The interaction between seminal plasma, sperm, and endometrium in inter- and intra-species breeding in equids.

In brief: In some instances, extra-species breeding in equids is more successful than intraspecies breeding; however, little is known about the immunomodulatory effect of donkey semen and seminal plasma on the mare's endometrium. This study compared the mare uterine inflammatory response during extra- and intraspecies breeding. Abstract: Anecdotal experience suggests horse mares have less post-breeding inflammation and better fertility when bred with donkeys. This study aimed to compare the post-breeding inflammatory response of mares exposed to donkey and horse semen and seminal plasma and evaluate the proteome and metabolome of donkey and horse sperm and seminal plasma. Uterine edema, intrauterine fluid accumulation, polymorphonuclear neutrophils on cytology, and concentrations of progesterone, and pro- and anti-inflammatory cytokines (IL1A, IL1B, IL4, IL6, CXCL8, IL10) were assessed pre- and post infusion of semen and seminal plasma (donkey and horse). The metabolome and proteome were analyzed by LC-MS/MS. Mare cycles bred with horse semen had a greater progesterone concentration than those bred with donkey semen at 8 days post ovulation (P = 0.046). At 6 h post infusion, the inflammatory response due to the donkey semen tended to be lower (P = 0.074). Donkey seminal plasma had anti-inflammatory properties compared to horse semen and seminal plasma, as determined by fewer neutrophils on uterine cytology (P < 0.05). Horse semen resulted in greater concentrations of IL6 and lesser concentrations of IL1B (P < 0.05). PGE1, PGE3, and lactoferrin concentrations were significantly more abundant in donkey sperm and seminal plasma. Prostaglandins play an important role in immunomodulation and might contribute to the response triggered in interspecies breeding. In conclusion, breeding horse mares with donkey semen induces similar post-breeding endometritis as observed with horse semen. Donkey seminal plasma results in a lower post-infusion inflammatory response compared to other combinations in the immediate post-breeding.

Single-nuclei analysis reveals depot-specific transcriptional heterogeneity and depot-specific cell types in adipose tissue of dairy cows.

Adipose tissue (AT) is an endocrine organ with a central role on whole-body energy metabolism and development of metabolic diseases. Single-cell and single-nuclei RNA sequencing (scRNA-seq and snRNA-seq, respectively) analyses in mice and human AT have revealed vast cell heterogeneity and functionally distinct subtypes that are potential therapeutic targets to metabolic disease. In periparturient dairy cows, AT goes through intensive remodeling and its dysfunction is associated with metabolic disease pathogenesis and decreased productive performance. The contributions of depot-specific cells and subtypes to the development of diseases in dairy cows remain to be studied. Our objective was to elucidate differences in cellular diversity of visceral (VAT) and subcutaneous (SAT) AT in dairy cows at the single-nuclei level. We collected matched SAT and VAT samples from three dairy cows and performed snRNA-seq analysis. We identified distinct cell types including four major mature adipocytes (AD) and three stem and progenitor cells (ASPC) subtypes, along with endothelial cells (EC), mesothelial cells (ME), immune cells, and pericytes and smooth muscle cells. All major cell types were present in both SAT and VAT, although a strong VAT-specificity was observed for ME, which were basically absent in SAT. One ASPC subtype was defined as adipogenic (PPARG+) while the other two had a fibro-adipogenic profile (PDGFRA+). We identified vascular and lymphatic EC subtypes, and different immune cell types and subtypes in both SAT and VAT, i.e., macrophages, monocytes, T cells, and natural killer cells. Not only did VAT show a greater proportion of immune cells, but these visceral immune cells had greater activation of pathways related to immune and inflammatory response, and complement cascade in comparison with SAT. There was a substantial contrast between depots for gene expression of complement cascade, which were greatly expressed by VAT cell subtypes compared to SAT, indicating a pro-inflammatory profile in VAT. Unprecedently, our study demonstrated cell-type and depot-specific heterogeneity in VAT and SAT of dairy cows. A better understanding of depot-specific molecular and cellular features of SAT and VAT will aid in the development of AT-targeted strategies to prevent and treat metabolic disease in dairy cows, especially during the periparturient period.

Tumor necrosis factor signaling during equine placental infection leads to pro-apoptotic and necroptotic outcomes.

Ascending placentitis is the leading cause of abortion in the horse. The pleiotropic cytokine tumor necrosis factor (TNF) is an upstream regulator of this disease, but little is understood regarding its function in pregnancy maintenance or placental infection. To assess this, RNA sequencing was performed on chorioallantois and endometrium of healthy pregnant mares at various gestational lengths (n = 4/gestational age), in addition to postpartum chorioallantois, and diestrus endometrium to assess expression of TNF, TNFR-1, and TNFR-2. Additionally, ascending placentitis was induced via trans-cervical inoculation of S. equi spp. zooepidemicus in pregnant mares (n = 6 infected / n = 6 control) and tissues and serum were collected to evaluate TNF-related transcripts. IHC was performed to confirm protein localization of TNFR-1 and TNFR-2. In healthy pregnancy, TNFR-1 appears to be the predominant TNF-related receptor. Following induction of disease, TNF concentrations increased in maternal serum, but expression did not alter at the tissue level. While both TNFR-1 and TNFR-2 increased following induction of disease, alterations in downstream pathways indicate that TNFR-1 is the dominant receptor in ascending placentitis, and is primarily activated within the chorioallantois, with minimal signaling occurring within the endometrium. In conclusion, TNF appears to be involved in the pathophysiology of ascending placentitis. An increase in this cytokine during disease progression is believed to activate TNFR-1 within the chorioallantois, leading to various pro-apoptotic and necroptotic outcomes, all of which may signal for fetal demise and impending abortion.

Transcriptional and Histochemical Signatures of Bone Marrow Mononuclear Cell-Mediated Resolution of Synovitis.

Osteoarthritis (OA) may result from impaired ability of synovial macrophages to resolve joint inflammation. Increasing macrophage counts in inflamed joints through injection with bone marrow mononuclear cells (BMNC) induces lasting resolution of synovial inflammation. To uncover mechanisms by which BMNC may affect resolution, in this study, differential transcriptional signatures of BMNC in response to normal (SF) and inflamed synovial fluid (ISF) were analyzed. We demonstrate the temporal behavior of co-expressed gene networks associated with traits from related in vivo and in vitro studies. We also identified activated and inhibited signaling pathways and upstream regulators, further determining their protein expression in the synovium of inflamed joints treated with BMNC or DPBS controls. BMNC responded to ISF with an early pro-inflammatory response characterized by a short spike in the expression of a NF-ƙB- and mitogen-related gene network. This response was associated with sustained increased expression of two gene networks comprising known drivers of resolution (IL-10, IGF-1, PPARG, isoprenoid biosynthesis). These networks were common to SF and ISF, but more highly expressed in ISF. Most highly activated pathways in ISF included the mevalonate pathway and PPAR-γ signaling, with pro-resolving functional annotations that improve mitochondrial metabolism and deactivate NF-ƙB signaling. Lower expression of mevalonate kinase and phospho-PPARγ in synovium from inflamed joints treated with BMNC, and equivalent IL-1ß staining between BMNC- and DPBS-treated joints, associates with accomplished resolution in BMNC-treated joints and emphasize the intricate balance of pro- and anti-inflammatory mechanisms required for resolution. Combined, our data suggest that BMNC-mediated resolution is characterized by constitutively expressed homeostatic mechanisms, whose expression are enhanced following inflammatory stimulus. These mechanisms translate into macrophage proliferation optimizing their capacity to counteract inflammatory damage and improving their general and mitochondrial metabolism to endure oxidative stress while driving tissue repair. Such effect is largely achieved through the synthesis of several lipids that mediate recovery of homeostasis. Our study reveals candidate mechanisms by which BMNC provide lasting improvement in patients with OA and suggests further investigation on the effects of PPAR-γ signaling enhancement for the treatment of arthritic conditions.

Transcriptomic analysis of equine chorioallantois reveals immune networks and molecular mechanisms involved in nocardioform placentitis.

Nocardioform placentitis (NP) continues to result in episodic outbreaks of abortion and preterm birth in mares and remains a poorly understood disease. The objective of this study was to characterize the transcriptome of the chorioallantois (CA) of mares with NP. The CA were collected from mares with confirmed NP based upon histopathology, microbiological culture and PCR for Amycolatopsis spp. Samples were collected from the margin of the NP lesion (NPL, n = 4) and grossly normal region (NPN, n = 4). Additionally, CA samples were collected from normal postpartum mares (Control; CRL, n = 4). Transcriptome analysis identified 2892 differentially expressed genes (DEGs) in NPL vs. CRL and 2450 DEGs in NPL vs. NPN. Functional genomics analysis elucidated that inflammatory signaling, toll-like receptor signaling, inflammasome activation, chemotaxis, and apoptosis pathways are involved in NP. The increased leukocytic infiltration in NPL was associated with the upregulation of matrix metalloproteinase (MMP1, MMP3, and MMP8) and apoptosis-related genes, such as caspases (CASP3 and CASP7), which could explain placental separation associated with NP. Also, NP was associated with downregulation of several placenta-regulatory genes (ABCG2, GCM1, EPAS1, and NR3C1), angiogenesis-related genes (VEGFA, FLT1, KDR, and ANGPT2), and glucose transporter coding genes (GLUT1, GLUT10, and GLUT12), as well as upregulation of hypoxia-related genes (HIF1A and EGLN3), which could elucidate placental insufficiency accompanying NP. In conclusion, our findings revealed for the first time, the key regulators and mechanisms underlying placental inflammation, separation, and insufficiency during NP, which might lead to the development of efficacious therapies or diagnostic aids by targeting the key molecular pathways.

Parental bias in expression and interaction of genes in the equine placenta.

Most autosomal genes in the placenta show a biallelic expression pattern. However, some genes exhibit allele-specific transcription depending on the parental origin of the chromosomes on which the copy of the gene resides. Parentally expressed genes are involved in the reciprocal interaction between maternal and paternal genes, coordinating the allocation of resources between fetus and mother. One of the main challenges of studying parental-specific allelic expression (allele-specific expression [ASE]) in the placenta is the maternal cellular remnant at the fetomaternal interface. Horses (Equus caballus) have an epitheliochorial placenta in which both the endometrial epithelium and the epithelium of the chorionic villi are juxtaposed with minimal extension into the uterine mucosa, yet there is no information available on the allelic gene expression of equine chorioallantois (CA). In the current study, we present a dataset of 1,336 genes showing ASE in the equine CA (https://pouya-dini.github.io/equine-gene-db/) along with a workflow for analyzing ASE genes. We further identified 254 potentially imprinted genes among the parentally expressed genes in the equine CA and evaluated the expression pattern of these genes throughout gestation. Our gene ontology analysis implies that maternally expressed genes tend to decrease the length of gestation, while paternally expressed genes extend the length of gestation. This study provides fundamental information regarding parental gene expression during equine pregnancy, a species with a negligible amount of maternal cellular remnant in its placenta. This information will provide the basis for a better understanding of the role of parental gene expression in the placenta during gestation.

Intrauterine Blood Plasma Platelet-Therapy Mitigates Persistent Breeding-Induced Endometritis, Reduces Uterine Infections, and Improves Embryo Recovery in Mares.

Microorganisms, including pathogenic or opportunistic bacteria and fungi, may gain access to the uterus during breeding, and infectious endometritis plays a major role in equine subfertility. This study aimed to assess the post-breeding inflammatory response, endometrial culture, and embryo recovery of mares susceptible to persistent breeding-induced endometritis (PBIE) treated with plasma-rich (PRP) or -poor (PPP) plasma. Mares (n = 12) susceptible to PBIE had three cycles randomly assigned to receive intrauterine infusions of lactate ringer solution (LRS, control), or autologous PRP or PPP pre- (-48 and -24 h) and post-breeding (6 and 24 h). Mares were bred with fresh semen from one stallion. Intrauterine fluid accumulation (IUF) and endometrial neutrophils were assessed every 24 h up to 96 h post-breeding. Uterine cytokines (Ilß, IL6, CXCL8, and IL10) were evaluated before (0 h), 6, and 24 h post-breeding, and endometrial culture three and nine days after breed. Embryo flushing was performed 8 days post-ovulation. Data were analyzed with mixed model, Tukey's post-hoc test, and multivariate regression. PRP treatment reduced endometrial neutrophils, post-breeding IUF, and pro-inflammatory cytokines when compared to control-assigned cycles, but not significantly different than PPP. Controls had a significantly higher percentage of positive bacterial cultures (33%) in comparison to PRP-assigned cycles (0%), whereas cycles treated with PPP were not significantly different from the other groups (25%). The PRP-assigned cycles had significantly greater embryo recovery rates (83%) than the control (33%), though not significantly different than PPP (60%). Plasma infusion reduced the duration and intensity of the post-breeding inflammatory response and improved embryo recovery in mares susceptible to PBIE. Platelets incrementally downregulate PBIE and appear to have a dose-dependent antimicrobial property.

Paternally expressed retrotransposon Gag-like 1 gene, RTL1, is one of the crucial elements for placental angiogenesis in horses†.

RTL1 (retrotransposon Gag-like 1) is an essential gene in the development of the human and murine placenta. Several fetal and placental abnormalities such as intrauterine growth restriction (IUGR) and hydrops conditions have been associated with altered expression of this gene. However, the function of RTL1 has not been identified. RTL1 is located on a highly conserved region in eutherian mammals. Therefore, the genetic and molecular analysis in horses could hold important implications for other species, including humans. Here, we demonstrated that RTL1 is paternally expressed and is localized within the endothelial cells of the equine (Equus caballus) chorioallantois. We developed an equine placental microvasculature primary cell culture and demonstrated that RTL1 knockdown leads to loss of the sprouting ability of these endothelial cells. We further demonstrated an association between abnormal expression of RTL1 and development of hydrallantois. Our data suggest that RTL1 may be essential for placental angiogenesis, and its abnormal expression can lead to placental insufficiency. This placental insufficiency could be the reason for IUGR and hydrops conditions reported in other species, including humans.

Equine cervical remodeling during placentitis and the prepartum period: a transcriptomic approach.

Cervical remodeling is a critical component in both term and preterm labor in eutherian mammals. However, the molecular mechanisms underlying cervical remodeling remain poorly understood in the mare. The current study compared the transcriptome of the equine cervix (cervical mucosa (CM) and stroma (CS)) during placentitis (placentitis group, n = 5) and normal prepartum mares (prepartum group, n = 3) to normal pregnant mares (control group, n = 4). Transcriptome analysis identified differentially expressed genes (DEGs) during placentitis (5310 in CM and 907 in CS) and during the normal prepartum period (189 in CM and 78 in CS). Our study revealed that cervical remodeling during placentitis was dominated by inflammatory signaling as reflected by the overrepresented toll-like receptor signaling, interleukin signaling, T cell activation, and B cell activation pathways. These pathways were accompanied by upregulation of several proteases, including matrix metalloproteinases (MMP1, MMP2, and MMP9), cathepsins (CTSB, CTSC, and CTSD) and a disintegrin and metalloproteinase with thrombospondin type 1 motifs (ADAMTS1, ADAMTS4, and ADAMTS5), which are crucial for degradation of cervical collagens during remodeling. Cervical remodeling during placentitis was also associated with upregulation of water channel-related transcripts (AQP9 and RLN), angiogenesis-related transcripts (NOS3, ENG1, THBS1, and RAC2), and aggrecan (ACAN), a hydrophilic glucosaminoglycan, with subsequent cervical hydration. The normal prepartum cervix was associated with upregulation of ADAMTS1, ADAMTS4, NOS3 and THBS1, which might reflect an early stage of cervical remodeling taking place in preparation for labor. In conclusion, our findings revealed the possible key regulators and mechanisms underlying equine cervical remodeling during placentitis and the normal prepartum period.

Interleukin-6 pathobiology in equine placental infection.

PROBLEM: Ascending placentitis is the leading cause of abortion in the horse. Interleukin (IL)-6 is considered predictive of placental infection in other species, but little is understood regarding its role in the pathophysiology of ascending placentitis. METHOD OF STUDY: Sub-acute ascending placentitis was induced via trans-cervical inoculation of S zooepidemicus, and various fluids/serum/tissues collected 8 days later. Concentrations of IL-6 were detected within fetal fluids and serum in inoculated (n = 6) and control (n = 6) mares. RNASeq was performed on the placenta (endometrium and chorioallantois) to assess transcripts relating to IL-6 pathways. IHC was performed for immunolocalization of IL-6 receptor (IL-6R) in the placenta. RESULTS: IL-6 concentrations increased in allantoic fluid following inoculation, with a trend toward an increase in amniotic fluid. Maternal serum IL-6 was increased in inoculated animals, while no changes were noted in fetal serum. mRNA expression of IL-6-related transcripts within the chorioallantois indicates that IL-6 is activating the classical JAK/STAT pathway, thereby acting as anti-inflammatory, anti-apoptotic, and pro-survival. The IL-6R was expressed within the chorioallantois, indicating a paracrine signaling pathway of maternal IL-6 to fetal IL-6R. CONCLUSION: IL-6 plays a crucial role in the placental response to induction of sub-acute equine ascending placentitis, and this could be noted in amniotic fluid, allantoic fluid, and maternal serum. Additionally, IL-6 is acting as anti-inflammatory in this disease, potentially altering disease progression, impeding abortion signals, and assisting with the production of a viable neonate.

The Effect of Mycobacterium Cell Wall Fraction on Histologic, Immunologic, and Clinical Parameters of Postpartum Involution in the Mare.

Maintaining yearly foal production is important for the economic success of the broodmare, and this requires breeding to occur as quickly postpartum as possible. The initial postpartum estrus occurs within 5-20 days postpartum, whereas the uterus is still undergoing repair from tissue alterations during pregnancy and parturition, a process known as involution. Attempts have been made to hasten this process, but with minimal success. Mycobacterium cell wall fraction (MCWF) is an immunomodulator that has been shown to reduce bacterial growth and alter aspects of the immune response to breeding, but it is unknown if MCWF hastens the process of involution. Therefore, the objectives of this study were to (1) investigate the effect of MCWF on tissue remodeling, (2) assess the effect of MCWF on the local immune system of the uterus, and (3) determine the optimal treatment interval needed for these processes to occur. We hypothesize that repeated treatments of MCWF postpartum will hasten the process of involution. To study this, 16 pregnant mares of mixed breeds were evaluated postpartum. Control mares (n = 4) received 1.5 mL lactated Ringer's solution intravenously on Day 1 (Day 0 = day of parturition) postpartum and again on Day 7, whereas treated mares either received 1.5 mL Settle intravenously on Day 1 and Day 7 (TX1; n = 6) or 1.5 mL Settle intravenously on Day 1 and then every 3 days until ovulation was detected (TX2; n = 6) and then evaluated until 15 days postpartum. Mares were assessed every 3 days for clinical, immunologic, and histologic parameters. Clinical parameters were assessed with transrectal ultrasonography and included ovarian activity, uterine fluid retention, and measurement of the uterine diameter, in addition to endometrial culture. Immunologic parameters included endometrial biopsies for quantitative polymerase chain reaction for expression of various cytokines (interleukin [IL]-1ß, IL-1RN, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor [TNF], interferon [IFN]-γ, and granulocyte-macrophage colony-stimulating factor) in addition to endometrial cytology. Formalin-fixed endometrial biopsies were histologically assessed for the retention of microcaruncles, dilation of endometrial glands, and inflammation of the mucosa, stratum compactum, and spongiosum. Statistics were performed using SAS 9.4, using a mixed model for repeated measures with mare and treatment as a random effect. All post-hoc analysis was done using a Tukey's honestly significant difference test. Involution was considered complete by Day 15 postpartum in all mares, and the day postpartum had a significant effect on almost all parameters investigated, indicating the immunologic process of involution. Treatment with MCWF decreased the magnitude of bacterial growth in addition to time to negative culture. In addition, MCWF increased the expression of IL-1ß, IFNγ, and TNF. Although minimal treatment effect was noted histologically, a decrease in mucosal inflammation was seen in MCWF-treated mares. In conclusion, involution appears to be influenced by the immune system. In addition, MCWF appears to have a bactericidal effect on the postpartum mare, and this may be because of an increase in proinflammatory cytokines. It is unknown if this bactericidal property will improve fertility on the first estrous cycle postpartum, and future studies are needed to determine this.

Biomarkers for placental disease in mares.

Placentitis is an important cause of abortion, stillbirth, and neonatal death in horses. The diagnosis of placentitis is based on occurrence of clinical signs (premature mammary gland development and vulvar discharge) and ultrasonography of the caudal placental pole. However, early and subtle cases can be missed. In the last few years, several studies have provided objective means of diagnosing placentitis in mares with single or serial measurements of blood markers. Among the markers evaluated the steroids produced by the fetoplacental unit have been shown to change in association with placentitis. Mares with chronic placentitis have an increase in peripheral progestogens; however, mares acutely infected will display a reduction in peripheral concentrations of progestogens. Estradiol-17ß (free- and conjugated form) concentrations are drastically reduced in plasma of mares with placentitis. Acute-phase proteins, particularly serum amyloid A, are increased in plasma of mares suffering from placentitis, and this increase is due to endometrial and chorioallantoic secretions, and minimally from the fetus. Alpha-fetoprotein, a protein expressed in the fetoplacental unit, was shown to be increased in plasma of mares suffering from placentitis. A plephora of microRNA have been identified in plasma and tissues of mares undergoing experimentally induced placentitis, but have not been tested in spontaneous cases. Unique proteomic signatures were found in the fetal fluids of mares undergoing experimentally induced ascending placentitis, making the fetal fluids potentially useful to diagnose placentitis in mares. However, currently the lack of use of transabdominal fetal fluid sampling prevents wide use of the fetal fluids as diagnostic techniques. This manuscript aimed to discuss recent discoveries regarding biomarkers for placentitis in mares.

Characterization of the placental transcriptome through mid to late gestation in the mare.

The placenta is a dynamic organ which undergoes extensive remodeling throughout pregnancy to support, protect and nourish the developing fetus. Despite the importance of the placenta, very little is known about its gene expression beyond very early pregnancy and post-partum. Therefore, we utilized RNA-sequencing to characterize the transcriptome from the fetal (chorioallantois) and maternal (endometrium) components of the placenta from mares throughout gestation (4, 6, 10, 11 m). Within the endometrium, 47% of genes changed throughout pregnancy, while in the chorioallantois, 29% of genes underwent significant changes in expression. Further bioinformatic analyses of both differentially expressed genes and highly expressed genes help reveal similarities and differences between tissues. Overall, the tissues were more similar than different, with ~ 95% of genes expressed in both tissues, and high similarities between the most highly expressed genes (9/20 conserved), as well as marked similarities between the PANTHER pathways identified. The most highly expressed genes fell under a few broad categories, including endocrine and immune-related transcripts, iron-binding proteins, extracellular matrix proteins, transport proteins and antioxidants. Serine protease inhibitors were particularly abundant, including SERPINA3, 6 and 14, as well as SPINK7 and 9. This paper also demonstrates the ability to effectively separate maternal and fetal components of the placenta, with only a minimal amount of chorioallantoic contamination in the endometrium (~8%). This aspect of equine placentation is a boon for better understanding gestational physiology and allows the horse to be used in areas where a separation of fetal and maternal tissues is essential. Overall, these data represent the first large-scale characterization of placental gene expression in any species and include time points from multiple mid- to late-gestational stages, helping further our understanding of gestational physiology.

The feto-maternal immune response to equine placentitis.

PROBLEM: Ascending placentitis is one of the leading causes of abortion in the horse. Minimal work has focused on its effect on fetal fluids or the antenatal immune response of the fetus. METHODOLOGY: Placentitis was induced via transcervical inoculation of Streptococcus equi ssp Zooepidemicus, and fluids/serum/tissues were collected 4-6 days later following euthanasia. Cytokine concentrations were detected using a multiplex immunoassay within fetal fluids (amniotic and allantoic) and serum (maternal and fetal) in inoculated and control mares. In addition, tissues from fetal (spleen, liver, lung, umbilicus, amnioallantois) and maternal (spleen, liver, lung, chorioallantois, endometrium) origin were analyzed in inoculated and control mares utilizing qPCR for expression of cytokines. RESULTS: No difference in cytokine concentrations in maternal or fetal serum was noted between inoculated and control mares. Concentrations of IL-1ß, IL-6, IL-10, and GRO were upregulated in the amniotic fluid following inoculation, with a trend toward higher IL-6 concentration in allantoic fluid. The amnioallantoic tissue separating the two fluids had higher expression of IL-1ß and IL-6 following inoculation, while chorioallantois and endometrium upregulated IL-1ß and IL-8 expression. IL-1ß was upregulated in the maternal spleen following inoculation. Fetal spleens were upregulated in expression of IL-1ß, GRO, and IL-6, while IL-6 was higher in fetal liver after inoculation than in controls. CONCLUSION: The maternal response to placentitis is primarily pro-inflammatory while the fetus appears to play a regulatory role in this inflammation. Additionally, amniotic fluid sampling may be more diagnostic of ascending placentitis than circulating cytokines.

Landscape of Overlapping Gene Expression in the Equine Placenta.

Increasing evidence suggests that overlapping genes are much more common in eukaryotic genomes than previously thought. These different-strand overlapping genes are potential sense-antisense (SAS) pairs, which might have regulatory effects on each other. In the present study, we identified the SAS loci in the equine genome using previously generated stranded, paired-end RNA sequencing data from the equine chorioallantois. We identified a total of 1261 overlapping loci. The ratio of the number of overlapping regions to chromosomal length was numerically higher on chromosome 11 followed by chromosomes 13 and 12. These results show that overlapping transcription is distributed throughout the equine genome, but that distributions differ for each chromosome. Next, we evaluated the expression patterns of SAS pairs during the course of gestation. The sense and antisense genes showed an overall positive correlation between the sense and antisense pairs. We further provide a list of SAS pairs with both positive and negative correlation in their expression patterns throughout gestation. This study characterizes the landscape of sense and antisense gene expression in the placenta for the first time and provides a resource that will enable researchers to elucidate the mechanisms of sense/antisense regulation during pregnancy.

Equine placentitis is associated with a downregulation in myometrial progestin signaling†.

The current study aimed to elucidate the mechanisms underlying myometrial activation during equine placentitis related to progestogens and the progesterone receptor signaling pathways. Placentitis was induced via intracervical inoculation with Streptococcus equi ssp zooepidemicus in mares at approximately 290 days of gestation (placentitis group; n = 6) with uninoculated gestationally matched mares as controls (n = 4). Mares in the placentitis and control groups were euthanized, and myometrial samples were collected from two regions: region 1-parallel to active placentitis lesion with placental separation in placentitis group (P1) or caudal pole of the placenta in control group (C1); and region 2-parallel to apparently normal placenta without separation in placentitis group (P2) or uterine body in control group (C2). In the current study, SRD5A1 and AKR1C23, which encode for the key P4 metabolizing enzymes, were downregulated in P1 in comparison to C1, C2, and P2, and this was associated with a decline (P < 0.05) in 5αDHP, allopregnanolone (3αDHP), and 20αDHP in P1 in comparison to C1. Further, myometrial expression of PR was downregulated (P < 0.05) in P1 in comparison to C1 and P2, and this was associated with activation of the inflammatory cascade as reflected by significant upregulation of IL-1ß and IL-8 in P1 in comparison to C1, C2, and P2, and supported by increased tissue leukocytes in P1 in comparison to C1. In conclusion, equine placentitis is associated with a localized withdrawal of progestins and a downregulation of the PR in the myometrium concomitant with upregulation of inflammatory cytokines and subsequent myometrial activation.

Small RNA (sRNA) expression in the chorioallantois, endometrium and serum of mares following experimental induction of placentitis.

Intrauterine infection and inflammation remain a major cause of preterm labour in women and mares, with little known about small RNA (sRNA) expression in tissue or circulation. To better characterise placental inflammation (placentitis), we examined sRNA expression in the endometrium, chorioallantois and serum of mares with and without placentitis. Disease was induced in 10 mares via intracervical inoculation of Streptococcus equi ssp. zooepidemicus, either with moderate or high levels of inoculum; three uninoculated gestationally matched mares were used as controls. Matched chorioallantois and endometrium were sampled in two locations: Region 1, gross inflammation near cervical star with placental separation and Region 2, gross inflammation without placental separation. In Region 1, 26 sRNAs were altered in chorioallantois, while 20 were altered in endometrium. Within Region 2, changes were more subdued in both chorioallantois (10 sRNAs) and endometrium (two sRNAs). Within serum, we identified nine significantly altered sRNAs. In summary, we have characterised the expression of sRNA in the chorioallantois, the endometrium and the serum of mares with experimentally induced placentitis using next-generation sequencing, identifying significant changes within each tissue examined. These data should provide valuable information about the physiology of placental inflammation to clinicians and researchers alike.

Equine fetal adrenal, gonadal and placental steroidogenesis

The authors apologize for errors in Figure 6 of their article published in the October 2017 issue of Reproduction (vol 154 iss 4 pp 445­454). The authors explain that the addition of data (Figure 6) on steroid concentrations in the chorioallantois to their manuscript on fetal adrenal and fetal gonadal steroids during development of the equine fetus was made in response to reviewer comments. However, in compiling, summarizing and graphing the data, the wrong units were used in the final figure. The manuscript as published represents the data in Figure 6 as "ng/g", when in fact they are "nmol/g". The authors very much regret having made the mistake and sincerely apologize for any confusion this might have caused.

Kinetics of the chromosome 14 microRNA cluster ortholog and its potential role during placental development in the pregnant mare.

BACKGROUND: The human chromosome 14 microRNA cluster (C14MC) is a conserved microRNA (miRNA) cluster across eutherian mammals, reported to play an important role in placental development. However, the expression kinetics and function of this cluster in the mammalian placenta are poorly understood. Here, we evaluated the expression kinetics of the equine C24MC, ortholog to the human C14MC, in the chorioallantoic membrane during the course of gestation. RESULTS: We demonstrated that C24MC-associated miRNAs presented a higher expression level during early stages of pregnancy, followed by a decline later in gestation. Evaluation of one member of C24MC (miR-409-3p) by in situ hybridization demonstrated that its cellular localization predominantly involved the chorion and allantoic epithelium and vascular endothelium. Additionally, expression of predicted target transcripts for C24MC-associated miRNAs was evaluated by RNA sequencing. Expression analysis of a subset of predicted mRNA targets showed a negative correlation with C24MC-associated miRNAs expression levels during gestation, suggesting the reciprocal control of these target transcripts by this miRNA cluster. Predicted functional analysis of these target mRNAs indicated enrichment of biological pathways related to embryonic development, endothelial cell migration and angiogenesis. Expression patterns of selected target mRNAs involved in angiogenesis were confirmed by RT-qPCR. CONCLUSION: This is the first report evaluating C24MC kinetics during pregnancy. The findings presented herein suggest that the C24MC may modulate angiogenic transcriptional profiles during placental development in the horse.

A comparison of progesterone assays for determination of peripheral pregnane concentrations in the late pregnant mare.

During the latter half of gestation in mares, there is a complex milieu of pregnanes in peripheral blood. Progesterone concentrations are often assessed by immunoassay during late gestation as a measure of pregnancy well-being; however, interpretation of results is complicated by the numerous cross-reacting pregnanes present in high concentrations during late gestation. Further, many mares are supplemented with an exogenous progestin, altrenogest, which may also cross-react with existing assays and further confound interpretation. The objectives of this study were: 1) to compare differences in pregnane concentrations determined with four immunoassays compared to LC-MS/MS and 2) to assess cross-reactivity observed with the same immunoassays, specifically considering pregnenolone (P5), progesterone (P4), 5α-dihydroprogesterone (DHP), allopregnanolone, and altrenogest. Blood samples from four healthy mares in late gestation were evaluated by immunoassay and by LC-MS/MS. Measured immuno-reactive progesterone (ir-progesterone) concentrations differed (p < 0.0001) between immunoassays, although results were highly correlated (r = 0.85-1.0; p < 0.001). Measured ir-progesterone concentrations by immunoassay were linearly associated (r2 = 0.68-0.76; p < 0.001) with concentrations of P5, P4, DHP, and allopregnanolone determined by LC-MS/MS. There was no detectable cross-reaction of altrenogest in any immunoassay, but varying degrees of cross-reactivity was observed with other pregnanes analyzed. These data confirm ir-progesterone concentrations during late gestation vary depending upon the assay used and the cross-reactivity to other pregnanes present in late gestation, although the synthetic progestin altrenogest did not affect the results of any immunoassay tested.