MicroRNAs in amniotic fluid and maternal blood plasma associated with sex determination and early gonad differentiation in cattle

MicroRNAs (miRNAs), as gene expression regulators, may play a critical role during the sex determination process. We hypothesised that the expression of miRNAs in amniotic fluid (AF) and maternal blood plasma (MP) during this process would be affected by the sex of the embryo. Amniotic fluid and MP were collected from six pregnant heifers (3 carrying a single male and 3 a single female embryo) following slaughter on Day 39 post insemination, coinciding with the peak of SRY expression. Samples (6 AF and 6 MP) were profiled using a miRNA Serum/Plasma Focus PCR Panel. Differentially expressed (DE) miRNAs were identified in AF (n = 5) and associated MP (n = 56) of male vs female embryos (P < 0.05). Functional analysis showed that inflammatory and immune response were amongst the 13 biological processes enriched by miRNAs DE in MP in the male group (FDR < 0.05), suggesting that these sex-dependent DE miRNAs may be implicated in modulating the receptivity of the dam to a male embryo. Further, we compared the downstream targets of the sex-dependent DE miRNAs detected in MP with genes previously identified as DE in male vs female genital ridges. The analyses revealed potential targets that might be important during this developmental stage such as SHROOM2, DDX3Y, SOX9, SRY, PPP1CB, JARID2, USP9X, KDM6A, and EIF2S3. Results from this study highlight novel aspects of sex determination and embryo-maternal communication in cattle such as the potential role of miRNAs in gonad development as well as in the modulation of the receptivity of the dam to a male embryo.

Pregnancy in a Patient With Mosaic Turner Syndrome: A Case Report

Turner Syndrome (TS) is a chromosomal sex disorder, phenotypically characterized by short stature, webbed neck, cubitus valgus, and rarely with slight intellectual disability. A majorityof TS patients (95%-98%) have infertility due to ovarian failure. Pregnancy in TS patients is an unusual case; however, pregnancy could rarely occur in mosaicism TS patients withoutany assistance. Pregnancy in such patients is associated with high risks of maternal mortality, spontaneous abortion, as well as the congenital and karyotype abnormalities of the fetus. A30-year-old pregnant woman has referred to our genetics lab with a history of polyabortivity. Her menarche occurred at the age of 13 years and her menstruation was claimed to be in aregular cycle. The patient’s two first pregnancies resulted in stillbirth, whereas the third one was delivered through caesarian surgery, but spoiled after 8 days. Our case was characterizedby mosaicism 45, X/45, XX, after referring as a multi-abortion case. The fourth pregnancy has happened at the age of 31 years and a healthy embryo with normal heart function wasdiagnosed by sonography in 17 weeks of gestation. The result of amniocentesis confirmed a healthy female embryo with 46, XX karyotype. Spontaneous pregnancy is regarded as aprecarious situation terrifying by abortion or malignancy; also, chromosomal abnormalities, like trisomy 21 and TS, are prevalent in offspring. Therefore, it is strongly recommended tohave cohort studies based on karyotype characterization to decrease the patient’s concerns as well as to follow more practical clinical approaches.

55 Identification of microRNAs associated with sex determination in bovine amniotic fluid and maternal blood plasma

In most eutherian mammals, sex determination is the process through which a bipotential gonad (also known as genital ridges) develops into a testis or ovary depending on the sex chromosome content of the embryo, specifically by the presence of the SRY/Sry gene (sex-determining region of the Y chromosome). MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression and are involved in diverse functional roles including development, differentiation, apoptosis, and immunity. We hypothesised that the expression of miRNAs in amniotic fluid (AF) and maternal blood plasma (MP) would be affected by the sex of the embryo around the time of sex determination. Amniotic fluid and MP were collected from 6 crossbred beef pregnant heifers (3 carrying a single male and 3 carrying a single female embryo) following slaughter on Day 39 (when the peak of SRY expression occurs in cattle). All heifers had been synchronized and inseminated with semen from the same beef bull. A total of 12 samples (6 AF and 6 MP) were profiled using the miRCURY LNA miRNA Serum/Plasma Focus PCR Panel (Qiagen; 179 assays targeting relevant miRNAs). Data were analysed by GeneGlobe Data Analysis Center (Qiagen). A threshold cycle cut-off of 35 was applied and data were analysed using an unpaired t-test. Gene ontology enrichment analysis was performed using the WebGestaltR package to explore the possible functions of differentially expressed (DE) miRNAs. In this study, DE miRNAs were identified in male vs. female AF (n=5; 3 upregulated and 2 downregulated; P<0.05) and MP (n=57; 54 upregulated and 3 downregulated; P<0.05). Although no enrichment was detected for DE miRNAs in AF (in either sex) or in MP in heifers carrying a female embryo, 37 biological processes were enriched by DE miRNAs in MP of heifers carrying a male embryo (false discovery rate<0.05). Interestingly, the top five most enriched biological processes were male gonad development, development of primary male sexual characteristics, signal transduction in absence of ligand, actomyosin structure organisation, and male sex differentiation, suggesting a potential role of these miRNAs in reproductive traits. Results from this study highlight unique aspects of sex determination in cattle such as the role of miRNAs in gonad development. Moreover, although it is well known that AF provides a protective space around the developing embryo/fetus that allows its movement and growth; here we provide evidence suggesting that its components may play important roles in fetal development. Finally, miRNAs in MP may offer new opportunities to investigate biomarkers for early prediction of embryo/fetal sex in commercial practice. This research was supported by the Science Foundation Ireland (13/IA/1983) and the European Union H2020 Marie Sklodowska-Curie Innovative Training Network project Biology and Technology of Reproductive Health - REP-BIOTECH - 675526.

Drug Repurposing of Bromodomain Inhibitors as Potential Novel Therapeutic Leads for Lymphatic Filariasis Guided by Multispecies Transcriptomics

ABSTRACT To better understand the transcriptomic interplay of organisms associated with lymphatic filariasis, we conducted multispecies transcriptome sequencing (RNA-Seq) on the filarial nematode Brugia malayi, its Wolbachia endosymbiont wBm, and its laboratory vector Aedes aegypti across the entire B. malayi life cycle. In wBm, transcription of the noncoding 6S RNA suggests that it may be a regulator of bacterial cell growth, as its transcript levels correlate with bacterial replication rates. For A. aegypti, the transcriptional response reflects the stress that B. malayi infection exerts on the mosquito with indicators of increased energy demand. In B. malayi, expression modules associated with adult female samples consistently contained an overrepresentation of genes involved in chromatin remodeling, such as the bromodomain-containing proteins. All bromodomain-containing proteins encoded by B. malayi were observed to be upregulated in the adult female, embryo, and microfilaria life stages, including 2 members of the bromodomain and extraterminal (BET) protein family. The BET inhibitor JQ1(+), originally developed as a cancer therapeutic, caused lethality of adult worms in vitro, suggesting that it may be a potential therapeutic that can be repurposed for treating lymphatic filariasis. IMPORTANCE The current treatment regimen for lymphatic filariasis is mostly microfilaricidal. In an effort to identify new drug candidates for lymphatic filariasis, we conducted a three-way transcriptomics/systems biology study of one of the causative agents of lymphatic filariasis, Brugia malayi, its Wolbachia endosymbiont wBm, and its vector host Aedes aegypti at 16 distinct B. malayi life stages. B. malayi upregulates the expression of bromodomain-containing proteins in the adult female, embryo, and microfilaria stages. In vitro, we find that the existing cancer therapeutic JQ1(+), which is a bromodomain and extraterminal protein inhibitor, has adulticidal activity in B. malayi.