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Cell-free DNA release following psychosocial and physical stress in women and men.

Author(s): Limberg AS, Berg F, Köper E, Lindgraf C, Gevers C, Kumsta R, Hummel EM, Moser DA

Publication: Transl Psychiatry, 2025, Vol. 15, Page 26

PubMed ID: 39863589 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of physical (exercise bike) and psychosocial (mock job interview and arithmetic test) stress on levels of cortisol, α-amylase, nuclear cfDNA and mtcfDNA in saliva and nuclear cfDNA and levels of mtcfDNA in plasma by comparing levels 2 min prior to each test with levels 2 min, 15 min and 45 min after the stressor. Responses were also compared between plasma from men, women not taking hormonal contraceptive and woman taking oral contraceptives. Capillary blood and saliva were collected from 15 healthy men, 15 healthy women not using hormonal contraceptives and 16 healthy women taking oral contraceptive (0.02–0.035 mg ethinylestradiol) 2 min before, and 2, 15 and 45 min after acute psychological stress or physical stress. Tests were conducted on women during the luteal phase. The acute psychological test consisted of a mock job interview (~15 min) and an arithmetic task. The physical test consisted of 4 minutes of a warm-up period on an exercise bicycle at 100 watts, then the resistance increased by 25 watts every 2 min until the participant could not maintain 65 revolutions per min or reached the maximum heart rate (220-age beats per min). The average duration of the physical test was 12 min and 53 seconds. At each time-point, saliva was collected prior to blood using a Salivette, centrifuged at 2500 g for 15 min at 4°C and stored at -20°C. Blood was collected from the fingertip using Microvette APT 250 K2E tubes, transferred into 0.5 mL tubes, and plasma was obtained by dual-centrifugation at 2500 g for 15 min at 4°C and stored at -80°C. Salivary cortisol was assayed using ELISA assays, and α-amylase was assayed using a colorimetric assay. cfDNA was quantified by real-time PCR amplification of long interspersed nuclear elements (LINE1) and mitochondrial DNA.

   Summary of Findings:

   Salivary cortisol and α-amylase levels were significantly affected by the timing of collection relative to the stress event (P<0.001, both), and for both there was a significant interaction between the timing relative to the stressor and the type of stress (P<0.01 and P<0.001, respectively). Salivary cortisol levels were 1.6- and 1.9-fold higher than baseline 2 min after psychosocial and physical stress, respectively, and both were higher than baseline levels 15 min after the stress.  In contrast, salivary α-amylase levels were 1.2-fold higher than baseline 2 min after physical stress but decreased by 20% in the 2 min following psychosocial stress and were lower than baseline 15 min after both physical and psychosocial stress.

   Plasma levels of nuclear and mitochondrial cfDNA were affected by the timing of collection relative to the stress event (P<0.001 and P= 0.006, respectively); nuclear cfDNA levels were also significantly affected by the type of stress (P=0.02). Further, analysis of plasma levels of nuclear and mitochondrial cfDNA showed an interaction between the timing of collection relative to stress and the type of stress (P<0.001 and P<0.004, respectively). There were differences in the levels of nuclear cfDNA in plasma between genders/oral contraceptive use for both stressors (P=0.04, both); but in all cases, levels were affected by the time of specimen collection relative to when the stress occurred.  Following physical stress, plasma levels of nuclear cfDNA initially rose, peaking at 2.2-fold higher than baseline 2 min following the stress (P<0.0001) but then declined steadily, such that levels were slightly lower than baseline by 45 min (P<0.01). A much smaller (<10%), but still significant, increase in plasma nuclear cfDNA levels was observed 2 min following psychosocial stress (P<0.05). Interestingly, mtcfDNA levels in plasma were lower (~75% of baseline) 2 min after physical stress (P<0.05) and then increased toward baseline, but mtcfDNA levels in plasma were higher than baseline 2 min and 45 min (1.6 fold) following psychosocial stress (P<0.01).

   Salivary nuclear cfDNA levels were significantly affected by the timing of collection relative to when the stress occurred (P=0.01) and was 1.3-fold higher 45 min following psychosocial stress (P<0.01) but was stable in the 45 min following physical stress. While mtcfDNA levels were 1.5-fold higher than baseline 45 min following psychosocial stress, no significant effect of the timing of collection relative to when the stress occurred were observed on mtcfDNA levels. No correlations between salivary cortisol or α-amylase levels and levels of nuclear cfDNA or mtcfDNA in plasma or saliva were observed.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood
   • Bodily Fluid - Saliva

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Steroid	ELISA
   DNA	Real-time qPCR
   Protein	ELISA

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Biospecimen location	Saliva Plasma
   Biospecimen Acquisition	Time of biospecimen collection	2 min prior to stress 2 min following physical stress 15 min following physical stress 45 min following physical stress 2 min following psychosocial stress 15 min following psychosocial stress 45 min following psychosocial stress
   Preaquisition	Patient gender	Female Male
   Preaquisition	Other drugs	Taking oral contraceptives (0.02–0.035 mg ethinylestradiol) Not taking hormonal contraceptives

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