MicroRNAs as Markers of Placental Health in the Mare

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Placentitis is a major cause of late-term abortion in mares, with up to 33% of abortions, stillbirths and perinatal losses being due to bacterial infections of the placenta. Complicating diagnosis, many cases of placentitis are silent with mares aborting with no premonitory signs. It is currently recommended that mares of high value be routinely evaluated for placentitis monthly beginning at the 7th month of pregnancy. Unfortunately, these examinations are time consuming and expensive without clearly improving outcomes.There is an enormous un-met need for a more efficient method of diagnosing placentitis in mares. MicroRNAs (miRNAs) have the potential to meet this need. MiRNAs are newly discovered non-coding RNAs which play an important role in regulating gene expression. As more research is done, it is becoming increasingly apparent that these small molecules have major implications in the future of both diagnostics and therapeutics. We have performed extensive preliminary research on the presence of miRNAs during equine pregnancy, as well as during placentitis. We have performed next-generation sequencing on both chorioallantois and serum from normal pregnant and non-pregnant mares. In preparation for this study, we have experimentally induced placentitis in five mares using a trans-cervical infusion of 5 x 106 colony forming units of Streptococcus equi zooepidemicus when the mares were between 281 and 295 days of gestation. Mares were monitored daily, including trans-rectal ultrasound to monitor the combined thickness of the uterus and placenta (CTUP) and separation of the placenta from the uterus. Serum was collected daily beginning on D0, the day of inoculation. Mares were euthanized when several conditions were met; 1) A significant increase in CTUP was seen (3+ mm); 2) A minimum of 25 mm of placental separation was visualized; and 3) A minimum of three days post-inoculation. An additional three mares undergoing daily ultrasounds and serum collection, but not receiving a trans-cervical inoculation, were euthanized as gestationally-matched controls. It was necessary to euthanize these mares because allowing the mares to spontaneously abort would significantly alter the miRNA profile of the tissues. As part of this project, we will sequence the chorioallantois and serum from the eight mares described above using next-generation sequencing, with funding provided by an AQHA Young Investigator Grant. Prior to submission for sequencing, miRNAs will be extracted from placental tissue and serum using Trizol® (Life Technologies, Carlsbad, CA). To extract RNA, tissue will be immersed in Trizol, homogenized, and RNA will be extracted per manufacturer's instructions. Final RNA concentration will be determined via Nanodrop (ThermoScientific, Wilminton, DE), and then stored at -80°C until use. Data generated from next-generation sequencing will be analyzed using the CLC Genomics Workbench (CLCbio, Waltham, MA). Quantitative PCR will be used to confirm differences in chorionic and serum miRNAs which are significantly different between mares with and without induced placentitis. Additionally, differential expression of target miRNAs will be evaluated in matching serum and chorion samples to determine correlation between circulating and chorionic miRNAs, and to assess the potential of specific miRNAs as biomarkers. MicroRNAs found to change significantly in serum during placentitis will be confirmed by qPCR initially using the serum samples submitted for next-generation sequencing. Assuming positive results, qPCR will be used to analyze concentrations of the select miRNAs within daily serum samples taken from mares with induced placentitis to determine how early in the disease process changes in miRNA concentrations can be detected. To confirm specificity of differentially regulated serum miRNAs, serum will be collected from mares with a wide range of inflammatory conditions occurring spontaneously on the UK research farm. If necessary, a collaboration will be made with local veterinarians to obtain a wider range of samples. Serum will be collected from a minimum of 20 patients with at least 5 diverse causes of infection, as well as matched controls. Serum from these animals will be evaluated for expression of miRNAs targeted above to determine specificity of aforementioned miRNAs in placentitis. Any miRNAs which appears to be specific to placentitis above will undergo further analysis to determine its expression in various tissues, including heart, liver, spleen, skeletal and cardiac muscle as well as small intestine. Specificity of these miRNAs to the placenta will further support their potential use as a biomarker for placentitis. Based on our preliminary data, we anticipate that mares will have significant changes in levels of circulating miRNAs during placentitis, some of which may hold the potential to be useful biomarkers for placentitis. We have identified a number of miRNAs which are exclusively present during pregnancy, as well as a separate subset which were present during diestrus but not pregnancy. This exclusivity suggests that we may find a subset of miRNAs which are exclusively present or absent in the serum of mares with placentitis. Once we have identified which miRNAs are present in the serum of mares with placentitis, we will assess how early in the disease process we are able to identify these changes. This will give us an idea as to how quickly miRNA populations change in response to infection. We will be able to assess the specificity of select miRNAs to placentitis vs other inflammatory conditions, as well as the specificity of miRNAs to the placenta itself. Although it is not clear that any of the miRNAs affected by placentitis will be specific to placentitis or even the placenta, preliminary data suggest that at least a subset will be specific to placentitis. Changes in circulating, placentitis-specific miRNAs would have a greater potential to be effective, specific biomarkers than any method currently on the market. By determining which chorion-specific miRNAs change significantly during the course of spontaneous cases of placentitis, as well as how early these changes occur, we hope to identify a highly specific panel of miRNAs for diagnostic purposes. MicroRNAs hold the potential to be a powerful diagnostic tool as well as an important therapeutic target; however, research has been largely neglected in the horse. Further research is sorely needed to determine the roles of these RNAs, as well as how they can be utilized to improve overall health and well-being. This study is primarily looking to characterize the population of miRNAs in the chorioallantois and serum during placentitis. While we hope to identify a sensitive, specific panel of miRNAs for use as a biomarker for the diagnosis of placentitis, the usefulness of this study is not limited to that single endpoint. Ultimately, regardless of the specific results, this study will help further the knowledge of the biology of placentitis in mares.
Effective start/end date4/1/173/31/18


  • Grayson Jockey Club Research Foundation Inc: $15,000.00


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