Parental methylome reprogramming in human uniparental blastocysts reveals germline memory transition.

Uniparental embryos derived from only the mother (gynogenetic [GG]) or the father (androgenetic [AG]) are unique models for studying genomic imprinting and parental contributions to embryonic development. Human parthenogenetic embryos can be obtained following artificial activation of unfertilized oocytes, but the production of AG embryos by injection of two sperm into one denucleated oocyte leads to an extra centriole, resulting in multipolar spindles, abnormal cell division, and developmental defects. Here, we improved androgenote production by transferring the male pronucleus from one zygote into another haploid androgenote to prevent extra centrioles and successfully generated human diploid AG embryos capable of developing into blastocysts with an identifiable inner cell mass (ICM) and trophectoderm (TE). The GG embryos were also generated. The zygotic genome was successfully activated in both the AG and GG embryos. DNA methylome analysis showed that the GG blastocysts partially retain the oocyte transcription-dependent methylation pattern, whereas the AG blastocyst methylome showed more extensive demethylation. The methylation states of most known imprinted differentially methylated regions (DMRs) were recapitulated in the AG and GG blastocysts. Novel candidate imprinted DMRs were also identified. The production of uniparental human embryos followed by transcriptome and methylome analysis is valuable for identifying parental contributions and epigenome memory transitions during early human development.

Publisher Correction: Chromatin analysis in human early development reveals epigenetic transition during ZGA.

In this Letter, the 'Open chromatin' label in Fig. 4a should have been centred above the first three columns, and the black horizontal line underneath the label should have been removed. In addition, there should have been a vertical black line between the last two sets of panels for consistency. Minor changes have also been made to Fig. 1 and to the legend of Fig. 3. These errrors have been corrected online, and see Supplementary Information to the accompanying Amendment for the original Fig. 4.

Chromatin analysis in human early development reveals epigenetic transition during ZGA.

Upon fertilization, drastic chromatin reorganization occurs during preimplantation development 1 . However, the global chromatin landscape and its molecular dynamics in this period remain largely unexplored in humans. Here we investigate chromatin states in human preimplantation development using an improved assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) 2 . We find widespread accessible chromatin regions in early human embryos that overlap extensively with putative cis-regulatory sequences and transposable elements. Integrative analyses show both conservation and divergence in regulatory circuitry between human and mouse early development, and between human pluripotency in vivo and human embryonic stem cells. In addition, we find widespread open chromatin regions before zygotic genome activation (ZGA). The accessible chromatin loci are readily found at CpG-rich promoters. Unexpectedly, many others reside in distal regions that overlap with DNA hypomethylated domains in human oocytes and are enriched for transcription factor-binding sites. A large portion of these regions then become inaccessible after ZGA in a transcription-dependent manner. Notably, such extensive chromatin reorganization during ZGA is conserved in mice and correlates with the reprogramming of the non-canonical histone mark H3K4me3, which is uniquely linked to genome silencing3-5. Taken together, these data not only reveal a conserved principle that underlies the chromatin transition during mammalian ZGA, but also help to advance our understanding of epigenetic reprogramming during human early development and in vitro fertilization.

Effective protection: the embryonic development and clinical outcomes of emergency vitrification of 1246 oocytes and Day 0-Day 5 embryos in a natural disaster.

STUDY QUESTION: Can emergency vitrification protect embryos and oocytes during natural disasters or other events that prevent normal practice to achieve satisfactory embryonic development and clinical outcomes at a later time? SUMMARY ANSWER: Emergency vitrification of oocytes and Day 0-Day 5 (D0-D5) embryos during disasters is a safe and effective protective measure. WHAT IS KNOWN ALREADY: When some destructive events such as floods, earthquakes, tsunamis, and other accidents occur, emergency vitrification in embryo laboratories to protect human embryos, oocytes, and sperm is one of the important measures of an IVF emergency plan. However, there are few detailed reports on emergency vitrification in a state of disaster, especially about oocytes and D0 zygotes. Therefore, the effectiveness and safety of emergency vitrification of oocytes and D0-D5 embryos in disaster states are still unclear. STUDY DESIGN, SIZE, DURATION: A retrospective study was made in the Reproductive Medicine Center of the First Affiliated Hospital of Zhengzhou University from January 2018 to November 2022. The record rainstorms in Zhengzhou, China, caused severe flooding, traffic disruptions, and power outages. From 17:30, 20 July 2021 to 17:30, 21 July 2021, 1246 oocytes and D0-D5 embryos of 155 patients were vitrified whilst the laboratory had only an emergency power supply. PARTICIPANTS/MATERIALS, SETTING, METHODS: As of 21 December 2021, 1149 emergency vitrified oocytes and D0-D5 embryos of 124 patients underwent frozen-thawed embryo transfer (FET). They were divided into the following four groups according to the days of embryo culture in vitro: oocyte group, Day 0-Day 1 (D0-D1) group, Day 2-Day 3 (D2-D3) group, and Day 4-Day 5 (D4-D5) group. Control groups for each were selected from fresh cycle patients who underwent IVF/ICSI from January 2018 to October 2021. Control and emergency vitrification patients were matched on criteria that included age, fertilization method, days of embryonic development, and number and grade of transferred embryos. A total of 493 control patients were randomly selected from the eligible patients and matched with the emergency vitrification groups in a ratio of 4:1. The results of assisted reproduction and follow-up of pregnancy were analyzed. The embryonic development, clinical outcomes, and birth outcomes in each group were statistically analyzed. MAIN RESULTS AND THE ROLE OF CHANCE: A significant difference was observed in fertilization rate (81% versus 72%, P = 0.022) between the oocyte group and the control group. Significant differences were also observed in the monozygotic twin pregnancy rate (10% versus 0%, P = 0.038) and ectopic pregnancy rate (5% versus 0%, P = 0.039) between the D0-D1 group and the control group. No significant differences (P > 0.05) were observed between vitrified oocytes/D0-D1 embryos/D2-D3 embryos and the control group on the number of high-quality embryos (3.17 ± 3.00 versus 3.84 ± 3.01, P = 0.346; 5.04 ± 3.66 versus 4.56 ± 2.87, P = 0.346; 4.85 ± 5.36 versus 5.04 ± 4.64, P = 0.839), the number of usable blastocysts (1.22 ± 1.78 versus 1.21 ± 2.03, P = 0.981; 2.16 ± 2.26 versus 1.55 ± 2.08, P = 0.090; 2.82 ± 3.23 versus 2.58 ± 3.32, P = 0.706), clinical pregnancy rate (56% versus 57%, P = 0.915; 55% versus 55%, P = 1.000; 40% versus 50%, P = 0.488), miscarriage rate (30% versus 15%, P = 0.496; 5% versus 11%, P = 0.678; 17% versus 20%, P = 1.000), and live birth rate (39% versus 49%, P = 0.460; 53% versus 50%, P = 0.772; 33% versus 40%, P = 0.635). No significant differences (P > 0.05) were observed between the D4-D5 group and the control group on clinical pregnancy rate (40% versus 55%, P = 0.645), miscarriage rate (0% versus 18%, P = 1.000), and live birth rate (40% versus 45%, P = 1.000). LIMITATIONS, REASONS FOR CAUTION: The retrospective study design is a limitation. The timing and extent of natural disasters are unpredictable, so the sample size of vitrified oocytes, zygotes, and embryos is beyond experimental control. WIDER IMPLICATIONS OF THE FINDINGS: This study is the first study analyzing embryonic development, clinical outcomes, and birth outcomes of large samples of oocytes, D0 zygotes, and D1-D5 embryos after emergency vitrification under the disaster conditions. The results show that emergency vitrification is a safe and effective protective measure applicable to oocytes and D0-D5 embryos. The embryology laboratories need to be equipped with an emergency uninterrupted power supply capable of delivering for 6-8 h at full load. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (grant 81871206). The authors declare that they have no conflicts of interest. All authors have completed the ICMJE Disclosure form. TRIAL REGISTRATION NUMBER: N/A.

High sperm DNA fragmentation increased embryo aneuploidy rate in patients undergoing preimplantation genetic testing.

RESEARCH QUESTION: Is high sperm DNA fragmentation (SDF) associated with a high embryonic aneuploidy rate in patients undergoing intracytoplasmic sperm injection (ICSI)-preimplantation genetic testing (PGT)? DESIGN: This was a retrospective study of 426 couples with normal karyotypes undergoing ICSI-PGT at the authors' centre from March 2017 to March 2021. SDF was assessed using the sperm chromatin structure assay. The population was divided into low and high SDF groups according to cut-off values found by the receiver operating characteristic (ROC) curve. A 1:1 ratio propensity score matching (PSM) method was used to control for potential confounding factors, and a generalized linear mixed model was established to evaluate the relationship between SDF and the embryonic aneuploidy rate. RESULTS: The ROC curve indicated a threshold of 30%. In total, 132 couples were included after PSM, and the high SDF group (>30%) had significantly higher SDF (40.74% ± 9.78% versus 15.54% ± 7.86%, P < 0.001) and a higher embryo aneuploidy rate (69.36% versus 53.96%, P < 0.001) compared with the low SDF group (≤30%). The two pronuclear fertilization rate, cleavage rate, rate of high-quality embryos at day 3 rate, blastocyst rate, biochemical pregnancy rate, clinical pregnancy rate, miscarriage rate, live birth rate, caesarean section rate, preterm birth rate, singleton rate and low birthweight rate were similar in both groups (P > 0.05). After PSM, SDF > 30% was significantly correlated with an increased embryo aneuploidy rate after adjusting for all confounding variables (adjusted odds ratio 1.70, 95% CI 1.00-2.88, P = 0.049). CONCLUSIONS: SDF > 30% was associated with an increased embryo aneuploidy rate in couples with normal karyotypes undergoing PGT, but did not affect embryonic and clinical outcomes after transfer of euploid embryos.

Pharmacognostic standardization and machine learning-based investigations on Akebia quinata and Akebia trifoliata.

Currently, Akebiae Caulis is being used in clinical practice, but there are few reseaches on its different varieties. To ensure the accuracy and effectiveness of clinical practice, this study distinguished the Akebia quinata (Thunb.) Decne. and Akebia trifoliata (Thunb.) Koidz, using organoleptic evaluation, microscopic observation, fluorescence reaction, physicochemical properties, thin-layer chromatography, IR spectroscopy, HPLC, four machine learning models, and in vitro antioxidant methods. Analysis of the powders of these two varieties using optical microscopy revealed the presence of starch granules, cork cells, crystal fibers, scalariform vessels, and wood fibers. Scanning electron microscopy revealed the presence of scalariform vessels, pitted vessels, wood fibers, and calcium oxalate crystals. Several tissues, including the cork layer, fiber population, cortex, phloem, pith, xylem, and ray, were found in the transverse section. In addition, thin-layer chromatography was used to identify two components: oleanolic acid and calceolarioside B; 11 common peaks were identified in 15 batches of SAQ and 5 batches of SAT by using HPLC. Support vector machine, BP neural networks, and GA-bp neural networks were able to predict 100% accurately of the different origins of stem of Akebia quinate (Thunb.) Decne (SAQ) and Akebia trifoliata (Thunb.) Koidz (SAT). Extreme learning machine achieved a correct rate of 87.5%. Meanwhile, Fourier-transform infrared spectroscopy fingerprint identified nine characteristic absorption peaks of the secondary metabolites of SAQ and SAT. 2,2-Diphenyl-1-1-picrylhydrazyl experiment revealed that the IC50 values of SAQ and SAT extracts were 155.49 and 128.75 µg/ml, respectively. For the 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) assay, the IC50 value of SAT extract was found to be 269.24 µg/ml, which was lower than that of SAQ extract (IC50 = 358.99 µg/ml). This study successfully used different methods to differentiate between A. quinata (Thunb.) Decne. and A. trifoliata (Thunb.) Koidz., to help decide on which type to use for clinical application.

The Effect of Chinese Agarwood Essential Oil with Cyclodextrin Inclusion against PCPA-Induced Insomnia Rats.

OBJECTIVE: To study the extraction process of agarwood active ingredients (AA) and investigate the safety and effectiveness of AA in the treatment of insomnia rats by nasal administration. METHOD: A ß-cyclodextrin (ß-CD) inclusion compound (a-ß-CD) was prepared from agarwood essential oil (AEO), and the preparation process was optimized and characterized. The safety of AA in nasal mucosa was evaluated through Bufo gargarizans maxillary mucosa and rat nasal mucosa models. Insomnia animal models were replicated by injecting p-chlorophenylalanine (PCPA), conducting behavioral tests, and detecting the expression levels of monoamine neurotransmitters (NE and 5-HT) and amino acids (GABA/Glu) in the rat hypothalamus. RESULTS: The optimum inclusion process conditions of ß-CD were as follows: the feeding ratio was 0.35:1.40 (g:g), the inclusion temperature was 45 °C, the inclusion time was 2 h, and the ICY% and IEO% were 53.78 ± 2.33% and 62.51 ± 3.21%, respectively. The inclusion ratio, temperature, and time are the three factors that have significant effects on the ICY% and IEO% of a-ß-CD. AA presented little damage to the nasal mucosa. AA increased the sleep rate, shortened the sleep latency, and prolonged the sleep time of the rats. The behavioral test results showed that AA could ameliorate depression in insomnia rats to a certain extent. The effect on the expression of monoamine neurotransmitters and amino acids in the hypothalamus of rats showed that AA could significantly reduce NE levels and increase the 5-HT level and GABA/Glu ratio in the hypothalamus of insomnia rats. CONCLUSION: The preparation of a-ß-CD from AEO can reduce its irritation, improve its stability, increase its curative effect, and facilitate its storage and transport. AA have certain therapeutic effects on insomnia. The mechanism of their effect on rat sleep may involve regulating the expression levels of monoamine neurotransmitters and amino acids in the hypothalamus.

Norcantharidin-Encapsulated C60-Modified Nanomicelles: A Potential Approach to Mitigate Cytotoxicity in Renal Cells and Simultaneously Enhance Anti-Tumor Activity in Hepatocellular Carcinoma Cells.

OBJECTIVE: The objective of this study was to examine the preparation process of DSPE-PEG-C60/NCTD micelles and assess the impact of fullerenol (C60)-modified micelles on the nephrotoxicity and antitumor activity of NCTD. METHOD: The micelles containing NCTD were prepared using the ultrasonic method and subsequently optimized and characterized. The cytotoxicity of micelles loaded with NCTD was assessed using the CCK-8 method on human hepatoma cell lines HepG2 and BEL-7402, as well as normal cell lines HK-2 and L02. Acridine orange/ethidium bromide (AO/EB) double staining and flow cytometry were employed to assess the impact of NCTD-loaded micelles on the apoptosis of the HK-2 cells and the HepG2 cells. Additionally, JC-1 fluorescence was utilized to quantify the alterations in mitochondrial membrane potential. The generation of reactive oxygen species (ROS) following micelle treatment was determined through 2',7'-dichlorofluorescein diacetate (DCFDA) staining. RESULTS: The particle size distribution of the DSPE-PEG-C60/NCTD micelles was determined to be 91.57 nm (PDI = 0.231). The zeta potential of the micelles was found to be -13.8 mV. The encapsulation efficiency was measured to be 91.9%. The in vitro release behavior of the micelles followed the Higuchi equation. Cellular experiments demonstrated a notable decrease in the toxicity of the C60-modified micelles against the HK-2 cells, accompanied by an augmented inhibitory effect on cancer cells. Compared to the free NCTD group, the DSPE-PEG-C60 micelles exhibited a decreased apoptosis rate (12%) for the HK-2 cell line, lower than the apoptosis rate observed in the NCTD group (36%) at an NCTD concentration of 75 µM. The rate of apoptosis in the HepG2 cells exhibited a significant increase (49%), surpassing the apoptosis rate observed in the NCTD group (24%) at a concentration of 150 µM NCTD. The HK-2 cells exhibited a reduction in intracellular ROS and an increase in mitochondrial membrane potential (ΔψM) upon exposure to C60-modified micelles compared to the NCTD group. CONCLUSIONS: The DSPE-PEG-C60/NCTD micelles, as prepared in this study, demonstrated the ability to decrease cytotoxicity and ROS levels in normal renal cells (HK-2) in vitro. Additionally, these micelles showed an enhanced antitumor activity against human hepatocellular carcinoma cells (HepG2, BEL-7402).

[Chromosomal aneuploidy and pregnancy outcomes for D5/D6 blastocysts from patients undergoing PGT-A treatment].

OBJECTIVE: To compare the prevalence of chromosomal aneuploidies and pregnancy outcomes of D5 and D6 blastocysts subjected to preimplantation genetic testing for aneuploidy (PGT-A). METHODS: Clinical and laboratory data of 268 couples who underwent PGT-A at the Reproductive Center of the First Affiliated Hospital of Zhengzhou University from September 2018 to September 2020 were collected. The prevalence of chromosomal aneuploidies and pregnancy outcomes of D5/D6 biopsied blastocysts were compared. RESULTS: Compared with D6 blastocysts, the euploidy rate of D5 blastocysts was significantly higher (49.1% vs. 41.1%, P = 0.001 1), whilst their aneuploidy rate was significantly lower (50.9% vs. 58.9%, P = 0.001 1). The rate of numerical abnormalities of D6 blastocysts was significantly higher than that of D5 blastocysts (27.9% vs. 20.2%, P = 0.000 5). For patients under 35 years old, the euploidy rate of D5 blastocysts was significantly higher than that of D6 blastocysts (53.8% vs. 44.3%, P = 0.001), whilst the numerical abnormality rate was significantly lower (16.3% vs. 23.9%, P = 0.001). For both D5 and D6 blastocysts, the euploidy rates for patients <= 35 were significantly higher than those for > 35. The elder group had the lowest rates for aneuploidies and live births. Compared with those receiving D6 blastocysts transplantation, the pregnancy rate, implantation rate and live birth rate for those receiving thawed D5 blastocysts transplantation were significantly higher (60.2% vs.37.0%, P = 0.000 3; 59.1% vs.37.0%, P = 0.000 6; 47.7% vs. 28.3%, P = 0.002). CONCLUSION: For patients undergoing PGT-A, the chromosomal euploidy rate for D5 blastocysts is higher than that for D6 blastocysts, and the clinical outcome of D5 blastocysts with normal signal is better than that of D6 blastocysts. Elder patients have a higher rate of aneuploidies.

Comparison of embryo implantation potential between time-lapse incubators and standard incubators: a randomized controlled study.

RESEARCH QUESTION: What are the potential clinical benefits of embryo culture and assessment in a time-lapse incubator compared with a standard incubator using static assessment? DESIGN: This large multicentre, single-blinded, randomized controlled study included 1224 participants randomly assigned (1:1) to the time-lapse or standard incubator group. In all patients one or two embryos were transferred on day 3. The primary outcome was the implantation rate in the first embryo transfer cycle. Secondary outcomes included the cumulative implantation rate, live birth rate in the first embryo transfer cycle and cumulative live birth rate. RESULTS: Among 1224 participants recruited, 1182 underwent embryo transfer. The number of successfully implanted embryos in the first transfer cycle was significantly higher in the time-lapse incubator group (time-lapse group: 52.35%, standard incubator group: 47.11%, P = 0.014). The implantation rate in the first embryo transfer cycle was still significantly higher in the time-lapse group than the standard incubator group after adjusting for age, body mass index, medical centre and embryo status (relative risk 1.11, 95% confidence interval 1.02-1.20, P = 0.020). However, the cumulative implantation rate, live birth rate in the first embryo transfer cycle and cumulative live birth rate were not statistically different between the groups. CONCLUSIONS: The implantation rate in the first embryo transfer cycle was significantly improved in the time-lapse group, but the effect of the time-lapse system on the cumulative implantation rate or cumulative live birth rate was not significant. The embryo assessment method offered by time-lapse systems rather than an undisturbed environment may play an important role in improving the implantation rate in the first embryo transfer cycle. These results are only applicable to young patients.

A novel monogenic preimplantation genetic testing strategy for sporadic polycystic kidney caused by de novo PKD1 mutation.

Autosomal dominant hereditary polycystic kidney disease (ADPKD) is the most common inherited kidney disease that causes end-stage renal disease and kidney failure. Preimplantation genetic testing for monogenic (PGT-M) can effectively prevent the transmission of genetic diseases from parents to the offspring before pregnancy. However, PGT-M currently adopts the single nucleotide polymorphism (SNP) linkage analysis for embryo's pathogenic gene carrying status and linkage analysis requires proband of the family. Here we report a new PGT-M strategy using single sperm SNP linkage analysis for male patient with sporadic ADPKD caused by de novo PKD1 mutation. We recruited five couples with male patient with ADPKD caused by de novo PKD1 mutation, and 39 embryos from six PGT-M cycles were detected. The five couples had at least one embryo that does not carry the PKD1 mutation. Within these five couples, the accuracy of carrier status of embryos was confirmed by amniotic fluid gene detection of two couples and two couples successfully delivered healthy fetuses. Therefore, the new PGT-M strategy of using single sperm SNP linkage analysis was proved to be feasible and effective for male patient with ADPKD caused by de novo PKD1 mutation.

Mechanism and optimization of supramolecular complexation-enhanced fluorescence spectroscopy for the determination of SN-38 in plasma and cells.

Quantitative detection of two different forms of SN-38 in biological samples is, currently, cumbersome and difficult. A revisit to the mechanism of supramolecular complexation-enhanced fluorescence spectroscopy helps to optimize the determination of SN-38 in plasma and the cellular pharmacokinetics in A549 cells based on the supramolecular complexation. Firstly, the inclusion mechanism dominated by thermodynamic constants was determined by measuring kinetic/thermodynamic parameters (kon , koff , ΔG, ΔH, ΔS). On this basis, the best effect of fluorescence sensitization was optimized through screening the interaction conditions (cyclodextrin species and concentrations, drug levels, temperature, pH of the buffer, and reaction time). Furthermore, the proportional relationship between the concentration of the inclusion complex and the fluorescence intensity was confirmed. Finally, a highly sensitive, selective spectrofluorimetric method was established and validated for quantitative analysis of the lactone and carboxylate molecular states of SN-38 plasma levels in rats and cell membrane transfer kinetics in A549 cell lines. The limits of detection for the lactone and carboxylate forms in plasma were found to be 0.44 ng·ml-1 and 0.28 ng·ml-1 , respectively. Precision and accuracy met the requirements of biological samples analysis. The proposed detection method provided a reference for elucidating the biodistribution of SN-38.

Mapping allele with resolved carrier status of Robertsonian and reciprocal translocation in human preimplantation embryos.

Reciprocal translocations (RecT) and Robertsonian translocations (RobT) are among the most common chromosomal abnormalities that cause infertility and birth defects. Preimplantation genetic testing for aneuploidy using comprehensive chromosome screening for in vitro fertilization enables embryo selection with balanced chromosomal ploidy; however, it is normally unable to determine whether an embryo is a translocation carrier. Here we report a method named "Mapping Allele with Resolved Carrier Status" (MaReCs), which enables chromosomal ploidy screening and resolution of the translocation carrier status of the same embryo. We performed MaReCs on 108 embryos, of which 96 were from 13 RecT carriers and 12 were from three RobT carriers. Thirteen of the sixteen patients had at least one diploid embryo. We have confirmed the accuracy of our carrier status determination in amniotic fluid karyotyping of seven cases as well as in the live birth we have thus far. Therefore, MaReCs accurately enables the selection of translocation-free embryos from patients carrying chromosomal translocations. We expect MaReCs will help reduce the propagation of RecT/RobT in the human population.

Live births following preimplantation genetic testing for dynamic mutation diseases by karyomapping: a report of three cases.

PURPOSE: The preimplantation genetic testing for monogenic defects (PGT-M) is a beneficial strategy for the patients suffering from a Mendelian disease, which could protect their offspring from inheriting the disease. The purpose of this study is to report the effectiveness of PGT-M based on karyomapping for three cases of dynamic mutation diseases with trinucleotide repeat expansion. METHODS: PGT-M was carried out on three couples, whose family members were diagnosed with Huntington's disease or spinocerebellar ataxias 2 or 12. The whole genome amplification was obtained using the multiple displacement amplification (MDA) method. Then, karyomapping was performed to detect the allele that is carrying the trinucleotide repeat expansion using single nucleotide polymorphism (SNP) linkage analyses, and the copy number variations (CNVs) of the embryos were also identified. Prenatal diagnosis was performed to validate the accuracy of PGT-M. RESULTS: PGT-M was successfully performed on the three couples, and they accepted the transfers of euploid blastocysts without the relevant pathogenic allele. The clinical pregnancies were acquired and the prenatal diagnosis of the three families confirmed the effectiveness of karyomapping. The three born babies were healthy and free of the pathogenic alleles HTT, ATXN2, or PPP2R2B corresponding to Huntington's disease, spinocerebellar ataxias 2 or 12, respectively. CONCLUSION: This study shows that karyomapping is a highly powerful and efficient approach for dynamic mutation detection in preimplantation embryos. In this work, we first report the birth of healthy babies that are free of the pathogenic gene for dynamic mutation diseases in patients receiving PGT-M by karyomapping.

Transcriptional profiling of long noncoding RNAs and their target transcripts in ovarian cortical tissues from women with normal menstrual cycles and primary ovarian insufficiency.

Previous studies have shown that long noncoding RNAs (lncRNAs) show a highly tissue- and disease-specific expression pattern and that they regulate the expression of neighboring genes. Because lncRNAs have been shown to be secreted into the general circulation, they may be used as diagnostic tools for some diseases. Primary ovarian insufficiency (POI) is a disease in which women have menstrual cessation before the age of 40, accompanied by elevated follicle stimulating hormone and decreased estrogen levels. In this study, ovarian cortical tissues from five women with normal menstrual cycles and from five POI patients were used for next-generation RNA sequencing. We found 20 differentially expressed lncRNAs with 12 upregulated and eight downregulated lncRNAs in cortical tissues of POI ovaries, compared with normal controls (fold change ≥ 2 and false discovery rate[FDR] ≤ 0.05). We also found 52 differentially expressed messenger RNA transcripts, with 33 upregulated and 19 downregulated ones (foldchange ≥ 2 and FDR ≤ 0.05). Functional annotation showed that these differentially expressed transcripts were associated with follicular development and granulosa cell function. Thirteen differentially expressed lncRNAs and their targeted neighboring transcripts were coregulated in ovarian cortical tissues, including lnc-ADAMTS1-1:1/ADAMTS1, lnc-PHLDA3-3:2/CSRP1, lnc-COL1A1-5:1/COL1A1, lnc-SAMD14-5:3/COL1A1, and lnc-GULP1-2:1/COL3A1. Furthermore, serum levels of these lncRNAs in POI patients were significantly different from those in normal patients ( p < 0.05), and expression differences were consistent with those in ovarian cortical tissues. This study showed that key lncRNAs were differentially expressed in both ovarian cortical tissues and serum samples between women with normal menstrual cycles and POI patients. Further studies on the regulation of ovarian lncRNAs during follicular development are critical in understanding the etiologies of POI. Analyses of lncRNA expression in serum samples might provide a basis for early diagnosis and treatment of POI.

Analysis of Related Causes for No Embryos Transferred and Corresponding Coping Measures in Assisted Reproductive Technology.

OBJECTIVE: To analyze the related causes for no embryos transferred in assisted reproductive technology (ART) in order to provide corresponding coping measures for infertile couples. STUDY DESIGN: The data of 607 couples who underwent ART and had no embryos transferred in our reproductive center between January 2010 and January 2014 were retrospectively analyzed. RESULTS: The cycles of no embryos transferred accounted for 3.99% (607/15,224) of total cycles. Of those, complete fertilization failure, oocyte retrieval failure, and complete abnormal fertilization accounted for 28.3% (172/607), 25.7% (156/607) and 22.24% (135/607), respectively. The incidence of complete abnormal fertilization was higher in IVF than in ICSI (p<0.05). In both IVF and ICSI cycles, the incidences of no embryos transferred were higher in the patients retrieving ≤3 oocytes than in the patients retrieving >3 oocytes (p<0.05). In IVF cycles the incidences of no embryos transferred were higher in the patients with primary infertility than in those with secondary infertility (p<0.05). CONCLUSION: The main causes of no embryos transferred are complete fertilization failure, oocyte retrieval failure, and complete abnormal fertilization. Retrieving adequate number of mature oocytes is the key to success of ART. Patients who experienced complete abnormal fertilization in IVF or the patients with primary infertility who experienced complete fertilization failure or normal fertilization without cleavage should receive ICSI in the next treatment.

Effects of Vitrification on Outcomes of In Vivo-Mature, In Vitro-Mature and Immature Human Oocytes.

BACKGROUND/AIMS: To observe the effects of vitrification on spindle, zona pellucida, embryonic aneuploidy and DNA injury in in vivo-maruted, in vitro-mature and immature human oocytes. METHODS: Between January 2009 and February 2015, 223 immature oocytes from 450 infertile patients, and 31 in vivo-mature oocytes from 3 infertile couples were collected. Of the 223 immature oocytes, 113 were used for in vitro culture before vitrification. Some oocytes were randomly divided into in vivo-mature group (group A, n = 15), in vitro-mature group (group B, n = 88) and immature group (group C, n = 85), and then the oocytes with spindle in these three groups after freezing-thawing were selected to use for Polscope imaging, embryonic aneuploidy screening and embryo development evaluation. Other oocytes were randomly divided into group A (n = 16), group B (n = 25) and group C (n = 25) for detecting DNA injury. RESULTS: After thawing, spindle occurrence rate, spindle Retardance value, and cleavage rate were significantly higher in groups A and B than in group C (all P < 0.05), but there were no statistical differences in fertility rate, high-quality embryo rate, blastulation rate and aneuploidy rate amongst the three groups (all P > 0.05). Zona pellucida density (ZPD) was significantly lower in group A than in groups B and C both before and after vitrification (all P < 0.05). ZPD was significantly higher after thawing than before vitrification (all P < 0.05), but zona pellucida thickness (ZPT) was not significantly changed in all the three groups (all P > 0.05). Rate of comet cells was significantly lower in group A than in groups B and C (all P < 0.01). Comet tail was significantly longer in group C than in groups B and A (all P < 0.05). CONCLUSION: In vivo- and in vitro-mature human oocytes are more suitable to vitrification than immature human oocytes. Spindle Retardance value has more predictive value for embryonic development potential than ZPD and ZPT.

[Percutaneous permeability of rhein at shenque acupoint in vitro].

To study rhein's permeative properties of acupoint and non-acupoint and different species' transdermal administration in vitro. Cumulative permeation amount and steady-state infiltration rate were taken as evaluative indexes to assess the permeability difference. The Valia-Chien diffusion cell method was used to conduct the permeability test, with fresh acupoint and non-acupoint skin of rat, rabbit and swine in vitro as permeation barriers, and blank 20% EtOH saline as absorption liquid. HPLC was used to determine the rhein. The absorption difference was observed by confocal laser scanning microscopy. The 24-hour cumulative permeation amount through acupoint skin in rats was (102.63±9.60) µg•cm⁻², the steady-state infiltration rate was 4.307 µg•cm⁻²â€¢h⁻¹, both were higher than that through non-acupoint skin. The thickness of acupoint skin in rat was thinner than that in rabbit and swine. The cumulative permeation amount and steady-state infiltration rate of rhein in acupoint of rat were signally higher than those in rabbit and swine. The absorption difference can be clearly observed through an accumulation of fluorescence. In conclusion, species and acupoint all affect the permeability of rhein in vitro. The permeation amount and rate of rhein on Shenque acupoint were better than that on non-acupoint skin, which could verify that treatment through Shenque acupoint is superior to that through non-acupoint. The preliminary mechanism may be the drug delivery through Shenque acupoint as a channel and carrier, which is a visual verification the specificity and superiority of clinical application through Shenque acupoint in treating diseases.

Role of PAFAH1B1 in human spermatogenesis, fertilization and early embryonic development.

Spermatogenesis, fertilization and subsequent embryonic development are complex processes that require tight regulation. The PAFAH1B1 gene plays important roles in these reproductive events in mice, but its expression and roles in human reproduction have not been investigated. Expression analysis of testicular tissue by reverse transcription quantitative PCR and immunohistochemistry revealed varied expression levels among samples of different spermatogenic abilities (as assessed by the Johnsen score), with protein expression restricted to spermatogonia, spermatocytes and spermatids. Immunofluorescence on spermatozoa showed expression over the acrosome and midpiece regions of ejaculated samples, whereas a high proportion of percutaneous epididymal sperm aspiration-derived spermatozoa showed expression restricted to the midpiece. Analysis for PAFAH1B1 mRNA also revealed different expression levels among unfertilized oocytes, zygotes, cleavage stage embryos and blastocysts, with protein localized at the membrane level in oocytes and zygotes, and gradually distributing within the cytoplasm of cleavage stage embryos and blastocysts. Interestingly, microinjection of PAFAH1B1 siRNA into zygotes significantly (P = 0.024) increased fragmentation formation rates in subsequent embryonic development stages. Altogether, these are the first results to support a role for PAFAH1B1 in human spermatogenesis and early embryonic development.

In vitro immunosuppressive and cytotoxic activities of Tripterygium wilfordii extract.

Tripterygium wilfordii Hook. f. (TW) is a traditional herbal medicine which has been widely used for the treatment of rheumatoid arthritis and other autoimmune diseases. However, adverse reactions of TW such as hepatotoxicity and nephrotoxicity have been frequently reported in clinic. With the aim to evaluate the potency and toxicity of TW, we collected eleven batches of TW from different localities across Chinese mainland, and investigated the inhibition of their methanol extracts on the proliferation of mouse spleen lymphocytes, normal human hepatocyte (L-02) cells and African green monkey kidney (COS-7) cells. TW extracts with three different concentrations were designed as the experimental groups. Our present findings provided consistent evidence that TW had significant concentration-dependent inhibitory action on lymphocytes, L-02 and COS-7 cells. At the concentrations of 0.75 and 1.5 mg/mL, most TW groups showed statistically significant inhibition of lymphocyte proliferation when compared with the control group (p < 0.01), and the inhibition of TW extract on lymphocytes was almost equal to 1.0 mg/mL aspirin (p > 0.05). In most test groups, significant toxicities were shown on L-02 cells at 0.6 and 3.0 mg/mL (p < 0.01), and on COS-7 cells at 3.0 mg/mL (p < 0.01). At 3.0 mg/mL, almost all TW groups exerted obvious toxicities toward L-02 and COS-7 cells which were equal to or even higher than 1.0 mg/mL aspirin. In view of these results, further studies are needed to elucidate the relations among the effective component, curative effect and toxicity of TW to ensure its effectiveness and safety for human consumption.