Laser-assisted selection of immotile spermatozoa has no effect on obstetric and neonatal outcomes of TESA-ICSI pregnancies.

BACKGROUND: Azoospermic patients have benefited from both epididymal and testicular spermatozoa intracytoplasmic sperm injection (ICSI) treatment and lasers have been used to identify viable, immotile spermatozoa before the procedure. There are limited studies on the safety of laser-assisted selection of immotile spermatozoa. The aim of this study was to investigate the impact of laser-assisted selection of immotile spermatozoa on the obstetric and neonatal outcomes after ICSI. METHODS: A retrospective comparative study was conducted on outcomes of ICSI cycles with testicular spermatozoa from June 2014 to June 2018. Of 132 cycles, 33 were allocated to the test group and oocytes were injected with immotile spermatozoa selected by laser, 99 cycles were allocated as control group. RESULTS: Compared with the control group, no significant differences were found in the pregnancy, implantation, miscarriage and live birth rates in the test group in either fresh or frozen transfer cycles. The cumulative live birth rate in the test group was 69.70%, which was slightly higher than in the control group (60.61%), but this was not statistically different. There were no differences in the average gestational age, premature birth rate, neonatal birth weight, and the malformation rate between the test and control groups (P > 0.05). In addition, the obstetric outcome between the two groups were not different (P > 0.05). CONCLUSIONS: No negative effect on perinatal and neonatal outcomes was seen by using laser-assisted selection of immotile spermatozoa for TESA-ICSI. This study endorses the use of laser-assisted selection of viable spermatozoa for ICSI cycles.

Nanocrystals based mucosal delivery system: research advances.

Nanocrystal technology is a new way to increase the solubility and bioavailability of poorly soluble drugs. As an intermediate preparation technology, nanocrystals are widely used in drug delivery for oral, venous, percutneous and inhalation administration, which exhibits a broad application prospect. By referring to the domestic anforeign literatures, this paper mainly reviews the preparation methods of nanocrystals for poorly soluble natural products and its application in the mucosal delivery for skin, eye, oral cavity and nasal cavity. This can provide the reference for the research and development of nanocrystal technology in natural product preparations.

Hypoxia enhances buffalo adipose-derived mesenchymal stem cells proliferation, stemness, and reprogramming into induced pluripotent stem cells.

Adipose tissue-derived mesenchymal stem cells (ASCs) from livestock are valuable resources for animal reproduction and veterinary therapeutics. Previous studies have shown that hypoxic conditions were beneficial in maintaining the physiological activities of ASCs. However, the effects of hypoxia on buffalo ASCs (bASCs) remain unclear. In this study, the effects of hypoxia on proliferation, stemness, and reprogramming into induced pluripotent stem cells (iPSCs) of bASCs were examined. The results showed that the hypoxic culture conditions (5% oxygen) enhanced the proliferation and colony formation of bASCs. The expression levels of proliferation-related genes, and secretion of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were significantly enhanced in hypoxia. Hypoxic culture conditions activated hypoxia-inducible factor-1α (HIF-1α), thereby contributing to the secretion of bFGF and VEGF, which in turn enhanced the expression of HIF-1α and promoted the proliferation of bASCs. Furthermore, in hypoxic culture conditions, bASCs exhibited the main characteristics of mesenchymal stem cells, and the expression levels of the pluripotent markers OCT4, NANOG, C-MYC, and the differentiation capacity of bASCs were significantly enhanced. Finally, bASCs were more efficiently and easily reprogrammed into iPSCs in hypoxic culture conditions and these iPSCs exhibited some characteristics of naïve pluripotent stem cells. These findings provide the theoretical guidance for elucidating the detailed mechanism of hypoxia on physiological activities of bASCs including proliferation, stemness maintenance, and reprogramming.

Molecular Cloning, Identification, and Expression Patterns of Myostatin Gene in Water Buffalo (Bubalus Bubalis).

Myostatin (MSTN), also named growth differentiation factor 8 (GDF8), is a transforming growth factor-ß (TGF-ß) family member with a key role in the negative regulation of skeletal muscle growth. However, its role in ovarian folliculogenesis remains unclear. To provide us with a basis for understanding this role, we cloned MSTN and examined its expression patterns in water buffalo (Bubalus bubalis). The complete ORF of the water buffalo MSTN gene is 1,128 nucleotides, which encode a 375 amino acid protein and sharing 99% identity at the deducted amino acid level with that of Bos taurus. Protein sequence analysis showed that MSTN is a weakly acerbic extracellular protein, consisting of signal peptides at 18-19 sites, a TGF-ß propeptide, and a TGF-ß domain. RT-PCR analyses demonstrated that water buffalo MSTN was expressed in multiple tissues but not limited to muscle. Immunohistochemistry staining confirmed the presence of MSTN in oocytes and granulosal cells. To our knowledge, this is the first study to confirm the expression of MSTN in the water buffalo ovary, suggesting an additional role of MSTN in water buffalo folliculogenesis, along with its role in skeletal muscle growth regulation. Further study of the regulatory mechanism of MSTN in water buffalo reproduction is warranted. ABBREVIATIONS: MSTN, myostatin; ORF, open reading frame.

Identification of Differentially Expressed mRNAs and miRNAs and Related Regulatory Networks in Cumulus Oophorus Complexes Associated with Fertilization.

Cumulus oophorus complexes (COCs) are the first extracellular barriers that sperm must pass through to fuse with oocytes, which have an important role in oocyte maturation and fertilization. However, little is known about the molecular mechanisms of COCs involved in fertilization. In this study, COCs were collected and then randomly divided into a test group that interacted with sperm and a control group that did not interact with sperm. Then, the total RNA was extracted; RNA transcriptome and small RNA libraries were prepared, sequenced, and analyzed. The results showed that 1283 differentially expressed genes (DEGs), including 560 upregulated and 723 downregulated genes. In addition, 57 differentially expressed miRNAs (DEMIs) with 35 upregulated and 22 downregulated were also detected. After the RNA-seq results were verified by RT-qPCR, 86 effective DEGs and 40 DEMIs were finally screened and a DEMI-DEG regulatory network was constructed. From this, the top ten hub target genes were HNF4A, SPN, WSCD1, TMEM239, SLC2A4, E2F2, SIAH3, ADORA3, PIK3R2, and GDNF, and they were all downregulated. The top ten hub DEMIs were miR-6876-5p, miR-877-3p, miR-6818-5p, miR-4690-3p, miR-6789-3p, miR-6837-5p, miR-6861-5p, miR-4421, miR-6501-5p, and miR-6875-3p, all of which were upregulated. The KEGG signaling pathway enrichment analysis showed that the effective DEGs were significantly enriched in the calcium, AMPK, and phospholipase D signaling pathways. Our study identified several DEGs and DEMIs and potential miRNA-mRNA regulatory pathways in COCs and these may contribute to fertilization. This study may provide novel insights into potential biomarkers for fertilization failure.

Mucus-penetrating dendritic mesoporous silica nanoparticle loading drug nanocrystal clusters to enhance permeation and intestinal absorption.

Multiple gastrointestinal barriers (mucus clearance and epithelium barrier) are the main challenges in the oral administration of nanocarriers. To achieve efficient mucus penetration and epithelial absorption, a novel strategy based on mesoporous silica nanoparticles with dendritic superstructure, hydrophilicity, and nearly neutral-charged modification was designed. The mPEG covalently grafted dendritic mesoporous silica nanoparticles (mPEG-DMSNs) had a particle size of about 200 nm and a loading capacity of up to 50% andrographolide (AG) as a nanocrystal cluster in the mesoporous structure. This dual strategy of combining with the surface topography structure and hydrophilic modification maintained a high mucus permeability and showed an increase in cell absorption. The mPEG-DMSN formulation also exhibited effective transepithelial transport and intestinal tract distribution. The pharmacokinetics study demonstrated that compared with other AG formulations, the andrographolide nanocrystals-loaded mPEG-DMSN (AG@mPEG-DMSN) exhibited much higher bioavailability. Also, AG@mPEG-DMSN could significantly improve the in vitro and in vivo anti-inflammatory efficacy of AG. In summary, mPEG-DMSN offers an interesting strategy to overcome the mucus clearance and epithelium barriers of the gastrointestinal tract.

Nanocrystals based pulmonary inhalation delivery system: advance and challenge.

Pulmonary inhalation administration is an ideal approach to locally treat lung disease and to achieve systemic administration for other diseases. However, the complex nature of the structural characteristics of the lungs often results in the difficulty in the development of lung inhalation preparations. Nanocrystals technology provides a potential formulation strategy for the pulmonary delivery of poorly soluble drugs, owing to the decreased particle size of drug, which is a potential approach to overcome the physiological barrier existing in the lungs and significantly increased bioavailability of drugs. The pulmonary inhalation administration has attracted considerable attentions in recent years. This review discusses the barriers for pulmonary drug delivery and the recent advance of the nanocrystals in pulmonary inhalation delivery. The presence of nanocrystals opens up new prospects for the development of novel pulmonary delivery system. The particle size control, physical instability, potential cytotoxicity, and clearance mechanism of inhaled nanocrystals based formulations are the major considerations in formulation development.

To Enhance Mucus Penetration and Lung Absorption of Drug by Inhalable Nanocrystals-In-Microparticles.

To effectively achieve the pulmonary delivery for curcumin (CN), novel inhalable mucus-penetrating nanocrystal-based microparticles (INMP) were designed. The D-Tocopherol acid polyethylene glycol 1000 succinate (TPGS) modified CN nanocrystals (CN-NS@TPGS) were prepared by high pressure homogenization and further converted into nanocrystal-based microparticles (CN-INMP@TPGS) using spray-drying. It was demonstrated that CN-NS@TPGS exhibited little interaction with the negatively charged mucin due to a strong electrostatic repulsion effect and PEG hydrophilic chain, and exhibited a much higher penetration ability across the mucus layer compared with poloxamer 407 modified CN-NS (CN-NS@P407) and tween 80 modified CN-NS (CN-NS@TW80). The aerodynamic results demonstrated that the CN-INMP with 10% TPGS acting as the stabilizer presented a high FPF value, indicating excellent deposition in the lung after inhalation administration. Additionally, in vivo bioavailability studies indicated that the AUC(0-t) of CN-INMP@TPGS (2413.18 ± 432.41 µg/L h) were 1.497- and 3.32-fold larger compared with those of CN-INMP@TW80 (1612.35 ± 261.35 µg/L h) and CN-INMP@P407 (3.103 ± 196.81 µg/L h), respectively. These results indicated that the CN-INMP@TPGS were absorbed rapidly after pulmonary administration and resulted in increased systemic absorption. Therefore, the inhalable CN-INMP could significantly improve the bioavailability of CN after inhalation administration. The developed mucus-penetrating nanocrystals-in-microparticles might be regarded as a promising formulation strategy for the pulmonary administration of poorly soluble drugs.

Fabrication and antibacterial evaluation of peppermint oil-loaded composite microcapsules by chitosan-decorated silica nanoparticles stabilized Pickering emulsion templating.

Novel peppermint oil (PO)-loaded composite microcapsules (CM) with hydroxypropyl methyl cellulose (HPMC)/chitosan/silica shells were effectively fabricated by PO Pickering emulsion, which were stabilized with chitosan-decorated silica nanoparticles (CSN). The surface modification of chitosan could improve the hydrophobicity of silica nanoparticles and favor their adsorption at the oil-water interface of PO Pickering emulsions. The microcapsule composite shells were formed dependent on the electrostatic adsorption of HPMC and CSN, and further subjected to spray-drying. The peppermint oil-loaded composite microcapsules with 100% HPMC as wall material (PO-CM@100%HPMC) seemed to be optimum formulation based on the prolonged release, acceptable entrapment efficiency (89.1%) and drug loading (25.5%). The PO-CM@100%HPMC could remarkably prolong the stability of PO. Moreover, the PO-CM@100%HPMC had a long-term antimicrobial activity (85.4%) against S. aureus and E. coli even after storage for 60 days. Therefore, the Pickering emulsions based microcapsules seemed to be a promising strategy for antibacterial application for PO.

Cellulose nanocrystals based clove oil Pickering emulsion for enhanced antibacterial activity.

An effective antibacterial system was developed by using clove essential oil Pickering emulsion (CO-PE). The carboxymethyl cellulose sodium modified cellulose nanocrystals (CNC) was used as the stabilizer of CO-PE, which were prepared by environmentally friendly approach of homogenization technology. The factors affecting the formation and stability of CO-PE were studied, such as CNC concentration, homogenization pressure, CO concentration and ionic concentration and pH. And the antibacterial performance of CO-PE against E. coli and S. aureus was investigated by determining the minimal inhibitory concentration (MIC). The results showed that 1% CNC stabilized CO-PE exhibited small droplet size and rough surface, and had good stability at high pH values or salt concentration, owing to the presence of CNC on interface of droplet. And the CNC-stabilized CO-PE exhibited higher antimicrobial activity at equivalent CO concentration, which might be attributed to efficiently adhere to bacterial membrane. Therefore, our research would provide new insights for antibacterial application of Pickering emulsions loading essential oils in the food and other industries.

Nose-to-brain delivery of drug nanocrystals by using Ca2+ responsive deacetylated gellan gum based in situ-nanogel.

The objective of this study is to use a carbohydrate polymer deacetylated gellan gum (DGG) as matrix to design nanocrystals based intranasal in situ gel (IG) for nose-to -brain delivery of drug. The harmine nanocrystals (HAR-NC) as model drug were prepared by coupling homogenization and spray-drying technology. The HAR-NC was redispersed in the (DGG) solutions and formed the ionic-triggered harmine nanocrystals based in situ gel (HAR-NC-IG). The crystal state of HAR remained unchanged during the homogenization and spray-drying. And the HAR-NC-IG with 0.5% DGG exhibited excellent in situ-gelation ability, water retention property and in vitro release behavior. The bioavailability in brain of intranasal HAR-NC-IG were 25-fold higher than that of oral HAR-NC, which could be attributed to nanosizing effect of HAR-NC and bioadhesive property of DGG triggered by nasal fluid. And the HAR-NC-IG could significantly inhibit the expression of acetylcholinesterase (AchE) and increase the content of acetylcholin (ACh) in brain compared with those of reference formulations (p < 0.01). The DGG based nanocrystals-in situ gel was a promising carrier for nose-to-brain delivery of poorly soluble drug, which could prolong the residence time and improve the bioavailability of poorly soluble drugs in brain.

Pregnancy Outcomes in Patients With Heart Disease in China.

To analyze pregnancy outcomes of patients with heart disease in a single center and to explore the risk factors of adverse outcomes. One thousand thirty-three pregnant women with heart disease were retrospectively included from 2010 to 2017. We collected data of maternal, obstetric, and fetal outcomes. Among 1,086 pregnancies, 295 (27.1%) with congenital heart disease, 244 (22.5%) with rheumatic heart disease, 387 (35.6%) with arrhythmia, and 55 (5.1%) with cardiomyopathy. There were 8 (0.7%) maternal deaths. Risk factors of mortality were New York Heart Association (NYHA) classification IV (p <0.001), cardiac surgery during pregnancy (p <0.001), and general anesthesia (p <0.001). Maternal cardiac complications occurred in 6.7% of women, with most in the cardiomyopathy (26.0%) and rheumatic heart disease (32.9%) groups. Multivariate logistic regression modeling was used to analyze the potential risk factors. NYHA classification III and IV independently predicted worse maternal outcomes. Peripartum intensive care unit admission rate was 10.2%. Admission to intensive care unit was associated with NYHA classification II/III/IV, modified World Health Organization (mWHO) classification II-III/III/IV, and cardiac surgery during pregnancy. In conclusion, pregnancy with heart disease is at higher risk of complications for both women and neonates. In our findings, maternal morbidity is associated with NYHA classification and mWHO classification.

Roles of maltodextrin and inulin as matrix formers on particle performance of inhalable drug nanocrystal-embedded microparticles.

The objective of this study was to investigate the influence of inulin (IL) and maltodextrin (MD) as matrix formers on the physical properties of drug nanocrystal-embedded microparticles (NEM) during spray-drying and storage. The redispersibility, aerodynamic performance and phase behaviour of NEM/MD and NEM/IL stored at different water activity (aw) values were evaluated. NEM with 2 g/g (relative to the weight of drug) of IL and MD exhibited the excellent performance after spray-drying. The water activity significantly influenced the redispersibility and aerodynamic performance of NEM/MD and NEM/IL. The NEM/MD presented a higher Tg at all aw values than did NEM/IL. The moisture-induced collapse of the amorphous glassy matrix of IL and MD could be responsible for the poor redispersibility and aerodynamic performance of NEM/IL and NEM/MD, respectively. The NEM/MD exhibited better aerodynamic performance at high aw (0.528) than did NEM/IL. Therefore, MD could be an excellent matrix former for inhalable NEM.

Isolation and characterization of buffalo (bubalus bubalis) amniotic mesenchymal stem cells derived from amnion from the first trimester pregnancy.

Amniotic mesenchymal stem cells (AMSCs) from livestock are valuable resources for animal reproduction and veterinary therapeutic. The purpose of this study is to explore a suitable way to isolate and culture the buffalo AMSCs (bAMSCs), and to identify their biological characteristics. Digestion with a combination of trypsin-EDTA and collagenase type I could obtain pure bAMSCs more effectively than trypsin-EDTA or collagenase type I alone. bAMSCs could proliferate steadily in vitro culture and exhibited fibroblastic-like morphology in vortex-shaped colony. bAMSCs were positive for MSC-specific markers CD44, CD90, CD105, CD73, ß-integrin (CD29) and CD166, and pluripotent markers OCT4, SOX2, NANOG, REX-1, SSEA-1, SSEA-4 and TRA-1-81, but negative for hematopoietic markers CD34, CD45 and epithelial cells specific marker Cytokeratin 18. In addition, bAMSCs were capable of differentiating into adipogenic, osteogenic, chondrogenic and neural lineages, with expression of FABP4, Ost, ACAN, COL2A1, Nestin and ß III-tubulin. Glycogen synthase kinase 3 inhibitor: kenpaullone promoted bAMSCs to differentiate into neural lineage. This study provides an effective protocol to obtain and characterize bAMSCs, which have proven useful as a cell resource for buffalo cell reprogramming studies and transgenic animal production.