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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.

High blood urea nitrogen (BUN) concentration is linked to low fertility in cows and ewes; however, this relationship has not been reported in mares. The study characterized the relationship between BUN and follicular fluid urea nitrogen (FUN) during follicle growth (Experiment 1) and the impact of BUN from embryo donors on the pregnancy outcome of recipient mares (Experiment 2). In experiment one, follicular fluid and blood samples were collected from mares during diestrus with growing follicles and during estrus with pre-ovulatory follicles (n = 16 and 10 mares, respectively). In experiment two, BUN concentrations of embryo donors were related to pregnancy outcome after embryo transfer. In experiment one, there was a strong positive correlation between BUN and FUN (R = 0.83; P 0.0001), with higher BUN in mares with growing follicles than with preovulatory follicles (P = 0.004) and higher FUN in growing follicles than in preovulatory follicles (P = 0.031). In experiment two, BUN was higher in donor mares that produced unsuccessful embryos compared to donor mares that produced embryos resulting in successful pregnancies at D14 (P 0.03). Additionally, there was an effect of age (P = 0.01) and interaction between age and lactation (P = 0.009) in donor mares for embryo survival after embryo transfer. Donor mares with unsuccessful embryos were older than donor mares with successful embryos. Therefore, these experiments showed that BUN was related to follicular fluid environment as well as to the survival of Day 7-8 embryos after transfer to recipient mares.

The endometrium, the inner uterine lining, is composed of cell layers that come in direct contact with an embryo during early pregnancy and later with the fetal placenta. The endometrium is responsible for signals associated with normal reproductive cyclicity as well as maintenance of pregnancy. In the mare, functionally competent in vitro models of the endometrium have not been successful. Furthermore, the ability to study various reproductive processes in vitro may allow critical evaluation of signaling pathways involved in the reproductive diseases of animals that cannot be handled frequently, such as various wildlife species. Here we report the establishment of organoids, 3D structures, derived from fresh and frozen–thawed equine endometrium (Equus ferus caballus and E. f. przewalskii). Although organoids from domestic mares responded to exogenous hormonal stimuli, organoids from Przewalski’s horse failed to respond to exogenous hormones. The present study represents a ‘first’ for any large animal model or endangered species. These physiologically functional organoids may facilitate improved understanding of normal reproductive mechanisms, uterine pathologies, and signaling mechanisms between the conceptus and endometrium and may lead to the development of novel bioassays for drug discovery.

Improved understanding of the molecular mechanisms underlying ascending equine placentitis holds the potential for the development of new diagnostic tools and therapies to forestall placentitis-induced preterm labor. The current study characterized the equine placental transcriptome (chorioallantois [CA] and endometrium [EN]) during placentitis (placentitis group, n = 6) in comparison to gestationally-matched controls (control group, n = 6). Transcriptome analysis identified 2953 and 805 differentially expressed genes in CA and EN during placentitis, respectively. Upstream regulator analysis revealed the central role of toll-like receptors (TLRs) in triggering the inflammatory signaling, and consequent immune-cell chemotaxis. Placentitis was associated with the upregulation of matrix metalloproteinase (MMP1, MMP2, and MMP9) and apoptosis-related genes such as caspases (CASP3, CASP4, and CASP7) in CA. Also, placentitis was associated with downregulation of transcripts coding for proteins essential for placental steroidogenesis (SRD5A1 and AKR1C1), progestin signaling (PGRMC1 and PXR) angiogenesis (VEGFA, VEGFR2, and VEGFR3), and nutrient transport (GLUT12 and SLC1A4), as well as upregulation of hypoxia-related genes (HIF1A and EGLN3), which could explain placental insufficiency during placentitis. Placentitis was also associated with aberrant expression of several placenta-regulatory genes, such as PLAC8, PAPPA, LGALS1, ABCG2, GCM1, and TEPP, which could negatively affect placental functions. In conclusion, our findings revealed for the first time the key regulators and mechanisms underlying placental inflammation, separation, and insufficiency during equine placentitis, which might lead to the development of efficacious therapies or diagnostic aids by targeting the key molecular pathways.Summary sentenceThis is the first study to characterize the equine placenta transcriptome to identify the key regulators and pathways underlying placental inflammation, separation, and insufficiency during the equine ascending placentitis.Graphical Abstract

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.

Background Granulosa cell tumours (GCT) are the most common ovarian tumours in mares. While the classical presentation may not represent diagnostic challenges, diagnosis is not easy in the early stages. Objectives Illustrate the variability in the presentation and serum biomarkers associated with ovarian abnormalities in the mare. Study design Retrospective case series. Methods Nonclassical cases of GCTs and other ovarian conditions were identified and behaviour, GCT endocrine results, palpation and ultrasonographic findings are described and the diagnostic value of each is discussed. Results Mares in this case series with GCTs had been presenting clinical signs ranging from no behavioural changes to behaviours including aggression, stallion-like and inability to work under saddle. Hormonal profiles of endocrinologically functional GCTs can be erratic and unpredictable. The clinical form and ultrasonographic appearance may also vary with time from an initially enlarged/anovulatory follicular structure that later develops a multicystic 'honeycomb' appearance. Mares with GCTs can also present with persistent anovulatory follicles or apparent luteal tissue that are unresponsive to treatment. If both ovaries are of relatively normal size and symmetry, but hormonal biomarkers are markedly increased (AMH >10 ng/mL, inhibin B and/or testosterone >100 pg/mL; 0.37 nmol/L), it is likely that a functional GCT is present. Still, it can be a challenge to decide which ovary to remove. Post-surgical endocrine testing can be helpful, especially if histopathology is not performed or a GCT is not found. Main limitations Cases limited to 14. Conclusions Granulosa cell tumours present with a wide variety of clinical signs that do not fit what is commonly described as 'classic'. Only if AMH, testosterone and inhibin B concentrations are markedly increased, and there is an abnormally enlarged ovary, the diagnosis of a GCT is more confident. In the presence of normal size ovaries, normal hormonal biomarkers and abnormal behaviour, it is more likely that the ovaries are not involved.

Introduction Hydrallantois is the excessive accumulation of fluid in the allantoic cavities during the last trimester of pregnancy, leading to abdominal wall hernias, cardiovascular shock, abortion, and dystocia. It has been postulated that hydrallantois is associated with structural and/or functional changes in the chorioallantoic membrane. In the present study, we hypothesized that angiogenesis is impaired in the hydrallantoic placenta. Method Capillary density in the hydrallantoic placenta was evaluated in the chorioallantois via immunohistochemistry for Von Willebrand Factor. Moreover, the expression of angiogenic genes was compared between equine hydrallantois and age-matched, normal placentas. Results In the hydrallantoic samples, edema was the main pathological finding. The capillary density was significantly lower in the hydrallantoic samples than in normal placentas. The reduction in the number of vessels was associated with abnormal expression of a subset of angiogenic and hypoxia-associated genes including VEGF, VEGFR1, VEGFR2, ANGPT1, eNOS and HIF1A. We believe that the capillary density and the abnormal expression of angiogenic genes leads to tissue hypoxia (high expression of HIF1A) and edema. Finally, we identified a lower expression of genes associated with steroidogenic enzyme (CYP19A1) and estrogen receptor signaling (ESR2) in the hydrallantoic placenta. Discussion Based on the presented data, we believe that formation of edema is due to disrupted vascular development (low number of capillaries) and hypoxia in the hydrallantoic placenta. The edema leads to further hypoxia and consequently, causes an increase in vessel permeability which leads to a gradual increase in interstitial fluid accumulation, resulting in an insufficient transplacental exchange rate and accumulation of fluid in the allantoic cavity.

Equine placentitis is associated with alterations in maternal peripheral steroid concentrations, which could negatively affect pregnancy outcome. This study aimed to elucidate the molecular mechanisms related to steroidogenesis and steroid-receptor signaling in the equine placenta during acute placentitis. Chorioallantois (CA) and endometrial (EN) samples were collected from mares with experimentally induced placentitis (n = 4) and un-inoculated gestationally age-matched mares (control group; n = 4). The mRNA expression of genes coding for steroidogenic enzymes (3βHSD, CYP11A1, CYP17A1, CYP19A1, SRD5A1, and AKR1C23) was evaluated using qRT-PCR. The concentration of these enzyme-dependent steroids (P5, P4, 5αDHP, 3αDHP, 20αDHP, 3β-20αDHP, 17OH-P, DHEA, A4, and estrone) was assessed using liquid chromatography-tandem mass spectrometry in both maternal circulation and placental tissue. Both SRD5A1 and AKR1C23, which encode for the key progesterone metabolizing enzymes, were downregulated (P P P P = 0.052). In the EN, AKR1C23 was also downregulated in the placentitis group compared to controls, and this downregulation was associated with a decline in EN concentrations of 3αDHP (P P P = 0.057) concentrations of estrone in CA. Moreover, ESR1 (steroid receptors) gene expression was downregulated (P = 0.057) in CA from placentitis mares. In conclusion, acute equine placentitis is associated with a local withdrawal of progestins in the placenta and tended to be accompanied with estrogen withdrawals in CA.

Introduction The tolerance of pregnancy by the maternal immune system is balanced between recognition and protection. In the human this is controlled by balancing helper T cell populations (Th1, Th2) in addition to immune suppression from the regulatory arm (Tregs), but this has not been evaluated in the horse. Methods RNA sequencing was performed on chorioallantois and endometrium of mares at 120, 180, 300 and 330 days of gestation (n = 4/stage), as well as 45-day chorioallantois (n = 4) and diestrus endometrium (n = 3). Transcripts were selected for relativity to Th1, Th2, or Treg-associated. qPCR and immunohistochemistry were used to confirm the results of select differentially expressed genes. Results In the endometrium, Th1 transcripts were highest in the diestrus mare and decreased as gestational length progressed. In contrast, Th2 transcripts were upregulated in comparison to the diestrus mare and highest in mid gestation. Treg transcripts were found increased in comparison to the diestrus mare, but decreased prepartum. In the chorioallantois no Th1 transcripts changed. The majority of Th2 transcripts increased from 45 to 300 days gestation, and then decreased prepartum. Treg-related transcripts trended down in the chorioallantois from 45 days to 120 days gestation, followed by an upregulation to 300 days and a secondary decline prepartum. Discussion The mare experiences a complex and evolving immune profile within the tissues of the feto-maternal interface. This consists of a balance between the Th1 and Th2 response, and a dynamic Treg response that is hypothesized to regulate overall events within the immune system.

A sufficient vascular network within the feto-maternal interface is necessary for placental function. Several pregnancy abnormalities have been associated with abnormal vascular formations in the placenta. We hypothesized that growth and expansion of the placental vascular network in the equine (Equus caballus) placenta is regulated by estrogens (estrogen family hormones), a hormone with a high circulating concentration during equine gestation. Administration of letrozole, a potent and specific inhibitor of aromatase, during the first trimester (D30 to D118), decreased circulatory estrone sulfate concentrations, increased circulatory testosterone and androstenedione concentrations, and tended to reduce the weight of the fetus (p &lt, 0.1). Moreover, the gene expression of CYP17A1 was increased, and the expression of androgen receptor was decreased in the D120 chorioallantois (CA) of letrozole-treated mares in comparison to that of the control mares. We also found that at D120, the number of vessels tended to decrease in the CAs with letrozole treatment (p = 0.07). In addition, expression of a subset of angiogenic genes, such as ANGPT1, VEGF, and NOS2, were altered in the CAs of letrozole-treated mares. We further demonstrated that 17β-estradiol increases the expression of ANGPT1 and VEGF and increases the angiogenic activity of equine endothelial cells in vitro. Our results from the estrogen-suppressed group demonstrated an impaired placental vascular network, suggesting an estrogen-dependent vasculogenesis in the equine CA during the first trimester.

Equine premature placental separation (PPS) is poorly understood and represents an important risk factor for fetal/neonatal hypoxia.To examine transcriptomic changes in the chorioallantois (CA) from mares with clinical PPS compared with the CA from normal foaling mares. Differential gene expression was determined and gene ontology as well as molecular pathways related to PPS were characterised.Retrospective case: control study.CA were collected from Thoroughbred mares with a clinical history of PPS (n = 33) and from control Thoroughbred mares (n = 4) with normal parturition for examination of transcriptional changes in the placenta associated with PPS. Transcriptomic changes in the villous CA near the cervical star were determined by Illumina® sequencing and subsequent bioinformatic analysis. PPS samples were divided by k-means clustering, and differentially expressed genes (DEGs) in each PPS cluster were identified by comparing to controls. Shared DEGs between PPS clusters were used for gene ontology analysis and pathway analysis.A total of 1204 DEGs were identified between PPS and control. Gene ontology revealed extracellular matrix (ECM) and cell adhesion, and pathway analysis revealed fatty acid, p-53, hypoxia and inflammation. Eleven key regulator genes of PPS including growth factors (IGF1, TGFB2, TGFB3), transcription factors (HIF1A, JUNB, SMAD3), and transmembrane receptors (FGFR1, TNFRSF1A, TYROBP) were also identified.The use of clinical history of PPS, in the absence of other criteria, may have led to misidentification of some cases as PPS.Transcriptomic analysis indicated that changes in ECM and cell adhesion were important factors in equine PPS. Key predicted upstream events include genes associated with hypoxia, inflammation and growth factors related to the pathogenesis of equine PPS.

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.

BACKGROUND Few studies have provided a longitudinal analysis of systemic concentrations of conjugated oestrogens (and androgens) throughout pregnancy in mares, and those only using immunoassay. The use of liquid chromatography tandem mass spectrometry (LC-MS/MS) will provide more accurate concentrations of circulating conjugated steroids. OBJECTIVES To characterise circulating concentrations of individual conjugated steroids throughout equine gestation by using LC-MS/MS. STUDY DESIGN Longitudinal study and comparison of pregnant mares treated with vehicle or letrozole in late gestation. METHODS Sulphated oestrogens and androgens were measured in mares throughout gestation and mares in late gestation (8-11 months) treated with vehicle or letrozole to inhibit oestrogen synthesis in late gestation. An analytical method was developed using LC-MS/MS to evaluate sulphated estrone, estradiol, testosterone and dehydroepiandrosterone (DHEAS) during equine gestation. RESULTS Estrone sulphate concentrations peaked by week 26 at almost 60 μg/mL, 50-fold higher than have been reported in studies using immunoassays. An increase in DHEAS was detected from 7 to 9 weeks of gestation, but concentrations remained consistently low (if detected) for the remainder of gestation and testosterone sulphate was undetectable at any stage. Estradiol sulphate concentrations were highly correlated with estrone sulphate but were a fraction of their level. Concentrations of both oestrogen sulphates decreased from their peak to parturition. Letrozole inhibited estrone and estradiol sulphate concentrations at 9.25 and 10.5 months of gestation but, no increase in DHEAS was observed. MAIN LIMITATIONS Limited number of mares sampled and available for analysis, lack of analysis of 5α-reduced and B-ring unsaturated steroids due to lack of available standards. CONCLUSIONS Dependent on methods of extraction and chromatography, and the specificity of primary antisera, immunoassays may underestimate oestrogen conjugate concentrations in blood from pregnant mares and may detect androgen conjugates (neither testosterone sulphate nor DHEAS were detected here by LC-MS/MS) that probably peak coincident with oestrogen conjugates between 6 and 7 months of equine gestation.

Granulosa cell tumor (GCT) is a sex-cord stromal tumor in mares and causes infertility. The objective of this study was to localize anti-Mullerian hormone (AMH) and its receptor, anti-Mullerian hormone receptor type 2 (AMHR2), in ovarian tissue samples obtained from 11 Thoroughbred mares diagnosed with GCT. Immunohistochemistry revealed positive immunostaining for both AMH and AMHR2 in the granulosa-like cells of GCT. Furthermore, double immunofluorescence staining revealed the presence of colocalization of AMH and AMHR2 in granulosa-like cells of GCT in mares. These findings suggest that granulosa-like cells of GCT are a target of AMH, suggesting that AMH may have paracrine and autocrine function on granulosa-like cells of GCT in mares. Moreover, this is the first report to demonstrate the colocalization of AMH and its receptor, AMHR2, in GCT-affected ovaries of mares by using double immunofluorescence staining.

The objectives of this study were to evaluate; 1) the stability of measured serum anti-Müllerian hormone (AMH) concentrations in samples after multiple freeze-thaw cycles, 2) the repeatability of AMH concentrations within mares during the same breeding season as well as across breeding seasons, and 3) the relationship between serum AMH concentrations and fertility (measured as first cycle pregnancy rates) in thoroughbred mares. For the first aim, AMH concentrations (n = 9) were examined across four freeze-thaw cycles with no significant change in measured AMH concentrations. For the second aim, serum AMH concentrations (n = 12) were examined over three successive estrous cycles and over two successive breeding seasons and AMH levels were significantly correlated for individual animals within (r; 0.71-82) or across breeding seasons (r = 0.81). For the third aim, Thoroughbred mares (n = 419) on farms in central Kentucky had blood samples taken during estrus. Pregnancy was determined with transrectal ultrasonography at Days 13-18 after mating and ovulation, and pregnancy outcome was recorded as open, pregnant or twins. The relationships between mare age, serum AMH concentrations and the interaction of age and AMH with pregnancy outcome was examined by nominal logistic regression, and the relationship between serum AMH concentrations and mare age, pregnancy outcome and the interaction of age and pregnancy outcome was examined by ANOVA. Data in this study were then stratified according to quartiles into lower (25%), mid-50% (second and third quartiles combined - 50%) and upper (25%) quartiles for age and serum AMH concentration for further analysis by logistic regression. There were significant effects of mare age and pregnancy outcome, but not their interaction on serum AMH concentrations which were higher (P = 0.04) in pregnant than in open mares (0.65 ± 0.03 vs 0.55 ± 0.04 ng/mL, respectively). Likewise, logistic regression revealed significant effect of mare age and AMH but not their interaction on pregnancy outcome on the first mated cycle. Mares in the lower AMH quartile were more likely to be open at Day 13-18 than mares in the middle (odds ratio (OR) = 1.87)=13 or upper quartile (OR = 2.62) for AMH concentrations. Mares in the mid-50% (OR = 3.91) or upper (OR = 4.97) age quartile were more likely to be open at Day 13-18 compared to mares in the young age quartile. Based upon a Chi-squared analysis, the proportion of pregnant mares differed across age quartiles (P 0.0001) and was greater (P 0.05) in the young mare quartile. The proportion of pregnant mares tended to differ across AMH quartile (P = 0.1), and when adjusted for age quartile using the Cochran-Mantel-Haenszel Test, the proportion of pregnant mares differed (P 0.05) across AMH quartile. In conclusion, mares with peripheral AMH concentrations in the lowest quartile had lower fertility compared to mares in the mid-50% or upper quartile.

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. Electronic supplementary material The online version of this article (10.1186/s12864-018-5341-2) contains supplementary material, which is available to authorized users.

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.

Placentitis is the leading cause of infectious abortion in the horse and contributes to roughly 19% of all abortions in the United States. A type of placental infection, nocardioform placentitis (NP) is associated with gram-positive branching actinomycetes localized within the ventral body of the feto-maternal interface to create a lymphoplasmacytic mucoid lesion. While the etiology of this disease is poorly described, this placental infection continues to cause episodic abortions in addition to weak and/or growth retarded neonates. The goal of the present study was to perform a comprehensive analysis of pregnancies associated with a nocardioform-affected placenta and make inferences into the epidemiology of this elusive disease. To do so, 264 mares were enrolled in the study, with 145 as having suspected disease (n = 145; NP) either based on pregnancy-related complications or postpartum placental evaluation, while an additional 119 were enrolled as healthy pregnancies (n = 119; CON). Following diagnosis as either NP or CON based on gross and histopathology at the University of Kentucky Veterinary Diagnostic Laboratory, information was gathered on the mares and neonates for comparisons between diseased and healthy pregnancies. Clinically, a significant portion of diseased mares had clinical indications of NP, including premature mammary gland development, thickening of the placenta noted on transrectal ultrasonography, and separation between the chorioallantois and endometrium noted on abdominal ultrasonography, while vulvar discharge was not commonly noted. Additionally, NP was correlated with increased mare age, decreased gestational length, and decreased neonatal weight, although neonatal IgG and WBC were comparable to CON. Incidence of NP was not correlated with last breeding date, pre- and post-breeding therapeutics, parity, prophylactic medications, or housing. Additionally, NP did not affect postpartum fertility. While NP was associated with a poor neonatal outcome (abortion and/or growth retarded neonate), this did not appear to be influenced by the bacteria isolated (Amycolatopsis spp. vs. Crossiella equi), and mares diagnosed with NP do not appear to be infectious to other pregnant mares nor have repetitive years of the disease. Interestingly, lesion size was positively correlated with last breeding date, as mares bred later in the breeding season correlating with a larger placental lesion. In conclusion, while the etiology of NP continues to elude researchers, the epidemiology of this disease has gained clarity, providing inferences into the management of suspect mares.

The goal of this study was to develop a safe, effective, and economical method for permanent sterilization of mares based upon tubo-ovarian ligation performed via colpotomy. In this study, we evaluated the application of a nylon cable tie (zip-tie) to the ovarian pedicle and oviduct of mares to induce ovarian ischemia and tubal ligation without removal of ovaries. Initially, efficiency of zip-ties on the ovarian pedicle was tested in vitro and in vivo. Based on the absence of leakage through the zip-tie ligated vessels in anatomic specimens, we confirmed the potential efficacy of the technique. Next, ligation of the ovarian pedicle via a standing colpotomy was conducted in five mares. Although the surgical procedure in these mares appeared to be quick and efficient, all five mares were noted to develop ovarian adhesions to surrounding abdominal viscera in either one or both ovaries postoperatively. Ovarian ischemia led to loss of ovarian activity based upon ultrasound examination, which was confirmed by a low plasma progesterone concentration in four of the five mares. During the postoperative period, four mares demonstrated clinical signs related to the ovarian adhesions and were euthanized. The postoperative complications associated with ovarian adhesions to abdominal viscera presented significant challenges, limiting the success of this study. While this technique resulted in ovarian ischemia and atrophy in four out of the five mares, we were unable to assess long-term effects on the health and reproduction of the mares due to the ovarian adhesions to the surrounding tissues and the potential for secondary complications. Although technically feasible, tubo-ovarian ligation via colpotomy does not appear to be a viable option for sterilization of mares using the described technique due to ovarian adhesions post procedure.

Serum amyloid A (SAA) and Haptoglobin (Hp) are acute phase proteins, produced during inflammation, such as placentitis. In horses, SAA and SAA1 are protein coding genes. Objectives were to analyze SAA and Hp concentrations and relative abundance of SAA, SAA1 and Hp mRNA transcript in maternal and fetal tissues after experimental induction of placentitis or mares of a control group. Serum Amyloid A family proteins were in marked abundance in the stroma of the endometrium and chorioallantois associated with inflammatory cells. Maternal plasma SAA concentrations were greater (P = 0.01) in mares with experimentally induced placentitis compared to those of the control group. Maternal Hp from the groups were not different, but fetal Hp concentrations of mares with experimentally induced placentitis were greater (P = 0.02). Maternal plasma SAA and Hp concentrations were greater than fetal plasma concentrations in mares with experimentally induced placentitis (P < 0.05). Relative abundance of SAA mRNA transcript was greater in the maternal, fetal liver and chorioallantois of mares with experimentally induced placentitis (P < 0.05) compared to those in the control group. Interestingly, relative abundance of SAA1 mRNA transcript was greater in the chorioallantois of mares with experimentally induced placentitis (P < 0.05). The SAA and Hp concentrations, therefore, were greater in mares with induced placentitis. Furthermore, relative abundance of SAA1 mRNA transcript is specifically greater in the chorioallantois of mares with placentitis, which warrants further studies to elucidate the immunological response of SAA1 in the chorioallantois of mares with placentitis.

Equine abortions are attributed to both infectious and noninfectious causes. Clinical extrapolations are often made from the experimental model for ascending placentitis towards other causes of fetal compromise, including various markers of inflammation, including the cytokines IL-2, 5, IL-6, IL-10, IFNγ, and TNF. It is unknown if these cytokine changes are noted under field conditions, or if they increase preceding other pregnancy related complications. To assess this, Thoroughbred mares (n = 702) had weekly blood obtained beginning in December 2013 and continuing until parturition. Fetal membranes were submitted to the UKVDL for complete gross and pathologic assessment and classified as either ascending placentitis (n = 6), focal mucoid placentitis (n = 6), idiopathic abortion (n = 6) or control (n = 20). Weekly serum samples were analyzed via immunoassay for concentrations of IL-2, IL-5, IL-6, IL-10, IFNγ, and TNF. For both focal mucoid placentitis and ascending placentitis, an increase (P.05) in the concentrations of IL-2, IL-5, IL-6, IL-10, IFNγ, and TNF was noted preceding parturition in comparison to controls. Cytokine profiles preceding idiopathic abortion did not differ from controls. In conclusion, serum cytokines may be considered potential biomarkers for the prediction of placental infection, while no changes in cytokine profiles were noted when noninfectious causes of abortion occurred. Additionally, this is the first study to report an increase in cytokines during the disease process of focal mucoid placentitis, the etiology of which includes Nocardioform placentitis.

Cases of nocardioform placentitis are characterized by focal, mucoid placentitis resulting in late-term abortion, premature birth, or small, full-term foals, occur sporadically, and are most commonly associated with Crossiella equi and Amycolatopsis spp. infection. The goal of this project was to develop an enzyme-linked immunosorbent assay (ELISA) for quantifying antibodies against Crossiella equi and Amycolatopsis spp. and utilize the ELISA to determine when exposure occurs. Serum samples collected during the 2020 foaling season from Crossiella equi (n = 8) and Amycolatopsis spp. (n = 32) infected mares, as well as nonaffected mares (n = 51 mares), were used to develop and optimize bacteria-specific ELISAs. Following development of the ELISAs, banked serum samples from a single, central Kentucky Thoroughbred farm collected during 2012 to 2013 (n = 104 mares) and 2013-14 (n = 82 mares) were analyzed. Differences in various groups were analyzed using one-way analysis of variance (ANOVA). Crossiella equi-infected mares had significantly higher ELISA unit (EU) values on the Crossiella equi ELISA near parturition when compared to the other two groups (P.001). Using the Amycolatopsis spp. ELISA, EU values were not significantly different between Amycolatopsis spp. infected and non-affected mares, suggesting this ELISA is not specific for Amycolatopsis spp. During 2013 to 2014, there were significant increases in EU values between June and late September for the Crossiella equi ELISA, suggesting exposure in the summer and early fall months. Data from the Crossiella equi ELISA may help provide a better understanding of the epidemiology of nocardioform placentitis, guide the development of a successful experimental challenge model, and allow for further refinement of these ELISAs.

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.

Ascending placentitis is a leading cause of abortion in the horse, but adaptive immune response to this disease is unknown. To evaluate this, sub-acute placentitis was experimentally-induced via trans-cervical inoculation of S. zooepidemicus, and endometrium and chorioallantois was collected 8 days later (n = 6 inoculated/n = 6 control). The expression of transcripts relating to Th1, Th2, Th17, and Treg maturation was assessed via RNASeq. IHC of transcription factors relating to each subtype in the same tissues (Th1: TBX21, Th2: GATA3, Th17: IRF4, Treg: FOXp3). An immunoassay was utilized to assess circulating cytokines (Th1: IFNg, IL-2; Th2: IL-4, IL-5; Th17: IL-17, IL-6; Treg: IL-10, GM-CSF). An increase in Th1 and Th17-related transcripts were noted in the chorioallantois, although no alterations were seen in the endometrium. Th2 and Treg-related transcripts altered in a dysregulated manner, as some transcripts increased in expression while others decreased. Immunolocalization of Th1, Th2, and Th17 cells was increased in diseased chorioallantois, while no Treg cells were noted in the diseased tissue. Secreted cytokines relating to Th1 (IFNg, IL-2), Th17 (IL-6), Th2 (IL-5), and Treg (IL-10) populations increased in maternal circulation eight days after inoculation. In conclusion, the Th1/Th17 response to ascending placentitis occurs primarily in the chorioallantois, indicating the adaptive immune response to occur in fetal derived placental tissue. Additionally, ascending placentitis leads to an increase in the helper T cell populations (Th1/Th17/Th2) while decreasing the Treg response. This increase in Th17-related responses alongside a diminishing Treg-related response may precede or contribute to fetal demise, abortion, or preterm labor.

Placenta-specific 8 (PLAC8) is one of the placenta-regulatory genes which is highly conserved among eutherian mammals. However, little is known about its expression in equine placenta (chorioallantois; CA and endometrium; EN) during normal and abnormal pregnancy. Therefore, the current study was designed to 1) elucidate the expression of PLAC8 in equine embryonic membranes during the preimplantation period, 2) characterize the expression profile of PLAC8 in equine CA (45d, 4mo, 6mo, 10 mo, 11 mo and postpartum) and EN (14d, 4mo, 6mo, 10 mo, and 11 mo) obtained from pregnant mares (n = 4/timepoint), as well as, d14 non-pregnant EN (n = 4), and 3) investigate the expression profile of PLAC8 in ascending placentitis (n = 5) and in nocardioform placentitis (n = 6) in comparison to normal CA. In the preimplantation period, PLAC8 mRNA was not abundant in the trophectoderm of d8 equine embryo and d14 conceptus, while it was abundant later in d 30, 31, 34, and 45 chorion. In normal pregnancy, PLAC8 mRNA expression in CA at 45 d gradually decline to reach nadir at 6mo before gradually increasing to its peak at 11mo and postpartum CA. The mRNA expression of PLAC8 was significantly upregulated in CA from mares with ascending and nocardioform placentitis compared to control mares. Immunohistochemistry revealed that PLAC8 is localized in equine chorionic epithelium and immune cells. Our results revealed that PLAC8 expression in equine chorion is dynamic during pregnancy and is regulated in an implantation-dependent manner. Moreover, PLAC8 is implicated in the immune response in CA during equine ascending placentitis and nocardioform placentitis.

Steroid production varies widely among species, with these differences becoming more pronounced during pregnancy. As a result, each species has its own distinct pattern of steroids, steroidogenic enzymes, receptors, and transporters to support its individual physiological requirements. Although the circulating steroid profile is well characterized during equine pregnancy, there is much yet to be explored regarding the factors that support steroidogenesis and steroid signaling. To obtain a holistic view of steroid-related transcripts, we sequenced chorioallantois (45 days, 4 months, 6 months, 10 months, 11 months, and post-partum) and endometrium (4 months, 6 months, 10 months, 11 months, and diestrus) throughout gestation, then looked in-depth at transcripts related to steroid synthesis, conjugation, transportation, and signaling. Key findings include: 1) differential expression of HSD17B isoforms among tissues (HSD17B1 high in the chorioallantois, while HSD17B2 is the dominant form in the endometrium) 2) a novel isoform with homology to SULT1A1 is the predominant sulfotransferase transcript in the chorioallantois; and 3) nuclear estrogen (ESR1, ESR2) and progesterone (PGR) expression is minimal to nonexistant in the chorioallantois and pregnant endometrium. Additionally, several hypotheses have been formed, including the possibility that the 45-day chorioallantois is able to synthesize steroids de novo from acetate and that horses utilize glucuronidation to clear estrogens from the endometrium during estrous, but not during pregnancy. In summary, these findings represent an in-depth look at equine steroid-related transcripts through gestation, providing novel hypotheses and future directions for equine endocrine research.

Reproductive steroids testosterone (T) and estrone sulfate (E1S) are used as diagnostic markers for cryptorchidism in horses. The human chorionic gonadotropin (hCG) stimulation test is used as a diagnostic aid because administration of this hormone results in greater incremental differences in circulating steroid concentrations. Thoughts regarding optimal sampling times following hCG administration, however, are inconsistent. Additionally, determination of half-life of these steroids is important in postsurgical samples to confirm complete removal of testicular tissue. Objectives of this study, therefore, were to determine optimal sampling periods for peak T and E1S after hCG administration and half-life of these steroids after castration. Eight pony stallions were randomly assigned to control or treatment groups (5000 IU hCG). Blood samples were collected following hCG administration. Subsequently, stallions were castrated and blood samples were collected post-castration. The T concentrations were greatest at 72 h after hCG and were greater (P0.02) in samples from hCG-treated than control animals: 9,903.4 ± 384 and 784.0 ± 192 pg/mL, respectively (Mean ± SEM). The T concentrations were also greater at 1, 12, 24, 48 and 96 h. The E1S concentrations did not change after administration of hCG. The T response to hCG administration was biphasic with a maximal response between 48-96 h after administration. Half-lives of T and E1S were 1.1 and 0.7 h, respectively, and concentration of T and E1S was similar to that of geldings at 24 h post-castration, which, therefore, should be considered an optimal time to ensure complete castration has occurred.

High blood urea nitrogen (BUN) in cows and ewes has a negative effect on embryo development; however, no comparable studies have been published in mares. The aims of the present study were to evaluate the effects of high BUN on blastocoele fluid, systemic progesterone and Day 14 equine embryos. When a follicle with a mean (±s.e.m.) diameter of 25±3mm was detected, mares were administered urea (0.4g kg−1) with sweet feed and molasses (n=9) or sweet feed and molasses alone (control; n=10). Blood samples were collected every other day. Mares were subjected to AI and the day ovulation was detected was designated as Day 0. Embryos were collected on Day 14 (urea-treated, n=5 embryos; control, n=7 embryos). There was an increase in systemic BUN in the urea-treated group compared with control (P

Historically, studies on the endocrinology of pregnancy and parturition in horses have made major contributions of relevance to mammals in general. Recent use of liquid chromatography mass spectrometry, measuring multiple steroid hormones simultaneously in blood, foetal and placental tissues throughout normal gestation, and in mares with experimentally induced placentitis, has advanced our current understanding of many of the unusual strategies seen during gestation and at foaling. This includes the stimulation of luteal steroidogeneisis by equine chorionic gonadotropin (eCG) from the endometrial cups, resulting in additional androgen and oestrogen secretion. Progesterone declines as the endometrial cups and eCG disappears, replaced by 5α-dihydroprogesterone (DHP), a potent equine progesterone receptor (PR) agonist, as the chorioallantoic placenta develops. Placental steroidogenesis thereafter is influenced by foetal pregnenolone and dehydroepiandrosterone secretion, providing substrate for 5α-pregnane and oestrogen synthesis, an unusual example of a ‘foeto-placental unit’. Foetal gonadal dehydroepiandrosterone fuels placental oestrone sulphate secretion, peaking at higher concentrations in mares than any other species known, declining steadily thereafter to term. Additional 5α-reduced (DHP) metabolites increase from mid-gestation to peak concentrations 3–5 days before foaling, declining prepartum, most likely as a result of selective loss of placental SRD5A1 (5α-reductase) expression and activity. Similar changes occur in mares with experimentally induced placentitis, which is also associated with a decreased ratio of equine PR-B:PR-A in myometrium, suggesting that progestin withdrawal is both systemic (pregnanes) and local (receptor-dependent) in mares. In addition, some steroids detected during equine pregnancy by immuno-assay are not detected by mass spectrometry, further illustrating the immense value of this technology.

The key event in placentitis-induced preterm labor is myometrial activation with the subsequent initiation of labor. However, the molecular mechanisms underlying myometrial activation are not fully understood in the mares. Therefore, the equine myometrial transcriptome was characterized during placentitis (290.0 ± 1.52 days of GA, n = 5) and the prepartum period (330 days of GA, n = 3) in comparison to normal pregnant mares (289.8 ± 2.18 days of GA, n = 4). Transcriptome analysis identified 596 and 290 DEGs in the myometrium during placentitis and the prepartum period, respectively, with 138 DEGs in common. The placentitis DEGs included eight genes (MMP1, MMP8, S100A9, S100A8, PI3, APOBEC3Z1B, RETN, and CXCL2) that are exclusively expressed in the inflamed myometrium. Pathway analysis elucidated that inflammatory signaling, Toll-like receptor signaling, and apoptosis pathways dominate myometrial activation during placentitis. The prepartum myometrium was associated with overexpression of inflammatory signaling, oxidative stress, and 5-hydroxytryptamine degradation. Gene ontology enrichment analysis identified several chemoattractant factors in the myometrium during placentitis and prepartum period, including CCL2, CXCL1, CXCL3, and CXCL6 in common. Upstream regulator analysis revealed 19 potential upstream regulators in placentitis dataset including transcription regulators (E2F1, FOXM1, HIF1A, JUNB, NFKB1A, and STAT1), transmembrane receptors (FAS, ICAM1, SELP, TLR2, and TYROBP), growth factors (HGF and TGFB3), enzymes (PTGS2 and PRKCP), and others (S100A8, S100A9, CD44, and C5AR1). Additionally, three upstream regulators (STAT3, EGR1, and F2R) were identified in the prepartum dataset. These findings revealed the key regulators and pathways underlying myometrial activation during placentitis, which aid in understanding the disease and facilitate the development of efficacious therapies.

Recently, anti-Mullerian hormone (AMH) has been reported as a useful diagnostic marker for equine granulosa cell tumors (GCTs). The aim of this study was to examine and compare serum AMH and immunoreactive (ir)-inhibin concentrations in mares with ovarian abnormalities other than GCT compared to mares with known GCT in order to determine more useful diagnostic criteria. Mares with a possible GCT based upon history were subsequently classified into those with GCT confirmed by histopathology and mares with other abnormal ovaries (AO) without a GCT based on follow-up fertility information. Median concentrations of AMH were 72.6 ng/mL (quartiles: 20.6–458.5 ng/mL) in GCT and 0.70 ng/mL (0.26–1.44 ng/mL) in AO mares, and median concentrations of ir-inhibin were 2.36 ng/mL (1.65–3.79 ng/mL) in GCT versus 0.93 ng/mL (0.69–1.46 ng/mL) in AO mares. The cutoff values based on receiver operating characteristic (ROC) analysis were 4.70 ng/mL for AMH and 1.65 ng/mL for ir-inhibin. These cutoff values were slightly higher than the traditional value based on the normal reference range. The sensitivity, specificity, accuracy, and area under the ROC curve of AMH were higher than those of ir-inhibin. In conclusion, we demonstrated the diagnostic criterion of serum AMH concentration for equine GCT. Anti-Mullerian hormone is a more useful marker for diagnosis of equine GCT than ir-inhibin.

Steroidogenic enzymes in placentas shape steroid hormone profiles in the maternal circulation of each mammalian species. These include 3β-hydroxysteroid dehydrogenase/Δ5-4 isomerase (3βHSD) and 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17) crucial for progesterone and androgen synthesis, respectively, as well as aromatase cytochrome P450 (P450arom) that converts Δ4-androgens to estrogens. 5α-reductase is another important enzyme in equine placentas because 5α-dihydroprogesterone (DHP) sustains pregnancy in the absence of progesterone in the second half of equine pregnancy. DHP and its metabolites decline dramatically days before foaling, but few studies have investigated placental enzyme activity before or at parturition in mares. Thus, key enzyme activities and transcript abundance were investigated in equine placentas at 300 days of gestation (GD300) and post-partum (term). Equine testis was used as a positive control for P450c17 activity. Substrates were incubated with microsomal preparations, together with enzyme inhibitors, and products were measured by liquid chromatography tandem mass spectrometry or radiometric methods (aromatase). Equine placenta expressed high levels of 3βHSD, 5α-reductase and aromatase, and minimal P450c17 activity at GD300 compared with testis (600-fold higher). At foaling, 3βHSD and aromatase activities and transcript abundance were unchanged but 5α-reductase (and P450c17) was no longer detectable (P

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 (r

Fertility control of feral equids is difficult. A 4-month pilot study was conducted with a hormone-free intrauterine device (iUPOD). There was evaluation of i) device retention; ii) contraceptive efficacy; iii) fertility following device removal; iv) effects of device on estrous cycle periodicity and; v) abundance of biofilm on devices after removal from the uterus. The iUPODs were inserted trans-cervically in eight mares at random stages of the estrous cycle. Mares were confined in a paddock with a stallion the following day and remained with the stallion for 120 days. Transabdominal detection of the iUPOD, using a non-invasive handheld magnetic detector wand, was performed weekly. Mares were examined using transrectal ultrasonography on days 0 (Time at insertion = day 0), 14, and 30, and subsequently every third week to assess number and size of follicles, corpora lutea, and whether there was intrauterine fluid (IUF) present. The mares and stallion were observed daily for mating behavior. Weekly samples were assayed for progesterone (P4) at day 0 and until 3 weeks subsequent to stallion removal. None of the mares became pregnant while fitted with the iUPOD. Two of four mares conceived within 30 days subsequent to iUPOD removal. Three of eight mares fitted with the device had periods greater than 14 days with P4 concentrations 1 ng/mL. There was a marked abundance of biofilm on devices of two mares at the time of device removal.

Multiparametric assessment of stallion sperm quality using flow cytometry can be a useful adjunct in semen evaluation; however, the availability of flow cytometers in veterinary practice is limited. The ability to preserve and transport sperm samples for later flow cytometric analysis using fixable probes would potentially facilitate this process. In the current study, we validated the combination of live/dead Zombie Green® (a fixable dye used to assess live and dead sperm) and MitoTracker Deep Red® (used to assess mitochondrial membrane potential). The assay was validated against classic, non-fixable, membrane assays (SYBR-14/PI). Our results demonstrated the feasibility of the assay. In conclusion, stained and fixed semen samples stored for 72 h obtained equivalent results to the exam on the same day; this new protocol shall facilitate the wider use of flow cytometry in stallion andrology in the future. © 2017 International Clinical Cytometry Society.

Equine pregnancy is characterized by very high circulating concentrations of estrogens. The physiological roles of estrogens during equine gestation are largely unknown, although some studies suggest a role in the regulation of uterine artery hemodynamics and a relationship between low circulating estrogen concentrations and late pregnancy loss. The objectives of this experiment were to evaluate the effects of estrogen suppression on uterine artery hemodynamics and on pregnancy outcome. Estrogen synthesis was suppressed using letrozole, a potent aromatase inhibitor. Twelve pregnant mares were randomly assigned to a control (n = 6) or treatment (n = 6; 500 mg letrozole orally every 4 days) group with treatment starting at 240 days of gestation and continuing until parturition. Weekly serum samples were analyzed to determine testosterone, dehydroepiandrosterone sulfate, estradiol, estrone sulfate, progestins, and prostaglandin F2α metabolite concentrations. Ultrasonographic examinations were performed biweekly and measurements included uterine artery hemodynamics (diameter, pulsatility, and resistance indices), fetal growth using the diameter of the fetal eye, and placental evaluation using the combined thickness of the uterus and placenta. At parturition, gestational length, foal weight, and neonatal viability were determined. Letrozole suppressed estrogen synthesis during gestation by approximately 90% compared to control values. This large reduction in circulating estrogens had no effect on uterine artery hemodynamics, normal placental development, maintenance of pregnancy, or neonatal viability. However, neonates from letrozole-treated mares had lower (P < 0.05) birth weights than controls, suggesting that estrogens may play a role in fetal growth that is not mediated through regulation of uterine blood flow.

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-1β, 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-1β and IL-6 following inoculation, while chorioallantois and endometrium upregulated IL-1β and IL-8 expression. IL-1β was upregulated in the maternal spleen following inoculation. Fetal spleens were upregulated in expression of IL-1β, 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.

Author(s): Fedorka, Carleigh Elizabeth; Ball, Barry A; Walker, Olivia F; Conley, Alan J; Corbin, Cynthia J; Lu, Kristina G; Hanneman, Jessica M; Troedsson, Mats HT; Adams, Amanda A | Abstract: ProblemProgestins are immunomodulatory in a variety of species. In the horse, the most commonly administered synthetic progestin is altrenogest (ALT), but its effect on the immune system of the non-pregnant mare is unknown.MethodsPeripheral blood mononuclear cells (PBMCs) from diestrous mares were incubated with varying concentrations of progesterone (P4) or ALT to assess intracellular production of IFNγ and the expression of select cytokines. Additionally, ten mares received either ALT or VEH daily utilizing a switchback design beginning on the day of ovulation and continuing for 7ndays. Circulating PBMCs and endometrial biopsies were obtained to assess the production and expression of the same cytokines.ResultsIn vitro, both P4 and ALT caused a dose-dependent decrease in intracellular IFNγ in PBMCs. P4 caused a dose-dependent decrease in the expression of IFNγ, IL-10 and IL-4, while ALT caused an increase in the expression of IL-6 and IL-1β in PBMCs. In vivo, ALT suppressed the intracellular levels of IFNγ in PBMCs on d6. While control mares experienced a decrease in IL-1β expression from d0 to d6, ALT-treated mares did not. In the endometrium, ALT increased the expression of IL-1RN and IFNγ in comparison with VEH-treated mares.ConclusionP4 and ALT appear to alter the immune system of the non-pregnant mare both systemically in addition to locally within the endometrium. Further research is necessary to determine the pathways through which this synthetic progestin functions on the immune system of the horse, and the consequences it may have.

High blood urea nitrogen (BUN) decreases fertility of several mammals, however, the mechanisms have not been investigated in mares. We developed an experimental model to elevate BUN, with urea and control treatments (7 mares/treatment), in a crossover design. Urea-treatment consisted of a loading dose of urea (0.03 g/kg of body weight (BW)) and urea injections over 6 hours (0.03 g/kg of BW/h). Control mares received the same volume of saline solution. Blood samples were collected to measure BUN. Uterine and vaginal pH were evaluated after the last intravenous infusion, then endometrial biopsies were collected for RNA-sequencing with a HiSeq 4000. Cuffdiff (2.2.1) was used to identify the differentially expressed genes (DEG) between urea and control groups (false discovery rate-adjusted p-value &lt, 0.1). There was a significant increase in BUN and a decrease of uterine pH in the urea group compared to the control group. A total of 193 genes were DEG between the urea and control groups, with five genes identified as upstream regulators (ETV4, EGF, EHF, IRS2, and SGK1). The DEG were predicted to be related to cell pH, ion homeostasis, changes in epithelial tissue, and solute carriers. Changes in gene expression reveal alterations in endometrial function that could be associated with adverse effects on fertility of mares.

Increasing evidence suggests that overlapping genes are much more common in eukaryotic genomes than previously thought. These different-strand overlapping genes are potential sense&ndash, 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.

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 5 alpha DHP, allopregnanolone (3 alpha DHP), and 20 alpha DHP 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-1 beta 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.

Characterisation of fetal fluids in healthy and disease states of pregnant mares can help to unravel the pathophysiology and to identify putative markers of disease. Thus, this study aimed to compare the protein composition of: (1) amniotic and allantoic fluids of healthy mares obtained immediately after euthanasia and (2) allantoic fluid harvested via centesis before and after experimental induction of placentitis via transcervical inoculation of Streptococcus equi ssp zooepidemicus in healthy mares. Fetal fluids were analysed with a high-throughput proteomic technique after in-gel digestion. Statistical comparisons were performed following normalisation of peptide spectral match. Global normalisation was performed to calculate relative expression. There were 112 unique proteins present in both allantoic and amniotic fluids. There were 13 and 29 proteins defined as amniotic- or allantoic-specific respectively that were present in at least two fluid samples. Another 26 proteins were present in both amniotic and allantoic fluids. Panther DB functional classification grouped fetal-fluid proteins as transfer carriers, signalling molecules, receptors, immunity, hydrolase, enzymes, membrane traffic, cytoskeleton, cell adhesion, calcium binding and extracellular matrix. Experimentally induced placentitis resulted in 10 proteins being upregulated and 10 downregulated in allantoic fluid. Newly identified proteins and changes in the fetal-fluid proteome provide clues about the physiology of pregnancy and pathogenesis of placentitis.