Cell-free mtDNA level and its biomarker potency for ART outcome are different in follicular fluid of PCOS and non-PCOS women

Introduction Lack of reliable biomarkers for estimating the outcome is one of the current gaps in ART. In this study, we assessed whether cell-free mitochondrial DNA within the follicular fluid (FF cf-mtDNA) of PCOS patients has biomarker applicability or not. Furthermore, probable involved mechanisms in the FF cf-mtDNA pathway were evaluated. Methods The level of FF cf-mtDNA was compared between 50 PCOS patients and 50 women without any certain reproductive disorder, and analyzed for correlations with ART outcome. The associations between levels of FF cf-mtDNA and TFAM, POLG, and RNase H1 genes expression in mural granulosa cells (MGCs), as well as IL-6, and TNFα in follicular fluid (FF) were assessed. Results We identified that FF cf-mtDNA level was significantly lower in PCOS women and was accompanied by a reduction in the expression of mtDNA biogenesis genes in MGCs of the patients. Although a significant association between FF cf-mtDNA level and ART outcome was observed in the control group, no correlation could be proved in the PCOS group. Moreover, the expression level of TFAM was negatively associated, while amounts of IL-6 and TNFα were positively correlated with FF cf-mtDNA level in both groups. Conclusion PCOS patients present a lower FF cf-mtDNA level in comparison with non-PCOS women. FF cf-mtDNA has biomarker applicability for ART outcome in women without any certain reproductive disorder, but not for those with PCOS. It seems that mtDNA packaging dysfunction results in elevated FF cf-mtDNA, and subsequent effects are triggered by increasing the inflammatory cytokines.</p

Analysis of the four bicistronic vectors for mAb and mRNA expression in established stable cell lines.

<p>Stably transfected pools were generated by transfection of CHO-S cells with various bicistronic vectors containing either IRES or F2A sequences and different promoters (CHEF or CMV). Levels of mAb and mRNA expression were measured by ELISA and qRT-PCR respectively. Black bars represent the mAb titer and gray bars represent mRNA fold induction. The error bars represent the standard deviation of three independent measurements.</p

Analysis of integrated transgene copy number in stable cell pools transfected by four bicistronic vectors.

<p>Antibody copy number based on light chain copy number was calculated by qRT-PCR. The error bars represent the standard deviation of three independent measurements.</p

Western blot analyses of purified mAb in triplicate stable cell pools transfected with vectors bearing F2A-mediated expression system.

<p>Light chains with various molecular weights (25, 28 & 30 kDa) were detected. Triplicates of each stable pool were shown with 1, 2 and 3. (A) Lane 1; CHEF-F2A (2), lane 2; CHEF-F2A (3), lane 3; protein molecular ladder. (B) Lane 1; protein molecular ladder, lane 2; CMV-F2A (1), lane 3; CMV-F2A (3).</p

Analysis of the stability of antibody expression over time by stable cell pools transfected with two vectors containing F2A sequences.

<p>Both stable cells were cultivated for 18 weeks upon removal of puromycin as a selection marker. Every 2 weeks, antibody expression was monitored and measured by ELISA. The other pools exhibited the same expression pattern; the data from one of them was represented.</p

Schematic illustration showing insertion of furin/2A sequences between light and heavy chain by overlap extension PCR.

<p>Schematic illustration showing insertion of furin/2A sequences between light and heavy chain by overlap extension PCR.</p

Oligonucleotides used for analysis of mRNA levels and relative gene copy numbers by quantitative real-time PCR.

<p>Oligonucleotides used for analysis of mRNA levels and relative gene copy numbers by quantitative real-time PCR.</p

SDS-PAGE and Western blot analysis of purified mAb in stable cell pools transfected with four bicistronic vectors.

<p>Supernatants of three different cell pools were purified using protein-A. Purified samples were analyzed with SDS-PAGE and western blot under reducing and non-reducing condition. Commercial IgG1 was used as a positive control. (A) SDS-PAGE profile of purified samples in reduced state, lane 1; Protein molecular ladder, 2; CHEF-F2A, 3; CMV-F2A, 4; CHEF-IRES, 5; CMV-IRES, 6; positive control. (B) SDS-PAGE profile of purified samples in non- reduced state, lane 1; CMV-IRES, 2; CHEF-IRES, 3; CMV-F2A, 4; CHEF-F2A, 5; protein molecular ladder. (C) Western blot analysis of samples under reducing condition, lane 1; protein molecular ladder, 2; CHEF-F2A, 3; CMV-F2A, 4; CHEF-IRES, 5; CMV-IRES, 6; positive control. (D) Western blot analysis of purified samples in non- reduced state, lane 1; CMV-IRES, 2; CHEF-IRES, 3; CMV-F2A, 4; CHEF-F2A, 5; protein molecular ladder.</p

Primers used for construction of the LC-furin-2A-HC gene.

<p>Primers used for construction of the LC-furin-2A-HC gene.</p