Dual suppression of follicle activation pathways completely prevents the cyclophosphamide-induced loss of ovarian reserve.

STUDY QUESTION: To what extent and how does combined administration of the follicle activation pathway suppressive agents temsirolimus (Tem) and c-terminus recombinant anti-Müllerian hormone (rAMH) protect against chemotherapy-induced ovarian reserve loss? SUMMARY ANSWER: Combined administration of Tem and rAMH completely prevents cyclophosphamide (Cy)-induced follicle depletion and protects the ovarian reserve in mice, primarily via primordial follicle (PMF) suppression of activation and to a lesser degree by reducing apoptosis. WHAT IS KNOWN ALREADY: There is conflicting evidence regarding the contributory roles of apoptosis and follicle activation in chemotherapy-induced PMF loss. Tem, a mammalian target of rapamycin (mTOR) inhibitor, reduces activity of the phosphoinositide 3-kinases-phosphatase and tensin homolog (PI3K-PTEN) pathway which provides intrinsic regulation of PMF activation. Anti-Müllerian hormone (AMH), secreted by early growing follicles, is an extrinsic regulator of PMF activation. STUDY DESIGN, SIZE, DURATION: Whole ovaries of 12-day-old mice were cultured ex vivo for 7 days in the presence of Cy ± rAMH or Tem. Eight-week-old mice were randomized into eight treatment groups: vehicle control/rAMH/Tem/Cy/Tem + rAMH/Cy + Tem/Cy + rAMH/Cy + Tem + rAMH. Twelve hours after treatment, ovaries were removed for DNA damage analysis, and 24 h after treatment either for analysis of PI3K pathway proteins or to be fixed and immunostained for analyses of proliferation and apoptosis. Three or 21 days following treatment, ovaries were fixed and sectioned for follicle counting. PARTICIPANTS/MATERIALS, SETTING, METHODS: Hematoxylin and eosin staining was used for differential follicle counts of primordial, primary, and secondary follicles in ex vivo (n = 16-18 ovaries per group) and in vivo ovaries (n = 8 mice per group). Histological analyses were carried out to measure proliferation by quantifying Ki-67-positive granulosa cells in primary follicles (n = 4 mice per group). DNA damage and apoptosis were measured by quantification of phosphorylated form of histone 2AX (γH2AX) and cleaved poly (ADP-ribose) polymerase (cPARP)-positive PMF oocytes, respectively (n = 8 mice per group). Protein extracts from whole ovaries were analyzed by western blotting. MAIN RESULTS AND THE ROLE OF CHANCE: In vivo experiments show that treatment with Cy alone caused significant loss of PMF reserve (32 ± 2.12 versus 144 ± 2.8 in control, P < 0.001), and this was significantly attenuated by treatment with either Tem (P < 0.001) or rAMH (P < 0.001). Combined cotreatment with Cy + Tem + rAMH provided complete protection of the PMF reserve, with no significant difference in numbers of PMF versus untreated animals. Similar results were demonstrated in the ex vivo experiments. Proliferation marker Ki-67 staining was significantly reduced in granulosa cells of primary follicles in the Cy + Tem + rAMH group compared with Cy alone group (after 24 h in vivo administration of Cy, 16% versus 65%, respectively; P < 0.001). Protein analysis demonstrated not significant increased phosphorylation of follicle activation proteins rpS6 and mTOR with in vivo administration of Cy alone (1.9 and 1.4 times the control ovaries, respectively), and this was reduced to below control levels in the Cy + Tem + rAMH group (P < 0.01). The Cy + Tem + rAMH combined cotreatment protected the follicle reservoir via inhibition of Cy-induced upregulation of the PI3K signaling pathway, together with replacement of AMH suppression of PMF activation with rAMH, implying a complementary effect of the two inhibitors. The DNA damage marker γH2AX was highly positive in PMF oocytes from Cy-treated ovaries 12 h after treatment, compared with controls (94% versus 59%, respectively, P < 0.001) and was significantly reduced to (69%) in Cy + Tem + rAMH cotreated ovaries (P < 0.001). However, only 22% of PMF oocytes of the Cy group showed apoptosis at 24 h, and this was significantly reduced (12%) in ovaries after treatment with Cy + Tem + rAMH (P < 0.01). This suggests that it is not possible to equate DNA damage with oocyte death, and also indicates that less than one-third of the total PMF loss can be attributed to apoptosis, implying that most of the PMF depletion results from PMF activation but that both mechanisms play a significant role. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The experimental design was limited by the selection of one time point for analysis of PMF activation and apoptosis (i.e. 24 h after Cy administration), although DNA damage was measured at 12 h after Cy administration and any impact on short-term follicle dynamics at 3 days after treatment. Protein analysis was conducted on whole ovary lysates therefore the protein changes identified cannot be localized to specific cells within the ovary. However, this complementary assay showed that there was activation in the ovary through massive reduction in the phosphorylation of key proteins in the PI3K cascade (rpS6 and mTOR), which is consistent with the sequence of events after Cy administration. WIDER IMPLICATIONS OF THE FINDINGS: Understanding the complementary nature of different follicle activation pathways and the impact of their suppression in prevention of chemotherapy-induced ovotoxic damage, as well as their involvement in DNA damage inhibition, provides an interesting direction for future research, and the potential for noninvasive pharmacological fertility preservation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a grant from the Morris Kahn Foundation. The authors declare no conflicts of interest.

FMR6 may play a role in the pathogenesis of fragile X-associated premature ovarian insufficiency.

The aim of this study was to evaluate whether long noncoding RNA accumulation play a role in the pathophysiology of fragile X-associated premature ovarian insufficiency (FXPOI). The study population consisted of 22 consecutive fragile X mental retardation 1 (FMR1) premutation carriers (CGGn 55-199 repeats) undergoing in vitro fertilization and pre-implantation genetic diagnosis (IVF-PGD) treatment. The control group consists of 11 patients, with <55 CGG repeats, undergoing IVF-ICSI for male factor infertility, matched by age, treated in the same period. After oocyte retrieval, granulosa cells from follicular fluid were washed and stored at -80 °C. RNA was transcribed to generate cDNA and the RNA levels were measured using RT-PCR. Transcripts levels in granulosa cells of long noncoding RNA's FMR4 and FMR6 were measured. In FMR1 premutation carriers there was a significant nonlinear association between the number of CGG repeats and the levels of FMR6 (p = 0.03), but not FMR4. The highest level of FMR6 was seen in women with mid-size CGG repeats (80-120). In addition, a significant negative linear correlation was observed between the number of oocytes retrieved and the RNA levels in granulosa cells of FMR6 (r = -0.53, p = 0.01) but not FMR4. Our study supports previous findings suggesting RNA toxic gain-of-function as one of the possible pathophysiologic mechanisms underlying FXPOI.

p53 regulates its own expression by an intrinsic exoribonuclease activity through AU-rich elements.

The onco-suppressor p53 protein plays also an important role in the control of various aspects of health and disease. p53 levels are low in normal cells and elevated under stress conditions. While low levels of p53 promote tumor formation, overactive p53 leads to premature aging and cell death. RNA degradation is a critical level of regulation contributing to the control of gene expression. p53, as an RNA-binding protein, exerts 3' → 5' exoribonuclease activity, mediating degradation of adenylate/uridylate-rich elements (ARE)-containing ssRNAs. The 3'-UTR of p53-mRNA, which is a target of p53 itself, harbors cis-acting AREs. Our results suggest that p53 controls its own expression through murine double-minute 2 (mdm2)-independent "RNA decay" function in cytoplasm. We demonstrate that p53 expresses an exoribonuclease activity through the binding to ARE sequences of p53-mRNA via translation-independent and translation-dependent polysome-associated pathways. Antagonistic interplay was detected between p53 levels and execution of its exoribonuclease function mirrored in low p53 levels in normal cells, due to the efficient exoribonuclease activity, and in the accumulation of p53 in cells exposed to p53-activating drugs in accordance with the reduced exoribonuclease activity. Apparently, p53, via control of its own mRNA stability and/or translation in cytoplasm, might act as a negative regulator of p53-mRNA levels. The observed connection between exoribonuclease activity and p53 abundance highlights the importance of this function affecting p53 expression, imperative for multiple functions, with implications for the steady-state levels of protein and for the p53 stress response. The modulation in expression of exoribonuclease activity would be translated into the alterations in p53 level. KEY MESSAGES: p53 controls its own expression through mdm2-independent "RNA decay" function in cytoplasm. p53 expresses an exoribonuclease activity through the binding to ARE sequences of p53-mRNA. Antagonistic interplay exists between stress-induced p53 and execution of its exoribonuclease function.

First delivery in a leukemia survivor after transplantation of cryopreserved ovarian tissue, evaluated for leukemia cells contamination.

OBJECTIVE: To describe a successful autologous ovarian tissue re-transplantation in a sterile leukemia survivor after evaluation for minimal residual disease and provide a review of the current literature. DESIGN: Presentation of a carefully designed workup taken to evaluate tissue for minimal residual disease, its limitations, and applicability to other patients. To date, there have not been any publications of auto-transplantations in leukemia survivors, owing to an estimated high risk for malignancy induction. SETTING: Large tertiary hospital. PATIENT(S): A 19-year-old acute myeloid leukemia patient underwent ovarian tissue cryopreservation during complete remission before bone marrow transplantation. After prolonged amenorrhea, the patient desired pregnancy. Laboratory tests showed antimüllerian hormone <0.1 ng/mL and FSH 116 mIU/mL. Ultrasound revealed no ovarian follicles. INTERVENTION(S): Ovarian tissue cryopreservation and auto-transplantation. Histology, immunohistochemistry, FISH, next-generation sequencing, and xenotransplantation were done to evaluate thawed tissue samples for the presence of leukemia cells. MAIN OUTCOME MEASURE(S): Evidence for leukemia cells in thawed ovarian tissue, reproductive outcomes and live birth after transplantation, and leukemia-free survival. RESULT(S): Histology was negative for leukemia cells. Three severe combined immunodeficiency mice, grafted with tissue fragments, were followed for 6 months and showed no macroscopic/microscopic signs for leukemia. Fluorescence in situ hybridization for disease-specific gene rearrangement resulted in a read below the probe's cut-off. A next-generation sequencing panel of genes implicated in myeloproliferative disorders did not reveal any significant molecular event. Transplantation was performed, followed by ovarian stimulation and IVF, resulting in the delivery of healthy newborn. More than 2 years have elapsed since transplantation, and the patient is leukemia free. CONCLUSION(S): Harvesting during complete remission, combined with intense tissue evaluation before transplantation, allowed a safe, successful transplantation in an acute myeloid leukemia survivor.

Elevated levels of FMR1 mRNA in granulosa cells are associated with low ovarian reserve in FMR1 premutation carriers.

AIM: To assess the role of mRNA accumulation in granulosa cells as the cause of low ovarian response among FMR1 premutation carriers undergoing pre-implantation genetic diagnosis (PGD). DESIGN: Case control study in an academic IVF unit. Twenty-one consecutive FMR1 premutation carriers and 15 control women were included. After oocyte retrieval the granulosa cells mRNA levels of FMR1 was measured using RT-PCR. RESULTS: In FMR1 premutation carriers, there was a significant non-linear association between the number of CGG repeats and the number of retrieved oocytes (p<0.0001) and a trend to granulosa cells FMR1 mRNA levels (p = 0.07). The lowest number of retrieved oocytes and the highest level of mRNA were seen in women with mid-size CGG repeats (80-120). A significant negative linear correlation was observed between the granulosa cells FMR1 mRNA levels and the number of retrieved oocytes (R2 linear = 0.231, P = 0.02). CONCLUSION: We suggest that there is a no-linear association between the number of CGG repeats and ovarian function, resulting from an increased granulosa cells FMR1 mRNA accumulation in FMR1 carriers in the mid-range (80-120 repeats).

Ribonuclease activity of p53 in cytoplasm in response to various stress signals.

The tumor suppressor p53 protein is expressed at low levels under normal conditions. The subcellular localization and functional activation of p53 are influenced by diverse stress signals. p53 in cytoplasm exerts intrinsic 3'→5' exonuclease activity with various RNA and DNA substrates. ssRNAs containing an adenosine and uridine-rich (ARE) element are permissive targets for p53-mediated degradation. The analysis of the exonuclease activity in cytoplasm with activated p53 induced by various drug treatments or following γ-irradiation revealed that the expression of p53 exonuclease activity in response to stress signals is heterogeneous. Various genotoxic and non-genotoxic agents upregulate p53 yet have different effects on expression of exonuclease activity with ARE RNA but not with DNA substrate. Ribonuclease activity is enhanced in cytoplasmic extracts of HCT116 (p53+/+) cells exposed to γ-irradiation or treated by the non-genotoxic drug AS101 but decreased following treatment by genotoxic (e.g., doxorubicin) or non-genotoxic (e.g., DFMO) agents, thus indicating that p53 exonuclease activity is dependent on the specific stress and nature of the substrate. Apparently, the disparity in expression of p53 ribonuclease activity after each treatment is attributable to the different post-treatment response and to two posttranscriptional events: the interaction of RNA-binding HuR protein with ARE RNA protects the substrate from degradation by p53 and/or decrease in p53 ARE RNA binding capacity due to phosphorylation at Ser392 leads to reduction in p5 ribonuclease activity. Our results provide new insights into p53 exonuclease function and into the mechanisms behind the regulation ARE-RNA degradation by p53 under different cellular conditions.