Novel PLCZ1 mutation caused polyspermy during in vitro fertilization.

ABSTRACT: Failure of oocyte activation, including polyspermy and defects in pronuclear (PN) formation, triggers early embryonic developmental arrest. Many studies have shown that phospholipase C zeta 1 ( PLCZ1 ) mutations cause failure of PN formation following intracytoplasmic sperm injection (ICSI); however, whether PLCZ1 mutation is associated with polyspermy during in vitro fertilization (IVF) remains unknown. Whole-exome sequencing (WES) was performed to identify candidate mutations in couples with primary infertility. Sanger sequencing was used to validate the mutations. Multiple PLCZ1 -mutated sperm were injected into human and mouse oocytes to explore whether PN formation was induced. Assisted oocyte activation (AOA) after ICSI was performed to overcome the failure of oocyte activation. We identified three PLCZ1 mutations in three patients who experienced polyspermy during IVF cycles, including a novel missense mutation c.1154C>T, p.R385Q. PN formation failure was observed during the ICSI cycle. However, injection of multiple PLCZ1 -mutated sperm induced PN formation, suggesting that the Ca 2+ oscillations induced by the sperm exceeded the necessary threshold for PN formation. AOA after ICSI enabled normal fertilization, and all patients achieved successful pregnancies. These findings expand the mutational spectrum of PLCZ1 and suggest an important role for PLCZ1 in terms of blocking polyspermy. Furthermore, this study may benefit genetic diagnoses in cases of abnormal fertilization and provide potential appropriate therapeutic measures for these patients with sperm-derived polyspermy.

Drimane-type sesquiterpenoids and their anti-inflammatory evaluation from Pyrrhoderma noxium HNNU0524.

Chemical investigation of a culture broth from the marine-derived fungus Pyrrhoderma noxium HNNU0524 yielded two new compounds including a drimane-type sesquiterpenoid named pyrrnoxin A (1) and a benzoic acid derivative, pyrrnoxin B (5), together with three related known analogues (2-4). The chemical structures of 1 and 5 were determined by detailed analysis of spectroscopic data, single-crystal X-ray crystallography, quantum mechanics-based DP4+ and ECD calculations. Compounds 2 and 3 moderately inhibited NO production of lipopolysaccharide-induced microglia cells BV2 with IC50 values of 26.6 and 60.5 µM, respectively.

Prolonged interval time between blastocyst biopsy and vitrification compromised the outcomes in preimplantation genetic testing.

This study aimed to evaluate to what extent the different interval times between trophectoderm (TE) biopsy and vitrification influence the clinical outcomes in preimplantation genetic testing (PGT) cycles. Patients who underwent frozen embryo transfer (FET) after PGT between 2015 and 2019 were recruited. In total, 297 cycles with single day 5 euploid blastocyst transfer were included. These cycles were divided into three groups according to the interval times: <1 h group, 1-2 h group, and ≥2 h group. Blastocyst survival, clinical pregnancy, miscarriage, and ongoing pregnancy rates were compared. The results showed that, in PGT-SR cycles, survival rate in the ≥2 h group (96.72%) was significantly lower than in the <1 h group (100%, P = 0.047). The clinical pregnancy rate in the ≥2 h group was 55.93%, significantly lower than in the <1 h group (74.26%, P = 0.017). The ongoing pregnancy rates in the 1-2 h group and the ≥2 h group were 48.28% and 47.46%, respectively, significantly lower than that in the <1 h group (67.33%, P < 0.05). The miscarriage rate in the 1-2 h group was 18.42%, significantly higher than that in the <1 h group (5.33%, P = 0.027). In PGT-A cycles, the clinical pregnancy and ongoing pregnancy rates in the <1 h group were 67.44% and 53.49%, respectively, higher than that in the 1-2 h group (52.94%, 47.06%, P > 0.05) and the ≥2 h group (52.63%, 36.84%, P > 0.05). In conclusion, vitrification of blastocysts beyond 1 h after biopsy significantly influences embryo survival and clinical outcomes and is therefore not recommended.

Amino acid ester-coupled caffeoylquinic acid derivatives as potential hypolipidemic agents: synthesis and biological evaluation.

Pandanus tectorius (L.) Parkins. (PTPs) is rich in caffeoylquinic acids and amino acids, especially some essential amino acids, such as valine, phenylalanine, and so forth. A series of novel amino acid ester-coupled caffeoylquinic acid derivatives have been designed and synthesized. Biological evaluation suggested that some amino acid ester-coupled derivatives exhibited varying degrees of lipid-lowering effects on oleic acid-elicited lipid accumulation in HepG2 liver cells. Particularly, derivatives 6c, 6d, 6e and 6f exhibited comparable potential lipid-lowering effect with the positive control simvastatin and chlorogenic acid. Further studies on the mechanism of 6c, 6d, 6e and 6f revealed that the lipid-lowering effects were related to their regulation of TG levels and mRNA levels of lipometabolic-modulating genes, and merit further investigation.

Shortened equilibration time can compromise clinical outcomes in human embryo vitrification.

Vitrification is an important way to cryopreserve human embryos and the recommended time of embryo exposure to the vitrification solution is 1 min. However, practically speaking, the duration of embryos exposure to equilibration solution can vary from 5 to 15 min. The purpose of this study was to investigate the effect of different equilibration times on the outcomes of frozen-thawed embryo transfer cycles. The data were collected from our medical records from January 2012 to June 2013 and a total of 517 cycles were included. These cycles were divided into four groups according to the equilibration time: (i) 5-6 min; (ii) 7-8 min; (iii) 9-10 min and (iv) 11-12 min. The results show that there were no differences in terms of survival rate and fully intact embryo rate among the four groups. However, lower clinical pregnancy, embryo implantation and live birth rates were observed in the 5-6 min exposure group (54.6%, 31.9% and 48.2%, respectively) compared with the three other groups. The corresponding rates in the 9-10 min group (73.5%, 47.6% and 64.7%) were the highest. This study indicated that different equilibration times influenced the clinical outcomes of human embryo vitrification and vitrification with shortened equilibration time compromised the clinical outcomes. Appropriate prolongation of the equilibrium time would probably improve the clinical outcomes.

[Risk factors for the failure of the InSure method in very preterm infants with respiratory distress syndrome].

OBJECTIVE: To study the risk factors for the failure of the InSure method in very preterm infants with respiratory distress syndrome (RDS). METHODS: Seventy-one very preterm infants with RDS treated with InSure method were enrolled. These infants were categorized into two groups: InSure success (42 cases) and InSure failure (29 cases). The differences in basic information were compared between the two groups, and logistic regression analysis was used to identify the risk factors for InSure failure. RESULTS: The failure rate of the InSure method was 41%. The failure group were much lower in the birth weight, the antenatal steroids utilization rate and the vaginal delivery rate than the success group (P<0.05). The incidence of patent ductus arteriosus in the failure group was significantly higher than in the success group (P<0.05). PaO2, PaO2/FiO2 and PaO2/PAO2 in the failure group were significantly lower than in the success group (P<0.05). PaCO2 in the failure group was much higher than in the success group (P<0.05). Further logistic regression analysis showed that birth weight <1 150 g (OR=22.240 95%CI=2.124-232.901), PaCO2>54 mm Hg(OR=9.360, 95%CI=1.958-44.741, and PaO2/FiO2 <195 (OR=6.570, 95%CI=1.027-42.003), were the independmend risk factors for InSure failure. Furthermore, the duration of oxygen therapy, the total time of hospitalization and the incidence of BPD in the failure group were much longer and higher than in the success group (P<0.05). CONCLUSIONS: Low birth weight, elevated PaCO2 and low PaO2/PiO2 ratio are the risk factors for the failure of the InSure method in very preterm infants.

The effects of different sperm preparation methods and incubation time on the sperm DNA fragmentation.

PURPOSE: To study the effect of different sperm preparation methods and incubation times post preparation on sperm DNA fragmentation. METHODS: Sperm DNA fragmentation was assessed by sperm chromatin dispersion (SCD) method on motile sperm prepared by gradient centrifugation or swim-up and incubated in IVF medium for up to 24 hours. Data were analyzed to discover the effect of preparation methods and incubation times on sperm DNA Fragmentation Index (DFI). RESULTS: There were no differences in DFI in sperm samples prepared by gradient centrifugation method or swim-up (3.87 ± 2.14 vs. 3.45 ± 1.83, p = 0.544). However, an increase was observed in DFI in samples prepared by swim-up after 6-8 hours compared with by gradient centrifugation (34° vs. 13°, p = 0.04). In the swim-up group, the DFI level at 4 hours was already significantly higher than it was initially. However, following gradient centrifugation, the DFI at 8 hours was significantly higher than the initial DFI level. CONCLUSION: Sperm samples prepared by gradient centrifugation may be more stable, in terms of DNA fragmentation, than samples prepared by swim-up.

[Expression of IL-8, SP-A and TGF-beta1 in bronchoalveolar lavage fluid of neonates with bronchopulmonary dysplasia].

OBJECTIVE: To investigate the expression of interleukin 8 (IL-8), surfactant protein-A (SP-A) and transforming growth factor beta1 (TGF-beta1) in bronchoalveolar lavage fluid (BALF) of neonates with bronchopulmonary dysplasia (BPD). METHODS: Thirty neonates with BPD and 30 gestational age-, gender-, and birth weight-matched neonates without BPD (control group) were enrolled from December 2007 to October 2009. Non-brochoscopic bronchoalveolar lavage was performed. The levels of IL-8, SP-A and TGF-beta1 in BALF were measured using ELISA. RESULTS: The levels of TGF beta1 (47+/-15 microg/mL vs 34+/-13 microg/mL) and IL-8 (54+/-16 microg/mL vs 28+/-13 microg/mL) in the BPD group were significantly higher than those in the control group (P<0.01). In contrast, the contents of SP-A in the BPD group were significantly lower than those in the control group (35+/-16 microg/mL vs 42+/-14 microg/mL;P<0.05). CONCLUSIONS: The increased expression of TGF-beta1 and IL-8 in BALF may be involved in abnormal lung development and maturation in neonates with BPD. The low expression of SP-A in the BPD group suggests that the exogenous SP-A administration may be an option for the treatment of BPD.

[Intra-amniotic administration of pulmonary surfactant improves lung ultrastructural pathology and enhances surfactant protein A expression in fetal rabbits with intrauterine infection].

OBJECTIVE: To observe the effect of intra-amniotic administration of pulmonary surfactant (PS) on the lung ultrastructure and expression of surfactant protein A (SP-A) in fetal rabbit with intrauterine infection. METHODS: Intra-amniotic PS injection was administered in a rabbit model of premature rupture of membrane and intrauterine infection induced by intrauterine colibacillus injection on the gestational days 24 and 26 days. The lung ultrastructural changes in the fetal rabbits were observed using electron microscope, and the expression of SP-A was measured with immunohistochemical staining and Western blotting 19.5 h after the PS administration. RESULTS: alveolar type I cell (AT I), alveolar type II cell (AT II). In fetal rabbits with intrauterine colibacillus injection, inflammatory cell infiltration was observed in the pulmonary alveolus, bronchus lumens and intracytoplasm irrespective of PS administration. Compared with those in normal fetal rabbits, the number of alveolar type II cells (AT II) in the lung tissue decreased in fetal rabbits with intrauterine infection, and vacuolization of the lamellar bodies occurred with evidence of cell apoptosis; PS administration resulted in increased number of the AT II cells and lamellar bodies and reduced the cell apoptosis. The expression of SP-A was significantly lower in the infection group than in normal control group (P<0.05), but comparable between the PS group and the control group (P>0.05). CONCLUSION: Changes in pulmonary alveolar ultrastructure and decreased expression of SP-A occur in fetal rabbits after intrauterine infection, and intra-amniotic administration of PS can alleviate these changes to promote lung maturation.

[Effect of basic fibroblast growth factor on expression of protein and mRNA of bone morphogenetic protein 4 in hypoxic-ischemic brain damage in newborn rats].

OBJECTIVE: To investigate the effect of basic fibroblast growth factor(bFGF) on expression of protein and mRNA of bone morphogenetic protein 4 in hypoxic-ischemic brain damage (HIBD) in newborn rats. METHOD: One hundred and twenty 7 days old neonatal rats were randomly divided into control group, hypoxic-ischemic brain damage and interventional group of bFGF, each having forty neonatal rats. After HIBD model was established, bFGF was given to interventional group by peritoneal injection for 5 continuous days. Every group was randomly divided into 7 days, 14 days, 21 days and 28 days group, according to the time of sacrifice. BMP4 protein in hippocampus was determined with immunohistochemical method. Messenger RNA of BMP4 were determined with in situ hybridization. Apoptosis of nerve cell was determined with TUNEL. Intergroup or intragroup comparisons were performed with analysis of variance. RESULT: On the days 7 and 14, expression of BMP4 protein in hippocampus was higher in interventional group of bFGF than in HIBD while expression of BMP4 protein in interventional group of bFGF and HIBD was lower on day 7 than on day 14. Expression of BMP4 protein on the days 21 and 28 had no significant difference among three groups. mRNA expression of BMP4 in interventional group of bFGF and HIBD was significantly higher in hippocampus than in control group. On the day 14, BMP4 mRNA in hippocampus widely expressed in HIBD while BMP4 mRNA only expressed in CA1 in interventional group of bFGF. Expression of BMP4 mRNA in hippocampus on the affected side decreased from the time of killing on 28th day while there was no significant change in interventional group of bFGF. Apoptosis of neural cells at the time of sacrifice on day 7 was lower in interventional group of bFGF than that in HIBD group (F=9.010, P<0.01). Apoptotic neural cells was higher in bFGF and HIBD groups at the time of killing on days 14, 21 and 28 than that on day 7 but that the bFGF group had less apoptotic neural cells than HIBD group (F=9.202, 7.932, 14.985, P<0.01). CONCLUSIONS: bFGF has a neurorestoration effect, which promotes expression of BMP4 protein and BMP4 mRNA in hippocampus of HIBD and inhibit apoptosis of neural cells.