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Comparison of rat fetal sex determination using placental gDNA and mRNA via qRT-PCR.

ABSTRACT: A growing need exists to consider fetal sex as a biological variable and accurately assess sex-specific effects. Among the multiple methods used to determine fetal sex, quantitative real-time polymerase chain reaction (qRT-PCR) of Sry (sex-determining region Y) with genomic DNA (gDNA) is commonly used in addition to use of methodologies such as transcriptomics and detection of Barr body. However, Sry messenger RNA (mRNA), a product of Sry gDNA, has not been previously assessed for sex determination. Using placental samples from timed-pregnant Wistar rats at gestational day (GD) 16, this study assessed the compatibility of Sry detection using gDNA versus mRNA to determine fetal sex. Samples used in this current study come from a larger study that investigated trichloroethylene (TCE) reproductive toxicity and potential modulation by N-acetyl-L-cysteine (NAC) and aminooxyacetic acid (AOAA). In 90 out of 91 samples, the sex classification determined by gDNA matched the sex classification determined by mRNA analyzing Sry (Sry/B2m) values. For both gDNA and mRNA, statistically significant differences in Sry/B2m values between males and females were observed with samples considered in totality and when samples were separated by treatment groups (all comparisons were p<0.01 or below, and all but two comparisons were p<0.001 or below). Finally, the validity of using Sry Cq values to determine fetal sex and the B2m reference gene were also discussed. Together, this study suggests that determination of fetal sex in Wistar rats can be accomplished using Sry measurements in gDNA or mRNA with highly compatible results.

SUBMITTER: Su AL

PROVIDER: S-EPMC7665159 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Comparison of rat fetal sex determination using placental gDNA and mRNA via qRT-PCR.

Su Anthony L AL Loch-Caruso Rita R

Journal of molecular biology and methods 20200705 2

A growing need exists to consider fetal sex as a biological variable and accurately assess sex-specific effects. Among the multiple methods used to determine fetal sex, quantitative real-time polymerase chain reaction (qRT-PCR) of Sry (sex-determining region Y) with genomic DNA (gDNA) is commonly used in addition to use of methodologies such as transcriptomics and detection of Barr body. However, Sry messenger RNA (mRNA), a product of Sry gDNA, has not been previously assess ...[more]

PMID: 33196062

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Project description:Parturition signals the end of immune tolerance in pregnancy. Term labour is usually a sterile inflammatory process triggered by damage associated molecular patterns (DAMPs) as a consequence of functional progesterone withdrawal. Activation of DAMPs recruits leukocytes and inflammatory cytokine responses in the myometrium, decidua, cervix and fetal membranes. Emerging evidence shows components of the inflammasome are detectable in both maternal decidua and placenta. However, the activation of the placental inflammasome with respect to mode of delivery has not been profiled. Placental chorionic villus samples from women delivering at term via unassisted vaginal (UV) birth, labouring lower segment caesarean section (LLSCS, emergency caesarean section) and prelabour lower segment caesarean section (PLSCS, elective caesarean section) underwent high throughput RNA sequencing (NextSeq Illumina) and bioinformatic analyses to identify differentially expressed inflammatory (DE) genes. DE genes (IL1RL1, STAT1, STAT2, IL2RB, IL17RE, IL18BP, TNFAIP2, TNFSF10 and TNFRSF8), as well as common inflammasome genes (IL1B, IL1R1, IL1R2, IL6, IL18, IL18R1, IL18R1, IL10, and IL33), were targets for further qPCR analyses and Western blotting to quantify protein expression. There was no specific sensor molecule-activated inflammasome which dominated expression when stratified by mode of delivery, implying that multiple inflammasomes may function synergistically during parturition. Whilst placentae from women who had UV births overall expressed pro-inflammatory mediators, placentae from LLSCS births demonstrated a much greater pro-inflammatory response, with additional interplay of pro- and anti-inflammatory mediators. As expected, inflammasome activation was very low in placentae from women who had PLSCS births. Sex-specific differences were also detected. Placentae from male-bearing pregnancies displayed higher inflammasome activation in LLSCS compared with PLSCS, and placentae from female-bearing pregnancies displayed higher inflammasome activation in LLSCS compared with UV. In conclusion, placental inflammasome activation differs with respect to mode of delivery and neonatal sex. Its assessment may identify babies who have been exposed to aberrant inflammation at birth that may compromise their development and long-term health and wellbeing.

Dataset Information

Comparison of rat fetal sex determination using placental gDNA and mRNA via qRT-PCR.

Publications

Comparison of rat fetal sex determination using placental gDNA and mRNA via qRT-PCR.

Similar Datasets

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