The first two blastomeres contribute unequally to the human embryo.

Retrospective lineage reconstruction of humans predicts that dramatic clonal imbalances in the body can be traced to the 2-cell stage embryo. However, whether and how such clonal asymmetries arise in the embryo is unclear. Here, we performed prospective lineage tracing of human embryos using live imaging, non-invasive cell labeling, and computational predictions to determine the contribution of each 2-cell stage blastomere to the epiblast (body), hypoblast (yolk sac), and trophectoderm (placenta). We show that the majority of epiblast cells originate from only one blastomere of the 2-cell stage embryo. We observe that only one to three cells become internalized at the 8-to-16-cell stage transition. Moreover, these internalized cells are more frequently derived from the first cell to divide at the 2-cell stage. We propose that cell division dynamics and a cell internalization bottleneck in the early embryo establish asymmetry in the clonal composition of the future human body.

Fingerprinting of anti-alpha enolase antibodies in systemic sclerosis.

OBJECTIVES: Anti-alpha enolase antibodies have been detected in systemic sclerosis (SSc), but little is known on their fine specificity and their predictive value on single disease manifestations. The aim of this work is to perform an epitope mapping of alpha enolase by means of truncated recombinant proteins and to analyse the clinico-serological correlations of anti-alpha enolase antibodies in SSc patients. METHODS: Thirty-eight SSc patients were recruited and fully clinically and serologically characterised. Plasmids encoding full length and truncated polypeptides of alpha enolase were generated; the polypeptides were purified under native conditions and used in dot blot to test sera from SSc patients and controls. The densitometric values obtained on all the polypeptides with anti-IgG subclass specific antibodies were analysed by cluster analysis and partial least square regression. RESULTS: Anti-alpha enolase antibodies (mostly IgG1 and IgG2) are detected in 47% of SSc patients. IgG1 target the amino terminal region of alpha enolase, while IgG2 are more restricted to the central portion of the molecule. Anti-alpha enolase antibodies are not associated with disease-specific antibodies or with interstitial lung disease and do not identify patients affected by the limited vs. diffuse form. CONCLUSIONS: Anti-alpha enolase antibodies are very frequent in SSc but are not associated with clinical or serological features of the disease. Further studies on larger cohorts of patients are necessary to define their possible contribution in defining specific subsets of the disease.

RIP-Chip analysis supports different roles for AGO2 and GW182 proteins in recruiting and processing microRNA targets.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNA molecules mediating the translational repression and degradation of target mRNAs in the cell. Mature miRNAs are used as a template by the RNA-induced silencing complex (RISC) to recognize the complementary mRNAs to be regulated. To discern further RISC functions, we analyzed the activities of two RISC proteins, AGO2 and GW182, in the MCF-7 human breast cancer cell line. METHODS: We performed three RIP-Chip experiments using either anti-AGO2 or anti-GW182 antibodies and compiled a data set made up of the miRNA and mRNA expression profiles of three samples for each experiment. Specifically, we analyzed the input sample, the immunoprecipitated fraction and the unbound sample resulting from the RIP experiment. We used the expression profile of the input sample to compute several variables, using formulae capable of integrating the information on miRNA binding sites, both in the 3'UTR and coding regions, with miRNA and mRNA expression level profiles. We compared immunoprecipitated vs unbound samples to determine the enriched or underrepresented genes in the immunoprecipitated fractions, independently for AGO2 and GW182 related samples. RESULTS: For each of the two proteins, we trained and tested several support vector machine algorithms capable of distinguishing the enriched from the underrepresented genes that were experimentally detected. The most efficient algorithm for distinguishing the enriched genes in AGO2 immunoprecipitated samples was trained by using variables involving the number of binding sites in both the 3'UTR and coding region, integrated with the miRNA expression profile, as expected for miRNA targets. On the other hand, we found that the best variable for distinguishing the enriched genes in the GW182 immunoprecipitated samples was the length of the coding region. CONCLUSIONS: Due to the major role of GW182 in GW/P-bodies, our data suggests that the AGO2-GW182 RISC recruits genes based on miRNA binding sites in the 3'UTR and coding region, but only the longer mRNAs probably remain sequestered in GW/P-bodies, functioning as a repository for translationally silenced RNAs.

Revisiting embryo assisted hatching approaches: a systematic review of the current protocols.

Zona pellucida (ZP) manipulation, termed "assisted hatching" (AH), has been introduced in order to favor embryo hatching and ultimately improve assisted reproductive technology success but with poor proofs of safety and biological plausibility. We herein provide a systematic review of clinical outcomes following the application of different methods of ZP manipulation on fresh or frozen/thawed embryos at different developmental stages in different groups of patients. Out of the 69 papers that compared the clinical outcomes deriving from hatched versus non-hatched embryos, only 11 considered blastocysts while the rest referred to cleavage stage embryos. The ZP thinning of fresh embryos either by chemical or laser approach was shown to provide very limited benefit in terms of clinical outcomes. Better results were observed with procedures implying a higher degree of zona manipulation, including zona removal. Studies comparing the mechanical or chemical procedures to those laser-mediated consistently reported a superiority of the latter ones over the former. Literature is consistent for a benefit of ZP breaching in thawed blastocysts. This review provides the current knowledge on the AH procedure in order to improve its efficacy in the appropriate context. Embryologists might benefit from the approaches presented herein in order to improve Assisted Reproduction Technologies (ART) outcomes.

A direct healthcare cost analysis of the cryopreserved versus fresh transfer policy at the blastocyst stage.

A cost analysis covering direct healthcare costs relating to IVF freeze-all policy was conducted. Normal- and high- responder patients treated with a freeze-all policy (n = 63) compared with fresh transfer IVF (n = 189) matched by age, body mass index, duration and cause of infertility, predictive factors for IVF (number of oocytes used for fertilization) and study period, according to a 1:3 ratio were included. Total costs per patient (€6952 versus €6863) and mean costs per live birth were similar between the freeze-all strategy (€13,101, 95% CI 10,686 to 17,041) and fresh transfer IVF (€15,279, 95% CI 13,212 to 18,030). A mean per live birth cost-saving of €2178 (95% CI -1810 to 6165) resulted in a freeze-all strategy owing to fewer embryo transfer procedures (1.29 ± 0.5 versus 1.41 ± 0.7); differences were not significant. Sensitivity analysis revealed that the freeze-all strategy remained cost-effective until the live birth rate is either higher or only slightly lower (≥-0.59%) in the freeze-all group compared with fresh cycles. A freeze-all policy does not increase costs compared with fresh transfer, owing to negligible additional expenses, i.e. vitrification, endometrial priming and monitoring, against fewer embryo transfer procedures required to achieve pregnancy.

Improving fertilization rate in ICSI cycles by adding myoinositol to the semen preparation procedures: a prospective, bicentric, randomized trial on sibling oocytes.

PURPOSE: To evaluate whether the in vitro incubation of spermatozoa with myoinositol may improve the fertilization rate in ICSI cycles. METHODS: This is a prospective, bicentric, randomized study on 500 MII sibling oocytes injected in 78 ICSI cycles performed between March and October 2013. Randomization of the oocytes into two groups was performed at the time of the denudation. Fertilization rates (per oocyte injected with spermatozoa treated with myoinositol versus per oocyte injected with spermatozoa treated with placebo) were measured as primary outcome and embryo morphology as secondary outcome. Clinical outcomes were also documented. RESULT (S): Fertilization rate (78.9 ± 28.6% vs 63.2 ± 36.7, P = 0.002) and percentage of grade A embryos on day 3 (59.8 ± 35.6% vs 43.5 ± 41.5, P = 0.019) were significantly higher when spermatozoa were treated in vitro with myoinositol versus placebo. No differences were found for the expanded blastocyst formation rate. CONCLUSION (S): In vitro treatment of spermatozoa with myoinositol may optimize ICSI outcomes by improving the fertilization rate and embryo quality on day 3. The improvement of the number and the quality of embryos available in an ICSI cycle may have clinical utility if these findings can be confirmed.

Trophectoderm biopsy protocols can affect clinical outcomes: time to focus on the blastocyst biopsy technique.

OBJECTIVE: To compare two different blastocyst biopsy protocols. DESIGN: Retrospective single-center cohort study. SETTINGS: Private in vitro fertilization center. PATIENT(S): The study included 1,670 frozen-thawed embryo transfers (FETs) with preimplantation genetic testing for aneuploidy (PGT-A). INTERVENTION: None. MAIN OUTCOME MEASURE(S): Survival rate (SR) after thawing, clinical pregnancy rate (CPR), ongoing implantation rate (IR), and live birth rate (LBR). RESULT(S): Eight hundred thirty-five FETs with PGT-A cycles including only embryos biopsied in the sequential blastocyst hatching and biopsy protocol paired with the ablation of one-fourth of the zona pellucida (ZP) were matched with 835 FETs with PGT-A cycles including only embryos biopsied in the day 3 prehatching protocol by female age (±1 year), number of embryos transferred, use of gestational carrier or egg donor, and day of blastocyst transfer. Only FETs with euploid blastocysts graded no lower than 4BB were included, and cycles with fewer than five oocytes were excluded. SR after thawing, CPR, ongoing IR, and LBR were significantly higher in the FET cycles with the embryos biopsied in the sequential hatching and biopsy protocol. Four cases of monozygotic twin pregnancies were reported with the day 3 prehatching protocol and none with the sequential hatching and biopsy protocol. CONCLUSION(S): Our results show, for the first time, that using different blastocyst biopsy protocols can affect clinical outcomes. Because the study was retrospective, our findings should be validated in a prospective trial.

The kelch protein NS1-BP interacts with alpha-enolase/MBP-1 and is involved in c-Myc gene transcriptional control.

Alpha-enolase is a key glycolytic enzyme that plays a functional role in several physiological processes depending on the cellular localization. The enzyme is mainly localized in the cytoplasm whereas an alternative translated form, named MBP-1, is predominantly nuclear. The MBP-1 protein has been characterized as a c-Myc promoter binding protein that negatively controls transcription. In the present study, we identified the kelch protein NS1-BP as one of the alpha-enolase/MBP-1 partners by using a yeast two-hybrid screening. Although NS1-BP has been originally described as a protein mainly localized in the nucleus, we provide evidence that NS1-BP also interacts with actin in human cells, as reported for most kelch-containing proteins. Here we showed that alpha-enolase and MBP-1 associate with NS1-BP in vitro and in vivo by GST pull-down assays and coimmunoprecipitation experiments; subsequent immunofluorescent staining confirmed colocalization of the proteins within the cells. Furthermore, functional analyses performed by cotransfection assays revealed that NS1-BP enhances the inhibitory effect exerted by MBP-1 on c-Myc promoter. In mammalian cells, the overexpression of both proteins resulted in an increased repression of basal c-Myc transcription and consistently affected the steady state levels of endogenous c-Myc mRNA. These findings further support the distinct roles of alpha-enolase and its MBP-1 variant in maintaining cell homeostasis. Moreover, our data suggest a novel function for NS1-BP in the control of cell proliferation.

Pro-invasive stimuli and the interacting protein Hsp70 favour the route of alpha-enolase to the cell surface.

Cell surface expression of alpha-enolase, a glycolytic enzyme displaying moonlighting activities, has been shown to contribute to the motility and invasiveness of cancer cells through the protein non-enzymatic function of binding plasminogen and enhancing plasmin formation. Although a few recent records indicate the involvement of protein partners in the localization of alpha-enolase to the plasma membrane, the cellular mechanisms underlying surface exposure remain largely elusive. Searching for novel interactors and signalling pathways, we used low-metastatic breast cancer cells, a doxorubicin-resistant counterpart and a non-tumourigenic mammary epithelial cell line. Here, we demonstrate by a combination of experimental approaches that epidermal growth factor (EGF) exposure, like lipopolysaccharide (LPS) exposure, promotes the surface expression of alpha-enolase. We also establish Heat shock protein 70 (Hsp70), a multifunctional chaperone distributed in intracellular, plasma membrane and extracellular compartments, as a novel alpha-enolase interactor and demonstrate a functional involvement of Hsp70 in the surface localization of alpha-enolase. Our results contribute to shedding light on the control of surface expression of alpha-enolase in non-tumourigenic and cancer cells and suggest novel targets to counteract the metastatic potential of tumours.

The ICSI procedure from past to future: a systematic review of the more controversial aspects.

BACKGROUND: ICSI is currently the most commonly used assisted reproductive technology, accounting for 70-80% of the cycles performed. This extensive use, even excessive, is partly due to the high level of standardization reached by the procedure. There are, however, some aspects that deserve attention and can still be ameliorated. The aim of this systematic review was to evaluate the results of available publications dealing with the management of specific situations during ICSI in order to support embryologists in trying to offer the best laboratory individualized treatment. METHODS: This systematic review is based on material obtained by searching PUBMED between January 1996 and March 2015. We included peer-reviewed, English-language journal articles that have evaluated ICSI outcomes in the case of (i) immature oocytes, (ii) oocyte degeneration, (iii) timing of the various phases, (iv) polar body position during injection, (v) zona-free oocytes, (vi) fertilization deficiency, (vii) round-headed sperm, (viii) immotile sperm and (ix) semen samples with high DNA fragmentation. RESULTS: More than 1770 articles were obtained, from which only 90 were specifically related to the issues developed for female gametes and 55 for the issues developed for male gametes. The studies selected for this review were organized in order to provide a guide to overcome roadblocks. According to these studies, the injection of rescue metaphase I oocytes should be discouraged due to poor clinical outcomes and a high aneuploidy rates; laser-assisted ICSI represents an efficient method to solve the high oocyte degeneration rate; the optimal ICSI timing and the best polar body position during the injection have not been clarified; injected zona-free oocytes, if handled carefully, can develop up to blastocyst stage and implant; efficient options can be offered to patients who suffered fertilization failure in previous conventional ICSI cycles. Most controversial and inconclusive are data on the best method to select a viable spermatozoa when only immotile spermatozoa are available for ICSI and, to date, there is no reliable approach to completely filter out spermatozoa with fragmented DNA from an ejaculate. However, most of the studies do not report essential clinical outcomes, such as live birth, miscarriage and fetal abnormality rate, which are essential to establish the safety of a procedure. CONCLUSIONS: This review provides the current knowledge on some controversial technical aspects of the ICSI procedures in order to improve its efficacy in specific contexts. Notwithstanding that embryologists might benefit from the approaches presented herein in order to improve ICSI outcomes, this area of expertise still demands a greater number of well-designed studies, especially in order to solve open issues about the safety of these procedures.

Cellular stress induces cap-independent alpha-enolase/MBP-1 translation.

Myc promoter-binding protein-1 (MBP-1) is a shorter protein variant of the glycolytic enzyme alpha-enolase. Although several lines of evidence indicate that MBP-1 acts as a tumor suppressor, the cellular mechanisms and signaling pathways underlying MBP-1 expression still remain largely elusive. To dissect these pathways, we used the SkBr3 breast cancer cell line and non-tumorigenic HEK293T cells ectopically overexpressing alpha-enolase/MBP-1. Here, we demonstrate that induced cell stresses promote MBP-1 expression through the AKT/PERK/eIF2α signaling axis. Our results contribute to shedding light on the molecular mechanisms underlying MBP-1 expression in non-tumorigenic and cancer cells.

Successful implantation and live birth of a healthy boy after triple biopsy and double vitrification of oocyte-embryo-blastocyst.

INTRODUCTION: Preimplantation genetic diagnosis and/or screening (PGD/PGS) allow the assessment of the genetic health of an embryo before transferring it into the uterus. These techniques require the removal of cellular material (polar bodies, blastomere(s) or trophectoderm cells) in order to perform the proper genetic analysis. We report the implantation and live birth outcome of a vitrified-warmed blastocyst developed after triple biopsy and double vitrification procedures at oocyte, cleavage embryo and blastocyst stage. CASE DESCRIPTION: An infertile couple, with family history of ß-thalassemia, searched for IVF procedure and PGD. First polar bodies biopsy with subsequent vitrification was uninformative due to meiotic crossing-over, so oocytes were inseminated after warming. Two embryos were obtained and blastomere biopsy was performed on day 3 with inconclusive results on their genetic status. Their culture resulted in one expanded blastocyst stage on day 7 that underwent trophectoderm biopsy and vitrification. This embryo showed to be normal. It was then warmed and transferred in an artificial cycle. DISCUSSION AND EVALUATION: Preconception genetic analysis by removal and analysis of the first polar body is technically possible, but the genetic information that we can obtain at this stage may be limited and the oocytes to be inseminated is not predictable. Compared to blastomere biopsy, trophectoderm biopsy has more diagnostic efficiency with respect to both chromosomal mosaicism and PCR accuracy, reducing the problems of amplification failure and allele drop out. Moreover, embryos biopsied at the cleavage stage seem to have lower implantation rate than biopsied blastocyst. CONCLUSIONS: This is the first case report of a live birth obtained from a three step biopsy and double vitrification procedures of a blastocyst. This case report seems also to suggest the harmlessness of all these procedures if carefully performed by a skilled biologist in an IVF lab with quality management system. Finally, our study highlight that blastocyst cryopreserved on day 7 have clinically important potential and embryos that not reach blastocyst stage on day 6 should not to be discharged because they may result in an ongoing pregnancy.

Sensitization of nanocrystalline TiO2 with black absorbers based on Os and Ru polypyridine complexes.

New metal complexes of the type [M(H3tcterpy)LY]+ (where M = Os(II) or Ru(II), L = substituted or unsubstituted bipyridine or pyridylquinoline, and Y = Cl-, I-, or SCN-) have been designed, synthesized, and characterized in view of their application for dye-sensitized solar cells (DSSCs). The Os dyes show a very broad absorption, and correspondingly, the DSSCs show an unprecedented spectral response in the NIR, with an onset at 1100 nm, reaching values of about 50% at 900 nm. The integrated photocurrent of some of such Os dyes is similar to that of the well-known [Ru(Htcterpy)(NCS)3](TBA) and superior to that of the [Ru(Hdcbpy)2(NCS)2](TBA)2 sensitizer. The Ru dyes show absorption and DSSC spectral response between those of the red and black dyes. Their advantage is their potential superior stability due to the reversible oxidative electrochemistry.