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SA1, SA2 and SIRT1 gene expression analysis

 

Clinical characteristics of all participants

A total of 50 IVF cycles in 50 women were included in this study. The main characteristics and hormone levels of patients are shown in Table I. When the enrolled patients were grouped according to the age, to the number of retrieved oocytes or to the BMI index, no signifi- cant differences were found in FSH, LH or E2 levels, or in E2 levels on the day of HCG injection.

SA1, SA2 and SIRT1 gene expression analysis

SA1 and SA2 expression levels were lower in women older than 38 years compared to women younger than 34 years, although the difference did not reach statistical significance (Fig. 1). However, SIRT1 mRNA levels in older patients were about twice as high as the level recorded in younger patients (P < 0.05) (Fig. 1). When the patients were clustered into three groups according to their body mass index (BMI), an interesting trend was highlighted, with the high-BMI group having a significant increase in both SA1, SA2 and SIRT1 expression compared to the low-BMI group (Fig. 1).

The same analysis was carried out taking into account the oocytes
retrieved. We demonstrated a significant increase in mRNA levels of
SA1, SA2 and SIRT1 in CCs purified from women producing more than 6
oocytes (high responders) when compared to women producing less
than 4 oocytes (poor responders) (P < 0.05) (Fig. 2). Moreover, statistical analysis of data showed that the number of oocytes retrieved strongly correlated with SA1 (r = −0.37; P < 0.05) and SA2 (r = −0.43; P < 0.05) expression profiling. Interesting insights come from the analysis of the relative telomere length in CCs as a function of SA1, SA2 and SIRT1 gene expression. As shown in Fig. 3, positive correlations between the relative telomere length and, respectively SA1 (r = 0.42; P < 0.001), SA2 (r = 0.36; P < 0.001) and SIRT1 (r = 0.36; P < 0.001) were, also, recorded. Finally, gene expression analysis indicated strong statistical cor- relations between SA1 and SA2 (r = 0.89; P < 0.05), SA1 and SIRT1 (r = 0.69; P < 0.001) and SA2 and SIRT1 (r = 0.78; P < 0.01) (Fig. 4).

To gain a clearer understanding of the higher mRNA SIRT1 transcripts disclosed in patients aged over 38 years, we also investigated SIRT1 protein expression in CCs.

As shown in Fig. 5 (upper panel), western blot analysis demonstrated that SIRT1 was present in CCs, and its expression was higher in older patients compared to younger patients, thus confirming the results from gene expression study. When the cellular localization was investigated by immunofluorescence, SIRT1 showed an intense cytoplasmic staining in older patients, whereas the fluorescence intensity appeared to be lower in younger women (Fig. 5; bottom panel). Sublingual NMN

 

Figure 1 qRT-PCR assessment of the expression of SA1, SA2 and SIRT1 in cumulus cells (CCs) as a function of mean age (upper panels) or BMI (low- er panels). SIRT1 mRNA levels significantly increases of about 100% in older patients (n = 24) when compared to younger (n = 22), whereas the differ- ences in SA1 and SA2 variations were not statistically significant. SA1, SA2 and SIRT1 expression was significant increased in patients having a BMI higher than 25 kg/m2 (n = 12) compared to the low-BMI group (<20 kg/m2) (n = 13). The relative gene expression, normalized by GAPDH reference gene, in comparison to the younger women or patients having BMI < 20 kg/m2 (considered equal to 1) is shown. Bars represent the mean ± SEM of the three technical repeats; *P < 0.05.

Figure 1 qRT-PCR assessment of the expression of SA1, SA2 and SIRT1 in cumulus cells (CCs) as a function of mean age (upper panels) or BMI (low- er panels). SIRT1 mRNA levels significantly increases of about 100% in older patients (n = 24) when compared to younger (n = 22), whereas the differ- ences in SA1 and SA2 variations were not statistically significant. SA1, SA2 and SIRT1 expression was significant increased in patients having a BMI higher than 25 kg/m2 (n = 12) compared to the low-BMI group (

Discussion

Cohesins SA1 and SA2 were originally identified for their role in chromatid cohesion, essential for the normal segregation of chromosomes, and for the DNA repair mediated by the homologous recombination (Nasmyth and Haering, 2009), however recent data assign to these proteins novel biological functions (Remeseiro et al., 2012a, 2012b; Linet al., 2016). Cohesin SA1 also drives DNA looping and gene regulation, whereas cohesin SA2 seems to be involved in mediating DNA double strand break repair in somatic cells (Kong et al., 2014). SA1 deficiency drives aneuploidy, embryo lethality and early onset tumorigenesis in mice as a result of defective telomere replication due to inefficient cohe- sion of telomere ends (Remeseiro et al., 2012a, 2012b). Interestingly in the present study, we report high transcription levels of cohesins SA1 and SA2 in the CCs and an evident decreasing trend with aging; this result confirms the reduced presence of meiotic cohesins in oocytes of aged women that has been previously reported (Tsutsumi et al., 2014).

Figure 2 qRT-PCR assessment of the expression of SA1, SA2 and SIRT1 in CCs as a function of the number of oocytes retrieved at the pick-up. In women producing more than 6 oocytes (high responders; n = 19), SA1, SA2 and SIRT1 expression significantly increases compared to that in women producing fewer than 4 oocytes (poor responders; n = 28). The relative gene expression, normalized by GAPDH reference gene, in comparison to the poor responders (considered equal to 1) is shown. Bars represent the mean ± SEM of the three technical repeats; *P < 0.05.

Figure 2 qRT-PCR assessment of the expression of SA1, SA2 and SIRT1 in CCs as a function of the number of oocytes retrieved at the pick-up. In women producing more than 6 oocytes (high responders; n = 19), SA1, SA2 and SIRT1 expression significantly increases compared to that in women producing fewer than 4 oocytes (poor responders; n = 28). The relative gene expression, normalized by GAPDH reference gene, in comparison to the poor responders (considered equal to 1) is shown. Bars represent the mean ± SEM of the three technical repeats; *P < 0.05.

Figure 5 Upper Panel. Western blot of SIRT1 in CC extracts prepared from younger (38 years) patients. β-actin (ACTB) was immunodetected to control for loading per lane. Computer-assisted semiquantitative analysis of the overall relative intensity of the bands is shown. The intensity was measured (pixel/mm2) and then normalized with respect to ACTB. Values are expressed as mean (+standard deviation). Bottom panel. Representative immunofluorescence of SIRT1 (red) in CCs prepared from a woman younger than 34 years (A) compared to a women older than 38 years (B). Nuclei were counterstained with DAPI (blue). Bar represents 25 μm. The graph reports the ImageJ based-analysis of SIRT1 intensity in CCs prepared from older in comparison with the younger patients, considered to be equal to 1. Data are represented as mean ± SD.

Figure 5 Upper Panel. Western blot of SIRT1 in CC extracts prepared from younger (38 years) patients. β-actin (ACTB) was immunodetected to control for loading per lane. Computer-assisted semiquantitative analysis of the overall relative intensity of the bands is shown. The intensity was measured (pixel/mm2) and then normalized with respect to ACTB. Values are expressed as mean (+standard deviation). Bottom panel. Representative immunofluorescence of SIRT1 (red) in CCs prepared from a woman younger than 34 years (A) compared to a women older than 38 years (B). Nuclei were counterstained with DAPI (blue). Bar represents 25 μm. The graph reports the ImageJ based-analysis of SIRT1 intensity in CCs prepared from older in comparison with the younger patients, considered to be equal to 1. Data are represented as mean ± SD.

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