Next-Generation Sequencing Is More Efficient at Detecting Mosaic Embryos and Improving Pregnancy Outcomes than Single-Nucleotide Polymorphism Array Analysis.

We compared chromosomal mosaicism, detected by next-generation sequencing (NGS), during preimplantation genetic testing (PGT) with that detected by single-nucleotide polymorphism (SNP) array-based PGT to assess the pregnancy outcomes associated with both platforms in a retrospective cohort study of patients undergoing in vitro fertilization in a single university-based assisted reproduction center. In total, 6427 blastocysts biopsied from 1513 patients who underwent 2833 oocyte retrievals from January 2017 to February 2019 were identified. The incidence of mosaicism was significantly higher in the NGS-based PGT group than in the SNP array-based PGT group. Furthermore, some aneuploid specimens were affected by mosaicism. The total mosaicism detection rate with NGS-based PGT (23.3%) was significantly higher than that with SNP array-based PGT (7.7%). Mosaicism rates were similar when stratified by maternal age or PGT type. The SNP array cohort showed a significantly higher spontaneous abortion rate than the NGS cohort (10.07% versus 6.33%; P = 0.0403). The ongoing pregnancy/live birth rate was higher in the NGS cohort (44.1%) than in the SNP array cohort (42.28%). Our results confirm that NGS-based PGT can detect mosaicism more frequently than SNP array-based PGT in trophectoderm specimens. Therefore, clinical application of NGS for PGT may improve pregnancy outcomes compared with that of SNP array-based PGT. More detailed blastocyst detection and classification is necessary to prioritize embryo transfers.

Hepatic Cyp1a2 Expression Reduction during Inflammation Elicited in a Rat Model of Intermittent Hypoxia.

BACKGROUND: Intermittent hypoxia (IH) is a key element of obstructive sleep apnea (OSA) that can lead to disorders in the liver. In this study, IH was established in a rat model to examine its effects on the expression of hepatic cytochrome P450 (CYP) and CYP regulators, including nuclear receptors. METHODS: Hematoxylin and eosin staining was conducted to analyze the general pathology of the liver of rats exposed to IH. The messenger RNA (mRNA) expression levels of inflammatory cytokines, CYPs, nuclear factor-κB (NF-κB), and nuclear factors in the liver were measured by quantitative reverse transcription polymerase chain reaction. RESULTS: We found inflammatory infiltrates in the liver of rats exposed to IH. The mRNA expression level of interleukin-1beta was increased in the liver of the IH-exposed rats (0.005 ± 0.001 vs. 0.038 ± 0.008, P = 0.042), whereas the mRNA expression level of Cyp1a2 was downregulated (0.022 ± 0.002 vs. 0.0050 ± 0.0002, P = 0.029). The hepatic level of transcription factor NF-κB was also reduced in the IH group relative to that in the control group, but the difference was not statistically significant and was parallel to the expression of the pregnane X receptor and constitutive androstane receptor. However, the decreased expression of the glucocorticoid receptor upon IH treatment was statistically significant (0.056 ± 0.012 vs. 0.032 ± 0.005, P = 0.035). CONCLUSIONS: These results indicate a decrease in expression of hepatic CYPs and their regulator GR in rats exposed to IH. Therefore, this should be noted for patients on medication, especially those on drugs metabolized via the hepatic system, and close attention should be paid to the liver function of patients with OSA-associated IH.

Impaired Capacity of Fibroblasts to Support Airway Epithelial Progenitors in Bronchiolitis Obliterans Syndrome.

BACKGROUND: Bronchiolitis obliterans syndrome (BOS) often develops in transplant patients and results in injury to the respiratory and terminal airway epithelium. Owing to its rising incidence, the pathogenesis of BOS is currently an area of intensive research. Studies have shown that injury to the respiratory epithelium results in dysregulation of epithelial repair. Airway epithelial regeneration is supported by stromal cells, including fibroblasts. This study aimed to investigate whether the supportive role of lung fibroblasts is altered in BOS. METHODS: Suspensions of lung cells were prepared by enzyme digestion. Lung progenitor cells (LPCs) were separated by fluorescence-activated cell sorting. Lung fibroblasts from patients with BOS or healthy controls were mixed with sorted mouse LPCs to compare the colony-forming efficiency of LPCs by counting the number of colonies with a diameter of ≥50 µm in each culture. Statistical analyses were performed using the SPSS 17.0 software (SPSS Inc., USA). The paired Student's t-test was used to test for statistical significance. RESULTS: LPCs were isolated with the surface phenotype of CD31-CD34-CD45- EpCAM+Sca-1+. The colony-forming efficiency of LPCs was significantly reduced when co-cultured with fibroblasts isolated from patients with BOS. The addition of SB431542 increased the colony-forming efficiency of LPCs to 1.8%; however, it was still significantly less than that in co-culture with healthy control fibroblasts (P < 0.05). CONCLUSION: The epithelial-supportive capacity of fibroblasts is impaired in the development of BOS and suggest that inefficient repair of airway epithelium could contribute to persistent airway inflammation in BOS.

Protective effect of autophagy inhibition on ischemia-reperfusion-induced injury of N2a cells.

Autophagy is a conserved and programmed catabolic process that degrades damaged proteins and organelles. But the underlying mechanism and functions of autophagy in the ischemia-reperfusion (IR)-induced injury are unknown. In this study, we employed simulated IR of N2a cells as an in vitro model of IR injury to the neurons and monitored autophagic processes. It was found that the levels of Beclin-1 (a key molecule of autophay complex, Beclin-1/class III PI3K) and LC-3II (an autophagy marker) were remarkably increased with time during the process of ischemia and the process of reperfusion after 90 min of ischemia, while the protein kinases p70S6K and mTOR which are involved in autophagy regulation showed delayed inactivation after reperfusion. Administration of 3-methyladenine (3MA), an inhibitor of class III PI3K, abolished autophagy during reperfusion, while employment of rapamycin, an inhibitor of mTORC1 (normally inducing autophagy), surprisingly weakened the induction of autophagy during reperfusion. Analyses of mitochondria function by relative cell viability demonstrated that autophagy inhibition by 3-MA attenuated the decline of mitochondria function during reperfusion. Our data demonstrated that there were two distinct dynamic patterns of autophagy during IR-induced N2a injury, Beclin-1/class III PI3K complex-dependent and mTORC1-dependent. Inhibition of over-autophagy improved cell survival. These suggest that targeting autophagy therapy will be a novel strategy to control IR-induced neuronal damage.

Effect of low-molecular-weight heparin and urokinase on pulmonary arteries involved in pulmonary embolism.

BACKGROUND: Pulmonary embolism (PE) is a common and often fatal disease. Early after pulmonary thromboembolism, inflammation and associated intimal hyperplasia occur within the pulmonary arteries, similar to what is observed with chronic thromboembolic pulmonary hypertension. This study tested the hypothesis that thrombolytic and anticoagulant agents would have anti-inflammatory effects or inhibit intimal hyperplasia of involved pulmonary arteries. METHODS: Seventy-two male New Zealand white rabbits were randomly divided into two groups (54 rabbits in the PE group and 18 in the sham group). Experimental PE was induced in 54 rabbits by femoral vein injection of autologous blood clots and confirmed with pulmonary angiography, and other 18 rabbits underwent sham operations. Fifty-four rabbits in the PE group were randomly divided into three groups: a control group (treated with normal saline), a low-molecular- weight heparin (LMWH) group (treated with LMWH), and a urokinase (UK) group (treated with UK). Arterial blood gas was analyzed at 2, 7, and 28 days (n = 6 per time point by random group division), then lung tissues were removed and were analyzed for pro-inflammatory cytokines and chemokines, and were stained for intimal hyperplasia. RESULTS: The overall survival of rabbits undergoing PE was 100%. PE distribution detected on digital signal angiography (DSA) and histopathology was shown in 67% of rabbits (36/54) in the bilateral low lobar pulmonary arteries (PAs). The results showed that alveolar-arterial partial pressure of oxygen (PO2) difference (PA-aO2) significantly increased and PO2 decreased in the control group compared with the sham group. Compared with controls, the UK group had a decreased level of PA-aO2 on day 2 (P < 0.05), however, there was no significant difference in the LMWH group. Compared with controls, the LMWH group had a decreased level of monocyte chemoattractant protein-1 (MCP-1) in affected tissue and serum samples on days 7 and 28 (P < 0.05), and the UK group had decreased levels on days 2 and 7 (P < 0.05). Compared with sham group, all PE groups had an increased level of interleukin-13 (IL-13) and transforming growth factor-ß (TGF-ß) in unaffected lung tissue samples at days 2 and 7. IL-13 in affected lung tissue in the LMWH group was decreased at all time points compared with controls (P < 0.05). However, TGF-ß in affected lung tissue of the LMWH and UK groups increased at day 28. There was less intimal hyperplasia in involved pulmonary arteries at days 7 and 28 in the LMWH group compared with controls; there was no statistical difference in the UK group compared with controls. CONCLUSIONS: UK treatment can rapidly improve the V/Q mismatch in PE and appears a short-term anti-inflammatory benefit. However, LMWH maybe inhibit the later local inflammatory reaction and reduce intimal hyperplasia.

Idiopathic pulmonary vein thrombosis extending to left atrium: a case report with a literature review .

Pulmonary vein thrombosis is a rare disease and is usually represented as a complication of atrial fibrillation, pulmonary tumors, and lobectomy. Although it is a potentially life threatening condition, the venous disease is easy to misdiagnose because of the non-specific symptoms. In this article, we present a 30-year-old patient who suffered from pulmonary vein thrombosis without any causes. He was diagnosed with other pulmonary disorders till the thrombus within the pulmonary vein extended into the left atrium. Left atrium mass resection and a left lower lobectomy were undertaken with relative urgency. The postoperative course was uneventful. The patient received a long course of oral anticoagulant therapy.

Does prohibitin expression regulate sperm mitochondrial membrane potential, sperm motility, and male fertility?

Prohibitin (PHB) is a highly conserved major sperm mitochondrial membrane protein whose absence in somatic cells is associated with mitochondrial membrane depolarization and increased generation of reactive oxygen species (ROS). Our recent findings suggest that high levels of oxidants in human semen may contribute to male infertility and that sperm motility could be the earliest and most sensitive indicator of oxidative damage. Based on PHB's roles in mitochondrial sub-compartmentalization and respiratory chain assembly, we examine sperm PHB expression and mitochondrial membrane potential (MITO) in infertile men with poor sperm motility (asthenospermia, A) and/or low sperm concentrations (oligoasthenospermia, OA). Here, we demonstrate that MITO is significantly lower in sperm from A and OA subjects than in normospermic (N) subjects; the decrease is more severe for OA than for A subjects. PHB expression is also significantly lower in sperm from A and OA subjects. Significantly positive correlations are found among PHB expression, MITO, and sperm motility in normospermic, asthenospermic, and oligoasthenospermic subjects. Collectively, our observations lead to the hypothesis that PHB expression is an indicator of sperm quality in infertile men, and that it regulates sperm motility via an alteration in MITO and increased ROS levels.

[Karyotype analysis of amniotic fluid cells and comparison of chromosomal abnormality rate during second trimester].

OBJECTIVE: To investigate the karyotypes of amniotic fluid cells and compare the incidence of chromosomal abnormality as well as to evaluate the clinical significance of abnormal karyotypes. METHODS: A total of 13 648 pregnant women came to Shanghai Jiai Genetics and IVF Institute, Obstetrics and Gynecology Hospital, Fudan University to do amniocentesis from September 1998 to November 2010, and 13 795 amniotic fluid specimens were successfully extracted and cultured, thus 13 795 fetuses received karyotype diagnosis. These fetuses were grouped according to different indications. If maternal age was ≥ 35, the fetuses were grouped into the advanced maternal age group (4065); and if maternal serum screening test revealed high-risk of trisomy 18 or trisomy 21, the fetuses were grouped into the high-risk serum screening group (6462); and those with abnormal signs of ultrasound screening were grouped into the abnormal ultrasound signs group (1539); and if either of the parents was with chromosome abnormalities, the fetus was grouped into the paternal/maternal abnormality group (108); whereas the remainder were grouped in other factors group (1621). The amniotic fluid cells were in-situ cultured on coverslips, harvested by conventional G-banded methods, and then analyzed by two doctors. In order to get rapid diagnosis, some pregnant women whose gestational age ≥ 26 weeks accepted fluorescense in situ hybridization (FISH). FISH was done on 78 uncultured amniotic fluid specimens using probes located at chromosome 13, 18, 21, X, Y. Some parents were required to analyze lymphocyte karyotype to help judging the origin of abnormal karyotype. RESULTS: (1) Classification and composition of abnormal karyotypes in each group: a total of 388 abnormal karyotypes were found among 13 795 fetuses, and the abnormal rate was 2.813% (388/13 795). Of the 388 fetuses, aneuploidy was the most common pattern which was up to 59.8% (232/388); autosomal structural abnormality rate was 24.7% (96/388); mosaicism was 12.4% (48/388). Other uncommon abnormal karyotypes included marker chromosome (5/388, 1.3%), sex chromosomal structural abnormality (4/388, 1.0%) and triploid (3/388, 0.8%). Aneuploidy was the most common in most groups except the paternal/maternal abnormality group. There were four cases of rare aneuploid in the advanced maternal age group, the high-risk serum screening group and the abnormal ultrasound signs group respectively. Every type of abnormality could be found in the abnormal ultrasound signs group, and autosomal structural abnormalities were concentrated in paternal/maternal abnormality group. Mosaicism mainly distributed in the high-risk serum screening group, accounting for 20.0% (29/145) of abnormalities in this group. (2) Abnormal types and the incidence: the most common type was trisomy 21 (138/388, 35.6%), followed by autosomal balanced structural rearrangements (80/388, 20.6%), mosaicism (48/388, 12.4%) and trisomy 18 (44/388, 11.3%). Others included non-balanced autosomal structural rearrangements (16/388, 4.1%), 45, X0 (16/388, 4.1%) and 47, XXY (15/388, 3.9%). (3) Lymphocyte karyotype analysis of the couples: parents of 153 fetuses were analyzed to determine the origin of abnormal karyotype. Fifty-eight familial and 95 de novo abnormalities were found. FISH results were the same with G-banding karyotype, and two of these were trisomy 21. CONCLUSIONS: Abnormal karyotype composition is different according to different maternal amniocentisis indications. There is a variety of abnormal karyotypes in the second trimester pregnancy, and the risk of fetal malformation is related with the kind of abnormal karyotype.

[Probe into sleep quality in the patients with irritable bowel syndrome].

OBJECTIVE: To investigate the sleep features in the patients with irritable bowel syndrome (IBS) and compare the sleep quality between those IBS patients who were with and without anxiety and depression. METHODS: Pittsburgh sleep quality index questionnaire (PSQI), self-rating anxiety scale (SAS) and self-rating depression scale (SDS) were measured in the 145 IBS patients and 59 regular physical examination volunteers. IBS patients were also divided into two subgroups--patients with or without anxiety and depression based on cutoff scores of SAS and SDS. Comparisons of sleep quality were made between subgroups, and between IBS patients and volunteer controls. RESULTS: Compared with the controls, the SAS raw score, SDS raw score and SAS positive incidence in IBS patients were shown statistically significant differences (P<0.05), while the SDS positive incidence had no statistically significant difference (P>0.05). PSQI total scores were significantly higher in the IBS patients without anxiety and depression (P<0.05), 3 domains (sleep quality, sleep disturbances and daytime function disorder) were also found statistically significant differences (P<0.05), compared with the controls. The IBS patients with anxiety and depression were statistically significantly different from the controls (P<0.05) in 6 domains (sleep quality, sleep latency, sleep efficiency, sleep disturbances, sleep time and daytime function disorder) and significantly higher PSQI total scores (P<0.05). Statistically significant differences (P<0.05) were also found in all 7 domains and with higher PSQI total scores in IBS patients with anxiety and depression, compared with IBS patients without anxiety and depression. CONCLUSIONS: IBS patients were more likely to have sleep abnormality, mainly in sleep quality, sleep disturbances and daytime function disorder and PSQI total scores. The abnormalities of these factors were independent of emotional disorder. However, emotional disorder worsened the sleep disorder in IBS patients.

[Eleven fatal cases with A H1N1 influenza in Tianjin].

OBJECTIVE: To describe the clinical characteristics of fatal cases with confirmed A H1N1 influenza so as to improve the diagnosis and treatment of this severe disease. METHODS: We retrospectively analyzed the medical records of 11 fatal cases with A H1N1 influenza in Tianjin between September 1 and November 4 2009. RESULTS: There were 6 males and 5 females with a median age of 25 (21 - 36) years old. Two cases were pregnant women and 3 patients suffered such concurrent disorders as dilated cardiomyopathy, multiple myeloma or atrophic gastritis. Over 3 lung fields were involved on chest imaging studies and the oxygenation index was less than 300 mm Hg at admission. The therapies of oseltamivir and methylprednisolone were administered and mechanical ventilation was initiated within 24 hours. Refractory hypoxemia and a higher level of lactate dehydrogenase were present during treatment. Pneumothorax or mediastinal emphysema occurred in 4 patients, acute renal failure in 1 and pneumopyothorax in 1. The culture of airway secretion at 3 - 7 days after mechanical ventilation showed Staphylococcus aureus in 4 patients and Aspergillus in 2. The progress of disease was so quick that the duration from onset of clinical symptoms to hospitalization was a median of 4 (3 - 6) days and the duration from onset of clinical symptoms to death a median of 12 days. CONCLUSION: The fatal cases with A H1N1 influenza in Tianjin occurred mostly in young individuals and pregnant women. This severe disease had a rapid progression. And bacterial co-infections were quite common. Refractory hypoxemia resulting in respiratory failure was the main mortality reason.

5-Azacytidine facilitates osteogenic gene expression and differentiation of mesenchymal stem cells by alteration in DNA methylation.

Mesenchymal stem cells (MSCs) are considered to be one of the most promising therapeutic cell sources as they encompass a plasticity of multiple cell lineages. The challenge in using these cells lies in developing well-defined protocols for directing cellular differentiation to generate a desired lineage. In this study, we investigated the effect of 5-azacytidine, a DNA demethylating agent, on osteogenic differentiation of MSCs. The cells were exposed to 5-azacytidine in culture medium for 24 h prior to osteogenic induction. Osteogenic differentiation was determined by several the appearance of a number of osteogenesis characteristics, including gene expression, ALP activity, and calcium mineralization. Pretreatment of MSCs with 5-azacytidine significantly facilitated osteogenic differentiation and was accompanied by hypomethylation of genomic DNA and increased osteogenic gene expression. Taking dlx5 as a representative, methylation alterations of the "CpG island shore" in the promoter caused by 5-azacytidine appeared to contribute to osteogenic differentiation.

[Application of bronchoscopic lung volume reduction using one-way flap device in sheep model of heterogeneous emphysema].

OBJECTIVE: To evaluate the effectiveness of bronchoscopic lung volume reduction (BLVR) using one way flap device in sheep model of heterogeneous emphysema. METHODS: Six 6-month sheep (weight: 20-30 kg) were treated with localized papain instillations to generate heterogeneous emphysema, subsequently underwent BLVR using one way-flap device at subsegment. Lung functional residual capacity (FRC) was analyzed before and 8 weeks after operation. Animals were euthanized at the 8-week time point. Lungs were removed en bloc and inflated with a super syringe to look for areas of gross collapse. Samples were collected from collapsed and non-collapsed areas, fixed in 10% buffered formalin, made paraffin section and stained with hematoxylin-eosin (HE) to observe morphologic change of bronchi. RESULTS: BLVR was well tolerated without complications, and it reduced lung volumes (change in residual volume 49.5%). There was no evidence of infection, abscess, or granuloma formation, or allergic reaction. Scar tissue, generated by BLVR, replaced hyperinflated lung,improved respiratory function safely and consistently. CONCLUSION: BLVR using one-way flap device is a minimally invasive procedure and the stimulus of the devices to walls of bronchi is slight, moreover, the technique may attain effectiveness of surgery. Therefore maybe it will be a perspective treatment of chronic obstructive pulmonary disease.