Single-cell RNA sequencing of human oocytes reveals a differential transcriptomic profile associated with agar-like zona pellucida.

BACKGROUND: Agar-like zona pellucida (ZP) is the most common type of abnormal ZP, and is one of the causes of low fertility or infertility. However, the molecular mechanism of agar-like ZP is unclear. Single-cell RNA-sequencing (scRNA-seq) analysis was used to assess the cellular and molecular landscape of oocytes with agar-like ZP. METHODS: Human metaphase I (MI) oocytes were collected from four patients with agar-like ZP and four healthy donors. Total RNA was isolated, cDNA was synthesized, and libraries were generated and subsequently sequenced on a HiSeq 2500 instrument. The scRNA-seq data were analyzed with R software. RESULTS: We identified 1320 genes that were differentially expressed between agar-like ZP oocytes and healthy donor oocytes. Gene Ontology term enrichment results showed that the genes downregulated in agar-like ZP oocytes were significantly related to extracellular matrix organization, while the genes upregulated in agar-like ZP oocytes were significantly related to the regulation of response to DNA damage stimulus. The Kyoto Encyclopedia of Genes and Genomes enrichment results showed that genes were enriched in the ECM-receptor interaction pathway and focal adhesion pathway. Other signaling pathways important in oocyte development were also enriched, such as PI3K-Akt. Differential expression analysis identified UBC, TLR4, RELA, ANXA5, CAV1, KPNA2, CCNA2, ACTA2, FYN and ITGB3 as genetic markers of oocytes with agar-like ZP. CONCLUSIONS: Our findings suggest that agar-like ZP oocytes exhibit significant downregulation of genes involved in the ECM-receptor interaction signaling pathway and focal adhesion pathway, which could lead to aberrant ZP formation, while the upregulated genes were significantly related to regulation of the response to DNA damage stimulus. Agar-like ZP formation may interfere with the normal exchange of signals between oocytes and perivitelline granulosa cells, thereby preventing cumulus cells from participating in oocyte DNA damage repair and causing MI arrest.

Comparison of extraction processes, characterization and intestinal protection activity of Bletilla striata polysaccharides.

We optimized the extraction process of Bletilla striata polysaccharides using orthogonal design, Box-Behnken design (BBD), and genetic algorithm-back propagation (GA-BP), then compared and evaluated them to confirm that the combination of BBD and GA-BP neural networks was capable of increasing polysaccharide yields and antioxidant activity. The optimal extraction parameters were as follows: liquid-to-solid ratio of 15 mL/g, extraction power of 450 W, and extraction time of 34 min. Under these conditions, the polysaccharide yield and antioxidant activity were 8.29 ± 0.50 % and 26.20 ± 0.28 (mM FE/mg). Subsequently, the polysaccharide was purified to obtain purified Bletilla striata polysaccharides 1 (pBSP1) with a Mw of 255.172 kDa. Scanning electron microscope (SEM), ultraviolet-visible detector (UV), fourier transform infrared spectrometer (FTIR), high performance liquid chromatography (HPLC), X-ray diffraction (XRD), nuclear magnetic resonance (NMR) and periodate oxidation were used to analyze the structure of pBSP1. The results showed pBSP1 had a smooth surface and a rough interior, with a composition of α-D conformation glucose (18.23 %) and ß-D conformation mannose (53.77 %), and an amorphous crystal structure. According to the results of thermogravimetric and rheological tests, pBSP1 exhibits good thermal stability and viscoelastic behavior. Furthermore, pBSP1 protected lipopolysaccharide (LPS)-induced GES - 1 and Caco2 cells, the results showed pBSP1(400 µg/mL) lowered TEER synthesis in Caco2 cells as well as apoptosis and reactive oxygen species (ROS) production in both cells, indicating that pBSP1 may have an intestine protective effect.

Combined immunochemotherapy achieving targeted co-delivery of chlorogenic acid and doxorubicin by sialic acid-modified liposomes enhances anti-cancer efficacy.

Malignant melanoma is a high-grade aggressive skin tumor with an increasing incidence and mortality rates worldwide. Chemotherapeutic drugs such as doxorubicin have limited efficacy against melanoma due to their poor sensitivity, severe side effects, and drug resistance. Recent studies have shown that combinations of immunotherapy and chemotherapy have a synergistic effect in enhancing the anti-tumor effect. Here, we have developed liposomes co-loaded with chlorogenic acid (CA) and doxorubicin (DOX), modified with sialic acid-octadecylamine conjugate (SA-ODA), designated CA-DOX-SAL, that facilitate drug delivery by recognizing Siglec-1 receptor on TAMs. The physicochemical studies revealed the particle size and zeta potential of CA-DOX-SAL as 128.3 ± 0.8 nm and - 4.33 ± 0.50 mV, respectively. In vitro, CA-DOX-SAL demonstrated robust cellular uptake through SA receptor-mediated tumor-associated macrophages (TAM) targeting and exerted greater cytotoxicity on tumor cells. In vivo, targeted liposomes were found to accumulate in the tumor area, leading to an improvement in anti-tumor efficacy. In addition, CA-DOX-SAL effectively inhibited B16F10 melanoma tumor growth by stimulating the transition from tumor-promoting M2-type to anti-tumor M1-type and directly killing tumor cells. Overall, the co-delivery of immunomodulatory CA and chemotherapeutic DOX presents a promising therapeutic strategy to enhance clinical outcomes in the treatment of melanoma.

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).

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.

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.

[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.

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.

Sanghuangporus vaninii ethanol extract alleviates hyperuricemic renal injury by regulating the uric acid transporters and inhibiting HK-2 apoptosis.

AIM OF THE STUDY: To investigate the acute toxicity of Sanghuangporus ethanol extract (SHEE) on ICR mice and the underlying mechanism of anti-hyperuricemic renal injury. MATERIALS AND METHODS: ICR mice were given a single gavage of 1250, 2500, and 5000 mg/kg SHEE, and the general behavior, mortality, body weight, dietary, and water intake were evaluated within 14 days to determine the acute toxicity level. The hyperuricemic kidney injury model in ICR mice was induced with potassium oxonate (PO) and adenine, and the mice were subsequently treated with SHEE (125, 250, 500 mg/kg). HE and hexamine silver staining (PASM) were used to observe the pathology of the kidney. Biochemical markers were tested by uric acid (UA), creatinine (Cr), blood urea nitrogen (BUN), xanthine oxidase (XOD), alanine transferase (ALT), and aspartate transaminase (AST) kits. An MTT assay was used to measure the effects of SHEE on the proliferation of HK-2 damaged by UA. Western blotting and RT-PCR were used to determine the expression of Bcl-2 family-related proteins and major UA transporters, including URAT1, GLUT9, OAT1, OAT3, and ABCG2, respectively. RESULTS: Firstly, the acute toxicity study data showed that the median lethal dose (LD50) of SHEE was above 5000 mg/kg, and its oral administration was nontoxic at 2500 mg/kg and below. In addition, SHEE alleviated HUA and its renal injury in ICR mice. SHEE reduced the contents of UA, Cr, BUN and XOD in blood and the contents of ALT and AST in the liver. Furthermore, SHEE inhibited the expression of URAT1 and GLUT9 and promoted the expression of OAT1, OAT3, and ABCG2. More importantly, SHEE could downregulate the apoptosis level and caspase-3 activity. CONCLUSIONS: Overall, an oral dose of SHEE below 2500 mg/kg is safe. SHEE inhibits HUA-induced kidney injury by regulating the UA transporters URAT1, GLUT9, OAT1, OAT3 and ABCG2 and inhibiting HK-2 apoptosis.

Artificial neural network model- and response surface methodology-based optimization of Atractylodis Macrocephalae Rhizoma polysaccharide extraction, kinetic modelling and structural characterization.

Atractylodis Macrocephalae Rhizoma (AMR) is the dried rhizome of Atractylodes macrocephala Koidz, which is widely used in the development of health products. AMR contains a large number of polysaccharides, but at present there are fewer applications for these polysaccharides. In this study, the effects of different extraction methods on the Atractylodis Macrocephalae Rhizoma polysaccharide (AMRP) yield were investigated, and the conditions for ultrasound-assisted extraction were optimized by response surface methodology (RSM) and three neural network models (BP neural network, GA-BP neural network and ACO-GA-BP neural network). The best conditions were a liquid-to-solid ratio of 17 mL/g, ultrasonic power of 400 W, extraction temperature of 72 °C, and extraction time of 40 min, which yielded 31.31% AMRP. The kinetic equation of AMRP was determined and compared with the results predicted by three neural network models. It was finally determined that the extraction conditions, kinetic processes and kinetic equation predicted by the GA-ACO-BP neural network were optimal. In addition, AMRP was characterized using SEM, FTIR, HPLC, UV, XRD, and NMR, and the structural study revealed that AMRP has a rough exterior and a porous interior; moreover, it contains high levels of glucose (5.07%), arabinose (0.80%), and galactose (0.74%). AMRP has three crystal structures, consisting of two ß-type monosaccharides and one α-type monosaccharide. Additionally, the effectiveness of AMRP as an antioxidant was demonstrated in an in vitro experiment.

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.

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.

Preparation and Evaluation of the Cytoprotective Activity of Micelles with DSPE-PEG-C60 as a Carrier Against Doxorubicin-Induced Cytotoxicity.

To deliver doxorubicin (DOX) with enhanced efficacy and safety in vivo, fullerenol-modified micelles were prepared with the amphiphilic polymer DSPE-PEG-C60 as a carrier, which was synthesized by linking C60(OH)22 with DSPE-PEG-NH2. Studies of its particle size, PDI, zeta potential, and encapsulation efficiency were performed. DOX was successfully loaded into the micelles, exhibiting a suitable particle size [97 nm, 211 nm, 260 nm, vector: DOX = 5:1, 10:1; 15:1 (W/W)], a negative zeta potential of around -30 mv, and an acceptable encapsulation efficiency [86.1, 95.4, 97.5%, vector: DOX = 5:1, 10:1; 15:1 (W/W)]. The release behaviors of DOX from DSPE-PEG-C60 micelles were consistent with the DSPE-PEG micelles, and it showed sustained release. There was lower cytotoxicity of DSPE-PEG-C60 micelles on normal cell lines (L02, H9c2, GES-1) than free DOX and DSPE-PEG micelles. We explored the protective role of DSPE-PEG-C60 on doxorubicin-induced cardiomyocyte damage in H9c2 cells, which were evaluated with a reactive oxygen species (ROS) assay kit, JC-1, and an FITC annexin V apoptosis detection kit for cellular oxidative stress, mitochondrial membrane potential, and apoptosis. The results showed that H9c2 cells exposed to DSPE-PEG-C60 micelles displayed decreased intracellular ROS, an increased ratio of red fluorescence (JC-1 aggregates) to green fluorescence (JC-1 monomers), and a lower apoptotic ratio than the control and DSPE-PEG micelle cells. In conclusion, the prepared DOX-loaded DSPE-PEG-C60 micelles have great promise for safe, effective tumor therapy.

Preparation, Characterization, and Evaluation of Breviscapine Nanosuspension and Its Freeze-Dried Powder.

As a biopharmaceutics classification system (BCS) class IV drug, breviscapine (Bre) has low solubility in water, poor chemical stability, a short biological half-life and rapid removal from plasma. This paper prepared a Bre nanosuspension (Bre-NS) by an ultrasound-assisted anti-solvent precipitation method. Characterization of Bre-NS was studied using a Box-Behnken design concerning drug concentration in DMSO, an anti-solvent-to-solvent ratio, and sonication time. Under the optimized conditions of 170 mg/mL for the drug concentration, a 1:60 solvent-to-anti-solvent ratio, and a 9 min sonication time, the particle size of Bre-NS was 303.7 ± 7.3 nm, the polydispersity index was 0.178 ± 0.015, and the zeta potential was -31.10 ± 0.26 mV. Combined with the results from differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), and Fourier transform-infrared spectroscopy (FT-IR), the findings indicated that the crystal form and chemical structure of Bre-NS did not change during the entire process. The optimized formulation displayed good stability, increased solubility, and better in vitro release. Therefore, the results of this study can be a reference for the delivery system design of insoluble active components and effective parts in traditional Chinese medicine.

Gene Spectrum and Clinical Traits of Nine Patients With Oocyte Maturation Arrest.

Background: Oocyte maturation arrest is a disease that produces immature oocytes and cannot be mature after culturing in vitro, which leads to female primary infertility. We aimed to summarize nine representative patients in our center to retrospectively analyze the genetic variants and clinical characteristics of oocyte maturation arrest. Methods: This study examined and analyzed nine families with oocyte maturation arrest. Whole-exome sequencing (WES) of the probands was performed to detect the pathogenic variants. Sanger sequencing verified the WES findings in patients and available parents. ExAC database was used to search the variant frequency. The variants were assessed by pathogenicity and conservational property prediction analysis and according to the American College of Medical Genetics and Genomics (ACMG). Phenotypes of oocytes were evaluated by a light microscopy, and the phenotype-genotype correlation was also evaluated. Results: Nine pathogenic variants in five genes were detected in nine patients, of which three were novel variants, including PATL2 [c.1374A > G (p. Ile458Met)] and [1289-1291del TCC (p. Leu430del)] and ZP2 [c.1543C > T (p. Pro515Ser)]. Nine variants were predicted to be pathogenic, resulting in different types of oocyte maturation arrest and clinical phenotypes. Conclusion: Three novel pathogenic variants were identified, enabling the expansion of the gene variant spectrum. The related pathogenic mutations of the PATL2, TUBB8, and ZP1∼3 genes were highly suggestive of being causative of oocyte maturation arrest.

Effect of ambient air pollutants on in vitro fertilization-embryo transfer pregnancy outcome in Zhengzhou, China.

With the acceleration of China's urbanization and industrialization, air pollution has become a major environmental problem. Retrospective data analysis of 6564 patients who underwent IVF-ET in the center for reproductive medicine of the First Affiliated Hospital of Zhengzhou University from 2015 to 2020. Different stages were selected from 90 days before oocyte retrieval to 35 days after transfer and divided into five exposure periods. Multivariate logistic regression was used to analyze the relationship between six ambient air pollutants (PM2.5, PM10, NO2, SO2, CO and O3) and the IVF-ET pregnancy outcome. The results showed that air pollutants can significantly affect the IVF pregnancy outcome. The harmful effects of ambient air pollutants are more obvious in the patients aged < 35 years, single embryo transfer and cleavage stage embryo transfer.

Network Pharmacology, Molecular Docking, and Molecular Dynamic-Based Investigation on the Mechanism of Compound Chrysanthemum in the Treatment of Asthenopia.

As a clinical empirical prescription for ophthalmology, compound chrysanthemum has been used gradually and has a good effect on eye fatigue. However, the detailed mechanisms of antiasthenopia have not been studied. In order to clarify the mechanisms of the compound chrysanthemum in the treatment of asthenopia, network pharmacology was combined with experimental study in this paper. A total of 593 genes and 39 active chemicals were identified, and both were considered to be essential to the advancement of asthenopia research. The results of the molecular docking analysis demonstrated a certain affinity between PRKACA, PRKCA, PRKCB, and their related compounds; molecular dynamic simulations assessed the stability of these receptors and ligands. The effects of compound chrysanthemum extract on ciliary muscle were studied in vitro and in vivo. By using the MTT assay, compound chrysanthemum extracts (50, 100, 200, 400, and 800 g·mL-1) showed no effect on the proliferation of rCSMCs for 24 and 48 hours. It raised nitric oxide and decreased Ca2+ in ciliary muscle cells isolated from the eyeballs of rats. Besides, compound chrysanthemum extract had a direct relaxing effect on the isolated gastric smooth muscle of rats by reducing the contractile tension. Furthermore, in vivo experiment results showed that, compared to the incandescent lamp-irradiated rats (model group), SD rats treated with compound chrysanthemum extracts (660 mg·kg-1 and 1320 mg·kg-1, orally) displayed considerably retracted pupils and increased NO content. It is also found that compound chrysanthemum extract can downregulate the mRNA expression of PKA and PKC in the calcium signaling pathway. Overall, our results suggested that compound chrysanthemum extract may lessen visual fatigue through multiple components, multiple targets, and multiple pathways.

Combination immunotherapy of chlorogenic acid liposomes modified with sialic acid and PD-1 blockers effectively enhances the anti-tumor immune response and therapeutic effects.

Melanoma is one of the most common malignant tumors. The anti-PD-1 antibody is used for the treatment of metastatic melanoma. Treatment success is only 35-40% and a range of immune-related adverse reactions can occur. Combination of anti-PD1 antibody therapy with other oncology therapies has been attempted. Herein, we assessed whether chlorogenic acid liposomes modified with sialic acid (CA-SAL) combined with anti-PD1 antibody treatment was efficacious as immunotherapy for melanoma. CA-SAL liposomes were prepared and characterized. In a mouse model of B16F10 tumor, mice were treated with an anti-PD1 antibody, CA-SAL, or combination of CA-SAL + anti-PD1 antibody, and compared with no treatment controls. The tumor inhibition rate, tumor-associated macrophages (TAMs) phenotype, T-cell activity, and safety were investigated. We observed a significant decrease in the proportion of M2-TAMs and CD4+Fop3+ T cells, while there was a significant increase in the proportion of M1-TAMs and CD8+ T cells, and in the activity of T cells, and thus in the tumor inhibition rate. No significant toxicity was observed in major organs. CA-SAL and anti-PD1 Ab combination therapy presented synergistic anti-tumor activity, which enhanced the efficacy of the PD-1 checkpoint blocker in a mouse model of melanoma. In summary, combination immunotherapy of CA-SAL and anti-PD1 Ab has broad prospects in improving the therapeutic effect of melanoma, and may provide a new strategy for clinical treatment.

Calcium Ionophore (A23187) Rescues the Activation of Unfertilized Oocytes After Intracytoplasmic Sperm Injection and Chromosome Analysis of Blastocyst After Activation.

Calcium is a crucial factor in regulating the biological behavior of cells. The imbalance of calcium homeostasis in cytoplasm will cause abnormal behavior of cells and the occurrence of diseases. In intracytoplasmic sperm injection (ICSI) cycle, the dysfunction of oocyte activation caused by insufficient release of Ca2+ from endoplasmic reticulum is one of the main reasons for repeated fertilization failure. Calcium ionophore (A23187) is a highly selective calcium ionophore, which can form stable complex with Ca2+ and pass through the cell membrane at will, effectively increasing intracellular Ca2+ levels. It has been reported that calcium ionophore (A23187) can activate oocytes and obtain normal embryos. However, there are few studies on unfertilized oocytes after calcium ionophore (A23187) rescue activation in ICSI cycle. The purpose of this study was to analyze the effects of calcium ionophore (A23187) rescue activation on the activation of unfertilized oocytes, embryonic development potential, embryonic development timing and chromosomal aneuploidy, and to compare and analyze the clinical data of patients with calcium ionophore (A23187) activation in clinical application. The results showed that a certain proportion of high-quality blastocysts with normal karyotype could be obtained after calcium ionophore (A23187) rescue activation of unfertilized oocytes, and it did not have a significant effect on the timing of embryo development. In clinical practice, direct activation with calcium ionophore (A23187) after ICSI was better than rescue activation the next day. In conclusions, the studies on the effectiveness and safety of calcium ionophore (A23187) rescue activation for oocytes with ICSI fertilization failure can enable some patients to obtain usable, high-quality embryos during the first ICSI cycle.