Validation of Non-Invasive Preimplantation Genetic Screening Using a Routine IVF Laboratory Workflow.

Embryo selection is needed to optimize the chances of pregnancy in assisted reproduction technology. This study aimed to validate non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) using a routine IVF laboratory workflow. Can niPGT-A combined with time-lapse morphokinetics provide a better embryo-selection strategy? A total of 118 spent culture mediums (SCMs) from 32 couples were collected. A total of 40 SCMs and 40 corresponding trophectoderm (TE) biopsy samples (n = 29) or arrested embryos (n = 11) were assessed for concordance. All embryos were cultured to the blastocyst stage (day 5 or 6) in a single-embryo culture time-lapse incubator. The modified multiple annealing and looping-based amplification cycle (MALBAC) single-cell whole genome amplification method was used to amplify cell-free DNA (cfDNA) from the SCM, which was then sequenced on the Illumina MiSeq system. The majority of insemination methods were conventional IVF. Low cfDNA concentrations were noted in this study. The amplification niPGT-A and conventional PGT-A was 67.7%. Based on this study, performing niPGT-A without altering the daily laboratory procedures cannot provide a precise diagnosis. However, niPGT-A can be applied in clinical IVF, enabling the addition of blastocysts with a better prediction of euploidy for transfer.

Use of Non-Destructive Measurements to Identify Cucurbit Species (Cucurbita maxima and Cucurbita moschata) Tolerant to Waterlogged Conditions.

Limited information is available regarding the physiology of squash plants grown under waterlogging stress. The objectives of this study were to investigate the growth and physiological performances of three cucurbit species, Cucurbita maxima cultivar (cv.) OK-101 (OK) and Cucurbita moschata cv. Early Price (EP) and Strong Man (SM), in response to waterlogging conditions, and to develop a precise, integrated, and quantitative non-destructive measurement of squash genotypes under stress. All tested plants were grown in a growth chamber under optimal irrigation and growth conditions for a month, and the pot plants were then subjected to non-waterlogging (control) and waterlogging treatments for periods of 1, 3, 7, and 13 days (d), followed by a 3-d post-waterlogging recovery period after water drainage. Plants with phenotypes, such as fresh weight (FW), dry weight (DW), and dry matter (DM) of shoots and roots, and various physiological systems, including relative water content (RWC), soil and plant analysis development (SPAD) chlorophyll meter, ratio of variable/maximal fluorescence (Fv/Fm), quantum photosynthetic yield (YII), normalized difference vegetation index (NDVI), and photochemical reflectance index (PRI) values, responded differently to waterlogging stress in accordance with the duration of the stress period and subsequent recovery period. When plants were treated with stress for 13 d, all plants exhibited harmful effects to their leaves compared with the control, but EP squash grew better than SM and OK squashes and exhibited stronger tolerance to waterlogging and showed less injury. Changes in the fresh weight, dry weight, and dry matter of shoots and roots indicated that OK plants suffered more severely than EP plants at the 3-d drainage period. The values of RWC, SPAD, Fv/Fm, YII, NDVI, and PRI in both SM and OK plants remarkably decreased at waterlogging at the 13-d time point compared with controls under identical time periods. However, the increased levels of SPAD, Fv/Fm, YII, NDVI, and PRI observed on 7 d or 13 d of waterlogging afforded the EP plant leaf with improved waterlogged tolerance. Significant and positive correlations were observed among NDVI and PRI with SPAD, Fv/Fm, and YII, indicating that these photosynthetic indices can be useful for developing non-destructive estimations of chlorophyll content in squashes when screening for waterlogging-tolerant plants, for establishing development practices for their cultivation in fields, and for enhanced cultivation during waterlogging in frequently flooded areas.

Upregulation of RelB in the miR-122 knockout mice contributes to increased levels of proinflammatory chemokines/cytokines in the liver and macrophages.

OBJECTIVE: MicroRNA-122 (miR-122) is the most abundant miRNA in the liver and it plays an important role in regulating liver metabolism and tumor formation. Previous studies also reveal an anti-inflammatory function of miR-122; however, relatively little is known about the mechanisms by which miR-122 suppresses inflammation. This study aims to search the effect of miR-122 on proinflammatory chemokines/cytokines production in mice. METHODS: Quantitative real-time PCR, Western blot analysis, and ELISA were performed to examine gene expression. TargetScan, miRanda, and microT v3.0 were used to search for possible miR-122 target sites in the 3'-untranslated regions (3'-UTR) of candidate genes. Luciferase reporter assay and site-directed mutagenesis were applied to verify miR-122 target sequences. LPS was applied to peritoneal macrophages and mice to evaluate inflammatory response. RESULTS: The expression of proinflammatory chemokines, including Ccl2, Ccl4, Ccl20, Cxcl2, and Cxcl10, and Relb in the livers of miR-122 knockout (KO) mice was increased. We identified Relb as a direct miR-122 target. Overexpressing RelB in the mouse liver increased the expression of Ccl2, Ccl4, Ccl20, Cxcl2, and Cxcl10. Peritoneal macrophages from miR-122 KO mice had a higher level of RelB, and they showed a stronger NF-κB activation and more TNF-α and IL-6 secretion after LPS stimulation. Overexpression of RelB in a macrophage cell line augmented LPS-induced TNF-α and IL-6 production. miR-122 KO mice showed a greatly increased mortality rate and generated a stronger and lasting inflammatory response to LPS. CONCLUSIONS: Deletion of miR-122 caused an upregulation of proinflammatory chemokines and RelB in the liver. Increased RelB may contribute to increases in these chemokine in the liver. Intriguingly, deletion of miR-122 also enhanced the sensitivity of macrophages and mice to LPS. Our results reveal that reducing RelB expression is a new mechanism by which miR-122 regulates inflammation.

Evaluation of the effect of the elective blastocyst-stage embryo transfer and freezing strategy on the abandonment of frozen embryos under the Taiwan National Assisted Reproduction Act.

PURPOSE: To evaluate the relationship between elective blastocyst transfer, freezing strategy, and the abandonment of frozen embryos with a storage time limit of 10 years as specified in the National Assisted Reproduction Act of Taiwan. METHODS: This two-phase retrospective cohort study was conducted at a single tertiary center, Kaohsiung Chang Gung Memorial Hospital (KCGMH), in 2019. Participants were selected from a data registry containing 4167 fresh IVF cycles, including phase 1 cycles from 1999 to 2009 and phase 2 cycles from 2010 to 2014, at KCGMH. RESULTS: In phase 1, embryo abandonment was associated with the production of more mature oocytes and embryos, the freezing of more embryos, young female age, blastocyst transfer, and positive pregnancy results. After adjustment for confounding factors, only positive pregnancy results (adjusted odds ratio [aOR] 4.38, 95% confidence interval [CI] 3.17, 6.04), the freezing of ≥ 2 embryos (aOR 3.68, 95% CI 3.10, 4.38), the production of ≥ 6 embryos (aOR 1.68, 95% CI 1.03, 2.73), and the use blastocyst transfer (aOR 2.46, 95% CI 1.64, 3.69) remained significantly associated with embryo abandonment. The factors associated with embryo abandonment or possible abandonment were similar in phase 2. CONCLUSION: For elective blastocyst stage transfer and a freezing strategy performed according to the Taiwan National Assisted Reproduction Act, a young female age ≤ 35 with positive pregnancy status due to the original IVF treatment, the production of ≥ 6 embryos, and the cryopreservation of ≥ 2 blastocysts may increase the likelihood of abandoning embryos in the future.

Particulate matter characteristics during agricultural waste burning in Taichung City, Taiwan.

Agricultural waste burning is performed after harvest periods in June and November in Taiwan. Typically, farmers use open burning to dispose of excess rice straw. PM(2.5) and PM(2.5-10) measurements were conducted at National Chung Hsing University in Taichung City using a dichotomous sampler. The sampling times were during straw burning periods after rice harvest during 2002-2005. Ionic species including SO(4)(2-), NO(3)(-), NH(4)(+), K(+), Ca(2+), Cl(-) and Na(+) and carbonaceous species (EC and OC) in PM(2.5) and PM(2.5-10) were analyzed. The results showed that the average PM(2.5) and PM(2.5-10) concentrations were 123.6 and 31.5 microg m(-3) during agricultural waste burning periods and 32.6 and 21.4 microg m(-3) during non-waste burning periods, respectively. The fine aerosol ionic species including Cl(-), K(+) and NO(3)(-) increased 11.0, 6.7 and 5.5 times during agricultural burning periods compared with periods when agricultural waste burning is not performed. K(+) was found mainly in the fine mode during agricultural burning. High nitrogen oxidation ratio was found during agricultural waste burning periods which might be caused by the conversion of Nitrogen dioxide (NO(2)) to NO(3)(-). It is concluded that agricultural waste burning with low dispersion often causes high PM(2.5) and gases pollutant events.