Epigenetic inhibition of lncRNA GMDS-AS1 by methyltransferase ESET promoted cell viability and metastasis of hepatocellular carcinoma

Background Long noncoding RNA (lncRNAs) GMDS-AS1 has been reported as a tumor regulator in tumor growth and metastasis, but its effect in hepatocellular carcinoma (HCC) remains unclear. ESET, a histone H3K9 methyl-transferase, is involved in epigenomic regulation of tumor progression in multiple cancers. However, the correlation between ESET and lncRNA in HCC is less reported. Methods Quantitative real-time PCR (qRT-PCR) was taken to determine the expression of ESET and GMDS-AS1. Western blot was taken to determine the target protein levels of ESET and GMDS-AS1. Online database and bioinformatics analysis were used to screen abnormally expressed genes. Luciferase assay was performed to confirm the binding of GMDS-AS1 and PSMB1. Ki67 and Edu were used for evaluated the proliferation of tumor cells. ChIP assay was performed to verify the relationship between H3K9me1 and lncRNA GMDS-AS1 promoter. Transwell was taken to determine the migration and invasion ability of tumor cells. CCK-8 was used for determining the viability of tumor cells. Flow cytometry was performed to detect the cell cycle of tumor cells. Results The expression of GMDS-AS1 was decreased and the expression of ESET was increased in HCC. GMDS-AS1 inhibition contributed to tumor development, and this effect was closely related to epigenetic inhibition of GMDS-AS1 by ESET. PSMB1, a downstream target of GMDS-AS1, promoted the tumor proliferation and was negatively regulated by GMDS-AS1. Conclusion Our result demonstrates anti-tumorigenic traits of lncRNA GMDS-AS1 in HCC and explains its pattern of regulation mediated by ESET. Our work unmasked an essential role of GMDS-AS1 in HCC progression and detected a novel pathway for ESET to promote HCC (AU)

Epigenetic inhibition of lncRNA GMDS-AS1 by methyltransferase ESET promoted cell viability and metastasis of hepatocellular carcinoma.

BACKGROUND: Long noncoding RNA (lncRNAs) GMDS-AS1 has been reported as a tumor regulator in tumor growth and metastasis, but its effect in hepatocellular carcinoma (HCC) remains unclear. ESET, a histone H3K9 methyl-transferase, is involved in epigenomic regulation of tumor progression in multiple cancers. However, the correlation between ESET and lncRNA in HCC is less reported. METHODS: Quantitative real-time PCR (qRT-PCR) was taken to determine the expression of ESET and GMDS-AS1. Western blot was taken to determine the target protein levels of ESET and GMDS-AS1. Online database and bioinformatics analysis were used to screen abnormally expressed genes. Luciferase assay was performed to confirm the binding of GMDS-AS1 and PSMB1. Ki67 and Edu were used for evaluated the proliferation of tumor cells. ChIP assay was performed to verify the relationship between H3K9me1 and lncRNA GMDS-AS1 promoter. Transwell was taken to determine the migration and invasion ability of tumor cells. CCK-8 was used for determining the viability of tumor cells. Flow cytometry was performed to detect the cell cycle of tumor cells. RESULTS: The expression of GMDS-AS1 was decreased and the expression of ESET was increased in HCC. GMDS-AS1 inhibition contributed to tumor development, and this effect was closely related to epigenetic inhibition of GMDS-AS1 by ESET. PSMB1, a downstream target of GMDS-AS1, promoted the tumor proliferation and was negatively regulated by GMDS-AS1. CONCLUSION: Our result demonstrates anti-tumorigenic traits of lncRNA GMDS-AS1 in HCC and explains its pattern of regulation mediated by ESET. Our work unmasked an essential role of GMDS-AS1 in HCC progression and detected a novel pathway for ESET to promote HCC.

Histone Methyltransferase SETDB1 Regulates the Development of Cortical Htr3a-Positive Interneurons and Mood Behaviors.

BACKGROUND: GABAergic (gamma-aminobutyric acidergic) interneurons (INs) are highly heterogeneous, and Htr3a labels a subpopulation of cortical INs originating from the embryonic caudal ganglionic eminence. SETDB1 is one of the histone H3K9 methyltransferases and plays an essential role in the excitatory neurons, but its role in regulating cortical inhibitory INs remains largely unknown. METHODS: In this study, we generated transgenic mice with conditional knockout of Setdb1 in neural progenitor cells (Setdb1-NS-cKO) and GABAergic neurons (Setdb1-Gad2-cKO). In addition, we performed RNA sequencing, ATAC-seq (assay for transposase-accessible chromatin with sequencing), chromatin immunoprecipitation sequencing, luciferase assay, chromatin conformation capture, and CRISPR (clustered regularly interspaced short palindromic repeats)/dCas9 to study the epigenetic mechanism underlying SETDB1-mediated transcriptional regulation of Htr3a. We also performed in situ hybridization and whole-cell recording to evaluate the functional properties of cortical Htr3a+ INs and behavioral tests for mood. RESULTS: We detected significant upregulation of Htr3a expression in the embryonic ganglionic eminence of Setdb1-NS-cKO and identified the endogenous retroviral sequence RMER21B as a new target of SETDB1. RMER21B showed enhancer activity and formed distal chromatin interaction with the promoter of Htr3a. In addition, we observed an increased number and enhanced excitability of Htr3a+ INs in the knockout cortex. Moreover, Setdb1-Gad2-cKO mice exhibited anxiety- and depressive-like behaviors, which were partially reversed by a 5-HT3 receptor antagonist. CONCLUSIONS: These findings suggest that SETDB1 represses Htr3a transcription via RMER21B-mediated distal chromatin interaction in the embryonic ganglionic eminence and regulates the development of cortical Htr3a+ INs and mood behaviors.

Clinical application of noninvasive chromosomal screening for elective single-blastocyst transfer in frozen-thawed cycles.

BACKGROUND: The objective of this study was to explore the clinical application of noninvasive chromosomal screening (NICS) for elective single-blastocyst transfer (eSBT) in frozen-thawed cycles. METHODS: This study retrospectively analysed the data of 212 frozen-thawed single-blastocyst transfers performed in our centre from January 2019 to July 2019. The frozen embryos were selected based on morphological grades and placed in preincubation for 6 h after warming. Then spent microdroplet culture media of frozen-thawed blastocysts were harvested and subjected to NICS. The clinical outcomes were evaluated and further stratified analysis were performed, especially different fertilization approaches. RESULTS: The clinical pregnancy, ongoing pregnancy, and live birth rates in the euploidy group were significantly higher than those in the aneuploidy group (56.2% versus 29.4%) but were nonsignificantly different from those in the chaotic abnormal/NA embryos group (56.2% versus 60.4%). Compared with day6 (D6) blastocysts, D5 blastocysts had a nonsignificantly different euploidy rate (40.4% versus 48.1%, P = 0.320) but significantly increased clinical pregnancy (57.7% versus 22.2%, P < 0.001), ongoing pregnancy (48.1% versus 14.8%, P < 0.001), and live birth rates (48.1% versus 13.0%, P < 0.001). The percentage of chaotic abnormal/NA embryos group was significantly higher among D5 embryos than among D6 embryos (30.1% versus 11.1%, P = 0.006). The percentage of aneuploid embryos was higher among the embryos with lower morphological quality(21.5% among 'good' embryos versus 34.6% among 'fair' embryos versus 46.0% among 'poor' embryos, P = 0.013); correspondingly, the overall clinical pregnancy, ongoing pregnancy and live birth rate rates showed similar declines. CONCLUSIONS: NICS combined with morphological assessment is an effective tool to guide frozen-thawed SBT. The optimal embryo for SBT is a 'euploid embryo with good morphology', followed sequentially by a 'chaotic abnormal/NA embryo with good morphology', 'euploid embryo with fair morphology', and 'chaotic abnormal/NA embryo with fair morphology'.

Preparation of the ubiquitination-triggered active form of SETDB1 in Escherichia coli for biochemical and structural analyses.

Trimethylation of histone H3 at K9 by the lysine methyltransferase, SET domain bifurcated histone lysine methyltransferase 1 (SETDB1) plays a pivotal role in silencing tissue-specific genes and retrotransposable elements. In mammalian cells, SETDB1 undergoes monoubiquitination in the insertion region of the SET domain in an E3 ubiquitin ligase-independent manner. This ubiquitination has been shown to enhance the histone H3-K9 methyltransferase activity of SETDB1; however, the molecular mechanism underlying SETDB1 activation by ubiquitination is unknown. In this study, we developed an Escherichia coli ubiquitination plasmid for the preparation of ubiquitinated SETDB1. Western blotting and mutational analyses showed that co-expression of the SET domain of SETDB1 with the proteins encoded by the ubiquitination plasmid led to site-specific monoubiquitination of the SET domain at K867. An in vitro histone H3 methylation assay demonstrated that the ubiquitinated SET domain of SETDB1 acquired enzymatic activity. Taken together, these findings demonstrate successful preparation of the active form of SETDB1 with the E.coli ubiquitination system, which will aid biochemical and structural studies of ubiquitinated SETDB1. Graphical Abstract.

Amino acid residue at the 165th position tunes EYFP chromophore maturation. A structure-based design.

For the whole GFP family, a few cases, when a single mutation in the chromophore environment strongly inhibits maturation, were described. Here we study EYFP-F165G - a variant of the enhanced yellow fluorescent protein - obtained by a single F165G replacement, and demonstrated multiple fluorescent states represented by the minor emission peaks in blue and yellow ranges (~470 and ~530 nm), and the major peak at ~330 nm. The latter has been assigned to tryptophan fluorescence, quenched due to excitation energy transfer to the mature chromophore in the parental EYFP protein. EYFP-F165G crystal structure revealed two general independent routes of post-translational chemistry, resulting in two main states of the polypeptide chain with the intact chromophore forming triad (~85%) and mature chromophore (~15%). Our experiments thus highlighted important stereochemical role of the 165th position strongly affecting spectral characteristics of the protein. On the basis of the determined EYFP-F165G three-dimensional structure, new variants with ~ 2-fold improved brightness were engineered.

More than the oocyte source, egg donors as patients: a national picture of United States egg donors.

PURPOSE: To characterize national oocyte donation practice patterns from the perspective of individual donors rather than of recipients. METHODS: Retrospective cohort including all donor oocyte retrievals and transfers reported to SARTCORS in 2016 and 2017 in the USA. Primary outcomes include characteristics of oocyte donors and of donor oocyte cycles. Secondary outcomes include overall pregnancy rates, elective single embryo transfer (eSET) rates, and perinatal outcomes among donor oocyte recipient transfers. RESULTS: During the study period, 49,193 donor oocyte retrievals were performed, of which the largest proportion were in the Western US. For all reported retrievals, there were 17,099 unique donors, each of whom underwent an average of 2.4 retrievals (range 1-22). Average donor age was 26.3 years (range 18-48). On average, 24.6 oocytes (SD 12.4) were retrieved each cycle, ranging from 0 to 102. Among 37,657 donor oocyte recipient transfers, 20,159 (53.5%) involved eSET, and 17,725 (47.1%) resulted in live birth. Miscarriage rates were 17.5%, and good perinatal outcome (GPO), defined as full-term normal birthweight delivery, was more likely among singleton (75.7%) than multiple (23.8%) pregnancies. CONCLUSION: The average number of retrievals that donors underwent and oocyte yield mirrored national guidelines; however outliers, exist that may unnecessarily increase donor risk. Additionally, among resultant donor transfers, 46.5% transferred more than one embryo despite national recommendations for eSET. The significantly higher likelihood of GPO among singleton pregnancies points to the need to further increase donor recipient eSET rates.

Extracellular vesicles, microRNA and the preimplantation embryo: non-invasive clues of embryo well-being.

Elective single embryo transfer is rapidly becoming the standard of care in assisted reproductive technology for patients under the age of 35 years with a good prognosis. Clinical pregnancy rates have become increasingly dependent on the selection of a single viable embryo for transfer, and diagnostic techniques facilitating this selection continue to develop. Current progress in elucidating the extracellular vesicle and microRNA components of the embryonic secretome is reviewed, and the potential for these findings to improve clinical embryo selection discussed. Key results have shown that extracellular vesicles and microRNAs are rapidly detectable constituents of the embryonic secretome. Evidence suggests that the vesicular population is largely exosomal in nature, secreted at all stages of preimplantation development and capable of traversing the zona pellucida. Both extracellular vesicle and microRNA concentrations within the secretome are elevated for blastocysts with diminished developmental competence, as indicated either by degeneracy or implantation failure, whereas studies have yet to firmly correlate individual microRNA sequences with pregnancy outcome. These emerging correlations support the viability of extracellular vesicles and microRNAs as the basis for a new diagnostic test to supplement or replace morphokinetic assessment.

The fibronectin type-III (FNIII) domain of ATF7IP contributes to efficient transcriptional silencing mediated by the SETDB1 complex.

BACKGROUND: The histone methyltransferase SETDB1 (also known as ESET) represses genes and various types of transposable elements, such as endogenous retroviruses (ERVs) and integrated exogenous retroviruses, through a deposition of trimethylation on lysine 9 of histone H3 (H3K9me3) in mouse embryonic stem cells (mESCs). ATF7IP (also known as MCAF1 or AM), a binding partner of SETDB1, regulates the nuclear localization and enzymatic activities of SETDB1 and plays a crucial role in SETDB1-mediated transcriptional silencing. In this study, we further dissected the ATF7IP function with its truncated mutants in Atf7ip knockout (KO) mESCs. RESULTS: We demonstrated that the SETDB1-interaction region within ATF7IP is essential for ATF7IP-dependent SETDB1 nuclear localization and silencing of both ERVs and integrated retroviral transgenes, whereas its C-terminal fibronectin type-III (FNIII) domain is dispensable for both these functions; rather, it has a role in efficient silencing mediated by the SETDB1 complex. Proteomic analysis identified a number of FNIII domain-interacting proteins, some of which have a consensus binding motif. We showed that one of the FNIII domain-binding proteins, ZMYM2, was involved in the efficient silencing of a transgene by ATF7IP. RNA-seq analysis of Atf7ip KO and WT or the FNIII domain mutant of ATF7IP-rescued Atf7ip KO mESCs showed that the FNIII domain mutant re-silenced most de-repressed SETDB1/ATF7IP-targeted ERVs compared to the WT. However, the silencing activity of the FNIII domain mutant was weaker than that of the ATF7IP WT, and some of the de-repressed germ cell-related genes in Atf7ip KO mESCs were not silenced by the FNIII domain mutant. Such germ cell-related genes are targeted and silenced by the MAX/MGA complex, and MGA was also identified as another potential binding molecule of the ATF7IP FNIII domain in the proteomic analysis. This suggests that the FNIII domain of ATF7IP acts as a binding hub of ATF7IP-interacting molecules possessing a specific interacting motif we named FAM and contributes to one layer of the SETDB1/ATF7IP complex-mediated silencing mechanisms. CONCLUSIONS: Our findings contributed to further understanding the function of ATF7IP in the SETDB1 complex, revealed the role of the FNIII domain of ATF7IP in transcriptional silencing, and suggested a potential underlying molecular mechanism for it.

Single embryo transfer: Why and how to identify the embryo with the best developmental potential.

Multiple pregnancies with higher risk of preterm birth and the associated higher morbidity have been a major obstacle from the early days of in vitro fertilization. A good strategy to avoid multiple pregnancies is elective single embryo transfer and cryopreservation of spare embryos. Important factors in adopting this strategy are good counselling of the patients and the selection of embryos with high implantation potential. Technical advances in embryo selection have been described during recent years, time lapse monitoring and genetic assessment of the embryos being the most important achievements. With these studies we have gained new information on early embryos. However, at present, there is insufficient evidence to recommend the routine use of these new techniques. The ultimate goal of infertility treatment is a healthy baby.

Guidance for elective single-embryo transfer should be applied to frozen embryo transfer cycles.

PURPOSE: To provide clinicians with data showing the benefits of transferring a single blastocyst in frozen embryo transfer (FET) cycles so that they may counsel their patients accordingly. METHODS: This is a closed cohort study of 678 FET cycles occurring between January 2011 and December 2017 in a private IVF laboratory and associated physicians' practice. Patients included in the analysis were less than 38 years of age at oocyte collection, had at least two vitrified blastocysts, and were undergoing their first autologous FET cycle. The patients were categorized into four groups after they had chosen either elective single-embryo transfer (eSET) or double-embryo transfer (eDET). Outcomes for eSET and eDET were compared within groups of patients having freeze-all IVF cycles (PGT-A patient vs. non-PGT-A patient) and fresh IVF transfer groups (negative outcome vs. pregnant/delivered in fresh cycle). Main outcome measures of the study were live birth, multiple pregnancy, and implantation rates. RESULTS: There were no statistically significant differences observed in live birth rates for eSET (54-62%) vs. eDET (54-66%) (P = 0.696-1.000) in the four patient groups evaluated. Multiple pregnancy rates were significantly decreased in all eSET groups (0-3%), compared with eDET groups (24-65%) (P = 0.0001-0.037). CONCLUSIONS: This data shows that transfer of a single vitrified-warmed blastocyst maintains live birth rates, while decreasing multiple pregnancies, and may become more acceptable to physicians and patients.

Time-lapse imaging of inner cell mass splitting with monochorionic triamniotic triplets after elective single embryo transfer: a case report.

RESEARCH QUESTION: Can detailed scrutiny of time-lapse imaging (TLI) of inner cell mass (ICM) splitting help to reduce the frequency of multiple pregnancies following elective single embryo transfer (eSET)? DESIGN: Retrospective analysis of time-lapse images of an embryo in vitro, which resulted in a monochorionic triamniotic pregnancy following eSET on Day 5 of development. RESULTS: A 37-year-old female patient underwent a frozen embryo transfer cycle whereby a single vitrified/warmed embryo was transferred at the hatching blastocyst stage. The subsequent pregnancy scan revealed a monochorionic triamniotic pregnancy. Because the blastocyst was cultured in an incubator incorporating TLI, retrospective scrutiny of the digital recordings demonstrated two distinct ICM structures splitting apart, which formed during the '8-shaped hatching'. CONCLUSIONS: Assisted reproductive techniques and in-vitro culture have been associated with an increased frequency of embryo splitting. This has been postulated to be linked to the in-vitro hatching method observed at the blastocyst stage of development. This case report highlights the need to objectively assess any splitting of the ICM, beyond standard grading of the quality of the ICM and the trophectoderm. Such assessments of ICM splitting should be routine practice in clinical embryology when selecting embryos for transfer.

The Histone Methyltransferase SETDB1 Controls T Helper Cell Lineage Integrity by Repressing Endogenous Retroviruses.

Upon activation, naive CD4+ T cells differentiate into distinct T cell subsets via processes reliant on epigenetically regulated, lineage-specific developmental programs. Here, we examined the function of the histone methyltransferase SETDB1 in T helper (Th) cell differentiation. Setdb1-/- naive CD4+ T cells exhibited exacerbated Th1 priming, and when exposed to a Th1-instructive signal, Setdb1-/- Th2 cells crossed lineage boundaries and acquired a Th1 phenotype. SETDB1 did not directly control Th1 gene promoter activity but relied instead on deposition of the repressive H3K9me3 mark at a restricted and cell-type-specific set of endogenous retroviruses (ERVs) located in the vicinity of genes involved in immune processes. Refined bioinformatic analyses suggest that these retrotransposons regulate Th1 gene cis-regulatory elements or act as Th1 gene enhancers. Thus, H3K9me3 deposition by SETDB1 ensures Th cell lineage integrity by repressing a repertoire of ERVs that have been exapted into cis-regulatory modules to shape and control the Th1 gene network.

Not all twins are monozygotic after elective single embryo transfer: analysis of 32,600 elective single embryo transfer cycles as reported to the Society for Assisted Reproductive Technology.

OBJECTIVE: To study the incidence and risk factors of multiple pregnancies after elective single ET. DESIGN: Historic cohort. SETTING: Not applicable. PATIENT(S): Women <35 years of age undergoing elective single ET entered in the SART CORS database from 2010 to 2013. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURE(S): Rate of sex discordant pregnancies. Rate of same sex pregnancies and risk factors for both same sex and sex discordant pregnancies. RESULT(S): A total of 32,600 cycles were reported to SART CORS during this time period. There were 15,143 pregnancies from which 14,888 were singletons (98.3%), 23 sex discordant (0.15%) multiple pregnancies, 226 (1.5%) sex concordant multiple pregnancies, and 6 (0.01%) pregnancies without sex information. When Weinberg's differential rule was applied, the rate of dizygotic pregnancies was calculated to be 18%. Unexplained infertility was found to be the biggest risk factor for sex discordant multiple pregnancies (adjusted odds ratio 4.33, 95% confidence interval 1.4-13.1), followed by elevated body mass index (BMI). The only risk factor found for sex concordant pregnancies was undergoing a fresh transfer (adjusted odds ratio 1.4, 95% confidence interval 1.02-1.95). CONCLUSION(S): Elective single ET improves, but does not completely eliminate the risk of multiple pregnancies. Patients should be counseled that there might be up to a ∼2% risk of multiple pregnancies, of which up to 18% can be dizygotic. Patients with elevated BMI and unexplained fertility may be at higher risk for sex discordant multiple pregnancies and patients undergoing fresh cycles may be at higher risk for sex concordant multiple pregnancies.

Antimüllerian hormone as a predictor of live birth following assisted reproduction: an analysis of 85,062 fresh and thawed cycles from the Society for Assisted Reproductive Technology Clinic Outcome Reporting System database for 2012-2013.

OBJECTIVE: To determine if serum antimüllerian hormone (AMH) is associated with and/or predictive of live birth assisted reproductive technology (ART) outcomes. DESIGN: Retrospective analysis of Society for Assisted Reproductive Technology Clinic Outcome Reporting System database from 2012 to 2013. SETTING: Not applicable. PATIENT(S): A total of 69,336 (81.8%) fresh and 15,458 (18.2%) frozen embryo transfer (FET) cycles with AMH values. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Live birth. RESULT(S): A total of 85,062 out of 259,499 (32.7%) fresh and frozen-thawed autologous non-preimplantation genetic diagnosis cycles had AMH reported for cycles over this 2-year period. Of those, 70,565 cycles which had embryo transfers were included in the analysis. Serum AMH was significantly associated with live birth outcome per transfer in both fresh and FET cycles. Multiple logistic regression demonstrated that AMH is an independent predictor of live birth in fresh transfer cycles and FET cycles when controlling for age, body mass index, race, day of transfer, and number of embryos transferred. Receiver operating characteristic (ROC) curves demonstrated that the areas under the curve (AUC) for AMH as predictors of live birth in fresh cycles and thawed cycles were 0.631 and 0.540, respectively, suggesting that AMH alone is a weak independent predictor of live birth after ART. Similar ROC curves were obtained also when elective single-embryo transfer (eSET) cycles were analyzed separately in either fresh (AUC 0.655) or FET (AUC 0.533) cycles, although AMH was not found to be an independent predictor in eSET cycles. CONCLUSION(S): AMH is a poor independent predictor of live birth outcome in either fresh or frozen embryo transfer for both eSET and non-SET transfers.

Physicians and patients' motivations to perform elective single or double-embryo transfers: A nationwide survey.

OBJECTIVES: To evaluate motivations to perform an elective single embryo transfer (e-SET). METHODS: Cross-sectional surveys to reproductive medicine specialists and to infertile patients undergoing assisted reproductive treatments. RESULTS: In the physician's survey (n = 278), we found that the main reasons for not offering e-SET were the physicians' belief that patients prefer optimizing the pregnancy rates regardless of the potential complications (57.1%). Regarding the decision making process, 76.7% of physicians thought that patients and doctors should make these decisions together and 93.3% would like to have a more formal decision-aid to help with counseling. In the patients' survey (n = 100), 21.3% chose e-SET, while 33% mentioned that complications associated to multiple pregnancies were insufficiently discussed. Among those patients, none chose to have e-SET, while 30% of those who had a full discussion selected e-SET (p = 0.05). CONCLUSIONS: Most physicians did not offer e-SET based on potential patients' negative feelings. Also, almost 30% take important decisions without the patient's participation. Patients that discussed more thoroughly this topic, more frequently selected e-SET. Almost all the physicians surveyed agreed that decision-aids could help in this important shared-decision process. PRACTICE IMPLICATIONS: Decision aids about e-SET vs DET are needed to help patients in the decision making process.

In silico probing and biological evaluation of SETDB1/ESET-targeted novel compounds that reduce tri-methylated histone H3K9 (H3K9me3) level.

ERG-associated protein with the SET domain (ESET/SET domain bifurcated 1/SETDB1/KMT1E) is a histone lysine methyltransferase (HKMT) and it preferentially tri-methylates lysine 9 of histone H3 (H3K9me3). SETDB1/ESET leads to heterochromatin condensation and epigenetic gene silencing. These functional changes are reported to correlate with Huntington's disease (HD) progression and mood-related disorders which make SETDB1/ESET a viable drug target. In this context, the present investigation was performed to identify novel peptide-competitive small molecule inhibitors of the SETDB1/ESET by a combined in silico-in vitro approach. A ligand-based pharmacophore model was built and employed for the virtual screening of ChemDiv and Asinex database. Also, a human SETDB1/ESET homology model was constructed to supplement the data further. Biological evaluation of the selected 21 candidates singled out 5 compounds exhibiting a notable reduction of the H3K9me3 level via inhibitory potential of SETDB1/ESET activity in SETDB1/ESET-inducible cell line and HD striatal cells. Later on, we identified two compounds as final hits that appear to have neuronal effects without cytotoxicity based on the result from MTT assay. These compounds hold the calibre to become the future lead compounds and can provide structural insights into more SETDB1/ESET-focused drug discovery research. Moreover, these SETDB1/ESET inhibitors may be applicable for the preclinical study to ameliorate neurodegenerative disorders via epigenetic regulation.

Comparación de resultados posterior a la transferencia electiva de embrión único euploide en ciclos frescos vs criopreservados / Results after elective single embryo transfer: Fresh euploid embryo transfer versus frozen-thawed transfer cycles

Resumen OBJETIVO: analizar las tasas de implantación y embarazo en ciclos de fertilización in vitro con transferencia electiva de un solo blastocisto, con control del factor embriónico mediante transferencia de embriones euploides. MATERIALES Y MÉTODOS: estudio retrospectivo de pacientes atendidas entre los años 2010 a 2015 en un centro privado, en protocolo de fertilización in vitro y que tuvieron, por lo menos, un embrión euploide disponible para transferencia. Para fines de estudio las pacientes se dividieron en dos grupos: 1) transferencia de embriones frescos y 2) embriones desvitrificados. Las variables categóricas se analizaron con χ2 y prueba exacta de Fisher; las variables continuas con t de Student. Se estableció significación estadística con un valor de p < 0.05. Para el análisis estadístico se usó SAS-STAT versión 9.4. RESULTADOS: se incluyeron 637 ciclos (frescos: 243 vs criopreservados: 394). La tasa de embarazo fue de 75.5% (n = 289) vs 66.3% (n = 159), embarazo clínico 62.5% (n = 235) vs 53.1% (n = 127) que fue estadísticamente significativo a favor de los ciclos criopreservados. Las tasas de embarazo múltiple fueron bajas (1.7 vs 1.6%) en ambas cohortes. CONCLUSIONES: la transferencia de un solo embrión disminuye significativamente la incidencia de embarazos múltiples y la morbilidad materna y neonatal. El mejor pronóstico en ciclos de fertilización in vitro homólogos se consigue con la transferencia de un solo embrión genéticamente equilibrado, en un ciclo posterior de preparación endometrial sintética o natural.

Abstract OBJECTIVE: To analyze the implantation and pregnancy rates in cycles of in vitro fertilization with elective transfer of a single blastocyst, with control of the embryonic factor by transfer of euploid embryos. MATERIALS AND METHODS: Retrospective analysis who included patients that underwent IVF and had at least one euploid embryo available for transfer between 2010 and 2015 on a single academic private practice. Cohorts were segregated in fresh embryo transfers (ET) vs frozen/thawed ET. Categorical variables were analyzed with χ2 and Fisher test when appropriate. Continuous variables were analyzed with Students t test. P value < 0.5 was established as statistically significant. SAS/STAT 9.4 was used for analysis. RESULTS: Six hundred and thirty-seven euploid SETs cycles (fresh cycle: n = 243; frozen/thaw cycle: n = 394) were identified. Pregnancy (75.5% (n=289) vs 66.3% (n = 159)) and clinical pregnancy rates (PR) (62.5% (n = 235) vs 53.1% (n = 127)) were statistically higher in the frozen/thaw cycles. Low rates of multiple pregnancies (1.7 and 1.6%) were observed in both cohorts. CONCLUSIONS: In one of the largest studies to date, a euploid SET during a frozen/thaw cycle showed significantly improved pregnancy and clinical PR compared to embryo transfer in fresh cycles. Single embryo transfer significantly reduces the incidence of multiple gestation and improves maternal and neonatal outcomes. An optimal outcome is achieved by the performance of a SET in FET cycles.

Canonical Wnt signalling regulates nuclear export of Setdb1 during skeletal muscle terminal differentiation.

The histone 3 lysine 9 methyltransferase Setdb1 is essential for both stem cell pluripotency and terminal differentiation of different cell types. To shed light on the roles of Setdb1 in these mutually exclusive processes, we used mouse skeletal myoblasts as a model of terminal differentiation. Ex vivo studies on isolated single myofibres showed that Setdb1 is required for adult muscle stem cells expansion following activation. In vitro studies in skeletal myoblasts confirmed that Setdb1 suppresses terminal differentiation. Genomic binding analyses showed a release of Setdb1 from selected target genes upon myoblast terminal differentiation, concomitant to a nuclear export of Setdb1 to the cytoplasm. Both genomic release and cytoplasmic Setdb1 relocalisation during differentiation were dependent on canonical Wnt signalling. Transcriptomic assays in myoblasts unravelled a significant overlap between Setdb1 and Wnt3a regulated genetic programmes. Together, our findings revealed Wnt-dependent subcellular relocalisation of Setdb1 as a novel mechanism regulating Setdb1 functions and myogenesis.

Elective single embryo transfer: Is frozen better than fresh?

OBJECTIVE: Single embryo transfer (SET) has been recommended to avoid multiple births following assisted reproductive technology (ART) procedures. Many studies have shown that frozen embryo transfer may yield better pregnancy rates than fresh embryo transfer. This study looked into pregnancy rates following fresh versus frozen single embryo transfer procedures in age-matched patients. METHODS: This retrospective case control study was carried out at a private clinic [NewLife Fertility Clinic, ON, Canada]. Patient groups included infertile women treated with IVF/ICSI and elective single embryo transfer (eSET) given either fresh or frozen embryos. Cycle outcomes were compared between patient groups matched by age. The primary endpoints were positive testing for ß-hCG and viable ongoing pregnancy. The secondary endpoints were live birth and miscarriage rates. RESULTS: A total of 583 eSET cycles (212 fresh transfer cycles and 371 frozen transfer cycles) were performed. Significantly higher pregnancy and live birth rates were observed among patients aged ≤ 39 years given frozen embryos. CONCLUSION: Frozen single embryo transfer was associated with higher pregnancy and live birth rates when compared to fresh single embryo transfer.