Inflammatory and anti-inflammatory cytokines in the endometrium of cows during anaphrodisia and estrous cycle

. Infertility and sexual cycle disorders in cows are an urgent and widespread problem in cattle breeding, causing significant losses to farms. At the same time, a detailed understanding of the cellular and molecular mechanisms in the bovine uterus is crucial to explain and avoid infertility in dairy cows. The research aims to determine the concentration of tumour necrosis factor (TNF α ), interleukin-1 (IL-1) and interlekin-4 (IL-4) in endometrial homogenates during oestrus, proestrus, corpus luteum and anaphrodisia caused by endometritis, as well as lactation. The research material was homogenates of cow endometrium taken from the horns of the uterus in its upper third. The methods used to determine the content of IL-1, IL-4 and TNF α in endometrial samples were enzyme-linked immunosorbent assay with a ratio (tissue homogenate-phosphate-salt buffer). The results of the study revealed changes in the content of IL-4, IL-1 and TNF-α in the functional layer of the endometrium during anaphrodisia and certain stages of the oestrous cycle. A significant increase in the content of TNF-α


INTRODUCTION
Maintaining and improving the reproductive performance of cows and heifers is one of the key parameters for the economic success of dairy farming.According to M. Bekara & N. Bareille (2019), infertility is a significant problem for dairy farming, as the associated extended calving intervals, reduced milk yields and increased culling rates contribute to huge economic losses.According to P. Lonergan et al. (2019), the causes of infertility are diverse and complex and, in addition to controlling the sexual cycle, efficiency of oestrus detection and proper breeding management, monitoring uterine health is a key factor in the proper reproductive performance of a dairy herd.
In the postpartum period in dairy cows, a high risk of clinical or subclinical diseases of the reproductive tract is present, which, following O. Pascottini & S. LeBlanc (2020), is accompanied by glucose intake, hypocalcaemia, lipid mobilisation from body fat, ketosis and the expression of proinflammatory cytokines that affect the immune response and change rapidly but differently in individual cows.At the same time, according to H. Pothmann et al. (2021), inflammatory processes and mRNA expression of cytokines at the end of the postpartum period can be considered as multifocal or widespread throughout the endometrium, regardless of the health of the uterus.
I. Sheldon et аl. (2019) analysed infectious factors and endometrial inflammation in the postpartum period in cows with clinical and subclinical endometritis to determine the main causes of deterioration of the structural and functional state of the uterine mucosa and showed that inflammatory processes that continue in the postpartum period can impair fertility, lead to anaphrodisia, and interfere with embryo development.N. Naim et al. (2020) have shown that the reproductive function is realised through the activation of cambial tissues, which include the endometrium, and the physiological restructuring of the latter depends on coordinated intercellular interaction through the cytokine system.
F. Pinaffi (2018) found an important role of gonadal hormones and cytokines in preparing the endometrium for implantation and activating the transformation of epithelial cells and stromal fibroblasts into decidual cells.The cytokines IL1, IL6 and TNFα are considered (Drillich & Wagener, 2018) as mediators of nonspecific inflammatory processes, but their activity is also physiologically enhanced during the early postpartum period with a high degree of mRNA expression of cytokines and acute phase proteins during the third week after delivery, regardless of the health status of the animals.
The cytokine system is quite informative about the intercellular interaction of the endometrium, in connection with the prediction of zygote nidulation.According to P. Abeysinghe et al. (2023), binding of interleukin-1 to the receptors of maternal placental cells ensures zygote nidulation, proliferation of placental barrier cells, embryogenesis, and an increase in the percentage of zygotes that reach the blastocyst stage.N. Nijkang (2019) points out that the main function of interleukin-4 is to regulate stem cell proliferation by inducing mitogen-activated protein kinases, which transmit intracellular signals that cause cell differentiation, metabolic changes, apoptosis, and stimulate luteal cells to secrete progesterone.Q. Huang (2021) has demonstrated that zygote nidulation occurs when proinflammatory cytokines are expressed, but subsequently, pregnancy proceeds physiologically in the event of a shift in the cytokine balance towards immunosuppressive cytokines (interleukin-4, interleukin-10), as the latter inhibits cellular immunity responses, whereas a significant increase in interleukin-4 indicates a violation of peripheral tolerance and a shift in the local immune response towards Th-1, which causes infertility, and the ratio of interleukin-1 to interleukin-4 is a prognostic indicator in assessing the course of fertilisation and pregnancy.
According to M. Lappas (2017), cytokines affect the regulation of numerous types of intercellular interactions: granulosa cell proliferation, expression of gonadotropin receptors, and, conversely, cytokine imbalance leads to desynchronisation of cell differentiation, implantation disorders and infertility.N. Naim et al. (2020) investigated the cytokine-regulated endothelial-haemostatic interaction during maternal placental remodelling, implantation, and pregnancy.Q. Huang et al. (2021) note that during endometrial proliferation and differentiation, cytokines stimulate the functional activity of cells and humoral regulation of intercellular and intersystemic connections.
Thus, the analysis of the cytokine profile of tissue extracts of the uterine mucosa of cows at different stages of the sexual cycle will make it possible to determine the fullness of sexual cyclicity, the prerequisites for the development of infertility and will help to optimise prognostic tests for the effectiveness of sexual function correction.The research aims to investigate the content of certain pro-and anti-inflammatory cytokines in cow endometrial tissue extracts at different stages of the sexual cycle and in anaphrodisia following endometritis and after partum retention.

MATERIALS AND METHODS
The research was carried out within the framework of the research work "Study of cellular, biochemical and molecular genetic mechanisms of infectious diseases, metabolic disorders and immunocompensatory processes of counteracting biotic and abiotic factors in obstetric-gynecological, andrological and surgical pathology in animals" (state registration number 0116U005121).
The selection of experimental samples of the uterine mucosa of cows was carried out in farms with different housing technologies in the period from 2016 to 2022: by the tethered method (Shorthorn cow breed, OJSC breeding plant "Mikhailovka", Lebedynsky district, Sumy region with a productivity of <6000 kg and SFH "Vitalia", Burynsky district, Sumy region, Simmental breed of cows with productivity of >6000 kg), as well as the untethered method (LLC AF "Vladana", Ukrainian Blackand-White cows and LLC AF "Lan", Sumy district, Sumy region, Holstein breed, with a productivity of >6000 kg).
The material for the study was fragments of endometrial tissue (2-6 grams) taken from the upper third of the uterine horns of euthanised cows from experimental farms.Endometrial tissue samples were taken from euthanised animals without gynaecological pathology of the reproductive organs, aged 3 to 10 years: in heat (n=5), during the corpus luteum (n=5), proestrus (n=5) and from cows that did not come into heat for more than 60 days after endometritis (n=5) and after delayed afterbirth (n=5).
The selected endometrial samples were washed with 0.9% sodium chloride saline with a theoretical osmolarity of 308 mosmol/l, pH 5.0-7.0.Subsequently, the washed samples were weighed on an electronic analytical balance of the 1st accuracy class AS 82/220 R2, labelled, packed in polystyrene containers, and subjected to cryopreservation at -20°C.To determine the concentration of cytokines IL-1, IL-4 and TNFα, endometrial samples were homogenised in the cold with the addition of 0.01 M phosphate-salt buffer (pH 7.4) containing 1% Triton X-100 solution in a ratio of 1:40, followed by placement in a +4°C refrigerator for 120 minutes ( Lazorenko & Izdepsky, 2012).
The endometrial homogenates were then centrifuged at 3000 rpm for 15 min.In the supernatant, the level of cytokines IL-1, IL-4 and TNFα was determined by enzyme-linked immunosorbent assay using diagnostic test kits manufactured by Peninsula Laboratories Inc, (USA).The actual content of IL-1, IL-4 and TNFα in endometrial samples was determined by considering the ratio (tissue homogenate-phosphate-salt buffer).
Experimental studies were performed by modern methodological approaches, following the requirements of DSTU ISO/IEC 17025:2005 (2006).The animals were kept, and all manipulations were carried out following the Order of the Ministry of Health of Ukraine No. 416/20729 "On Approval of the Procedure for Conducting Animal Tests in Research Institutions" (Law of Ukraine No. 249, 2012) and the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (1986).The digital material obtained in the research was processed by variational statistical methods using statistical techniques (STATISTICA 10.0 for Windows) with the determination of the arithmetic mean (M), the statistical error of the arithmetic mean (m), and the probability of difference (p) between two variation series according to the Student's criterion with values of p≤0.05, p≤0.01, and p≤0.001.

RESULTS AND DISCUSSION
The complex pathogenetic aspects of the interaction of anti-inflammatory and pro-inflammatory cytokines during endometrial remodelling and post-morbid state in cows are not well understood.Therefore, the studies that detail the above processes are new and relevant.The data presented in Figure 1 indicate that during oestrus, the concentration of TNFα in the endometrium is the lowest, amounting to 5.03±2.16pg/ml, while during proestrus and the corpus luteum, its level increases, compared to the oestrous phase, by almost 4.2-4.3times (21.15±3.08 pg/ml and 21.63±4.69pg/ml, p<0.01), respectively.

Proestrus Endometritis cases
Litter retention cases pg/ml

Figure 1. TNF-α content in cow endometrial homogenates, pg/ml Source: compiled by the authors
The study of TNF-α content in endometrial homogenates of cows, during anaphrodisia and after endometritis and retained litter, indicates high expression, with the accumulation of this cytokine in the endometrium.In particular, the concentration of TNF-α in endometrial homogenates was significantly higher in patients with endometritis -2.6 times to 13.07±1.98pg/ ml (p<0.01) and 3.6 times after afterbirth to 18.21±3.99pg/ml (p<0.01),compared to the index during oestrus.
At the same time, the level of endometrial TNFα decreased by 1.7 and 1.2 times in cows with endometritis and after delayed afterbirth, respectively, compared to the same indicator in the corpus luteum, respectively.Concerning the proestrus, the content of TNFα in the endometrium decreased by 1.6 times in animals with endometritis and by 1.2 times after delayed birth, respectively.The data shown in Figure 2 demonstrate an increase in the level of IL-1 in cow endometrial homogenates during oestrus (8325.46±69.18pg/ml), almost 2-fold, compared to the corpus luteum (4153.82±74.23 pg/ ml) and proestrus (4092.57±476.05pg/ml), p<0.001, respectively.The absence of a significant difference in the content of endometrial IL-1 in proestrus compared to corpus luteum is obviously due to the attenuation of the temporary inflammatory response inherent in the endometrium in oestrus at this period of the sexual cycle and a shift in the cytokine balance in favour of immunosuppressive cytokines, which creates optimal conditions for zygote nidulation.The concentration of IL-1 in the endometrium of anaphroditic cows suffering from endometritis and afterbirth retention was 2.5 times lower than that of cows in heat, 3351.71±1422.42pg/ ml and 3412.43±430.12pg/ ml, respectively, p<0.01, respectively, which is associated with the inhibition of the transformation of stromal fibroblasts and epithelial cells into decidual cells, and the impossibility of endometrial remodelling necessary for implantation.Thus, studies have shown that the key proinflammatory cytokine in the endometrium of cows during oestrus is IL-1, while from 7 to 18 days of the sexual cycle its concentration decreases significantly, almost reaching the level of animals that do not show sexual cyclicity, and TNFα takes over the role of the dominant proinflammatory cytokine, the expression of which increases significantly during this period.As can be seen from the data presented in Figure 3, the level of IL-4 in endometrial homogenates during oestrus reaches its maximum value -407.07±45.78pg/ ml, while during the corpus luteum it begins to show a tendency to decrease by almost 1.4 times (295.31±42.16pg/ml), and during proestrus it undergoes a significant decrease by 3.4 times (118.56±32.34pg/ml), p<0.001, respectively.At the same time, a significant difference in the concentration of endometrial IL-4 was found between days 7-8 and 17-18 of the sexual cycle, amounting to 2.5 times (p<0.01),respectively.The high concentration of this cytokine during oestrus is probably a consequence of the inhibitory effect of IL-4 on proinflammatory cytokines, the content of which at this time significantly increases, and in particular, the level of IL-1.The level of IL-4 in the endometrial homogenates of cows in the state of anaphrodisia and suffering from endometritis (121.03±52.36pg/ml) and afterbirth retention (129.58±58.28pg/ml) underwent a significant decrease compared to the indicator during the oestrous phase of the cycle by 3.4 and 3.2 times, p<0.001, respectively.
To assess the cytokine imbalance between proinflammatory IL-1 and anti-inflammatory IL-4, the ratio between IL-1 and IL-4 was calculated as an index reflecting the balance between both interleukins and the balance of cytokine expression.The ratio of IL-1 to IL-4 is an objective criterion for predicting the type of tissue regeneration, since in the event of a local imbalance of these cytokines, the regeneration mechanism undergoes a pathophysiological shift and causes tissue remodelling defects and infertility (Fig. 4) The highest index of the ratio between IL-1 and IL-4 was found to be during proestrus (34.53), while during oestrus it was 20.45, and during the corpus luteum, this ratio had a minimum value of 14.08.The obtained results demonstrate the role of IL-1 and IL-4 in the process of remodelling since changes in concentration confirm the activation of physiological endometrial remodelling during proestrus and inhibition of remodelling during corpus luteum.The index of the ratio between IL-1 and IL-4 in animals with endometritis and retained litter was 27.69 and 26.35, respectively, remaining significantly lower than the same index in the period of proestrus.The abovementioned changes indicate the inhibition of regenerative and restorative reactions due to the extinction of the metabolic and secretory activity of the postmorbid endometrium.
It is likely that in the present study, the increase in TNFα during active endometrial remodelling during days 7(8) to 17 (18) of the sexual cycle is due to increased reproduction of endometrial components.In particular, studies (Abeysinghe et al., 2023) have shown that TNFα initiates the production of other cytokines and prostaglandins, which causes stimulation, proliferation, and differentiation through the pleiotropic effect of different cell types.Furthermore, according to N. Nijkang (2019), TNFα can induce apoptosis of immunocompetent cells, connective tissue cells, as well as circulating and exudative neutrophils through the activation of cellular caspases that destroy intracellular components.TNF-α, like IL-1 and IL-6, is a powerful stimulator of acute-phase protein synthesis in the event of tissue damage, so even a slight increase in TNF-α levels causes mobilisation of energy reserves and stimulates the body's defence responses.
Thus, the increase in TNFα content in the endometrium from day 7 to day 18 of the sexual cycle indicates the influence of this cytokine on the differentiation and active proliferation of cells of the functional layer of the uterine mucosa, and the activation of humoral regulation of intercellular connections and intersystemic cellular associations.According to M. Segura-Benítez et al. (2022), this finding details the peculiarities of intercellular matrix remodelling during the stages of the oestrous cycle and clarifies the optimisation of implantation conditions.There is no characteristic cell proliferation and differentiation in the postmorbid state, as evidenced by a decrease in TNFα.S. Warashina et al. (2022) note that this cytokine is a powerful stimulator of acute phase protein synthesis and, in the pathological process, even a slight increase in it causes mobilisation of energy reserves and stimulates the body's defence responses.At the same time, a significant increase in TNFα content in the case of reproductive dysfunction, according to H. Ragsdale et al. (2019), indicates its involvement in the regulation of reproductive processes, as high levels of TNFα activate the biosynthesis of oestrogens by cytotoxic and proliferative cell receptor types.
According to F. Almughlliq et al. (2019), this illustrates the disorganisation of the connective tissue matrix of the postmorbid endometrium, since anaphrodisia results in a significant decrease in the concentration of connective tissue markers -glycoproteins and glycosaminoglycans, which is a consequence of the decrease in secretory cells due to the induction of their apoptosis under conditions of TNF-α expression.It is known that IL-1 exerts its biological effect during intercellular contact through specific and non-specific receptors on the surface of target cells involved in the process of implantation and pregnancy.Several studies (Kuhla et al., 2019;Koh et al., 2020), that record an increase in IL-1 and TNFα levels during the corpus luteum, i.e., at the beginning of the inhibition stage, are contradictory to other studies (Abeysinghe et al., 2023), who indicate a maximum increase in IL-1 in endometrial cells during the follicular phase compared to the luteal phase.These circumstances give rise to the need to detail these studies, as they do not reflect the overall concentration of cytokines in endometrial tissue.
In the present study, an increase in endometrial IL-1 was detected during hunting.This trend, according to researchers (Huang et al., 2021), is likely due to the ability of this cytokine to stimulate the expression of release factor genes that enhance the production of gonadotropic hormones, followed by the induction of the synthesis of estrogens and other gonadal hormones, activation of the transformation of stromal fibroblasts and epithelial cells into decidual cells, which in general details the process of intercellular matrix remodelling and ensures nidulation.
Studies conducted by M. Lappas (2017), and H. Ragsdale et al. (2019) indicate that IL-1 stimulates the transformation of endometrial epithelial cells and stromal fibroblasts into decidual cells, the expression of releasing factor genes, and can affect homeostasis by increasing corticotropin-releasing factor, adrenocorticotropic hormone, and others.At the same time, N. Nijkang et al. (2019) point out that the release of hypothalamic monoamines and neuropeptides under the influence of endogenous IL-1 disrupts the synthesis of gonadotropic and sex hormones, thereby causing anaphrodisia.
Opposite to proinflammatory IL-1 and TNFα is IL-4, a pleiotropic cytokine produced by various immune cells during intercellular interaction and involved, on the one hand, in enhancing tissue alteration and destruction, and, on the other hand, in limiting the inflammation focus, increasing tissue barrier functions, activating regeneration, forming the body's defence response, and inhibiting proinflammatory cytokines.According to N. Nijkang et al. (2019) and N. Turner et al. (2022), it activates the attenuation of the immune response, and catalyses the endometrium's tolerance to the inflammatory process, thereby including the regeneration phase and restoration of the function of the maternal part of the placenta.The results of our research are consistent with the findings of other scientists.
In particular, as shown in studies (Taylor et al., 2021), interleukin-4 is found in endometrial stromal phagocytes and actively regulates stem cell proliferation by inducing mitogen-activated protein kinases.The latter transmits an intracellular signal that causes cell differentiation, metabolic changes, and apoptosis.In addition, IL-4 stimulates luteal cells to increase progesterone.The findings of a significant decrease in IL-4 in endometrial tissue are consistent with the results of studies (Rasheed & Hamid, 2020) and may indicate a lack of cell proliferation and differentiation, and as a result, the inability of postmorbid endometrium to nidulation.
Individual studies (Sheldon et al., 2020) have shown that IL-4 levels in physiological pregnancy decrease compared to pregnancies that are pathological and end in abortion.A significant increase in IL-4 levels indicates a violation of peripheral tolerance and a shift in the local immune response towards IL-1, which causes infertility.Assessment of serum cytokine levels (proinflammatory IL-1 and regulatory IL-4, which is an inducer of activation of the immune system cellular chain) reflects the dynamics of intercellular interaction and the nature of the pathological process (Salas-Huetos, et al., 2020).According to J. Wisler et al. (2022), excessive levels of IL-1 destroy intercellular interactions, causing infertility, due to the excessive development of the endometrial inflammatory response during the stage of excitation.The results of studies obtained by M. Segura-Benítez et al. (2022) indicate that physiological zygote nidulation is possible under conditions of a balance between the immunomodulatory and immunosuppressive effects of the endometrium, in the implementation of which components of the cytokine system are directly involved.
M. Zargar et аl.(2020) point to a specific balance between the anti-inflammatory components of the immune system and pro-inflammatory cytokines.It is this balance of inflammatory and anti-inflammatory cytokines that determines the course of inflammation, the development of possible complications, and organ and tissue dysfunctions.F. Almughlliq et al. (2019) proved that full endometrial regeneration is possible under conditions of a balanced ratio of IL-1, TNF-α and their antagonist IL-4, which play an important role in the formation of regenerative and repair reactions.In the case of their local imbalance, the regeneration mechanism acquires a pathophysiological shift and causes tissue remodelling defects.The index of the ratio of IL-1 to IL-4 in cows with endometritis and afterbirth retention was significantly lower compared to the index of proestrus.
The cytokine ratio of IL-1, TNF-α and IL-4 is an objective criterion for predicting the type of tissue regeneration.I. Sheldon et al. (2020), having studied the ratio of IL-1 and IL-4, proved that cytokine balance is a prognostic indicator in assessing the course of pregnancy.An excessive increase in the concentration of IL-1 against a sharp decrease in IL-4 leads to abortion.Whereas, according to P. Abeysinghe et al. (2023), the correction of the above ratio towards a decrease in IL-1 concentration against the background of an increase in IL-4 ensures a physiological course of pregnancy.Thus, further research on a detailed understanding of the cellular and molecular mechanisms occurring in the bovine endometrium under physiological and pathological conditions is crucial to explain and avoid infertility in dairy cows.
The concentration of IL-1 in the endometrium of animals in the state of anaphrodisia and suffering from endometritis and retention of afterbirth is 2.5 times lower than that of cows in heat, 2.5 times (3351.71±1422.42pg/ml and 3412.43±430.12pg/ ml), p<0.01, respectively.The content of IL-4 in endometrial homogenates of cows during the heat reaches maximum values -407.07±45.78pg/ml, while during the oestrus it begins to show a tendency to decrease by almost 1.4 times (295.31±42.16pg/ml), and during proestrus it undergoes a significant decrease by 3.4 times (118.56±32.34pg/ml), p<0.001, respectively.
The concentration of IL-4 in endometrial homogenates of anaphroditic cows suffering from endometritis (121.03±52.36pg/ml) and retention of afterbirth (129.58±58.28pg/ml) undergoes a significant decrease compared to the indicator during the oestrous phase of the cycle by 3.4 and 3.2 times, p<0.001, respectively.The highest index of the ratio between IL-1 and IL-4 was found to be during proestrus -34.53, while during oestrus it was 20.45, and during the corpus luteum, this ratio was 14.08, while in animals with endometritis and retention of the litter, it was 27.69 and 26.35, respectively, remaining significantly lower than the same index of proestrus.Thus, the prospect of further research is the need to use the data obtained to develop reasonable methods for correcting the sexual cyclicity of cows in the post morbid state.

Figure 2 .
Figure 2. IL-1 content in cow endometrial homogenates, pg/ml Source: compiled by the authors

Figure 3 .
Figure 3. IL-4 content in cow endometrial homogenates, pg/ml Source: compiled by the authors