Gene expression analysis.

<p>Gene expression analysis.</p

Apoptosis quantification by TUNEL analysis.

<p>(A) cell nuclei stained with DAPI (blue) to perform the TUNEL assay. The sample from the G3 group. The TUNEL + signal was found in the stoma cells (red) from G3; (B) note the primordial follicle in the G2 sample. The TUNEL + signal was found in the stroma, but note that it was absent in the oocyte and GC from the follicles of the G2 group; (C) the G6 sample. Primordial follicles surrounded by flattened shaped GC and stroma nuclei stained with DAPI (blue). TUNEL-positive cells stained in red in the stroma. Follicles were negative for DNA damage in the G6 sample; (D) the G5 sample that contained two primordial follicles, cell nuclei stained with DAPI. No TUNEL + signal was detected in oocyte and GC, but was positive in the ovarian stroma; (E) cell death index. Quantification of the follicles with a positive TUNEL signal in oocytes or GC. No differences were detected between activated and non activated samples; (F) TUNEL-positive stromal area quantification. No differences were detected for follicles when the TUNEL+ area was compared between the activated and non activated groups. This finding suggests that the cell damage found in the stroma from all the groups was due to the culture process.</p

Follicular densities and percentages.

<p>Follicular densities and percentages.</p

Short-Term PTEN Inhibition Improves <i>In Vitro</i> Activation of Primordial Follicles, Preserves Follicular Viability, and Restores AMH Levels in Cryopreserved Ovarian Tissue From Cancer Patients

<div><p>Introduction</p><p><i>In vitro</i> activation and growth of primordial dormant follicles to produce fertilizable oocytes would provide a useful instrument for fertility preservation. The employment of Phosphatase and TENsin homolog (PTEN) inhibitors, in combination with Protein kinase B (Akt) stimulating molecules, has been previously employed to increase follicular activation through the stimulation of the PTEN-Akt pathway.</p><p>Methods</p><p>We aim to establish improved <i>in vitro</i> activation also for cancer patients whose ovarian tissue has already been cryopreserved. Fresh and previously cryopreserved human ovarian cortex were exposed to short-term, low-concentration and ovary-specific treatment with only a PTEN inhibitor.</p><p>Results</p><p>Our <i>in vitro</i> activation protocol enhances the activation mechanisms of primordial follicles in both fresh and cryopreserved samples, and enlarges growing populations without inducing apoptosis in either follicles or the surrounding stroma. Treatment augments estradiol secretion and restores the expression levels of the previously diminished Anti-Müllerian hormone by means of cryopreservation procedures. Genomic modulation of the relative expression of <i>PTEN</i> pathway genes was found in treated samples.</p><p>Conclusion</p><p>The <i>in vitro</i> activation protocol offers new alternatives for patients with cryopreserved tissue as it increases the pool of viable activated follicles available for <i>in vitro</i> growth procedures. The combination of ovarian tissue cryopreservation and <i>in vitro</i> activation of primordial follicles, the main ovarian reserve component, will be a major advancement in fertility preservation.</p></div

Analysis of the relative expression of the PTEN pathway genes.

<p>(A) fold change of the <i>PTEN</i> gene. Significant differences were obtained when ∆Ct were analyzed and showed the PTEN inhibition produced by bpv(pic) at 100 μM in fresh (G1 <i>vs</i>. G2 p = 0.01) and in previously cryopreserved ovarian tissues (G4 <i>vs</i>. G5 p = 0.04); (B) fold change of the <i>FOXO3</i> gene. A significant decrease was detected only in the fresh tissues that underwent IVA treatment when ∆Ct were analyzed (G1 <i>vs</i>. G2 p = 0.015).</p

Activation, proliferation and hormone production induced by IVA.

<p>(A) and (B); FOXO3 detection and localization to monitor follicular activation. Follicles from the G2 and G5 samples are shown, respectively. Note that the activated follicles from the G2 and G5 groups present a FOXO3 nuclear extrusion (arrow) and a positive signal in GC; (C) the percentage of primordial follicular activation was assessed in fresh ovarian tissues, and a significant increase was observed in G2 when compared to G1 (*p = 0.03); (D) when the activated primordial follicular percentage was compared in the previously cryopreserved-thawed group, follicle activation was enhanced in G5 vs. G4 (**p = 0.03); (E) fresh activated sample (G2), primordial follicle showing Ki-67 staining in GC and oocyte nuclei; F) the Ki-67-positive signal in the oocytes of the primordial follicles from the cryopreserved-activated sample (G5); (G) quantification of the AMH expression in GC. An increase in the AMH expression was observed in the GC from the follicles in both G2 (§ p = 0.006) and G5 (§§ p = 0.008) as a result of the <i>in vitro</i> activation procedure with 100 μM of bpv(pic); (H) estradiol secretion to culture media. As the graph depicts, <i>in vitro</i> activation procedures increased E2 secretion in the <i>in vitro</i> activated fresh (G2 <i>vs</i>. G3 *p = 0.036) and cryopreserved (G5 <i>vs</i>. G6 **p = 0.001) samples when compared to their own controls.</p