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

Follicular densities and percentages.

<p>Follicular densities and percentages.</p

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

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

Models of the TSA-treated hESC and decidual hESCs.

<p><b>A,</b> In control endometrial stromal cells, trophoblast invasion is massive due to low levels of TIMPs and high levels of MMPs and uPA proteases. <b>B,</b> TSA-treated hESCs inhibits HDAC and promotes histone acetylation at the promoters of TIMP-1 and TIMP-3 by increasing the transcription of these genes. In contrast, the levels and activities of MMPs and uPA decrease. These ECM modulators changes reduced the motility of hESCs and the invasion of trophoblast cells. <b>C</b>, In <i>in vitro</i> decidualized hESCs, the intracellular cAMP levels increase and promote histone acetylation at the TIMP-1 and TIMP-3 gene promoters. Moreover, the activity and level of MMPs and uPA decreases and, together, precisely control the invasion of trophoblast cells.</p

miRNA Signature and Dicer Requirement during Human Endometrial Stromal Decidualization <em>In Vitro</em>

<div><p>Decidualization is a morphological and biochemical transformation of endometrial stromal fibroblast into differentiated decidual cells, which is critical for embryo implantation and pregnancy establishment. The complex regulatory networks have been elucidated at both the transcriptome and the proteome levels, however very little is known about the post-transcriptional regulation of this process. miRNAs regulate multiple physiological pathways and their de-regulation is associated with human disorders including gynaecological conditions such as endometriosis and preeclampsia. In this study we profile the miRNAs expression throughout human endometrial stromal (hESCs) decidualization and analyze the requirement of the miRNA biogenesis enzyme Dicer during this process. A total of 26 miRNAs were upregulated and 17 miRNAs downregulated in decidualized hESCs compared to non-decidualized hESCs. Three miRNAs families, miR-181, miR-183 and miR-200, are down-regulated during the decidualization process. Using miRNAs target prediction algorithms we have identified the potential targets and pathways regulated by these miRNAs. The knockdown of Dicer has a minor effect on hESCs during <em>in vitro</em> decidualization. We have analyzed a battery of decidualization markers such as cell morphology, Prolactin, IGFBP-1, MPIF-1 and TIMP-3 secretion as well as HOXA10, COX2, SP1, C/EBPß and FOXO1 expression in decidualized hESCs with decreased Dicer function. We found decreased levels of HOXA10 and altered intracellular organization of actin filaments in Dicer knockdown decidualized hESCs compared to control. Our results provide the miRNA signature of hESC during the decidualization process <em>in vitro</em>. We also provide the first functional characterization of Dicer during human endometrial decidualization although surprisingly we found that Dicer plays a minor role regulating this process suggesting that alternative biogenesis miRNAs pathways must be involved in human endometrial decidualization.</p> </div

TSA reduces hESC motility and limits trophoblast outgrowth and invasion.

<p><b>A</b>, Wound healing assay was performed in 48 hour-treated hESCs at two different TSA concentrations (0.1 µM and 1 µM) or a vehicle (control). The % of migrating cells was calculated as the area covered by cells and is represented as an average of three independent experiments. Statistical analysis ** p<0.01. <b>B,</b> Invasion assays of the TSA-treated hESCs were conducted in collagen transwells. hESCs were allowed to invade for 48 h after being previously treated with a vehicle (control), or with 0.1 µM and 1 µM TSA. The histograms show a percentage of invading cells, with the invasion of the control cells designated as 100%. Representative bright field images of the cells invading the underside of the filter are shown below the histograms. Data represent the mean of three independent experiments. Statistical analysis,* p<0.05 and ** p<0.01. <b>C</b>, TSA-treated hESCs limit mouse embryos invasion potential. hESCs and the mouse embryo were marked by Vimentin (red) and E-Cadherin (green), respectively. Nuclei were marked by DAPI (blue). The invasion area was identified by the absence of stromal cells (red) and outlined by a white line. Bar size is 50 µm. <b>D</b>, 1 µM TSA-treated hESCs significantly decreased the area of embryo spreading. Data represent the mean areas of at least 8 embryos in three independent experiments. Statistical analysis, ** p<0.01. <b>E,</b> Schematic representation of the collagen transwell invasion assay used to measure the effect of the TSA-treated hESCs conditioned medium on human trophoblast cells. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030508#s2" target="_blank">Material and Methods</a> for procedure description. <b>F,</b> Histograms show the percentage of the invading cells, with invasion of the control cells designated as 100%. Representative bright field images are shown below the histograms. Representative bright field images of the cells invading the underside of the filter are shown below the histograms. Data represent the mean of three independent experiments.</p

miR-181, miR-183 and miR-200 miRNAs families members are similarly regulated during in vitro decidualization.

<p><b>A</b>, miR-95, miR-135b and miR-96 expression levels by quantitative PCR in hESCs treated with 17β-estradiol (E), progesterone (P), or both (E+P), for 9 days. <b>B</b>, miR-181 <b>C</b>, miR-200 and <b>D</b>, miR-183 family members’ expression in the E+P decidualized hESCs for 9 days if compared to the non decidualized control hESCs. <b>A</b>, <b>B</b>, <b>C</b>, and <b>D</b>, Data represent the mean of three independent experiments. Error bars represent the standard error of the mean (SEM). Statistical analysis,* p<0.05. The molecular pathways potentially regulated by the mir-181, miR-200 and miR-183 families with the potential target genes are listed below the histograms.</p