Total and endothelial cell-derived cell-free DNA in blood plasma does not change during menstruation.
Author(s): Yuwono NL, Henry CE, Ford CE, Warton K
Publication: PLoS One, 2021, Vol. 16, Page e0250561
PubMed ID: 33901234 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to investigate if menstruation impacts total cell-free DNA (cfDNA) concentration, cfDNA integrity, or endothelial cell-derived cfDNA concentration in plasma from healthy women.
Conclusion of Paper
Case-matched blood specimens obtained on menstruating and non-menstruating days had comparable levels of the 115 and 247 bp fragment of ALU, integrity (ratio of the 115 to 247 bp fragment), and concentration of endothelial cell-derived cfDNA. Although average cfDNA levels quantified by the 115 bp ALU fragment were not correlated in peripheral blood collected on menstruating and non-menstruating days, weak-modest correlations were observed in the 247 bp amplified fragment, the ratio of the 115 to 247 bp fragment, and the concentration of endothelial cell-derived cfDNA relative to total cfDNA.
Studies
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Study Purpose
The purpose of this study was to investigate if menstruation impacts total cfDNA concentration, cfDNA integrity, or endothelial cell-derived cfDNA concentration in plasma from healthy women. K2EDTA blood was collected from 40 women one or two days after the start of menstruation and on a non-menstruating day (follicular or luteal phase). The majority (80%) of specimens were collected over the course of a single cycle and all specimens were collected within 3 cycles. Within 3 h of blood collection, plasma was separated by centrifugation at 2,500 x g for 10 min at 4°C followed by 3,500 x g for 10 min at 4°C. Plasma was stored at -80°C until cfDNA extraction using the QIAamp Circulating Nucleic Acid Kit. cfDNA was quantified by real-time PCR amplification of 115 and 247 bp fragments of the ALU repeat. cfDNA was bisulfite converted using the Epitect Bisulfite Kit and methylation of the CDH5 region was evaluated in a reaction designed to only amplify unmethylated CDH5. Endothelial cell-derived cfDNA was quantified using the unmethylated CDH5 specific real-time PCR assay.
Summary of Findings:
Average cfDNA levels were comparable in case-matched blood specimens obtained on menstruating and non-menstruating days when measured by amplification of the 115 bp (1.68 versus 1.79 ng/mL plasma) or 247 bp (0.64 versus 0.66 ng/mL plasma) fragment of ALU. cfDNA levels differed by up to 7.7-fold between blood draws. Average cfDNA levels quantified by the 115 bp product in peripheral blood collected on menstruating and non-menstruating days were not correlated (r=0.262, P=0.0516) and only a weak correlation occurred when quantified by amplification of the 247 bp fragment (r=0.333, P=0.0179). Importantly, cfDNA integrity (as assessed by the ratio of 115 to 247 bp fragments of ALU) was also comparable in specimens obtained on menstruating and non-menstruating days (2.69 versus 2.84) and these ratios were modestly correlated (r=0.548, P=0.0001). The concentration of endothelial cell-derived cfDNA was also comparable between specimens obtained on menstruating and non-menstruating days (1.07 versus 1.01 ng/mL plasma). Finally, the concentration of endothelial cell-derived cfDNA relative to total cfDNA (quantified by 115 bp fragment) was comparable between specimens obtained on menstruating and non-menstruating days and the values were modestly correlated (r = 0.4008; P = 0.0052).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Bisulfite conversion assay DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Diagnosis/ patient condition Menstruating
Non-menstruating
Biospecimen Acquisition Time of biospecimen collection During menstruation
During follicular or luteal phase
Real-time qPCR Specific Length of gene fragment 115 bp
247 bp
Real-time qPCR Specific Targeted nucleic acid ALU
Unmethylated CDH4