Ongoing pregnancy rates in women with low and extremely low AMH levels. A multivariate analysis of 769 cycles.

BACKGROUND: The ideal test for ovarian reserve should permit the identification of women who have no real chance of pregnancy with IVF treatments consequent upon an extremely reduced ovarian reserve. The aim of the current study was to evaluate pregnancy rates in patients with low AMH levels (0.2-1 ng/ml) and extremely low AMH levels (<0.2 ng/ml) and to determine the cumulative pregnancy rates following consecutive IVF treatments. METHODS: We conducted an historical cohort analysis at a tertiary medical center. Serum AMH levels were measured at initial clinic visit and prior to all following treatment cycles in 181 women (769 cycles) with an initial AMH level ≤1 ng/ml, undergoing IVF-ICSI. Main outcome measures were laboratory outcomes and pregnancy rates. RESULTS: Seventy patients undergoing 249 cycles had extremely low AMH levels (≤0.2 ng/ml), whereas 111 patients undergoing 520 cycles had low AMH levels (0.21-1.0 ng/ml). Number of oocytes retrieved per cycle, fertilized oocytes and number of transferred embryos were significantly lower in the extremely low AMH levels group compared to the low AMH levels (P<0.003). Crude ongoing pregnancy rates were 4.4% for both groups of patients. Among 48 cycles of women aged ≥42 with AMH levels of ≤0.2 ng/ml no pregnancies were observed. But, in patients with AMH levels of 0.2-1.0 ng/ml, 3 ongoing pregnancies out of 192 cycles (1.6%) were observed. However, in a multivariate regression analysis adjusted for age and cycle characteristics, no significant differences in ongoing pregnancy rates per cycle between the two groups were evident. Cumulative pregnancy rates of 20% were observed following five cycles, for both groups of patients. CONCLUSIONS: Patients with extremely low AMH measurements have reasonable and similar pregnancy rates as patients with low AMH. Therefore, AMH should not be used as the criterion to exclude couples from performing additional IVF treatments

The TSC-mTOR Pathway Mediates Translational Activation of TOP mRNAs by Insulin Largely in a Raptor- or Rictor-Independent Manner▿ ‡

The stimulatory effect of insulin on protein synthesis is due to its ability to activate various translation factors. We now show that insulin can increase protein synthesis capacity also by translational activation of TOP mRNAs encoding various components of the translation machinery. This translational activation involves the tuberous sclerosis complex (TSC), as the knockout of TSC1 or TSC2 rescues TOP mRNAs from translational repression in mitotically arrested cells. Similar results were obtained upon overexpression of Rheb, an immediate TSC1-TSC2 target. The role of mTOR, a downstream effector of Rheb, in translational control of TOP mRNAs has been extensively studied, albeit with conflicting results. Even though rapamycin fully blocks mTOR complex 1 (mTORC1) kinase activity, the response of TOP mRNAs to this drug varies from complete resistance to high sensitivity. Here we show that mTOR knockdown blunts the translation efficiency of TOP mRNAs in insulin-treated cells, thus unequivocally establishing a role for mTOR in this mode of regulation. However, knockout of the raptor or rictor gene has only a slight effect on the translation efficiency of these mRNAs, implying that mTOR exerts its effect on TOP mRNAs through a novel pathway with a minor, if any, contribution of the canonical mTOR complexes mTORC1 and mTORC2. This conclusion is further supported by the observation that raptor knockout renders the translation of TOP mRNAs rapamycin hypersensitive

Reassessment of the role of TSC, mTORC1 and microRNAs in amino acids-meditated translational control of TOP mRNAs.

TOP mRNAs encode components of the translational apparatus, and repression of their translation comprises one mechanism, by which cells encountering amino acid deprivation downregulate the biosynthesis of the protein synthesis machinery. This mode of regulation involves TSC as knockout of TSC1 or TSC2 rescued TOP mRNAs translation in amino acid-starved cells. The involvement of mTOR in translational control of TOP mRNAs is demonstrated by the ability of constitutively active mTOR to relieve the translational repression of TOP mRNA upon amino acid deprivation. Consistently, knockdown of this kinase as well as its inhibition by pharmacological means blocked amino acid-induced translational activation of these mRNAs. The signaling of amino acids to TOP mRNAs involves RagB, as overexpression of active RagB derepressed the translation of these mRNAs in amino acid-starved cells. Nonetheless, knockdown of raptor or rictor failed to suppress translational activation of TOP mRNAs by amino acids, suggesting that mTORC1 or mTORC2 plays a minor, if any, role in this mode of regulation. Finally, miR10a has previously been suggested to positively regulate the translation of TOP mRNAs. However, we show here that titration of this microRNA failed to downregulate the basal translation efficiency of TOP mRNAs. Moreover, Drosha knockdown or Dicer knockout, which carries out the first and second processing steps in microRNAs biosynthesis, respectively, failed to block the translational activation of TOP mRNAs by amino acid or serum stimulation. Evidently, these results are questioning the positive role of microRNAs in this mode of regulation

Cumulative ongoing pregnancy rates for patients with AMH level of 0.2–1 ng/ml or ≤0.2 ng/ml.

<p>Cumulative ongoing pregnancy rates for patients with AMH level of 0.2–1 ng/ml or ≤0.2 ng/ml.</p

769 cycles parameters in 181 women with low serum AMH levels (0.2–1 ng/ml) and extremely low serum AMH levels (≤0.2 ng/ml).

<p>769 cycles parameters in 181 women with low serum AMH levels (0.2–1 ng/ml) and extremely low serum AMH levels (≤0.2 ng/ml).</p

Multivariable analysis of ongoing pregnancy rates (34 ongoing pregnancies in 769 cycles, 181 women).

<p>Multivariable analysis of ongoing pregnancy rates (34 ongoing pregnancies in 769 cycles, 181 women).</p

Analysis of pregnancy rates per age.

<p>Analysis of pregnancy rates per age.</p

Patients characteristics of 181 women with low serum AMH levels (0.2–1 ng/ml) and extremely low serum AMH levels (≤0.2 ng/ml).

<p>Patients characteristics of 181 women with low serum AMH levels (0.2–1 ng/ml) and extremely low serum AMH levels (≤0.2 ng/ml).</p

Rapamycin represses the translation of TOP mRNAs in an FKBP-12-dependent fashion.

<p>(A) HEK293 cells were amino acid-starved for 3 h and then refed for 3 h in the absence or presence of rapamycin (20 nM), FK506 (20 mM), or both. Cytoplasmic proteins were subjected to Western blot analysis. (B) HEK293 cells were amino acid-starved for 3 h (−AA), refed for 3 h (−AA→+AA) in the absence or presence of rapamycin (20 nM), FK506 (20 mM) or both. Cytoplasmic extracts were subjected to polysomal analysis.</p

mTOR mediates amino acid-induced translational activation of TOP mRNAs.

<p>(A) Kinetics of the effect of rapamycin on mTORC1 activity. 293 cells were amino acid-starved for 2 h and then refed for the indicated time in the presence or absence of 20 nM rapamycin, after which cells were harvested. The cytoplasmic proteins were subjected to Western blot analysis with anti-rpS6 or anti-Phospho-rpS6 antibodies. The chemiluminescent signals of phospho rpS6 were quantified and normalized to those obtained for rpS6 within the same protein extract. The results are numerically presented relative to those obtained for amino acid-starved cells (time zero), which were arbitrarily set at 1. (B) Kinetics of the effect of rapamycin on polysomal association of TOP mRNAs. HEK293 cells were amino acid-starved for 3 h (time zero), and then refed in the absence (open symbols) or presence (filled symbols) of 20 nM rapamycin (rapa). At the indicated times cells were harvested and cytoplasmic extracts were subjected to polysomal analysis. The percentage of mRNA in polysomes at each time point is presented as an average of at least 2 measurements. (C) HEK293 cells were infected with viruses expressing HcRed (Red) shRNA or mTOR shRNA1. Cells were amino acid-starved for 3 h followed by 3 h amino acid stimulation on day 4 post-infection. The abundance of mTOR and its activity were monitored by Western blot analysis of cytoplasmic proteins with the indicated antibodies. (D) Cytoplasmic extracts from cells described in (C) were subjected to polysomal analysis. (E) and (F) HEK293 were transiently transfected with plasmid-based vectors expressing either wild-type (WT) mTOR or enhanced (En) mTOR. 48 h later cells were amino acid-starved for 3 h and harvested. Cytoplasmic proteins were subject to Western blot analysis (E) and cytoplasmic extracts to polysomal analysis (F). The percentage of mRNA in polysomes is presented as an average ± SEM of three experiments.</p