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Objective of the study was to assess whether there are some differences in biometric measurements of the reproductive organs using mechanical sector or linear array ultrasound probe in comparison to the macroscopic measurements. The results revealed no significant differences between ultrasonographic (USG) images in comparison to macroscopic features. High correlations between post - mortem biometric measurements of examined structures and monitored via USG in conscious animals using both probes were found (P

Contents Sex steroids in synergy with prostaglandins (PG) are involved in the regulation of cyclic ovarian function. In this study, we investigated the mRNA expression of three genes involved in arachidonic acid (AA) metabolism and hence PG production in domestic cats: PG-endoperoxide synthase ( PTGS2), PGF2α synthase ( PGFS) and PGE2 synthase ( PGES). Feline endometria (n = 16) were collected at oestrus and mid and late phases of pseudopregnancy. In addition, the effects of E2 and/or P4 on PG secretion and gene expression on endometrial explants were studied in an in vitro culture system. Expression levels of all examined genes were up-regulated at the mid phase of pseudopregnancy. The effects of E2 and/or P4 treatment on both PG secretion and expression of the genes were observed after 12 h of culture. Expression of PGES was significantly up-regulated by E2 plus P4 at oestrus and the mid phase of pseudopregnancy and was also up-regulated by a single treatment with P4 at late pseudopregnancy (p < 0.05). Simultaneous incubation with E2 and P4 up-regulated PTGS2 gene expression at oestrus and mid-luteal phase (p < 0.05). Progesterone plus E2 significantly increased PGE2 secretion at oestrus and the mid phase of pseudopregnancy. However, treatment with E2 and/or P4 affected neither PGF2α secretion nor PGFS expression at any phase after 12 h of culture. The overall findings indicate that genes involved in PG synthesis are up-regulated at the mid phase of pseudopregnancy. An increase in PGE2 secretion and up-regulation of PGES and PTGS2 are the main responses of the endometrium to treatment with E2 and P4 at oestrus and the mid phase of pseudopregnancy in the cat. These data support the hypothesis that ovarian sex steroids via endometrial PGE2 are involved in endocrine homoeostasis, especially at oestrus and the mid, but not the late, phase of pseudopregnancy in cats. [ABSTRACT FROM AUTHOR]

Contents Previous in vitro studies demonstrated that bovine endometrium has the capacity to convert inactive cortisone to biologically active cortisol (Cr) and that Cr inhibits cytokine-stimulated prostaglandin F2α (PGF) production. This study was carried out to test the hypothesis that bovine reproductive tract has the capacity to convert cortisone to Cr in vivo and to evaluate the effects of intravaginal application of exogenous cortisone on uterine PGF secretion during the late luteal stage. The temporal relationships between PGF and Cr levels in uterine plasma were also determined. Catheters were inserted into jugular vein (JV), uterine vein (UV), vena cava caudalis (VCC) and aorta abdominalis (AA) of six cows on Day 15 of the oestrous cycle (ovulation = Day 0) for frequent blood collection. On Day 16, the cows were divided randomly into two groups and infused intravaginally with vaseline gel (10 ml; control; n = 3) or cortisone dissolved in vaseline gel (100 mg; n = 3). Blood samples were collected at −2, −1, −0.5, 0, 0.5, 1, 1.5, 2, 3, 4, 5 and 6 h after treatments (0 h). Intravaginal application of cortisone increased plasma concentrations of Cr between 0.5 and 1.5 h in UV, at 0.5 h in VCC, at 1 h in JV and at 1.5 h in AA. The plasma concentrations of PGF in UV and of PGF metabolite in JV were greater at 0.5 and 1 h in the cortisone-treated animals than in control animals. The levels of PGF in UV blood plasma decreased after Cr reached its highest levels. The overall findings suggest that the female reproductive tract has the capacity to convert cortisone to Cr in vivo. Based on the temporal changes of PGF and Cr levels in the uterine plasma, a biphasic response in PGF secretion was found to be associated to the Cr increase induced by the cortisone treatment at the late luteal stage in non-pregnant cows. [ABSTRACT FROM AUTHOR]

Contents Prostaglandin F2α (PGF) is considered to be the main luteolysin in cattle. We have previously demonstrated that cortisol (Cr) suppresses PGF production in non-pregnant bovine endometrium. This study was carried out to test whether exogenous PGF increases ovarian and/or uterine PGF production and to determine the temporal relationship between PGF and Cr in ovarian and uterine circulations during PGF-induced luteolysis in cows. Catheters were inserted into the ovarian vein (OV), uterine vein (UV) and jugular vein (JV) of 10 cows on Day 9 of the oestrous cycle (Ovulation = Day 0) for frequent blood collection. On Day 10, the cows were divided randomly into two groups and treated with a luteolytic dose of a PGF analogue (cloprostenol) or saline solution. Blood samples were collected at −0.25, 0, 0.25, 0.5, 1 and 2 h and then at 2-h intervals until 12 h after treatment (0 h). The basal concentrations of PGF and Cr in OV and UV plasma were not significantly different. Injection of a PGF analogue induced more than twofold increases in the levels of PGF between 0.25 and 1 h in UV plasma, but not in OV plasma. PGF increased (p < 0.05) the concentrations of Cr in OV, UV and JV plasma between 0.5 and 1 h. The Cr levels in OV, UV and JV plasma were similar. The PGF levels in UV plasma decreased after Cr reached its highest levels. The overall results suggest that the uterus rather than the ovary increases PGF production in response to PGF injection. Based on the temporal changes of PGF and Cr in the ovarian and uterine circulations, Cr may act to reduce uterine PGF production in non-pregnant cows in vivo. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine leukotrienes (LTs) functions in the bovine corpus luteum (BCL) during the oestrous cycle. In steroidogenic CL cells we examined the effect of luteotropic [LH, prostaglandin E (PGE)] and luteolytic (PGF, cytokines) factors on: the levels of LTB and C, the expression of 5-lipoxygenase (LO), LT receptors type I (LTR-I) and LTR-II, and the effects of LTB and C stimulations on the levels of progesterone (P4), PGE, F and nitric oxide (NO) metabolites. Both luteolytic and luteotropic factors stimulated 5-LO expression on days 2-4 and 17-19 of the cycle. Leukotriene receptors type I expression increased after PGE and tumour necrosis factor α with interferon γ (TNF/IFN) stimulation on days 2-4 of the cycle. Leukotriene receptor type II expression increased after PGE and TNF/IFN stimulation on days 2-4 and 17-19 of the cycle, and LTR-II expression on days 8-10 of the cycle was unchanged after cell stimulation with any factor. Leukotriene B level increased after BSC incubation with luteotropic factors during all examined days of the cycle and after cytokine stimulation at early- and mid-luteal stages, whereas luteolytic factors stimulated LTC secretion over the entire cycle. Leukotriene B stimulated P4 secretion at the mid-luteal stage and stimulated NO secretion during all examined phases. Leukotriene B stimulated PGE secretion at the early- and mid-luteal stage. Leukotriene C inhibited P4 secretion at the mid- and regressing-luteal stage, stimulated NO (entire cycle) and PGF at mid- and regressing-luteal phases. Leukotrienes modulate steroidogenic cells functions, depending on the stage of the cycle. Leukotriene B plays a luteotropic role stimulating P4 and PGE secretions; LTC stimulates the secretion of luteolytic factors and enhances the luteolytic cascade within BCL. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine which cells are the source of production and target for leukotriene (LTs) action within the bovine ovary. Luteal (CL, days 14-16 of the oestrous cycle), steroidogenic cells (LSC) and endothelial cells (LEC) of the bovine corpus luteum (CL), and granulosa cells (GC) were isolated enzymatically, cultured in a monolayer and incubated with LTC, LTB, Azelastine (an antagonist of LTC) or Dapsone (an antagonist of LTB). Then cells were collected for determination of mRNA expression for LT receptors ( LTRs) and 5-lipoxygenase ( 5-LO) by real time RT-PCR, and media were collected for determination of prostaglandin (PG)E, F, progesterone (P4; LSC only), endothelin-1 (ET-1; LEC only) and 17-β oestradiol (E2; GC only). The greatest mRNA expression for LTR-II and 5-LO were found in LEC, whereas LTR-I mRNA expression did not differ among cell types. The level of PGE increased after LTs treatment in each type of ovarian cell, excluding LTC treatment in LEC. The secretion of PGF was also increased by LTs, but decreased after LTB treatment of LSC. In GC cultures, both LTs stimulated E2 secretion; in LEC cultures, LTB stimulated whereas LTC inhibited P4 secretion; in LEC cultures, LTC stimulated but LTB inhibited ET-1 secretion. The results show that LTs are produced locally and are involved in PGs production/secretion in all examined cells (LSC, LEC and GC) of bovine ovary. Leukotriene treatment modulate secretion of E2, by GC, P4 by LSC and ET-1 by LEC, which indicates that LTs are involved in regulation of ovarian secretory functions. [ABSTRACT FROM AUTHOR]

Background: Endometrosis (chronic degenerative endometritis) results in morphological changes in the equine endometrium and impairs its secretory function. However, the effect of this condition on the myometrium remains unclear. Lysophosphatidic acid (LPA) may affect female reproductive function and embryo transport by influencing uterine contractility through its receptors (LPARs). The objective of this study was to determine myometrial LPAR1-6 mRNA transcription, and the effects of LPA on myometrial contractions in mares with endometrosis during the mid-luteal and follicular phases of the estrous cycle., Results: A reduction in myometrial LPAR1 mRNA transcription was observed in mares with endometrosis during the mid-luteal phase, in comparison to those with category I endometria (P < 0.05). While, upregulation of myometrial LPAR3 or LPAR6 mRNA transcription was observed in mares with category III or IIB endometria; respectively (P < 0.05). An increase in myometrial LPAR1, LPAR3 and LPAR5 mRNA transcription was observed during the follicular phase in mares with category IIA endometrium in comparison to their expression in category I endometrium (P < 0.05). During endometrosis progression LPA reduced the force of myometrial contractions in both phases of the estrous cycle (P < 0.05). However, in mares with category IIA endometrium during the follicular phase, LPA was found to increase the force of contraction of myometrial strips in comparison to mares with category I endometrium (P < 0.01)., Conclusion: In the course of endometrosis in mares, a disruption in the myometrial mRNA transcription of LPARs has been observed. This is the first study to examine the impact of LPA on myometrial contractility at diffrent stage of endometrosis. However, it is essential to consider that multiple factors may contribute to this process. Alternations in contractile activity and changes in myometrial LPARs mRNA transcription may indicate impaired LPA-signaling mechanisms in equine myometrium during endometrosis., Competing Interests: Declarations. Ethics approval and consent to participate: All material collection procedures were approved by the Local Ethics Committee for Experiments on Animals in Olsztyn, Poland (Agreements No. 51/2011). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Introduction: Prostaglandins (PG) are important regulators of the myometrial contractility in mammals. Endometrosis, a condition characterized by morphological changes in the equine endometrium, also affects endometrial secretory function. However, it remains unclear whether and how endometrosis affects myometrial function., Methods: This study investigated: (i) mRNA transcription of genes encoding specific enzymes responsible for PG synthesis, such as prostaglandin-endoperoxide synthase ( PTGS2 ), PGE 2 synthase ( PTGES ), PGF 2α synthase ( PTGFS ) and PG receptors : PGE 2 receptors ( PTGER1- 4 ), and PGF 2α receptor ( PTGFS ) in equine myometrium and, (ii) the effects of PGE 2 and PGF 2α on myometrial contractile activity, during endometrosis in mares. The myometria used in experiments 1 and 2 were collected from mares in the mid-luteal ( n = 23) and follicular ( n = 20) phases of the estrous cycle, according to the histological classification of the endometrium (Kenney and Doig categories I, IIA, IIB, and III)., Results: In experiment 1, changes in mRNA transcription of PG synthase or PG receptors in the myometrium during the course of endometrosis were determined using qPCR. During the mid-luteal phase, myometrial mRNA transcription of PTGES increased in mares with endometrial category IIB compared to category I. However, myometrial mRNA transcription of PTGER1 decreased during the progression of endometrosis compared to category I. During the follicular phase, mRNA transcription of PTGER1 and PTGER2 increased in mares with endometrial categories III or IIA, respectively. In addition, mRNA transcription of PTGFS increased in mares with endometrium category IIA compared to category I. In experiment 2, the force of myometrial contractions was measured using an isometric concentration transducer. In the follicular phase, PGE 2 decreased the force of contractions in mares with endometrial categories IIA, IIB, and III compared to the respective control groups. Prostaglandin F 2α increased the force of myometrial contractions in mares with category IIA endometrium, whereas it decreased in category IIB compared to the respective control groups., Discussion: We concluded that in the progression of endometrosis there are changes in the myometrial transcription of mRNA encoding PG synthases and receptors , particularly PTGER1 and PTGER2 . Mares with endometrosis had abnormal myometrial contractile responses to PG. These findings suggest that myometrial function may be compromised during the progression of endometrosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Piotrowska-Tomala, Szóstek-Mioduchowska, Drzewiecka, Jonczyk, Wójtowicz, Wrobel, Ferreira-Dias and Skarzynski.)

The path to fertility in the mare requires an understanding of the hormonal influences, the immune response, genetics, and epigenetic mechanisms involved not only in physiological reproductive processes, but also such pathologies as endometritis and endometrosis. Endometritis may lead to endometrosis establishment. In the presence of endometritis, neutrophils arrive at the mare endometrium, and form neutrophil extracellular traps. While NETosis plays pivotal roles, prolonged inflammation can lead to chronic endometritis, endometrosis, and fertility issues. Matrix metalloproteinases and epigenetic changes influence the course of endometrosis. Inhibitors of specific enzymes involved in NETosis and epigenetic inhibitors have shown potential in reducing pro-fibrotic effects. Collagen type III (COL3) has emerged as a putative biomarker, correlating with endometrosis and useful in fertility assessment. Thus, COL3 may offer a non-invasive diagnostic tool, as a complement to histopathological methods. Epigenetic modifications and miRNA expressions offer new avenues for therapeutic strategies, emphasizing the importance of understanding the cellular mechanisms at play in mare endometrial fibrosis., Competing Interests: >Conflicts of interest: The authors have no conflict of interest to declare., (Copyright © The Author(s).)

Differences in structural and functional properties between oocytes and cumulus cells (CCs) may cause low vitrification efficiency for cumulus-oocyte complexes (COCs). We have suggested that the disconnection of CCs and oocytes in order to further cryopreservation in various ways will positively affect the viability after thawing, while further co-culture in vitro will contribute to the restoration of lost intercellular gap junctions. This study aimed to determine the optimal method of cryopreservation of the suspension of CCs to mature GV oocytes in vitro and to determine the level of mRNA expression of the genes ( GJA1 , GJA4 ; BCL2 , BAX ) and gene-specific epigenetic marks ( DNMT3A ) after cryopreservation and in vitro maturation (IVM) in various culture systems. We have shown that the slow freezing of CCs in microstraws preserved the largest number of viable cells with intact DNA compared with the methods of vitrification and slow freezing in microdroplets. Cryopreservation caused the upregulation of the genes Cx37 and Cx43 in the oocytes to restore gap junctions between cells. In conclusion, the presence of CCs in the co-culture system during IVM of oocytes played an important role in the regulation of the expression of the intercellular proteins Cx37 and Cx43, apoptotic changes, and oocyte methylation. Slow freezing in microstraws was considered to be an optimal method for cryopreservation of CCs.

The establishment of the fetomaternal interface depends on precisely regulated communication between the conceptus and the uterine environment. Recent evidence suggests that microRNAs (miRNAs) may play an important role in embryo-maternal dialogue. This study aimed to determine the expression profile of endometrial miRNAs during days 26-28 of equine pregnancy. Additionally, the study aimed to predict target genes for differentially expressed miRNAs (DEmiRs) and their potential role in embryo attachment, adhesion, and implantation. Using next-generation sequencing, we identified 81 DEmiRs between equine endometrium during the pre-attachment period of pregnancy (day 26-28) and endometrium during the mid-luteal phase of the estrous cycle (day 10-12). The identified DEmiRs appear to have a significant role in regulating the expression of genes that influence cell fate and properties, as well as endometrial receptivity formation. These miRNAs include eca-miR-21, eca-miR-126-3p, eca-miR-145, eca-miR-451, eca-miR-491-5p, members of the miR-200 family, and the miRNA-17-92 cluster. The target genes predicted for the identified DEmiRs are associated with ion channel activity and sphingolipid metabolism. Furthermore, it was noted that the expression of mucin 1 and leukemia inhibitory factor, genes potentially regulated by the identified DEmiRs, was up-regulated at day 26-28 of pregnancy. This suggests that miRNAs may play a role in regulating specific genes to create a favorable uterine environment that is necessary for proper attachment, adhesion, and implantation of the embryo in mares., (© 2024. The Author(s).)

Background: Intrauterine devices (IUD) are used in the veterinary practice as the non-pharmacological method of oestrus suppression in mares. When placed in the uterus, IUD create a physical contact with the endometrium that mimics the presence of an equine embryo. However, the mechanism of their action has not been fully elucidated. The objective of the present study was to examine the effect of mechanical stimulation of IUD on mare`s endometrium in both in vitro and in vivo study. For this purpose, we demonstrated the effect of IUD on prostaglandin (PG) F 2α and PGE 2 secretion, and mRNA transcription of genes involved in PG synthesis pathway in equine endometrial cells in vitro. In the in vivo study, we aimed to compare short-term effect of IUD inserted on day 0 (oestrus) with day 5-6 post-ovulation (the specific time when embryo reaches uterus after fertilization) on PG secretion from equine endometrium. To determine the long-term effect on PG synthase mRNA transcription, a single endometrial biopsy was taken only once within each group of mares at certain time points of the estrous cycle from mares placement with IUD on days 0 or 5-6 post-ovualtion., Results: We showed for the first time that the incubation of the endometrial cells with the presence of IUD altered the pattern of PG synthase mRNA transcription in equine epithelial and stromal endometrial cells. In vivo, in mares placement with IUD on day 0, PGE 2 concentrations in blood plasma were upregulated between 1 and 6, and at 10 h after the IUD insertion, compared with the control mares (P < 0.05). Moreover, the decrease of PTGFS mRNA transcription on day 16- 18, associated with an elevation in PTGES mRNA transcription on day 20 -21 of the estrous cycle in endometrial biopsies collected from mares placement with IUD on days 5-6 suggest an antiluteolytic action of IUD during the estrous cycle., Conclusion: We conclude that the application of IUD may mimic the equine conceptus presence through the physical contact with the endometrium altering PG synthase transcription, and act as a potent modulator of endometrial PG secretion both in vitro and in vivo., (© 2024. The Author(s).)

In the current study, transcriptome profiles of mare endometrium, classified into categories I, IIA, and IIB according to Kenney and Doig, were compared using RNA sequencing, analyzed, and functionally annotated using in silico analysis. In the mild stage (IIA) of endometrosis compared to category I endometrium, differentially expressed genes (DEGs) were annotated to inflammation, abnormal metabolism, wound healing, and quantity of connective tissue. In the moderate stage (IIB) of endometrosis compared to category I endometrium, DEGs were annotated to inflammation, fibrosis, cellular homeostasis, mitochondrial dysfunction, and pregnancy disorders. Ingenuity pathway analysis (IPA) identified cytokines such as transforming growth factor (TGF)-β1, interleukin (IL)-4, IL-13, and IL-17 as upstream regulators of DEGs associated with cellular homeostasis, metabolism, and fibrosis signaling pathways. In vitro studies showed the effect of these cytokines on DEGs such as ADAMTS1, -4, -5, -9, and HK2 in endometrial fibroblasts at different stages of endometrosis. The effect of cytokines on ADAMTS members' gene transcription in fibroblasts differs according to the severity of endometrosis. The identified transcriptomic changes associated with endometrosis suggest that inflammation and metabolic changes are features of mild and moderate stages of endometrosis. The changes of ADAMTS-1, -4, -5, -9, in fibrotic endometrium as well as in endometrial fibroblast in response to TGF-β1, IL-4, IL-13, and IL-17 suggest the important role of these factors in the development of endometrosis., (© 2023. The Author(s).)

Mare endometrial fibrosis (endometrosis), is one of the main causes of equine infertility. Despite the high prevalence, both ethology, pathogenesis and the nature of its progression remain poorly understood. Recent studies have shown that microRNAs (miRNAs) are important regulators in multiple cellular processes and functions under physiological and pathological circumstances. In this article, we reported changes in miRNA expression at different stages of endometrosis and the effect of transforming growth factor (TGF)-β1 on the expression of the most dysregulated miRNAs. We identified 1, 26, and 5 differentially expressed miRNAs (DEmiRs), in categories IIA (mild fibrosis), IIB (moderate fibrosis), and III (severe fibrosis) groups compared to category I (no fibrosis) endometria group, respectively (P adjusted  < 0.05, log2FC ≥ 1.0/log2FC ≤  - 1.0). This study indicated the potential involvement of miRNAs in the regulation of the process associated to the development and progression of endometrosis. The functional enrichment analysis revealed, that DEmiRs target genes involved in the mitogen-activated protein kinases, Hippo, and phosphoinositide-3-kinase (PI3K)-Akt signalling pathways, focal adhesion, and extracellular matrix-receptor interaction. Moreover, we demonstrated that the most potent profibrotic cytokine-TGF-β1-downregulated novel-eca-miR-42 (P < 0.05) expression in fibroblasts derived from endometria at early-stage endometrosis (category IIA)., (© 2023. Springer Nature Limited.)

Endometrosis negatively affects endometrial function and fertility in mares, due to excessive deposition of type I (COL1) and type III (COL3) collagens. The pro-fibrotic transforming growth factor (TGF-β1) induces myofibroblast differentiation, characterized by α-smooth muscle actin (α-SMA) expression, and collagen synthesis. In humans, fibrosis has been linked to epigenetic mechanisms. To the best of our knowledge, this has not been described in mare endometrium. Therefore, this study aimed to investigate the in vitro epigenetic regulation in TGF-β1-treated mare endometrial fibroblasts and the use of 5-aza-2'-deoxycytidine (5-aza-dC), an epigenetic modifier, as a putative treatment option for endometrial fibrosis. Methods and Results: The in vitro effects of TGF-β1 and of 5-aza-dC on DNA methyltransferases ( DNMT1 , DNMT3A, and DNMT3B ), COL1A1 , COL3A1 , and α-SMA transcripts were analyzed in endometrial fibroblasts, and COL1 and COL3 secretion in a co-culture medium. TGF-β1 upregulated DNMT3A transcripts and collagen secretion. In TGF-β1-treated endometrial fibroblasts, DNA methylation inhibitor 5-aza-dC decreased collagen transcripts and secretion, but not α-SMA transcripts. Conclusion: These findings suggest a possible role of epigenetic mechanisms during equine endometrial fibrogenesis. The in vitro effect of 5-aza-dC on collagen reduction in TGF-β1-treated fibroblasts highlights this epigenetic involvement. This may pave the way to different therapeutic approaches for endometrosis., Competing Interests: The authors declare no conflicts of interest.

In mammals, the corpus luteum (CL) is a transient organ that secretes progesterone (P4). In the absence of pregnancy, the CL undergoes regression (luteolysis), which is a crucial preparation step for the next estrous cycle. Luteolysis, initiated by uterine prostaglandin F 2α (PGF) in cattle, is usually divided into two phases, namely functional luteolysis characterized by a decline in P4 concentration and structural luteolysis characterized by the elimination of luteal tissues from the ovary. Programmed cell death (PCD) of luteal cells, including luteal steroidogenic cells (LSCs) and luteal endothelial cells (LECs), plays a crucial role in structural luteolysis. The main types of PCD are caspase-dependent apoptosis (type 1), autophagic cell death (ACD) via the autophagy-related gene (ATG) family (type 2), and receptor-interacting protein kinase (RIPK)-dependent programmed necrosis (necroptosis, type 3). However, these PCD signaling pathways are not completely independent and interact with each other. Over the past several decades, most studies on luteolysis have focused on apoptosis as the principal mode of bovine luteal cell death. Recently, ATG family members were reported to be expressed in bovine CL, and their levels increased during luteolysis. Furthermore, the expression of RIPKs, which are crucial mediators of necroptosis, is reported to increase in bovine CL during luteolysis and is upregulated by pro-inflammatory cytokines in bovine LSCs and LECs. Therefore, apoptosis, ACD, and necroptosis may contribute to bovine CL regression. In this article, we present the recent findings regarding the mechanisms of the three main types of PCD and the contribution of these mechanisms to luteolysis.

Endometrium type I (COL1) and III (COL3) collagen accumulation, periglandular fibrosis and mare infertility characterize endometrosis. Metalloproteinase-2 (MMP-2), MMP-9 and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) are involved in collagen turnover. Since epigenetic changes may control fibroproliferative diseases, we hypothesized that epigenetic mechanisms could modulate equine endometrosis. Epigenetic changes can be reversed and therefore extremely promising for therapeutic use. Methylation pattern analysis of a particular gene zone is used to detect epigenetic changes. DNA methylation commonly mediates gene repression. Thus, this study aimed to evaluate if the transcription of some genes involved in equine endometrosis was altered with endometrial fibrosis, and if the observed changes were epigenetically modulated, through DNA methylation analysis. Endometrial biopsies collected from cyclic mares were histologically classified (Kenney and Doig category I, n = 6; category IIA, n = 6; category IIB, n = 6 and category III, n = 6). Transcription of COL1A1, COL1A2, COL3A1, MMP2, MMP9, TIMP1 , and TIMP2 genes and DNA methylation pattern by pyrosequencing of COL1A1, MMP2, MMP9, TIMP1 genes were evaluated. Both MMP2 and MMP9 transcripts decreased with fibrosis, when compared with healthy endometrium (category I) ( P < 0.05). TIMP1 transcripts were higher in category III, when compared to category I endometrium ( P < 0.05). No differences were found for COL1A1, COL1A2, COL3A1 and TIMP2 transcripts between endometrial categories. There were higher methylation levels of (i) COL1A1 in category IIB ( P < 0.05) and III ( P < 0.01), when compared to category I; (ii) MMP2 in category III, when compared to category I ( P < 0.001) and IIA ( P < 0.05); and (iii) MMP9 in category III, when compared to category I and IIA ( P < 0.05). No differences in TIMP1 methylation levels were observed between endometrial categories. The hypermethylation of MMP2 and MMP9 , but not of COL1A1 genes, occurred simultaneously with a decrease in their mRNA levels, with endometrial fibrosis, suggesting that this hypermethylation is responsible for repressing their transcription. Our results show that endometrosis is epigenetically modulated by anti-fibrotic genes ( MMP2 and MMP9 ) inhibition, rather than fibrotic genes activation and therefore, might be promising targets for therapeutic use., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Alpoim-Moreira, Fernandes, Pimenta, Bliebernicht, Rebordão, Castelo-Branco, Szóstek-Mioduchowska, Skarzynski and Ferreira-Dias.)

Collagen pathological deposition in equine endometrium (endometrosis) is responsible for infertility. Kenney and Doig's endometrial biopsy histopathological classification is the gold standard method for endometrosis evaluation, whereby blood biomarkers identification would be less invasive and could provide additional information regarding endometrosis diagnosis and fertility prognosis. This study aimed to identify blood biomarkers for endometrosis diagnosis (42 mares were used in experiment 1), and fertility assessment (50 mares were used in experiment 2). Reproductive examination, endometrial biopsy histopathological classification (Kenney and Doig) and blood collection were performed. Endometrium and serum collagen type I (COL1) and type III (COL3), and hydroxyproline concentrations were measured (ELISA). Serum COL3 cut-off value of 60.9 ng/mL allowed healthy endometria (category I) differentiation from endometria with degenerative/fibrotic lesions (categories IIA, IIB or III) with 100% specificity and 75.9% sensitivity. This cut-off value enabled category I + IIA differentiation from IIB + III (76% specificity, 81% sensitivity), and category III differentiation from others (65% specificity, 92.3% sensitivity). COL1 and hydroxyproline were not valid as blood biomarkers. Serum COL3 cut-off value of 146 ng/mL differentiated fertile from infertile mares (82.4% specificity, 55.6% sensitivity), and was not correlated with mares' age. Only COL3 may prove useful as a diagnostic aid in mares with endometrial fibrosis and as a fertility indicator.

We examined the effect of prostaglandin (PG) E 2 on the secretory function of equine corpus luteum (CL), according to the application site: intra-CL injection vs. an intrauterine (intra-U) administration. Moreover, the effect of intra-CL injection vs. intra-U administration of both luteotropic factors: PGE 2 and human chorionic gonadotropin (hCG) as a positive control, on CL function was additionally compared. Mares were assigned to the groups ( n = 6 per group): (1) an intra-CL saline injection (control); (2) an intra-CL injection of PGE 2 (5 mg/ml); (3) an intra-CL injection of hCG (1,500 IU/ml); (4) an intra-U saline administration (control); (5) an intra-U administration of PGE 2 (5 mg/5 ml); (6) an intra-U administration of hCG (1,500 IU/5 ml). Progesterone (P 4 ) and PGE 2 concentrations were measured in blood plasma samples collected at -2, -1, and 0 (pre-treatment), and at 1, 2, 3, 4, 6, 8, 10, 12, and 24 h after treatments. Moreover, effects of different doses of PGE 2 application on the concentration of total PGF 2α (PGF 2α and its main metabolite 13,14-dihydro-15-keto-prostaglandin F 2α - PGFM) was determined. The time point of PGE 2 , hCG, or saline administration was defined as hour "0" of the experiment. An intra-CL injection of PGE 2 increased P 4 and PGE 2 concentrations between 3 and 4 h or at 3 and 12 h, respectively ( p < 0.05). While intra-U administration of PGE 2 elevated P 4 concentrations between 8 and 24 h, PGE 2 was upregulated at 1 h and between 3 and 4 h ( p < 0.05). An intra-CL injection of hCG increased P 4 concentrations at 1, 6, and 12 h ( p < 0.05), while its intra-U administration enhanced P 4 and PGE 2 concentrations between 1 and 12 h or at 3 h and between 6 and 10 h, respectively ( p < 0.05). An application of PGE 2 , dependently on the dose, supports equine CL function, regardless of the application site, consequently leading to differences in both P 4 and PGE 2 concentrations in blood plasma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Piotrowska-Tomala, Jonczyk, Szóstek-Mioduchowska, Żebrowska, Ferreira-Dias and Skarzynski.)

We investigated the effects of different doses of dinoprost injected directly into the bovine corpus luteum (CL) on (i) concentrations of progesterone (P 4 ) and oxytocin (OT) in peripheral blood and (ii) mRNA levels of steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (P450scc), hydroxy-delta-5-steroid dehydrogenase, 3 β- and steroid delta-isomerase 1 (HSD3B), and receptor-interacting protein kinases 1 and 3 (RIPK1, RIPK3) in CL tissue. Moreover, we examined the effects of dinoprost, injected intra-CL or administered intramuscularly (IM), on CL secretory function and on indicators of CL vascular network status: luteal tissue area (LTA), CL blood flow (CLBF), and the CLBF:LTA ratio (Adj. CLBF), in cows at the early and mid-luteal phases. In the Experiment 1, cows (day 10 of the cycle) were allocated to (i) an intra-CL injection of saline (control; n = 3); (ii) an intra-CL injection of dinoprost (1.25 mg; 2.5 mg, or 5 mg; n = 3 for each dose); (iii) an IM administration of saline (control; n = 3); or (iv) an IM administration of dinoprost (25 mg; positive control; n = 3). Concentrations of OT and P 4 were measured in plasma samples. The mRNA expression of steroidogenesis- or necroptosis-related factors was determined in CL tissue 4 h after treatments. In Experiment 2, cows on day 4 ( n = 12) or day 10 ( n = 12) were allocated to (i) an intra-CL injection of dinoprost (2.5 mg/0.5 ml; n = 6), or (ii) IM administration of dinoprost (25 mg/5 ml; n = 6). Concentrations of P 4 were measured in plasma samples. Luteal tissue area, CLBF, and Adj. CLBF were assessed based on color Doppler ultrasonography. An intra-CL injection of dinoprost increased OT and decreased P 4 levels in the jugular vein (JV) in a dose-dependent manner in cows at the mid-luteal phase. Increased CLBF and Adj. CLBF, accompanied by reduced P 4 levels, were observed 2 h after intra-CL dinoprost injection in middle-stage CL. Decreased STAR and increased RIPK1 and RIPK3 mRNA levels confirmed that 2.5 mg dinoprost injected directly into CL is the minimum dose that induces luteolytic cascade. Injection of dinoprost directly into the CL (at a dosage lower than recommended for peripheral application) results in a pattern similar to IM dinoprost administration., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jonczyk, Piotrowska-Tomala and Skarzynski.)

Corpus luteum (CL), a transitory gland, undergoes rapid growth in a limited time to produce progesterone (P4) followed by its regression. A complex molecular signaling is involved in controlling luteal P4 production. In the present study, 2D gel electrophoresis-based proteomics and in silico functional analysis were used to identify changes in key proteins and pathways in CL along the different stages of the estrous cycle as its development progresses from early (Day 3) to mid-luteal phase (Day 9), effective functioning (Day 12) followed by regression (Day 15) or, in the case of pregnancy, rescue of function (Day 15). A total of 273 proteins were identified by MALDI-MS/MS analysis that showed significant changes in abundances at different stages of CL development or regression and rescue. Functional annotation of differentially abundant proteins suggested enrichment of several important pathways and functions during CL development and function maintenance including cell survival, endocytosis, oxidative stress response, estradiol metabolism, and angiogenesis. On the other hand, differentially abundant proteins during CL regression were associated with decreased steroid synthesis and metabolism and increased apoptosis, necrosis, and infiltration of immune cells. Establishment of pregnancy rescues CL from regression by maintaining the expression of proteins that support steroidogenesis as pathways such as the super-pathway of cholesterol biosynthesis, RhoA signaling, and functions such as fatty acid metabolism and sterol transport were enriched in CL of pregnancy. In this study, some novel proteins were identified along CL development that advances our understanding of CL survival and steroidogenesis.

Cathepsin G (CAT) is a protease released by neutrophils when forming neutrophil extracellular traps that was already associated with inducing type I collagen (COL1) in equine endometrium in vitro. Endometrosis is a fibrotic condition mainly characterized by COL1 deposition in the equine endometrium. The objective was to evaluate if noscapine (an alkaloid for cough treatment with anti-neoplastic and anti-fibrotic properties) would reduce COL1A2 transcription (evaluated by qPCR) and COL1 protein relative abundance (evaluated by western blot) induced by CAT in equine endometrial explants from follicular and mid-luteal phases treated for 24 or 48 h. The explants treated with CAT increased COL1 expression. Noscapine decreased COL1A2 transcription at both estrous cycle phases, but COL1 relative protein only at the follicular phase, both induced by CAT. Additionally, the noscapine anti-fibrotic action was found to be more effective in the follicular phase. The CAT treatment caused more fibrosis at the longest period of treatment, while noscapine acted better at the shortest time of treatment. Our results showed that noscapine could act as an anti-fibrotic drug in equine endometrosis by inhibiting CAT in vitro. Noscapine offers a new promising therapeutic tool for treating fibrosis as a single non-selective agent to be considered in the future.

Glucocorticoids (GCs) are known to play an important role in maintaining basal and stress-related homeostasis by interacting with endocrine mediators and prostaglandins (PGs). Although a growing body of evidence shows that GCs exert their regulatory action at a multitude of sites in the reproductive axis through corticosteroid receptors, little is known about the direct role of cortisol, an active form of GCs, in the equine endometrium. Thus, the study aimed to determine the effect of cortisol on PGF 2α synthesis in the endometrial tissue and cells in vitro. In Exp.1, the immunolocalization and the expression of the glucocorticoid receptor (GCR) in the endometrium throughout the estrous cycle were established. In Exp. 2 and 3, the effects of cortisol on PGF 2α secretion and transcripts associated with the arachidonic acid (AA) cascade in endometrial tissues, and cells were defined. Endometrial tissues obtained from the early, mid, and late luteal phases and the follicular phase of the estrous cycle were exposed to cortisol (100, 200, and 400 nM) for 24 h. Endometrial epithelial and stromal cells (early phase of estrous cycle) were exposed to cortisol (100 nM) for 24 h. Then, PGF 2α secretion and transcripts associated with the AA cascade (PLA2G2A, PLA2G4A, PTGS2, and PGFS) were assessed. GCR was expressed in the cytoplasm and the nucleus in the luminal and glandular epithelium as well as in the stroma. Endometrial GCR protein abundance was up-regulated at the late luteal phase compared to the mid-luteal phase of the estrous cycle. Cortisol dose-dependently decreased PGF 2α secretion, PLA2G2A and PLA2G4A transcripts in endometrial tissues. Additionally, cortisol treatment decreased PGF 2α secretion from endometrial epithelial and stromal cells. Moreover, it affected PLA2G2A, PLA2G4A, and PTGS2 transcripts in endometrial stromal cells. These findings suggest that cortisol suppresses the synthesis of PGF 2α by affecting the AA cascade in the equine endometrium during the estrous cycle., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Endometrosis, a fibrotic disease of mare endometrium, impairs uterine function. Prostaglandins (PG), despite modulating reproductive physiological functions, may also cause local pathological collagen deposition (fibrogenesis). We have previously shown that neutrophil extracellular traps (NETs) may also favor mare endometrosis. The aim of this study was to investigate the effect of enzymes present in NETs on PGF 2α -pathway activation. Kenney and Doig's type I/IIA and IIB/III mare endometria, from follicular phase (FLP) and mid-luteal (MLP) phase, were cultured in vitro in the presence of NETs enzymes (elastase, cathepsin-G or myeloperoxidase). Production of PGF 2α (EIA) and transcription (qPCR) of its synthases ( PTGS2 , AKR1C3 ) and receptor ( PTGFR ) genes were evaluated. PGF 2α and PTGFR were influenced by endometrial category and estrous cycle phase. In FLP endometrium, NETs enzymes induced both high PGF 2α production and/or PTGFR transcription. In MLP type I/IIA tissues, down-regulation of PTGFR transcripts occurred. However, in MLP type IIB/III endometrium, high levels of PTGFR transcripts were induced by NETs enzymes. As PGF2α-pathway activation facilitates fibrogenesis in other tissues, PGF2α may be involved in endometrosis pathogenesis. In the mare, the endocrine microenvironment of healthy and pathological endometrium might modulate the PGF 2α pathway, as well as fibrosis outcome on endometrium challenged by NETs enzymes.

Introduction: Since 2015, the Ann and Robert H. Lurie Children's Hospital Emergency Department (ED) has improved the recognition and treatment of pediatric sepsis and septic shock. Despite existing clinical care guidelines, the ED had not yet achieved the Surviving Sepsis Campaign timeliness goals for fluid and antibiotic administration., Methods: The team conducted a multidisciplinary Kaizen event to evaluate clinical workflows and identify opportunities to improve sepsis care adherence. Using rigorous quality improvement methodology, frontline providers mapped workflows to identify barriers and prioritize emerging solutions., Results: Thirty-seven staff members across 17 disciplines participated. Nurses and physicians identified communication gaps at pathway initiation. Access to supplies, inadequate task delegation, and a lack of urgency for a subset of pathway patients delayed treatment. Prioritized interventions included scripted communication tools, a delineated response plan, and standardized reassessment processes. Revisions to the key driver diagram were made after the improvement event, guiding future plan-do-study-act cycles., Conclusions: Frontline provider participation in the Kaizen event uncovered barriers to care and identified the root causes of ineffective communication and system process inefficiencies. Engaging key stakeholders from multiple care areas in a candid context was a novel approach to process improvement within our department. The Kaizen methodology is fundamental to developing sustainable quality improvement practices, creating momentum for a continuous improvement culture to engrain quality improvement in practice. The success of Kaizen will shape the format of future ED improvement projects., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Endometrosis is a reproductive pathology that is responsible for mare infertility. Our recent studies have focused on the involvement of neutrophil extracellular traps enzymes, such as elastase (ELA), in the development of equine endometrosis. Noscapine (NOSC) is an alkaloid derived from poppy opium with anticough, antistroke, anticancer, and antifibrotic properties. The present work investigates the putative inhibitory in vitro effect of NOSC on collagen type I alpha 2 chain ( COL1A2 ) mRNA and COL1 protein relative abundance induced by ELA in endometrial explants of mares in the follicular or mid-luteal phases at 24 or 48 h of treatment. The COL1A2 mRNA was evaluated by qPCR and COL1 protein relative abundance by Western blot. In equine endometrial explants, ELA increased COL 1 expression, while NOSC inhibited it at both estrous cycle phases and treatment times. These findings contribute to the future development of new endometrosis treatment approaches. Noscapine could be a drug capable of preventing collagen synthesis in mare's endometrium and facilitate the therapeutic approach.

In this paper, newly discovered mechanisms of atresia and cell death processes in bovine ovarian follicles are investigated. For this purpose the mRNA expression of receptor interacting protein kinases 1 and 3 ( RIPK1 and RIPK3 ) of the granulosa and theca cells derived from healthy and atretic follicles are studied. The follicles were assigned as either healthy or atretic based on the estradiol to progesterone ratio. A statistically significant difference was recorded for the mRNA expression of a RIPK1 and RIPK3 between granulosa cells from healthy and atretic follicles. To further investigate this result a systems biology approach was used. The genes playing roles in necroptosis, apoptosis and atresia were chosen and a network was created based on human genes annotated by the IMEx database in Cytoscape to identify hubs and bottle-necks. Moreover, correlation networks were built in the Cluepedia plug-in. The networks were created separately for terms describing apoptosis and programmed cell death. We demonstrate that necroptosis (RIPK-dependent cell death pathway) is an alternative mechanism responsible for death of bovine granulosa and theca cells. We conclude that both apoptosis and necroptosis occur in the granulosa cells of dominant follicles undergoing luteinisation and in the theca cells from newly selected follicles.

The oviduct presents the ideal conditions for fertilization and early embryonic development. In this study, (i) vascularization pattern; (ii) microvascular density; (iii) transcripts of angiogenic factors ( FGF1 , FGF2 , VEGF ) and their receptors- FGFR1 , FGFR2 , KDR , respectively, and (iv) the relative protein abundance of those receptors were assessed in cyclic mares' oviducts. The oviductal artery, arterioles and their ramifications, viewed by means of vascular injection-corrosion, differed in the infundibulum, ampulla and isthmus. The isthmus, immunostained with CD31, presented the largest vascular area and the highest number of vascular structures in the follicular phase. Transcripts (qPCR) and relative protein abundance (Western blot) of angiogenic factors fibroblast growth factor 1 ( FGF1) and 2 ( FGF2) and vascular endothelial growth factor ( VEGF) , and their respective receptors ( FGFR1 , FGFR2 , VEGFR2 = KDR ), were present in all oviduct portions throughout the estrous cycle. Upregulation of the transcripts of angiogenic receptors FGF1 and FGFR1 in the ampulla and isthmus and of FGF2 and KDR in the isthmus were noted. Furthermore, in the isthmus, the relative protein abundance of FGFR1 and KDR was the highest. This study shows that the equine oviduct presents differences in microvascular density in its three portions. The angiogenic factors VEGF, FGF1, FGF2 and their respective receptors are expressed in all studied regions of the mare oviduct, in agreement with microvascular patterns.

The roles of fibroblast growth factor 2 (FGF2) in the corpus luteum (CL) function and its modulatory effect on prostaglandin (PG) F 2α during the bovine estrous cycle were studied using the following design of in vivo and in vitro experiments: (1) effects of FGF2 and FGF receptor 1 inhibitor (PD173074) on bovine CL function in the early (PGF 2α -resistant) and mid (PGF 2α -responsive) luteal stage in vivo, (2) the modulatory effect of FGF2 on PGF 2α action during the luteal phase in vivo and (3) effects of FGF2 and PD173074 on bovine CL secretory function in vitro. Cows were treated by injection into the CL with: (1) saline (control), (2) FGF2, (3) PD173074, (4) FGF2 followed by intramuscular (i.m.) PGF 2α , (5) PD173074 followed by i.m. PGF 2α and (6) i.m. PGF 2α as a positive control. For in vitro experiments, CL explants were treated with the aforementioned factors. Progesterone (P 4 ) concentrations of blood samples or culture media were determined by radioimmunoassay. Relative mRNA expressions of the genes involved in angiogenesis and steroidogenesis were determined by quantitative real-time PCR. Although FGF2 treatment on day 4 of the estrous cycle did not change the cycle length, FGF2 with PGF 2α decreased the P 4 concentrations observed during the estrous cycle compared to the control group (P < 0.001). Moreover, FGF2 treatment on day 10 prolonged CL function as indicated by a significantly greater concentration of P 4 on day 21 compared to the control group. In the in vitro study, FGF2 decreased cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and hydroxy-delta-5-steroid dehydrogenase (HSD3B1) mRNA expression (P < 0.01) and decreased P 4 production in the early-stage CL (P < 0.001). However, FGF2 + PGF 2α or PGF 2α alone resulted in an elevation of steroidogenic acute regulatory protein and CYP11A1 mRNA expression and P 4 secretion in the early-stage CL (P < 0.01). In the mid-luteal phase, FGF2 upregulated CYP11A1 and HSD3B1 mRNA expression (P < 0.01), while FGF2 + PGF 2α increased only HSD3B1 mRNA expression (P < 0.001). In conclusion, FGF2 seems to play a modulatory role in CL development or luteolysis, differentially regulating steroidogenesis and angiogenic factors as well as PGF 2α actions., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Although proteases found in neutrophil extracellular traps (NETs) have antimicrobial properties, they also stimulate collagen type 1 (COL1) production by the mare endometrium, contributing for the development of endometrosis. Cathepsin G (CAT), a protease present in NETs, is inhibited by specific inhibitors, such as cathepsin G inhibitor I (INH; β-keto-phosphonic acid). Matrix metallopeptidases (MMPs) are proteases involved in the equilibrium of the extracellular matrix. The objective of this study was to investigate the effect of CAT and INH (a selective CAT inhibitor) on the expression of MMP-2 and MMP-9 and on gelatinolytic activity. In addition, the putative inhibitory effect of INH on CAT-induced COL1 production in mare endometrium was assessed. Endometrial explants retrieved from mares in follicular phase or midluteal phase were treated for 24 or 48 h with CAT, inhibitor alone, or both treatments. In explants, transcripts (quantitative polymerase chain reaction) of COL1A2, MMP2 , and MMP9 , as well as the relative abundance of COL1 protein (Western blot), and activity of MMP-2 and MMP-9 (zymography) were evaluated. The protease CAT induced COL1 expression in explants, at both estrous cycle phases and treatment times. The inhibitory effect of INH was observed on COL1A2 transcripts in follicular phase at 24-h treatment, and in midluteal phase at 48 h ( P < 0.05), and on the relative abundance of COL protein in follicular phase and midluteal phase explants, at 48 h ( P < 0.001). Our study suggests that MMP-2 might also be involved in an earlier response to CAT, and MMP-9 in a later response, mainly in the follicular phase. While the use of INH reduced CAT-induced COL1 endometrial expression, MMPs might be involved in the fibrogenic response to CAT. Therefore, in mare endometrium, the use of INH may be a future potential therapeutic means to reduce CAT-induced COL1 formation and to hamper endometrosis establishment., (Copyright © 2020 Amaral, Fernandes, Morazzo, Rebordão, Szóstek-Mioduchowska, Lukasik, Gawronska-Kozak, Telo da Gama, Skarzynski and Ferreira-Dias.)

An increasing number of studies have shown that prostaglandins (PGs) exert multiple regulatory actions in the processes associated to tissue remodeling and fibrosis. Extracellular matrix (ECM) turnover is mediated by matrix metallopeptidases (MMPs). The knowledge about the regulation of their expression in mare endometrium is still limited. Thus, the aim of this study was to investigate whether: (i) profibrotic transforming growth factor (TGF)-β1 modulates PG production in equine endometrium; and (ii) PGE 2 and PGF 2α modulate MMPs, their tissue inhibitors (TIMPs), and collagen 1 (COL1) expression. In experiment 1, the effect of TGF-β1 (5 ng/mL) on PG secretion and PG synthases mRNA transcription, after 24 and 48 h treatment of mare endometrial fibroblast and epithelial cells was investigated using ELISA and qPCR. In experiment 2, the effects of PGE 2 and PGF 2α  in doses 10 -7 M and 10 -8 M on secretion and MMP1, 2, 9, 13, TIMP1, 2, and COL1A1 mRNA transcription in mare endometrial fibroblasts were assessed. Transforming growth factor-β1 treatment decreased secretion of PGF 2α by endometrial fibroblasts (P < 0.05) and PGF 2α and PGE 2 by endometrial epithelial cells (P < 0.05). Prostaglandin E 2 increased MMP-2 and MMP-9, and decreased MMP-13 secretion by endometrial fibroblasts (P < 0.05). Additionally, PGF 2α treatment increased MMP-2, MMP-13 and COL1, but decreased MMP-1 secretion by endometrial fibroblasts (P < 0.05). Prostaglandins may be involved in the processes associated to pathological endometrial remodeling by their effect on MMP expression. The effect of PGF 2α on COL1 secretion from fibroblasts suggests its profibrotic role in pathological endometrial remodeling., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Prostaglandins (PGs) play crucial roles in the regulation of the oestrus cycle and establishment of pregnancy in animals. Luteinizing hormone (LH) and ovarian steroids are involved in regulating endometrial PG production in many species. Their effects on PG production and associated pathways in the mare myometrium and endometrium are the subjects of our interest. This study aimed to evaluate the specific effects of LH and ovarian steroids on equine myometrial and endometrial tissues on (i) PGE 2 and PGF 2α secretion and (ii) transcription of genes encoding specific enzymes responsible for PG synthesis, such as prostaglandin-endoperoxide synthase (PTGS2), PGE 2 synthases (PGES), PGF 2α synthases (PGFS), and PGI 2 synthases (PGIS), using equine myometrial and endometrial explants. Equine myometrial and endometrial tissues were collected at the mid-luteal (n = 6) and follicular (n = 6) phases of the oestrus cycle and were exposed to: (1) vehicle (control), (2) arachidonic acid (AA, 50 ng/mL, positive control), (3) LH (10 ng/mL), (4) progesterone (P 4 , 10 -7 M) and (5) 17-β oestradiol (E 2 , 10 -9 M) for 24 h. After exposure, PGF 2α and PGE 2 concentrations were determined using direct enzyme immunoassays. Alterations in PG synthase mRNA expression were determined using RT-qPCR. After 24 h, LH and P 4 increased PGE 2 and PGF 2α secretion by myometrial tissues at the mid-luteal phase (P < 0.05), whereas PG secretion was augmented by LH and E 2 during the follicular phase (P < 0.01). In contrast, LH and E 2 increased PGE 2 and PGF 2α secretion by endometrial tissues during the mid-luteal phase (P < 0.05), while E 2 enhanced PGE 2 secretion during the follicular phase of the oestrus cycle (P < 0.01). These results indicate that LH and ovarian steroids modulate PG production in equine myometrial and endometrial tissues and affect PG synthase expression at the mRNA level. We conclude that the equine myometrium is an alternative source of PG production and participates in the regulation of uterus function during the oestrus cycle., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The innate and adaptive immune mechanisms are key components of regulation of reproductive physiological function and uterine disorders in equine uterus. The predominant immunological response in equine endometrium, characterized by an innate immune response, occurs under estrogens influence, in the follicular phase. Although, the increase in immune-related genes in equine endometrium during estrus has been suggested to play a role in uterine clearance after mating, immune cells and their product, i.e. cytokines play also mandatory role in the luteal development and maintenance, regression of equine corpus luteum, as well as in early pregnancy. Innate immune response is nonspecific and acts as the first line of defense against pathogens, foreign stimuli that include constituents of seminal fluid and local infections (endometritis). It has been recently established that a phagocytosis-independent mechanism to restrain bacteria, by means of neutrophil extracellular traps (NETs) formation, is involved in pathogenesis of in mare endometrial fibrosis (endometrosis). Moreover, persistent macrophages and mast cell activation could also have pro-fibrotic roles by secreting great amounts of pro-fibrotic factors and lead to fibrosis. This review will highlight the involvement of immune key components of the innate and adaptive immune system and their products in equine uterus and their contribution to reproductive physiological function and uterine disorders., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020. Published by Elsevier Inc.)

Neutrophil extracellular traps (NETs) fight endometritis, and elastase (ELA), a protease found in NETs, might induce collagen type I (COL1) accumulation in equine endometrium. Metallopeptidases (MMPs) are involved in extracellular matrix balance. The aim was to evaluate the effects of ELA and sivelestat (selective elastase inhibitor) on MMP-2 and MMP-9 expression and gelatinolytic activity, as well as the potential inhibitory effect of sivelestat on ELA-induced COL1 in equine endometrium. Endometrial explants from follicular (FP) and mid-luteal (MLP) phases were treated for 24 or 48 h with ELA, sivelestat, and their combination. Transcripts of COL1A2 , MMP2, and MMP9 were evaluated by qPCR; COL1 protein relative abundance by Western blot, and MMP-2 and MMP-9 gelatinolytic activity by zymography. In response to ELA treatment, there was an increase in MMP2 mRNA transcription (24 h) in active MMP-2 (48 h), both in FP, and in MMP9 transcripts in FP (48 h) and MLP (24 h) ( p < 0.05). Sivelestat inhibited ELA-induced COL1A2 transcripts in FP (24 h) and MLP (24 h, 48 h) ( p < 0.05). The sivelestat inhibitory effect was detected in MMP 9 transcripts in FP at 48 h ( p < 0.05), but proteases activity was unchanged. Thus, MMP-2 and MMP-9 might be implicated in endometrium fibrotic response to ELA. In mare endometrium, sivelestat may decrease ELA-induced COL1 deposition and hinder endometrosis development., Competing Interests: The authors declare that there are no conflict of interest.