 µm (c, d, g, h). Staining: H&E.

Following implantation, the maternal caruncular structures and fetal cotyledons unite to form placentomes. Trophoblastic cells arising after embryonic development play a role in this connection between the maternal endometrium and the fetal embryo (Figure 1d).

Samples taken from the placentomal region showed mononuclear trophoblast cells (MTC) and binucleated trophoblast cells (BTC) in the early period. As pregnancy progressed, the presence of large binucleated trophoblast cells became noticeable (Figure 1h). Primary villi from the chorionic plate of the placentome branched to form secondary and tertiary villi (Figure 1b). An increase in the number of secondary and tertiary villi was observed in the later stages of pregnancy compared to the early stages (Figure 1j). These chorionic villi contained nested fetal and maternal tissues (Figure 1j). Within the fetal stroma of the placentome, fetal mesenchyme composed of fibrocytes and fibroblasts was present (Figure 1f).

When the embryo began to attach to the uterine caruncular regions, the columnar epithelial cells of the uterus transformed into syncytial plaques (Figure 1h). The placenta barrier between maternal and fetal tissues consisted, on the fetal side, of fetal mesenchyme, fetal vascular endothelium, and fetal trophoblastic epithelium, while on the maternal side it consisted of maternal connective tissue, maternal uterine epithelium, and maternal vascular endothelium. During the second and third trimesters, an increase in the number of mononuclear and binucleated trophoblast cells embedded in the trophoblastic epithelium was observed. Mononuclear trophoblast cells had round, euchromatic stained nuclei and columnar or cuboidal cells. Binucleate trophoblast cells were round or oval in shape with two nuclei (Figure 1h).

Immunohistochemical Localization of Irisin in Interplacentomal and Placentome Region: In the interplacental region of uterine samples obtained from early, mid, and late pregnancy, irisin was detected in the cytoplasmic and nuclear compartments of luminal and glandular epithelial cells, as well as in stromal and smooth muscle cells (Figure 2). When the immunoreactivity of irisin in these cells was evaluated according to histoscores, no statistically significant difference was observed among the three distinct gestational periods (p<0.05, Table 1).

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Table 1: Distribution of irisin protein expression in the placenta and uterus of hair goats during early (0?66 days), mid (67-100 days), and late (100-150 days) pregnancy based on immunostaining intensity scores (IS). All values are presented as mean ± standard deviation

Analysis of irisin positivity in the cellular components of the interplacental region revealed particularly strong staining around the glandular structures (Figure 2c-2g-2k). Furthermore, an increase in reactivity was noted in the later stages of pregnancy; however, this increase was not statistically significant (p>0.05, Table 1). Irisin immunoreactivity was detected in the luminal epithelium of the uterus throughout all pregnancy stages, and similar histoscores were observed across different gestational periods. Irisin also exhibited positive staining in the cytoplasm of smooth muscle cells located in the interplacental region (Figure 2a-2e).

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Figure 2: Immunolocalisation of irisin in goat placentomal and interplacentomal part, in the first period of pregnancy early (0-66 days) (a-d), mid (67-100 days) (e-h), and late (100-150 days) (i-l) stages of gestation Fe: Fetal Epithelial, FS: Fetal Stroma, , ME: Maternal Epithelium, MS: Maternal Stroma, LE: Luminal Epithelium, S: Stroma, Gl: Gland Epithelium, M:Miyometrium, hollow arrow(⇒): positive cell. Scale bars: 200 µm (a, b, e, f, g, ı, j, k); 50 µm (c, d, h, l). Counterstain: Mayer's hematoxylin.

In the fetal portion of the placenta, irisin demonstrated both cytoplasmic and nuclear immunoreactivity in fetal stromal cells as well as in mononuclear and binuclear trophoblasts (Figure 2d-2l).

This immunoreactivity was higher in the first trimester and decreased in later stages; however, this change was not statistically significant (p>0.05, Table 1).

Evaluation of the maternal compartment of the placenta showed that maternal stroma was more intense in the first trimester (Figure 2d). A statistically significant difference was detected between groups (p<0.05, Table 1). Irisin reactivity was generally absent in the maternal epithelium. No positivity was observed in the negative control preparations.

The placentome is the structure formed by the combination of the caruncular structure of the mother and the cotyledonary structure of the offspring shaped by glandless endometrium areas²⁴. In goats^17,25 and sheep^26,27 cotyledon and caruncular tissues form in the placentome and are intertwined with each other. Consistent with the literature, our study showed the formation of fetal cotyledon tissues consisting of the chorionic plate and the maternal caruncular structure consisting of the basal plate. In the early stages of pregnancy, the cotyledons were not yet well defined; as gestation progressed, they fused with the caruncles to form the placentomes.

After uterine implantation, the trophoblast cells that form as a result of divisions first become mononuclear cells (single-nucleated trophoblast cells), and then become specialized into binuclear cells (double-nucleated trophoblast cells) ²⁴. Mononucleate trophoblast cells and binucleate trophoblast cells were observed in the fetal trophoblastic epithelium, as in West African Dwarf goats²⁴ and Yankasa and Balami sheep²⁶. In early pregnancy, mononuclear trophoblasts predominate, whereas binuclear trophoblasts increase in number as gestation progresses. This increase and feto-maternal communication are important for placental development and hormone production.

Meeting the increased nutrient and energy needs of mothers and offspring during pregnancy is important for the healthy development of both mother and offspring. Irisin was first discovered by Boström et al. ⁷ and is known to be associated with energy metabolism by activating numerous pathways in muscle and fat cells^7,16,28-30. In addition to its energy-regulating effects, irisin also supports the blood-placental barrier by promoting the proliferation and development of trophoblast cells and supporting vascular structure through its regulating properties of endothelial cells^12,31. Irisin is expressed in the ovary, placenta^9, and neonatal cord serum³² and plays a role in embryonic development that occurs during pregnancy³³.

The precursor of irisin, FNDC5, is expressed in the placenta of pregnant women, and one study reported that maternal serum irisin levels were higher in pregnant women than in non-pregnant women throughout pregnancy^9,10. In the study by Yuksel et al. ^34, maternal serum irisin levels were found to be significantly lower in women with gestational diabetes, while umbilical cord blood irisin levels did not differ between the GDM and control groups.

This is the first study to describe irisin expression in the interplacentomal and placentomal regions of ruminant goats. The present study investigated the immunoexpression and tissue distribution of irisin at different stages of pregnancy in goats. The detection of irisin immunoexpression in the interplacentomal and placentomal regions suggests a potential association with metabolic regulation, energy homeostasis, or trophoblast functions.

Irisin expression was observed to increase throughout pregnancy in the luminal and glandular epithelium interplacental region. The increased number of irisin-positive cells in the glandular epithelial region of the interplacentomal zone, in relation to meeting energy needs during pregnancy, suggested in previous studies that increased trophoblast activity via AKT-AMPK¹¹ signaling may contribute to elevated irisin levels. This finding supports the presence of an increasing immune response during pregnancy, consistent with previous reports in the literature⁹.

As for the irisin expression in the placentomal region, higher irisin expression was determined in the maternal and fetal portions of the placenta during the early stages of pregnancy. This suggests that irisin may contribute to placental development and implantation. The high irisin expression observed in the maternal stroma during early pregnancy (p<0.05), and its subsequent decrease as pregnancy progresses, further suggests that irisin may play a role in the interactions between trophoblast and endometrial tissues during implantation in ruminant animals. Increased oxidative stress in the early stages of pregnancy, increased inflammatory signals, and the high energy requirements of the fetus for placentation, despite its small size, may have increased irisin expression. This increase may be an adaptive mechanism to support placental development. In the second and third trimesters of pregnancy, the largely formed placenta, resulting in decreased trophoblastic cell invasion, and subsequent more balanced hormones may have led to a more stable irisin expression.

This study presents the first immunohistochemical evidence of irisin expression in the interplacental and placental regions of ruminant goats. Irisin manifested as increased expression in the glandular, luminal, stromal, and muscle components of the interplacental region throughout pregnancy. The higher expression observed in early pregnancy and the increase in maternal stromal tissues suggest that irisin may play a role in implantation and the interaction between trophoblast and endometrial tissues. Furthermore, the persistence of irisin expression in later stages may indicate a potential role in supporting placental function and contributing to metabolic and immunological processes, but further studies are needed to confirm these relationships. Overall, these findings highlight irisin as a possible modulator of placental development and maternal-fetal communication in goats, however, the study was conducted with a limited number of slaughterhouse materials and samples; this can be considered a limitation of the study. However, this forms a basis for future studies with larger sample sizes that will investigate their functional roles in the reproduction of ruminant animals.

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