Weiran Chai

Roles of Purinergic Receptor P2Y, G Protein–Coupled 12 in the Development of Atherosclerosis in Apolipoprotein E–Deficient Mice

Objective—The aim of the study was to evaluate the role of purinergic receptor P2Y, G protein–coupled 12 (P2Y12), an ADP receptor, in the development of atherosclerotic lesions. Methods and Results—Apolipoprotein E–null mice were crossed with P2y12−/− mice to generate double knockout mice. The double knockout mice and the control apolipoprotein E–null mice were fed a high-fat diet for 20 weeks. Assessment of the atherosclerotic lesions in the control and double knockout mice demonstrated that P2Y12 deficiency caused a diminished lesion area, an increased fibrous content at the plaque site, and decreased monocyte/macrophage infiltration of the lesions. Polymerase chain reaction studies revealed that white blood cells do not express significant levels of P2Y12. Bone marrow transplantation experiments confirmed that P2Y12 expressed on platelets is a key factor responsible for atherosclerosis, but do not exclude a role of smooth muscle cell P2Y12. Supernatant fluid from activated P2y12+/+ but not P2y12−/− platelets was capable of causing monocyte migration. In vitro studies showed that platelet P2Y12 deficiency suppressed platelet factor 4 secretion and P-selectin expression. Further work demonstrated that platelet P2Y12, through inhibition of the cAMP/protein kinase A pathway, critically regulates the release of platelet factor 4, and thereby affects monocyte recruitment and infiltration. Conclusion—These results demonstrate that P2Y12 modulates atherogenesis, at least in part by augmenting inflammatory cell recruitment via regulation of platelet &agr;-granule release.

Integrin αIIb-Mediated PI3K/Akt Activation in Platelets

Integrin αIIbβ3 mediated bidirectional signaling plays a critical role in thrombosis and haemostasis. Signaling mediated by the β3 subunit has been extensively studied, but αIIb mediated signaling has not been characterized. Previously, we reported that platelet granule secretion and TxA2 production induced by αIIb mediated outside-in signaling is negatively regulated by the β3 cytoplasmic domain residues R724KEFAKFEEER734. In this study, we identified part of the signaling pathway utilized by αIIb mediated outside-in signaling. Platelets from humans and gene deficient mice, and genetically modified CHO cells as well as a variety of kinase inhibitors were used for this work. We found that aggregation of TxA2 production and granule secretion by β3Δ724 human platelets initiated by αIIb mediated outside-in signaling was inhibited by the Src family kinase inhibitor PP2 and the PI3K inhibitor wortmannin, respectively, but not by the MAPK inhibitor U0126. Also, PP2 and wortmannin, and the palmitoylated β3 peptide R724KEFAKFEEER734, each inhibited the phosphorylation of Akt residue Ser473 and prevented TxA2 production and storage granule secretion. Similarly, Akt phosphorylation in mouse platelets stimulated by the PAR4 agonist peptide AYPGKF was αIIbβ3-dependent, and blocked by PP2, wortmannin and the palmitoylated peptide p-RKEFAKFEEER. Akt was also phosphorylated in response to mAb D3 plus Fg treatment of CHO cells in suspension expressing αIIbβ3-Δ724 or αIIbβ3E724AERKFERKFE734, but not in cells expressing wild type αIIbβ3. In summary, SFK(s) and PI3K/Akt signaling is utilized by αIIb-mediated outside-in signaling to activate platelets even in the absence of all but 8 membrane proximal residues of the β3 cytoplasmic domain. Our results provide new insight into the signaling pathway used by αIIb-mediated outside-in signaling in platelets.

Protective Effect of Quercetin on the Development of Preimplantation Mouse Embryos against Hydrogen Peroxide-Induced Oxidative Injury

Quercetin, a plant-derived flavonoid in Chinese herbs, fruits and wine, displays antioxidant properties in many pathological processes associated with oxidative stress. However, the effect of quercetin on the development of preimplantation embryos under oxidative stress is unclear. The present study sought to determine the protective effect and underlying mechanism of action of quercetin against hydrogen peroxide (H2O2)-induced oxidative injury in mouse zygotes. H2O2 treatment impaired the development of mouse zygotes in vitro, decreasing the rates of blastocyst formation and hatched, and increasing the fragmentation, apoptosis and retardation in blastocysts. Quercetin strongly protected zygotes from H2O2-induced oxidative injury by decreasing the reactive oxygen species level, maintaining mitochondrial function and modulating total antioxidant capability, the activity of the enzymatic antioxidants, including glutathione peroxidase and catalase activity to keep the cellular redox environment. Additionally, quercetin had no effect on the level of glutathione, the main non-enzymatic antioxidant in embryos.

Antibodies against AT1 Receptors Are Associated with Vascular Endothelial and Smooth Muscle Function Impairment: Protective Effects of Hydroxysafflor Yellow A

Ample evidence has shown that autoantibodies against AT1 receptors (AT1-AA) are closely associated with human cardiovascular disease. The aim of this study was to investigate mechanisms underlying AT1-AA-induced vascular structural and functional impairments in the formation of hypertension, and explore ways for preventive treatment. We used synthetic peptide corresponding to the sequence of the second extracellular loop of the AT1 receptor (165–191) to immunize rats and establish an active immunization model. Part of the model received preventive therapy by losartan (20 mg/kg/day) and hyroxysafflor yellow A (HSYA) (10 mg/kg/day). The result show that systolic blood pressure (SBP) and heart rate (HR) of immunized rats was significantly higher, and closely correlated with the plasma AT1-Ab titer. The systolic response of thoracic aortic was increased, but diastolic effects were attenuated markedly. Histological observation showed that the thoracic aortic endothelium of the immunized rats became thinner or ruptured, inflammatory cell infiltration, medial smooth muscle cell proliferation and migration, the vascular wall became thicker. There was no significant difference in serum antibody titer between losartan and HSYA groups and the immunized group. The vascular structure and function were reversed, and plasma biochemical parameters were also improved significantly in the two treatment groups. These results suggest that AT1-Ab could induce injury to vascular endothelial cells, and proliferation of smooth muscle cells. These changes were involved in the formation of hypertension. Treatment with AT1 receptor antagonists and anti oxidative therapy could block the pathogenic effect of AT1-Ab on vascular endothelial and smooth muscle cells.

The pregnancy outcome of progestin‐primed ovarian stimulation using 4 versus 10 mg of medroxyprogesterone acetate per day in infertile women undergoing in vitro fertilisation: a randomised controlled trial

To investigate the clinical outcome and endocrinological characteristics of progestin‐primed ovarian stimulation (PPOS) using 4 versus 10 mg of medroxyprogesterone acetate (MPA) per day in infertile women with normal ovary reserve.

Eight-Shaped Hatching Increases the Risk of Inner Cell Mass Splitting in Extended Mouse Embryo Culture.

Increased risk of monozygotic twinning (MZT) has been shown to be associated with assisted reproduction techniques, particularly blastocyst culture. Interestingly, inner cell mass (ICM) splitting in human ‘8’-shaped hatching blastocysts that resulted in MZT was reported. However, the underlying cause of MZT is not known. In this study, we investigated in a mouse model whether in vitro culture leads to ICM splitting and its association with hatching types. Blastocyst hatching was observed in: (i) in vivo developed blastocysts and (ii–iii) in vitro cultured blastocysts following in vivo or in vitro fertilization. We found that ‘8’-shaped hatching occurred with significantly higher frequency in the two groups of in vitro cultured blastocysts than in the group of in vivo developed blastocysts (24.4% and 20.4% versus 0.8%, respectively; n = 805, P < 0.01). Moreover, Oct4 immunofluorescence staining was performed to identify the ICM in the hatching and hatched blastocysts. Scattered and split distribution of ICM cells was observed around the small zona opening of ‘8’-shaped hatching blastocysts. This occurred at a high frequency in the in vitro cultured groups. Furthermore, we found more double OCT4-positive masses, suggestive of increased ICM splitting in ‘8’-shaped hatching and hatched blastocysts than in ‘U’-shaped hatching and hatched blastocysts (12.5% versus 1.9%, respectively; n = 838, P < 0.01). Therefore, our results demonstrate that extended in vitro culture can cause high frequencies of ‘8’-shaped hatching, and ‘8’-shaped hatching that may disturb ICM herniation leading to increased risk of ICM splitting in mouse blastocysts. These results may provide insights into the increased risk of human MZT after in vitro fertilization and blastocyst transfer.

Polymorphism in the Alternative Donor Site of the Cryptic Exon of LHCGR: Functional Consequences and Associations with Testosterone Level

Selective splicing is a feature of luteinizing hormone receptor (LHCGR). A cryptic exon (LHCGR-exon 6A) was found to be derived from alternative splicing in intron 6 of the LHCGR gene, which including two transcripts LHCGR-exon 6A-long and LHCGR-exon 6A-short. We addressed the functional consequences of SNP rs68073206, located at the +5 position of an alternative 5′ splice donor site, and observed its association with male infertility in the subjects with azoospermia, oligoasthenozoospermia and normozoospermia. The translation product of splicing variant LHCGR-exon 6A was expressed in the cytoplasm and exhibited no affinity with [125I]-hCG. No dominant negative effect was observed in cells co-expressed with LHCGR-exon 6A and wild-type LHCGR. The long transcript (LHCGR-exon 6A-long) was significantly elevated in the granulosa cells with G/G genotypes, which could be reproduced in vitro by mini-gene construct transfection. Genotyping analysis showed no association between rs68073206 and male infertility. However, this polymorphism was significantly associated with testosterone levels in normozoospermic subjects (n = 210). In conclusion, SNP rs68073206 in the splicing site of the cryptic exon 6A of the LHCGR gene affect the splicing pattern in the gene, which may play a role in the modulation of the LHCGR sensitivity in the gonads.

Effect of Natural Cycle Endometrial Preparation for Frozen-Thawed Embryo Transfer in Patients with Advanced Endometriosis

Background The aim of this study was to investigate the effect of natural cycle (NC) endometrial preparation for frozen-thawed embryo transfer (FET) in women with advanced endometriosis. Material/Methods This retrospective study included 179 patients with stage III–IV endometriosis who underwent 233 FET cycles at a tertiary care academic reproductive medical center between March 2011 and August 2013 (group A). The control group included 258 patients with tubal factor infertility who underwent 300 FET cycles (group B). Both groups were prepared for FET using a NC protocol. Rates of implantation, clinical pregnancy, live birth, ongoing pregnancy, miscarriage, and pregnancy complication were recorded. Results The implantation rate (A: 36.0%, B: 30.4%, P=0.06), the pregnancy rate (A: 50.2%, B: 45.3%, P=0.263), and the live birth rate (A: 39.91%, B: 39.0%, P=0.428) were similar between the stage III–IV endometriosis and tubal factor infertility groups. No differences were observed in ongoing rates of pregnancy, miscarriage, and pregnancy complications, independent of endometriosis severity. No congenital birth defects were found. When high-quality embryos are transferred, pregnancy results were not affected by active endometriosis. Although severe endometriosis did not affect birth rate, higher frequencies of premature delivery (mean gestational age A: 37 weeks, B: 38.3 weeks, P=0.044) and low birth weight were observed (<2500 g A: 26.4%, B: 16.6%, P=0.047). Conclusions There was no difference in pregnancy outcomes between patients with endometriosis and those with tubal infertility. Pregnancy outcomes in patients with endometriosis were not affected by endometriosis severity. Pregnancy outcomes were not affected by active endometrial cyst.

The role of combining medroxyprogesterone 17-acetate with human menopausal gonadotropin in mouse ovarian follicular development

Medroxyprogesterone 17-acetate (MPA) combined with human menopausal gonadotropin (hMG) has been effectively used for ovarian stimulation in clinical practice. However, the molecular mechanism of MPA + hMG treatment in follicular development is poorly described. Here we performed a study to investigate the impact of MPA + hMG on ovarian stimulation utilizing a mouse model in vivo. Forty female BALB/C mice were randomly divided into four groups of 10 each and treated during ciestrus stage and continued for 5 days: control group, MPA group, hMG group, and MPA + hMG group. Morphological and molecular biology methods were used for detecting serum hormones and ovarian function. MPA + hMG group exhibited increasing follicle stimulating hormone (FSH), antral follicle, FSH receptor (FSHR) and phosphorylated mammal target of rapamycin (p-mTOR), and decreasing luteinizing hormone (LH), estradiol (E2), progesterone (P), corpus luteum, phosphoinositide 3-kinase (PI3K), Akt and mTOR compared with control group. In contrast, MPA + hMG group showed reduced FSH, LH, E2, P, corpus luteum, LH receptor (LHR), and activated PI3K,/Akt/mTOR pathway compared with hMG group (P < 0.05). Collectively, these data definitively established that MPA plus hMG may modulate the hormone, hormone receptor and PI3K/Akt/mTOR signaling pathway to influence follicular development in the mouse ovary. Our study provides overwhelming support for MPA + hMG as an effective treatment for infertility in women.

Use of medroxyprogesterone acetate in women with ovarian endometriosis undergoing controlled ovarian hyperstimulation for in vitro fertilization

This study investigated the use of medroxyprogesterone acetate (MPA) or a short protocol for controlled ovarian hyperstimulation (COH) in patients with advanced endometriosis who have normal ovarian function, and to compare cycle characteristics and pregnancy outcomes after frozen-thawed embryo transfer (FET). This was a retrospective case-control study of 244 patients with advanced endometriosis undering COH. The patients were allocated to three groups: the surgery group with MPA COH (62 patients, 71 IVF/ICSI cycles, 78 FET cycles); the aspiration group with MPA COH (85 patients had ovarian “chocolate” cysts (>3 cm) aspirated, 90 IVF/ICSI cycles, 76 FET cycles); and the short protocol group (97 patients, 101 IVF/ICSI cycles, 51 FET cycles). The results showed that higher rates of mature oocyte, D3 high quality embryo, hMG dose were observed in the two study groups using MPA compared with the short protocol. The number of >10–14 mm follicles on the trigger day, D3 top-quality embryos, viable embryos, rates of cancellation, fertilization, implantation, pregnancy outcomes were similar among the three groups. The oocytes, embryos, and pregnancy outcomes were not influenced by endometrioma surgery or presence of endometrioma. MPA COH could be effective for women with ovarian advanced endometriosis who had normal ovarian function.