Liraglutide improves lipid metabolism by enhancing cholesterol efflux associated with ABCA1 and ERK1/2 pathway.

BACKGROUND: Reverse cholesterol transport (RCT) is an important cardioprotective mechanism and the decrease in cholesterol efflux can result in the dyslipidemia. Although liraglutide, a glucagon like peptide-1 analogue, has mainly impacted blood glucose, recent data has also suggested a beneficial effect on blood lipid. However, the exact mechanism by which liraglutide modulates lipid metabolism, especially its effect on RCT, remain undetermined. Hence, the aim of the present study was to investigate the potential impacts and potential underlying mechanisms of liraglutide on the cholesterol efflux in both db/db mice and HepG2 cells. METHODS: Six-week old db/db mice with high fat diet (HFD) and wild type mice were administered either liraglutide (200 µg/kg) or equivoluminal saline subcutaneously, twice daily for 8 weeks and body weight was measured every week. After the 8-week treatment, the blood was collected for lipid evaluation and liver was obtained from the mice for hematoxylin-eosin (HE) staining, red O staining and Western blotting. Cholesterol efflux was assessed by measuring the radioactivity in the plasma and feces after intraperitoneal injection of 3H-labeled cholesterol. HepG2 Cells were treated with different concentrations of glucose (0, 5, 25, and 50 mmol/L) with or without liraglutide (1000 nmol/L) for 24 h. The intracellular cholesterol efflux was detected by BODIPY-cholesterol fluorescence labeling. Real-time PCR or Western blotting was used to examine the expression levels of ABCA1, ABCG1 and SR-B1. RESULTS: Liraglutide significantly decreased blood glucose, serum total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C). It also reduced liver lipid deposition in db/db mice fed with HFD. Moreover, the movement of 3H-cholesterol from macrophages to plasma and feces was significantly enhanced in db/db mice fed with HFD after liraglutide adminstration. In vitro study, liraglutide could promote the cholesterol efflux of HepG2 cells under high glucose, and also increase the expression of ABCA1 by activating the ERK1/2 pathway. CONCLUSIONS: Liraglutide could improve lipid metabolism and hepatic lipid accumulation in db/db mice fed with HFD by promoting reversal of cholesterol transport, which was associated with the up-regulation of ABCA1 mediated by the ERK1/2 phosphorylation.

The Anti-Allergic Rhinitis Effect of Traditional Chinese Medicine of Shenqi by Regulating Mast Cell Degranulation and Th1/Th2 Cytokine Balance.

Shenqi is a traditional Chinese polyherbal medicine has been widely used for the treatment of allergic rhinitis (AR). The aim of this study was to investigate the anti-allergic rhinitis activity of Shenqi and explore its underlying molecular mechanism. Ovalbumin (OVA)-induced allergic rhinitis rat model was used to evaluate the anti-allergic rhinitis effect of Shenqi. The effect of Shenqi on IgE-mediated degranulation was measured using rat basophilic leukemia (RBL-2H3) cells. Primary spleen lymphocytes were isolated to investigate the anti-allergic mechanism of Shenqi by detecting the expression of transcription factors via Western blot and the level of cytokines (IL-4 and IFN-γ) via ELISA. In OVA-induced AR rat models, Shenqi relieved the allergic rhinitis symptoms, inhibited the histopathological changes of nasal mucosa, and reduced the levels of IL-4 and IgE. The results from the in vitro study certified that Shenqi inhibited mast cell degranulation. Furthermore, the results of GATA3, T-bet, p-STAT6, and SOCS1 expression and production of IFN-γ and IL-4 demonstrated that Shenqi balanced the ratio of Th1/Th2 (IFN-γ/IL-4) in OVA-stimulated spleen lymphocytes. In conclusion, these results suggest that Shenqi exhibits an obvious anti-allergic effect by suppressing the mast cell-mediated allergic response and by improving the imbalance of Th1/Th2 ratio in allergic rhinitis.

Aberrant DNA methylation of imprinted loci in human spontaneous abortions after assisted reproduction techniques and natural conception.

STUDY QUESTION: Do assisted reproduction techniques (ARTs) affect DNA methylation of imprinted genes and does aberrant methylation of imprinted genes account for the incidence of human spontaneous abortion (SA)? SUMMARY ANSWER: Our results show that imprinting errors of imprinted genes may contribute to human SA, and the occurrence of aberrant methylation of imprinted genes in ART pregnancies was comparable with that in natural pregnancies. WHAT IS KNOWN ALREADY: Animal data and human studies demonstrated that in vitro culture of embryos can cause methylation defects in individual genes, which might affect subsequent embryonic development and contribute to SA. However, our previous studies showed an abnormal methylation pattern of PEG1 in human aborted chrionic villus samples (CVS) but an increased occurrence of aberrant methylation in CVS from ART-derived pregnancies was not observed. STUDY DESIGN, SIZE AND DURATION: CVS were collected from women who underwent abortion procedures in the Department of Gynecology and Obstetrics in Nanfang Hospital from May 2008 to July 2011. Muscle samples (MS) were obtained from aborted fetuses and stillbirths. The samples were divided into four experimental groups: (A) SA/stillbirth after ART (n = 75), (B) multi-fetal reduction after ART (n = 73), (C) SA/stillbirth of natural pregnancies (n = 90) and (D) induced abortion (IA) of natural pregnancies (n = 82). PARTICIPANTS/MATERIALS, SETTING AND METHODS: The mean ± SD age of patients was 31.0 ± 4.1 (range: 18-45 years). The DNA methylation patterns of one paternally methylated (H19) and two maternally methylated (LIT1 and SNRPN) genes were analyzed in CVS and MS using pyrosequencing and bisulfite sequencing PCR. MAIN RESULTS AND THE ROLE OF CHANCE: Clear hypo-methylation (<10%) or hyper-methylation (>90%) were not detected in LIT1 and SNRPN but two regions of hyper-methylation (91.7 and 91.4%) were observed in H19. The mean percentage of methylation in the SA samples (groups A and C) was higher than that in the IA samples (groups B and D; P<0.05). Box plot analyses showed that in the 165 SA samples, methylation values for 40/495 (8.1%) differentially methylated regions of the three genes represented outliers. The incidence of outlier was highest for LIT1 (13.3%, 22/165). In contrast, no outliers were found in the 155 IA samples. The receiver operating characteristic curve analyses showed a positive correlation between percentage methylation of all three genes and incidence of SA (P<0.05). In addition, the conception modes (natural versus ART) and the fertilization methods used in ART (IVF and ICSI) did not affect the methylation patterns of the imprinted genes. No increase in the rate of abnormal methylation was found in the ART samples. LIMITATIONS AND REASONS FOR CAUTION: The studied loci represent only a small fraction of developmentally important genes. Further studies are needed to evaluate changes in the expression and the methylation status of other genes that may lead to SA. WIDER IMPLICATIONS OF THE FINDINGS: The findings provide new insights into the etiology of human SA. The possibility that the abnormal methylation seen is a consequence of the defect that led to the SA cannot be excluded. STUDY FUNDING/COMPETING INTEREST(S): None of the authors has any competing interest. This study was supported by National Natural Science Foundation of China (81170574), The National Key Basic Research Development Plan of China (973 Program) (2007CB948104), Comprehensive strategic sciences cooperation projects of Guangdong Province and Chinese Academy (04020416) and Guangzhou Science and Technology Program key projects (11C22120737).

MBOAT1 homozygous missense variant causes nonobstructive azoospermia.

Nonobstructive azoospermia (NOA) is a common cause of infertility and is defined as the complete absence of sperm in ejaculation due to defective spermatogenesis. The aim of this study was to identify the genetic etiology of NOA in an infertile male from a Chinese consanguineous family. A homozygous missense variant of the membrane-bound O-acyltransferase domain-containing 1 (MBOAT1) gene (c.770C>T, p.Thr257Met) was found by whole-exome sequencing (WES). Bioinformatic analysis also showed that this variant was a pathogenic variant and that the amino acid residue in MBOAT1 was highly conserved in mammals. Quantitative polymerase chain reaction (Q-PCR) analysis showed that the mRNA level of MBOAT1 in the patient was 22.0% lower than that in his father. Furthermore, we screened variants of MBOAT1 in a broader population and found an additional homozygous variant of the MBOAT1 gene in 123 infertile men. Our data identified homozygous variants of the MBOAT1 gene associated with male infertility. This study will provide new insights for researchers to understand the molecular mechanisms of male infertility and will help clinicians make accurate diagnoses.

Berberine attenuates atherosclerotic lesions and hepatic steatosis in ApoE-/- mice by down-regulating PCSK9 via ERK1/2 pathway.

BACKGROUND: It has been demonstrated that berberine (BBR), a kind of alkaloid derived from Chinese herbal medicine, has multiple pharmacological effects on human's diseases including anti-atherosclerosis action. However, although the previous studies showed that the beneficial impact of BBR on atherosclerosis might be associated with proprotein convertase subtilisin/kexin type 9 (PCSK9), the exact underlying mechanism are not fully determined. The present study aimed to investigate potential mechanisms of anti-atherosclerosis by BBR using ApoE-/- mice. METHODS: The eight-week mice were divided into five groups: group 1 (wild type C57BL/6J mice with normal diet), group 2 (ApoE-/- mice with normal diet), group 3 [ApoE-/- mice with high-fat diet (HFD)], group 4 (ApoE-/- mice with HFD, and treatment with low dose BBR of 50 mg/kg/d), and group 5 (ApoE-/- mice with HFD, and treatment with high dose BBR of 100 mg/kg/d). After a 16-week treatment, the blood sample, aorta and liver were collected for lipid analysis, hematoxylin-eosin (HE) or oil red O staining, and Western blotting respectively. Besides, HepG2 Cells were cultured and treated with different concentrations of BBR (0, 5, 25 and 50 µg/mL) for 24 hours. Subsequently, cells were collected for real-time PCR or western blotting assays. Finally, the expression levels of PCSK9, LDL receptor (LDLR), ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), and scavenger receptor class B type I (SR-BI) were examined. RESULTS: Fifty mg/kg/d and 100 mg/kg/d of BBR decreased total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL) cholesterol (LDL-C), and increased high-density lipoprotein cholesterol (HDL-C) level. Moreover, BBR reduced aorta atherosclerotic plaque, and ameliorated lipid deposition in ApoE-/- mice fed with HFD. Finally, in vitro study showed that BBR promoted intracellular cholesterol efflux, up-regulated LDLR and down-regulated PCSK9 expression via the ERK1/2 pathway in cultured HepG2 cells. CONCLUSIONS: Data indicated that BBR significantly attenuated lipid disorder, reduced aortic plaque formation, and alleviated hepatic lipid accumulation in ApoE-/- mice fed with HFD, which was associated with down-regulation of PCSK9 through ERK1/2 pathway.

Combined analysis of endometrial thickness and pattern in predicting outcome of in vitro fertilization and embryo transfer: a retrospective cohort study.

OBJECTIVE: To evaluate the combined effect of endometrial thickness and pattern on clinical outcome in patients undergoing in vitro fertilization/intracytoplasmic sperm injection and embryo transfer (IVF/ICSI-ET). METHODS: Cycles of IVF/ICSI-ET conducted between January 2003 and December 2008 at a university-based reproductive center were reviewed retrospectively. Endometrial ultrasonographic characteristics were recorded on the day of hCG administration. In the combined analysis, endometrial thickness groups (group 1: equal or <7 mm; group 2: 7-14 mm; group 3: >14 mm) were subdivided into two endometrial patterns (pattern A: triple-line; pattern B: no-triple line). Clinical pregnancy rate (CPR) and early miscarriage rate in different groups were analyzed. RESULTS: A total of 2896 cycles were reviewed. Clinical pregnancy rate (CPR) was 24.4% in group 1-A. There were no second trimester pregnancies in group 1-B. Miscarriage rate in group 2-A was significantly lower compared to group 2-B (P < 0.01), although CPR did not show any significant differences between the groups. A no-triple line endometrial pattern with moderate endometrial thickness (7-14 mm) had a detrimental effect on pregnancy outcome, but not the occurrence of pregnancy. In group 3, there was no difference in CPR and miscarriage rates between the two patterns; adequate endometrial thickness (>14 mm) seemed to mitigate the detrimental impact (high miscarriage rate) of pattern B. CONCLUSION: Combined analysis of endometrial thickness and pattern on the day of hCG administration was a better predictor of the outcome of IVF/ICSI-ET and may be more helpful for patient counseling than the separate analyses.

cDNA sequence and tissues expression analysis of lipoprotein lipase from common carp (Cyprinus carpio Var. Jian).

A full-length cDNA coding lipoprotein lipase (LPL) was cloned from liver of adult common carp (Cyprinus carpio Var. Jian) by RT-PCR and rapid amplification of cDNA ends (RACE) approaches. The cDNA obtained was 2,411 bp long with a 1,524 bp open reading frame (ORF) encoding 507 amino acids. This amino acid sequence contains two structural regions: N-terminus (24-354 residues) and C-terminus (355-507 residues). Before N-terminus, 1-23 residues is signal peptide, 6-23 residues is transmembrance helix. At N-terminus, some conversed functional sites were found, including two N-linked glycosylation sites Asn(41) and Asn(88); one catalytic triad Ser(174), Asp(198) and His(283); one conserved heparin-binding site Arg(321) to Arg(324) (RKNR); eight cysteines residues Cys(69) and Cys(82), Cys(258) and Cys(281), Cys(306) and Cys(325), Cys(317) and Cys(320) which are involved in four disulfide bridges; one polypeptide "lid" that participates in substrate specificity. At C-terminus, Asn(401) is another N-linked glycosylation site, and Trp(434) and Trp(435) (WW) is lipid-binding site. The amino acid sequence has a high similarity, and shows similar structural features to LPL of other species. Tissue distribution of LPL mRNA in liver, head kidney, mesenteric adipose tissue, heart and white muscle of common carp was analyzed by semi-quantitative RT-PCR method using beta-actin gene as internal control. The result showed that the expressions of LPL mRNA were detected in all examined tissues of common carp. The expression levels of LPL in the mesenteric adipose tissue was highest among these tissues, following in liver and head kidney, and the lowest expression was found in heart and white muscle.

Advanced oxidation protein products promote inflammation in diabetic kidney through activation of renal nicotinamide adenine dinucleotide phosphate oxidase.

The involvement of inflammatory processes has been recognized in development and/or progression of diabetic nephropathy. However, the mechanisms involved in the pathogenesis of renal inflammation have not been completely understood. In this study, we tested the hypothesis that accumulation of advanced oxidation protein products (AOPPs), which occurs in diabetes, may promote inflammatory responses in diabetic kidney. Streptozotocin-induced diabetic rats were randomized to iv injection of vehicle, native rat serum albumin (RSA), and AOPPs-modified RSA (AOPPs-RSA) in the presence or absence of oral administration of apocynin. A control group was followed concurrently. Compared with RSA- or vehicle-treated diabetic rats, AOPPs-RSA-treated animals displayed significant increase in renal macrophage infiltration and overexpression of monocyte chemoattractant protein-1 and TGF-beta1. This was associated with deteriorated structural and functional abnormalities of diabetic kidney, such as glomerular hypertrophy, fibronectin accumulation, and albuminuria. AOPP challenge significantly increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent superoxide generation in renal homogenates and up-regulated membrane expression of renal NADPH oxidase subunits p47(phox) and gp91(phox). All these AOPPs-induced perturbations in diabetic kidney could be prevented by the NADPH oxidase inhibitor apocynin. These data suggest that chronic accumulation of AOPPs may promote renal inflammation in diabetes probably through activation of renal NADPH oxidase.

Study of DNA methylation patterns of imprinted genes in children born after assisted reproductive technologies reveals no imprinting errors: A pilot study.

Assisted reproductive technology (ART) including in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) have been shown to be associated with abnormal genomic imprinting, thus increasing the incidence of imprinting disorders such as Beckwith-Wiedemann syndrome (BWS) and Angelman syndrome (AS) in ART-conceived children. Furthermore, a recent study described abnormal DNA methylation in clinically normal children conceived by ART. However, data from different studies are conflicting or inconclusive. This study examined DNA methylation patterns of multiple imprinted genes in children born after ART to primarily evaluate the impact of ART on genomic imprinting. A total of 101 newborns conceived by ART (40 ICSI and 61 IVF) and 60 naturally conceived newborns were involved in our study. After obtaining the approval of the Institutional Ethics Committee, umbilical cord blood was collected from each infant. Genomic DNA was isolated from each blood sample and treated using sodium bisulfite. Subsequently, using methylation-specific PCR (MS-PCR), we analyzed six differentially methylated regions (DMRs) including KvDMR1, SNRPN, MEST, MEG3, TNDM and XIST. Meanwhile, information regarding twin pregnancies, gestational age, and birth weight of the neonates was documented. None of the cases presented with phenotypic abnormalities. Children conceived by ART were more likely to have low birth weight and to be born before term, compared with children conceived spontaneously. However, 7 months to 3 years of clinical follow-up showed that none of the children had clinical symptoms of any imprinting diseases. Furthermore, the MS-PCR results showed that all 161 children had normal DNA methylation patterns at six DMRs despite the different mode of conception. Our data did not indicate a higher risk of DNA-methylation defects in children born after ART. However, further studies using quantitative methods are needed to confirm these results.

Assisted reproductive technologies do not increase risk of abnormal methylation of PEG1/MEST in human early pregnancy loss.

OBJECTIVE: To evaluate the epigenetic risk linked to assisted reproductive technology (ART) by analyzing the methylation patterns of imprinted PEG1 gene in aborted human chorionic villus. DESIGN: Experimental research study. SETTING: Research laboratory. PATIENT(S): Four patients groups were tested: spontaneous abortion after ART (n = 44), multifetal reduction after ART (n = 22), spontaneous abortion of natural pregnancies (n = 45), and induced abortion of natural pregnancies (n = 47). INTERVENTION(S): Methylation patterns of PEG1 in the aborted chorionic villus were determined. MAIN OUTCOME MEASURE(S): The DNA methylation patterns were analyzed using pyrosequencing and bisulfite sequencing polymerase chain reaction. The percentage of methylation was compared in chorionic villus from the four experimental groups. RESULT(S): Regardless of conception method, the PEG1 methylation percentage in chorionic villus from spontaneous abortions was significantly higher than in villus from induced abortions and multifetal reduction. In the spontaneous abortions groups, the percent methylation of PEG1 was similar in the villus derived from ART and from natural pregnancies. The two fertilization methods (IVF and intracytoplasmic sperm injection) did not show significant differences either. However, receiver operating characteristic curve analysis revealed a significant positive correlation between PEG1 methylation percentage and rate of early spontaneous abortions. CONCLUSION(S): As some studies have suggested, imprinting errors of PEG1 may contribute to spontaneous abortion, but ART procedures might not increase the occurrence of aberrant PEG1 methylation patterns.

[Prediction of ovarian reserve, poor response and pregnancy outcome based on basal antral follicle count and age in patients undergoing in vitro fertilization-embryo transfer].

OBJECTIVE: To study the value of basal antral follicle count (AFC) and age in predicting ovarian response and clinical outcome of in vitro fertilization-embryo transfer (IVF-ET). METHODS: A total of 1319 oocyte retrieval cycles in women with an AFC≤10 and complete IVF/ICSI cycles were analyzed retrospectively. According to the AFC, the patients were divided into groups A, B, and C with AFC≤4, of 5-7, and of 8-10, respectively, and each was further divided into <38 years old group and ≥38 years old group. The oocytes retrieved, ovarian response, implantation rate, cancellations, pregnancy, pregnancy loss, and live births were evaluated. RESULTS: As the AFC increased, the total gonadotrophin (Gn) dose increased and the follicles aspirated and oocytes retrieved decreased significantly (P<0.001). Patients below 38 years of age had a lower total Gn dose and more follicles aspirated and oocytes retrieved than older patients. An AFC>7 and age≥38 years was associated with significantly lower total Gn dose, greater number of follicles aspirated and oocytes retrieved, and lower pregnancy rate than an AFC≤7 and age<38 years (P<0.05). Bivariate correlation and linear regression analysis identified AFC as the best single predictor of ovarian response in IVF. The pregnancy rate differed significantly between the 3 groups, and older patients (≥38 years) had higher early miscarriage rate. CONCLUSION: Antral follicle count≤7 or age≥38 years old with AFC≤10 is the suitable threshold of diminished ovarian reserve in controlled ovarian stimulation for infertile women. Combination of AFC and age is the best predictor of ovarian response in IVF. Age has a better predictive value of pregnancy rate than AFC. AFC influences mainly the oocytes quantity, while age also affects oocyte quality.

[Impact of elevated basal follicle-stimulating hormone on the quantity and quality of oocytes and embryos and pregnancy outcomes in young women].

OBJECTIVE: To evaluate the effect of elevated basal follicle-stimulating hormone (FSH) on both the quantity and quality of oocytes and embryos and the clinical outcomes of pregnancy in women under 35 years of age. METHODS: A retrospective analysis was conducted for inspecting 294 in vitro fertilization-embryo transfer (IVF-ET) cycles in women under 35 years of age. According to the basal FSH levels, the women were divided into groups A, B, and C with basal FSH of 10-14.99, 15-19.99 and ≥20 IU/L, respectively, to compare the average number of oocytes retrieved, morphologies of the oocytes and embryos, and clinical outcomes of pregnancy. RESULTS: Group A showed greater average numbers of oocytes collected, total embryos and good-quality embryos with a lower gonadotrophin dose required to achieve follicular maturity than groups B and C. The 3 groups showed no significant differences in the percentage of metaphase II oocytes, optimal embryos-blastomere number, normal fertilization rate, cleavage rate, good-quality embryo rate, implantation rate, pregnancy rates, live birth rate or miscarriage rate, but the pregnancy rates and live birth rate tended to decrease in women with basal FSH ≥15 U/L. CONCLUSION: In women below 35 years of age, an elevated serum FSH (especially one ≥15 U/L) indicates diminished ovarian reserve and reduced numbers of oocyte and embryo but not poor oocyte or embryos quality, and good clinical pregnancy rate can still be expected.

Aberrant DNA methylation of imprinted H19 gene in human preimplantation embryos.

DNA methylation patterns of the H19 differentially methylated region were successfully determined in 32 of 50 human day 3 embryos produced and discarded after assisted reproductive technology procedures. We found methylation patterns similar to those of somatic cells in 81.3% of embryos and demethylation or hypomethylation patterns in 18.7% of embryos.

[Clinical outcomes and birth defects resulting from intracytoplasmic sperm injection using sperms of different origins and parameters].

OBJECTIVE: To explore the quality of the embryos, clinical outcomes and birth defects resulting from intracytoplasmic sperm injection (ICSI) treatment using sperms of different origins and parameters. METHODS: A total of 980 ICSI-ET (embryo-transfer) cycles were divided into 4 groups, namely normal sperm or mild oligozoospermia group (group A), severe oligozoospermia group (group B), epididymal aspirates group (group C) and testicular biopsies group (group D). The cleavage rate, embryo quality, fertilization rate, clinical pregnancy rate, and rates of birth defects were compared between the groups. RESULTS: The fertilization rate, cleavage rate and good-quality embryo rate were not significantly different among the 4 groups (P>0.05), and the embryo implantation rate and clinical pregnancy rate were significantly higher in group C than in groups A and B (P<0.05). Groups A, B and C showed no significant differences in the rates of birth defects (P>0.05), and no birth defects occurred in group D. CONCLUSIONS: Sperms of different parameters and origins used in ICSI treatment can achieve similar fertilization rate, good-quality embryo rate and delivery rate. The embryo implantation rate and clinical pregnancy rate of epididymal sperm group are higher than those of ejaculated groups, possibly due to the younger age of the patients and a greater number of oocytes retrieved in group C than in groups A and B.

[In vitro fertilization versus intracytoplasmic sperm injection for primary and secondary infertility using sibling oocytes: clinical analysis of the outcomes].

OBJECTIVE: To evaluate the clinical outcomes of intracytoplasmic sperm injection (ICSI) and conventional in vitro fertilization (IVF) using sibling oocytes for treatment of primary and secondary infertility. METHODS: A total of 149 cycles of IVF and ICSI were conducted between January, 2003 and December, 2008 in our center, including 98 cycles in patients with primary infertility and 51 in those with secondary infertility. According to the embryos derived from ICSI, IVF and their combination, the clinical pregnancy rate, delivery rate and birth defect of the 3 groups were analyzed. RESULTS: The fertilization failure rate of IVF was significantly higher in primary infertility group than in secondary infertility group (10.2% vs 3.9%, P<0.05). No fertilization failure occurred in ICSI group. The fertilization rates and good quality embryo rates in ICSI group were significant higher than those in IVF group, and the abnormal fertilization rate was significantly lower in ICSI group (P<0.05). No significant difference were found in the implantation rates, clinical pregnancy rates, delivery rates or the rates of birth defects of the offsprings between IVF, ICSI and IVF+ICSI groups. CONCLUSION: IVF combined with ICSI may result in increased fertilization rate and avoid total fertilization failure with favorable clinical outcomes in patients with long-term infertility, and ICSI may not increase the birth defects of the offspring in these patients.

Molecular cloning and tissue distribution of lipoprotein lipase full-length cDNA from Pengze crucian carp (Carassius auratus var. Pengze).

A full-length cDNA coding lipoprotein lipase (LPL) was cloned from liver of adult Pengze crucian carp (Carassius auratus var. Pengze) by RT-PCR and rapid amplification of cDNA ends (RACE) approaches. The cDNA obtained was 1877 bp long with a 1524 bp open reading frame (ORF) encoding 507 amino acids, including a putative signal peptide of 23 amino acids long. The deduced amino acid sequence has a high similarity and shows similar structural features to LPL of other species. The LPL protein has a calculated molecular mass of 57.7 kDa and isolectric point of 7.85. Tissue distribution of LPL mRNA in mesenteric adipose tissue, liver, heart, head kidney and white muscle of adult Pengze crucian carp was analyzed by semi-quantitative RT-PCR method using beta-actin gene as internal control, the result showed that this gene was ubiquitously expressed in all tissues tested with the highest abundance in mesenteric adipose tissue, following in head kidney and liver, and the lowest expression was found in heart and white muscle.