Clinical re-biopsy of segmental gains-the primary source of preimplantation genetic testing false positives.

PURPOSE: Does re-biopsy of blastocysts classified as abnormal (ABN) due to segmental aneuploidy (SA) have clinical utility? METHODS: The live birth (LB) outcomes of mosaic SAs, compared to other categories, were determined after transfer of 3084 PGT-A tested blastocysts. An initial 12-month trial thawed 111 blastocysts classified as ABN due to a SA for clinical re-biopsy, with an additional 58 from a subsequent 16-month revised protocol. Where re-biopsy failed to corroborate the original classification, blastocysts were reported as mosaic and suitable for clinical use. RESULTS: Segmental mosaics had a LB rate (54.1%) which was indistinguishable from that of euploid (53.7%). Numeric mosaics had statistically significant (P < 0.05) reduced LB rates compared to euploid, with high-level numerics (19.2%) also exhibiting a significant reduction compared to low level (42.3%). Of the initial 111 blastocysts with SAs, 85 could be re-biopsied. Segmental gains became suitable for re-biopsy at a high rate (90.9%), with 84.2% (16/19) of these reclassified as mosaic. Only 73.0% of deletions and complex changes were suitable for re-biopsy, of which 73.0% (46/63) were confirmed ABN. The subsequent 16-month period primarily focused on gains, confirming the high rate at which they can be reclassified as clinically useable. CONCLUSIONS: Blastocysts harboring mosaic segmental duplications, rather than SAs in general, are the primary source of false-positive PGT-A results and represent a category with a LB rate similar to that of euploid. A high degree of confidence in the reliability of PGT-A results can be maintained by performing confirmatory clinical TE biopsies.

Moonlighting glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is required for efficient hepatitis C virus and dengue virus infections in human Huh-7.5.1 cells.

Hijacking of cellular biosynthetic pathways by human enveloped viruses is a shared molecular event essential for the viral lifecycle. In this study, the accumulating evidence of the importance of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the host secretory pathway led us to hypothesize that this moonlighting enzyme could play a key role in the lifecycle steps of two important Flaviviridae members, hepatitis C virus (HCV) and dengue virus (DENV). We used short interfering RNA (siRNA)-mediated knockdown of human GAPDH in Huh-7.5.1 cells- both pre- and post-HCV infection- to demonstrate that GAPDH is a host factor for HCV infection. siRNA-induced GAPDH knockdown performed pre-HCV infection inhibits HCV core production in infected cells and leads to a decrease in infectivity of the HCV-infected cell supernatants. siRNA-induced GAPDH knockdown performed post-HCV infection does not have an effect on HCV core abundance in infected cells, but does lead to a decrease in infectivity of the HCV-infected cell supernatants. Exogenous expression of V5-tagged human GAPDH, pre- and post-infection, increases the infectivity of HCV-infected cell supernatants, suggesting a role for GAPDH during HCV post-replication steps. Interestingly, siRNA-induced GAPDH knockdown in HCV replicon-harbouring cells had no effect on viral RNA replication. Importantly, we confirmed the important role of GAPDH in the HCV lifecycle using Huh-7-derived stable GAPDH-knockdown clones. Finally, siRNA-induced GAPDH knockdown inhibits intracellular DENV-2 E glycoprotein production in infected cells. Collectively, our findings suggest that the moonlighting enzyme, GAPDH, is an important host factor for HCV infection, and they support its potential role in the DENV lifecycle.

Clinical use of monopronucleated zygotes following blastocyst culture and preimplantation genetic screening, including verification of biparental chromosome inheritance.

In assisted reproduction, embryos derived from monopronucleated (1PN) zygotes are considered abnormal and unsuitable for clinical use. Outcomes of 1PN-derived embryos designated for preimplantation genetic screening (PGS) were analysed. These embryos, especially from intracytoplasmic sperm injection (ICSI), were found to have a low developmental potential; 1PN and 2PN day 5 blastocyst development for IVF was 14.8% versus 36.4% (P < 0.0001), and for ICSI, 6.6% versus 34.0% (P < 0.0001), respectively. With the use of comparative genomic hybridization or next-generation sequencing, PGS was successfully carried out for 74 IVF and 32 ICSI 1PN-derived blastocysts, revealing adjusted abnormality rates of 39.7% and 40.6%, respectively. Additionally, 24 female 1PN-derived blastocysts underwent testing for biparental inheritance, with one ICSI-derived embryo demonstrating paternal only contribution, thus presenting a risk for complete hydatidiform molar pregnancy. Single embryo transfer of 20 IVF and six ICSI 1PN-derived blastocysts with no detectable abnormalities resulted in nine clinical pregnancies. Six have been delivered and three are ongoing, with no anomalies reported to date. The limitation of this study is that pronuclear status was determined through one static observation. The results suggest that 1PN-derived embryos, in which euploidy and biparental inheritance have been established, can provide a source of clinically useful embryos.

The original Beckman Coulter Generation II assay significantly underestimates AMH levels compared with the revised protocol.

PURPOSE: Anti-Müllerian hormone (AMH) is used as a marker for ovarian reserve. Since 2011, the standard test for AMH has been the Beckman Coulter Generation (Gen) II assay. However, in July 2013, the protocol was revised due to falsely low readings. The aim of this study was to compare AMH levels measured with the original and revised Gen II assay and to establish a fertile female reference range for the revised protocol. METHODS: Serum AMH levels were measured for 492 natural conception first trimester pregnant women using the original and revised Gen II assay. RESULTS: The original protocol significantly underestimated AMH levels compared with the revised protocol (p < 0.001), the median being 8.4 and 14.2 pmol/L, respectively. In all samples with detectable AMH levels, the revised protocol yielded a higher concentration compared with the original protocol, the magnitude shift ranging from 3.4 to 283.3 % (median 68.0 %). AMH levels measured with the revised protocol were collated to generate an age-specific reference range, with median levels peaking at 27 years then declining with advancing age. The median AMH concentration for ages 20-24 was 17.3 pmol/L, ages 25-29 was 20.5 pmol/L, ages 30-34 was 17.8 pmol/L, ages 35-39 was 10.8 pmol/L, and ages 40-44 was 6.1 pmol/L. CONCLUSIONS: Our study demonstrated that the original Gen II assay significantly underestimated AMH levels, suggesting caution is required when interpreting literature and testing results achieved with this assay. We also established the revised Gen II assay reference range for AMH in women with unassisted proven fertility.

Embryo vitrification using a novel semi-automated closed system yields in vitro outcomes equivalent to the manual Cryotop method.

STUDY QUESTION: Can the equilibration steps prior to embryo vitrification be automated? SUMMARY ANSWER: We have developed the 'Gavi' system which automatically performs equilibration steps before closed system vitrification on up to four embryos at a time and gives in vitro outcomes equivalent to the manual Cryotop method. WHAT IS KNOWN ALREADY: Embryo cryopreservation is an essential component of a successful assisted reproduction clinic, with vitrification providing excellent embryo survival and pregnancy outcomes. However, vitrification is a manual, labour-intensive and highly skilled procedure, and results can vary between embryologists and clinics. A closed system whereby the embryo does not come in direct contact with liquid nitrogen is preferred by many clinics and is a regulatory requirement in some countries. STUDY DESIGN, SIZE, DURATION: The Gavi system, an automation instrument with a novel closed system device, was used to equilibrate embryos prior to vitrification. Outcomes for embryos automatically processed with the Gavi system were compared with those processed with the manual Cryotop method and with fresh (non-vitrified) controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: The efficacy of the Gavi system (Alpha model) was assessed for mouse (Quackenbush Swiss and F1 C57BL/6J x CBA) zygotes, cleavage stage embryos and blastocysts, and for donated human vitrified-warmed blastocysts. The main outcomes assessed included recovery, survival and in vitro embryo development after vitrification-warming. Cooling and warming rates were measured using a thermocouple probe. MAIN RESULTS AND THE ROLE OF CHANCE: Mouse embryos vitrified after processing with the automated Gavi system achieved equivalent in vitro outcomes to that of Cryotop controls. For example, for mouse blastocysts both the Gavi system (n = 176) and manual Cryotop method (n = 172) gave a 99% recovery rate, of which 54 and 50%, respectively, progressed to fully hatched blastocysts 48 h after warming. The outcomes for human blastocysts processed with the Gavi system (n = 23) were also equivalent to Cryotop controls (n = 13) including 100% recovery for both groups, of which 17 and 15%, respectively, progressed to fully hatched blastocysts 48 h after warming. The cooling and warming rates achieved with the Gavi system were 14 136°C/min and 11 239°C/min, respectively. LIMITATIONS, REASONS FOR CAUTION: Testing of the Gavi system described here was limited to in vitro development of embryos from two mouse strains and a limited number of human embryos. Validation of Gavi system advanced production models is now required to confirm the success of semi-automated vitrification, including clinical evaluation of pregnancy outcomes from the transfer of Gavi vitrified-warmed human embryos. WIDER IMPLICATIONS OF THE FINDINGS: The Gavi system has the potential to revolutionize and standardize vitrification of embryos and oocytes. The success of the Gavi system shows that it is possible to semi-automate complicated labour-intensive ART methods and processes, and opens up the possibility for further improvements in clinical outcomes and efficiencies in the ART clinic. STUDY FUNDING/COMPETING INTERESTS: This study was funded by Genea Ltd. S.B., N.M.T., T.T.P., S.J.M., M.C.B. and T.S. are shareholders of Genea Ltd. E.V., C.H., C.L., S.R.L. and S.M.D. are shareholders of Planet Innovation Pty Ltd. The remaining authors are employees of either Genea Ltd. or Planet Innovation Pty Ltd.

Spatiotemporal resolution in high-speed atomic force microscopy for studying biological macromolecules in action.

High-speed atomic force microscopy (HS-AFM) is a unique approach that allows direct real-time visualization of biological macromolecules in action under near-physiological conditions, without any chemical labeling. Typically, the temporal resolution is sub-100 ms, and the spatial resolution is 2-3 nm in the lateral direction and ∼0.1 nm in the vertical direction. A wide range of biomolecular systems and their dynamic processes have been studied by HS-AFM, providing deep mechanistic insights into how biomolecules function. However, the level of mechanistic detail gleaned from an HS-AFM experiment critically depends on the spatiotemporal resolution of the system. In this review article, we explain the principle of HS-AFM and describe how the resolution is determined. We also discuss recent attempts to improve the resolution of HS-AFM to further extend the observable range of biological phenomena.

Adult chronic kidney disease: neurocognition in chronic renal failure.

In recent years there has been a keen interest in the neurocognitive sequelae of renal failure and subsequent end-stage renal disease given its increasing prevalence and incidence. This review article summarizes the relevant information on cognitive functioning in chronic kidney disease in adults before the initiation of dialysis, after the initiation of dialysis, and after renal transplantation. In general, compared to pre-dialysis, there is an improvement in cognitive function after the institution of dialysis and further improvement after renal transplantation. Throughout the paper an attempt is made to highlight the importance of considering disease related variables in the neuropsychological assessment of individuals with chronic kidney disease. The paper concludes with a discussion of future avenues of research.

The First Asian, Single-Center Experience of Blastocyst Preimplantation Genetic Diagnosis with HLA Matching in Thailand for the Prevention of Thalassemia and Subsequent Curative Hematopoietic Stem Cell Transplantation of Twelve Affected Siblings.

RESULTS: In 221 cycles from 138 patients (104 cycles requiring HLA matching), 90.5% had embryo(s) biopsied for genetic testing. There were 119 embryo transfers for thalassemia (76) and thalassemia-HLA cases (43), respectively, resulting in overall clinical pregnancy rates of 54.6%, implantation rates of 45.7%, and live birth rates of 44.1%. Our dataset included fifteen PGD-HLA live births with successful HSCT in twelve affected siblings, 67% using umbilical cord blood stem cells (UCBSC) as the only SC source. CONCLUSIONS: We report favorable thalassemia PGD and PGD-HLA laboratory and clinical outcomes from a single center. The ultimate success in PGD-HLA is of course the cure of a thalassemia-affected sibling by HSCT. Our PGD-HLA HSCT series is the first and largest performed entirely in Asia with twelve successful and two pending cures and predominant UCBSC use.

What next for preimplantation genetic screening (PGS)? Experience with blastocyst biopsy and testing for aneuploidy.

Blastocysts more commonly have a normal karyotype than cleavage-stage embryos do. Moreover, blastocysts have also made a metabolic transition from catabolism and recycling of the oocyte's reserves and resources, processes that fuel the first 3 days of cleavage. Although not all blastocysts are karyotypically equal, it is still to be determined to what extent a mosaic karyotype might be a normal feature among embryos, both at the cleavage stage and the blastocyst stage--and when looking for karyotypic abnormalities by embryo biopsy might help the chance of implantation rather than harm it. It is also still impractical to look at all the chromosomes that can, through their aneuploidy, stand in the way of successful embryonic and fetal development. We report a randomized clinical trial of blastocyst biopsy followed by preimplantation genetic screening (PGS) for aneuploidy using 5-colour FISH. The trial was suspended and then terminated early when we were unable to show an advantage for PGS. If we are correct in assuming that mitotic non-disjunction is common by the stage of the blastocyst (and that it is much less ominous than meiotic non-disjunction), then further studies of effective PGS of blastocysts for aneuploidy require methods of analysis that cover all the chromosomes and can differentiate the triallelic and monoallelic states of meiotically derived aneuploidies from the biallelic state of mitotic aneuploidies.

Impact of multiple blastocyst biopsy and vitrification-warming procedures on pregnancy outcomes.

OBJECTIVE: To assess the impact of multiple blastocyst biopsy and vitrification-warming procedures on clinical outcomes. DESIGN: Retrospective study. SETTING: Private fertility clinic. PATIENT(S): Preimplantation genetic diagnosis (PGD) patients undergoing comprehensive chromosome screening, including monogenic disorder and chromosome rearrangement cases. INTERVENTION(S): Warming and transfer of euploid blastocysts biopsied and vitrified-warmed once (group 1 [G1, control]; n = 2,130), biopsied once but vitrified-warmed twice (group 2 [G2]; n = 34), or biopsied and vitrified-warmed twice (group 3 [G3]; n = 29). MAIN OUTCOME MEASURE(S): Thaw (for transfer) survival rate and clinical pregnancy rate (CPR). RESULT(S): The thaw survival rates were 98.4% for G1, 97.3% for G2, and 93.3% for G3, with once biopsied and vitrified-warmed embryos being significantly higher than twice biopsied and vitrified-warmed embryos (G1 vs. G3; P=.032). There was a slight reduction in CPR with an additional vitrification-warming (G1 54.3% vs. G2 47.1%) and larger reduction with an additional embryo biopsy (G2 47.1% vs. G3 31.0%), but neither difference was statistically significant. However, the combined effect of both additional biopsy and vitrification-warming resulted in a significantly reduced CPR (G1 54.3% vs. G3 31.0%; P=.013). CONCLUSION(S): This study indicates that blastocysts biopsied and vitrified-warmed twice have reduced clinical outcomes compared with blastocysts biopsied and vitrified-warmed once. PGD patients should be advised that performing a second biopsy and vitrification-warming in cases of failure to obtain a result from initial biopsy will reduce the chance of pregnancy. Patients with inherited disorders may elect to proceed with the second biopsy and vitrification to avoid transfer of embryos with the genetic condition.

Human Subtilisin Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P) regulates cytoplasmic lipid droplet abundance: A potential target for indirect-acting anti-dengue virus agents.

Viral hijacking and manipulation of host-cell biosynthetic pathways by human enveloped viruses are shared molecular events essential for the viral lifecycle. For Flaviviridae members such as hepatitis C virus and dengue virus (DENV), one of the key subsets of cellular pathways that undergo manipulation is the lipid metabolic pathways, underlining the importance of cellular lipids and, in particular, lipid droplets (LDs) in viral infection. Here, we hypothesize that targeting cellular enzymes that act as key regulators of lipid homeostasis and LD formation could represent a powerful approach to developing a novel class of broad-spectrum antivirals against infection associated with all DENV serotypes (1-4) circulating around the world. Using PF-429242, an active-site-directed inhibitor of SKI-1/S1P, we demonstrate that inhibition of SKI-1/S1P enzymatic activity in human hepatoma Huh-7.5.1 cells results in a robust reduction of the LD numbers and LD-positive areas and provides a means of effectively inhibiting infection by DENV (1-4). Pre-treatment of Huh-7.5.1 cells with PF-429242 results in a dose-dependent inhibition of DENV infection [median inhibitory dose (EC50) = 1.2 microM; median cytotoxic dose (CC50) = 81 microM; selectivity index (SI) = 68)] and a ~3-log decrease in DENV-2 titer with 20 microM of PF-429242. Post-treatment of DENV-2 infected Huh-7.5.1 cells with PF-429242 does not affect viral RNA abundance, but it does compromise the assembly and/or release of infectious virus particles. PF-429242 antiviral activity is reversed by exogenous oleic acid, which acts as an inducer of LD formation in PF-429242-treated and non-treated control cells. Collectively, our results demonstrate that human SKI-1/S1P is a potential target for indirect-acting pan-serotypic anti-DENV agents and reveal new therapeutic opportunities associated with the use of lipid-modulating drugs for controlling DENV infection.

A Parallel Synthesis Approach to the Identification of Novel Diheteroarylamide-Based Compounds Blocking HIV Replication: Potential Inhibitors of HIV-1 Pre-mRNA Alternative Splicing.

A 256-compound library was evaluated in an anti-HIV screen to identify structural "mimics" of the fused tetracyclic indole compound 1 (IDC16) that conserve its anti-HIV activity without associated cytotoxicity. Four diheteroarylamide-type compounds, containing a common 5-nitroisobenzothiazole motif, were identified as active. In subsequent screens, the most potent compound 9 (1C8) was active against wild-type HIV-1IIIB (subtype B, X4-tropic) and HIV-1 97USSN54 (subtype A, R5-tropic) with EC50's of 0.6 and 0.9 µM, respectively. Compound 9 also inhibited HIV strains resistant to drugs targeting HIV reverse transcriptase, protease, integrase, and coreceptor CCR5 with EC50's ranging from 0.9 to 1.5 µM. The CC50 value obtained in a cytotoxicity assay for compound 9 was >100 µM, corresponding to a therapeutic index (CC50/EC50) of approximately 100. Further comparison studies revealed that, whereas the anti-HIV activity for compound 9 and the parent molecule 1 are similar, the cytotoxic effect for compound 9 was, as planned, markedly suppressed.

Moving to blastocyst biopsy for preimplantation genetic diagnosis and single embryo transfer at Sydney IVF.

Developments at Sydney IVF in the successful culture of blastocysts, combined with day 5 or 6 blastocyst biopsy and blastocyst cryostorage after biopsy, permit up to five or six cells to be genetically tested, leaving the inner cell mass intact and enabling embryos to be electively transferred one at a time. These advantages can be obtained without diminishing the chance of pregnancy compared with cleavage-stage biopsy and testing.

Single-embryo transfer of vitrified-warmed blastocysts yields equivalent live-birth rates and improved neonatal outcomes compared with fresh transfers.

OBJECTIVE: To compare pregnancy and neonatal outcomes after fresh and vitrified-warmed single-blastocyst transfers. DESIGN: Retrospective study. SETTING: Private in vitro fertilization (IVF) clinic. PATIENT(S): 1,209 infertile patients who underwent a total of 1,157 fresh and 645 vitrified-warmed embryo transfers. INTERVENTION(S): Day-5 single-blastocyst transfers using fresh or vitrified-warmed (Cryotop method) grade I and grade II embryos. MAIN OUTCOME MEASURE(S): Fetal heart pregnancy rate, live-birth rate, gestational age, and live-birth weight. RESULT(S): The overall blastocyst thaw survival rate was 94.4% and was not significantly different between blastocyst grades or developmental stages. Similar clinical outcomes were achieved for fresh and vitrified-warmed blastocyst transfers; for example, grade I blastocysts had a live-birth rate of 52.8% versus 55.3%, respectively, and grade II blastocysts had a rate of 34.9% versus 30.4%, respectively. Significantly improved neonatal outcomes were evident for vitrified-warmed blastocyst transfers for gestational age, being on average 0.3 weeks longer, and for live-birth weight with babies born on average 145 g heavier (3,296 g versus 3,441 g for fresh and vitrified-warmed groups, respectively), as compared with fresh transfers. CONCLUSION(S): Embryo transfer of vitrified-warmed blastocysts yields equivalent live-birth rates and improved neonatal outcomes compared with fresh transfers.

The genetic screening of preimplantation embryos by comparative genomic hybridisation.

Comparative genomic hybridization (CGH) is an indirect DNA-based test which allows for the accurate analysis of aneuploidy involving any of the 24 types of chromosomes present (22 autosomes and the X and Y sex chromosomes). Traditionally, embryos have been screened using fluorescence in situ hybridization (FISH)--a technique that was limited in the number of chromosomes able to be identified in any one sample. Early CGH reports on aneuploidy in preimplantation embryos showed that any of the 24 chromosomes could be involved and so FISH methods were going to be ineffective in screening out abnormal embryos. Our results from routine clinical application of array CGH in preimplantation genetic diagnosis (PGD) patients confirm previous reports on patterns of chromosomal contribution to aneuploidy. The pregnancy outcomes following embryo transfer also indicate that despite the requirement to freeze embryos, rates are encouraging, and successful ongoing pregnancies can be achieved.

Pregnancies and live births after trophectoderm biopsy and preimplantation genetic testing of human blastocysts.

OBJECTIVE: To compare multiple-cell trophectoderm biopsy for preimplantation genetic diagnosis (PGD) from day-5 blastocysts with previously published experience with day-3 cleavage-stage embryos. DESIGN: Retrospective review of laboratory and clinical experience. SETTING: Sydney IVF, a private clinic in Australia. PATIENT(S): Preimplantation genetic diagnosis (PGD) patients age < 44 years with at least one IVF blastocyst suitable for biopsy, recruited from January 2002 through August 2004. INTERVENTION(S): Biopsy of trophectoderm from blastocysts on day 5 or 6, with same-day PGD for mutation testing, translocation testing, aneuploidy screening or sex selection. Spare, normal biopsied blastocysts were cryostored for possible later transfer. MAIN OUTCOME MEASURE(S): Fetal heart-positive pregnancy rate and accumulating live birth rate after adding results from biopsied fresh and frozen blastocysts for particular couples. RESULT(S): In 231 started PGD treatment cycles, unambiguous results were obtained from 974 of 1,050 biopsied blastocysts (93%); all blastocysts survived the biopsy procedure by reconstitution of their blastocele. One hundred nineteen women (median age, 36 years) have had 127 blastocysts transferred fresh (fetal heart-positive implantation rate, 41%). Of 146 blastocysts cryostored, 27 have been thawed (all with > 50% cell survival) and 24 transferred (implantation rate, 26%). To date, 53 pregnancies have been delivered or are ongoing, with an additional 4 clinical miscarriages (7%) and 6 subclinical miscarriages (total miscarriage rate, including biochemical pregnancies, 16%). There were no twin pregnancies. CONCLUSION(S): With technically appropriate blastocyst culture and freezing, blastocyst biopsy and cryostorage and later transfer of biopsied blastocysts is shown to be a practical and probably preferable path to preimplantation genetic testing of embryos compared with cleavage-stage embryo biopsy, being accompanied by a high implantation rate (and hence more conducive to elective single embryo transfer) and by a low rate of twinning and miscarriage.