Enhancement of the efficiency of oocyte vitrification through regulation of histone deacetylase 6 expression.

OBJECTIVE: Successful oocyte vitrification (OV) is critical for cryopreservation of the oocytes from female patients with infertility, polycystic ovaries, and gynecologic cancers. Recent evidence suggests that relatively low levels of histone acetylation are critical for maintenance of the maturation capacity of cryopreserved oocytes. However, previous studies have only demonstrated a key role of histone deacetylases (HDAC) 1 and 2 in the cryopreservation of oocytes. METHODS: In this study, we investigated the role of HDAC6 in these settings. We found that mouse oocytes with low HDAC6 levels decreased survival rate, cleavage rate, and blastocyst rate after OV. Bioinformatics analyses were used to predict HDAC6-targeting microRNAs (miRNAs), while the functional binding of miRNAs to HDAC6 mRNA was evaluated by a dual luciferase reporter assay. RESULTS: Among all HDAC6-targeting miRNAs, we detected expression of miR-558, miR-527, and miR-762 in mouse oocytes. Specifically, we found that only miR-762 significantly inhibited protein translation of HDAC6 via binding to the 3'-UTR of the HDAC6 mRNA. Transfection of oocytes with HDAC6 or antisense of miR-762 significantly increased the survival rate, the cleavage rate, and blastocyst rate after OV. CONCLUSION: As a result, our data suggest that induction of HDAC6 levels by miR-762 suppression may improve the current protocol for OV.

Melatonin Reduces Androgen Production and Upregulates Heme Oxygenase-1 Expression in Granulosa Cells from PCOS Patients with Hypoestrogenia and Hyperandrogenia.

BACKGROUND/AIMS: Polycystic ovary syndrome (PCOS) is an endocrine disorder characterized by abnormal hormone levels in peripheral blood and poor-quality oocytes. PCOS is a pathophysiological syndrome caused by chronic inflammation and oxidative stress. The aim of this study was to investigate the mechanism of melatonin regulation on androgen production and antioxidative damage in granulosa cells from PCOS patients with hypoestrogenia and hyperandrogenia. METHODS: Cumulus-oocyte complexes were collected from PCOS patients who had low levels of estrogen in follicular fluids. RESULTS: Melatonin triggered upregulation of cytochrome P450 family 19 subfamily A member 1 (CYP19A1) expression via the extracellular signal-regulated kinase pathway in luteinized granulosa cells. As a result, conversion of androgen to 17ß-estradiol was accelerated. We also found that melatonin significantly reduced the levels of inducible nitric oxide (NO) synthetase and NO in luteinized granulosa cells. Levels of transcripts encoding NF-E2-related factor-2 and its downstream target heme oxygenase-1 were also increased, leading to anti-inflammatory and antioxidant effects. We also found that melatonin could improve oocyte development potential. CONCLUSION: Our preliminary results showed that melatonin had a positive impact on oocyte quality in PCOS patients with hypoestrogenia and hyperandrogenia.

Establishment of porcine and human expanded potential stem cells.

We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro. Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine.

A novel method for cryopreservation of individual human spermatozoa.

The purpose of this study is to develop a novel method for the cryopreservation and efficient post-thaw recovery of individual or small numbers of human spermatozoa. Spermatozoa equilibrated in cryoprotectant buffer were injected with an intracytoplasmic sperm injection (ICSI) needle into a droplet of cryoprotectant on a homemade cryoleaf. The droplet was of cryoprotectant and seminal plasma at a ratio of 1:1. The sperm-loaded cryoleaf was slowly lowered over and stored in liquid nitrogen. Spermatozoa were thawed in a 37°C oil bath without dilution and centrifugation. To test the fertilizing ability of these spermatozoa, the recovered spermatozoa were injected by ICSI into 1-d-old or in vitro-matured human oocytes. Fresh spermatozoa from the same semen samples served as controls. The trials were performed in two separate experiments. In the first set of experiments, 92 spermatozoa were thawed and carefully investigated. The spermatozoa from percutaneous epididymal sperm aspiration had a motility recovery of 92.9% (13/14); ejaculated spermatozoa had a motility recovery of 61.5% (48/78), and only 1.3% (1/78) was lost. Together in the first and second set of experiments, the fertilization rates for the fresh and frozen-thawed spermatozoa were 67.6% (25/37) and 60.6% (40/66), respectively (P = 0.052). The mean embryo cleavage rates in the fresh and frozen-thawed groups were 88% (22/25) and 85% (34/40), respectively (P = 0.990). This cryopreservation method for individual or small numbers of human spermatozoa was efficient and simple. These findings make this method a promising technique for the clinical application of ejaculated sperm from oligozoospermic patients.

Optimal surgical timing and treatment of thumb duplication in children / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the optimal surgical timing and treatment of congenital duplication of the thumb in children.</p><p><b>METHODS</b>A clinical study was performed on 154 fingers of the 132 patients (including 85 males, 47 females;mean age (1.53 +/- 2.47) years; ranged from 2 months to 13 years). Duplicated thumbs were surgically treated from December 2007 to February 2012. All patients underwent detailed physical examination and radiological assessment. Surgical methods should be selected according to the age, Wassel classification and deformity. The operation steps included resection of the most hypoplastic thumb, followed by skin flap plasty, tendon shift (transplantation) , reconstruction of collateral ligament and articular capsule. 11 cases over the age of 6 underwent osteotomy discretion as appropriate.</p><p><b>RESULTS</b>A total of 117 fingers of the 104 patients were followed up for an average 36. 7 months (range,6-55 months). We adopted upgrade Tada standard to evaluate the follow-up results as excellent in 77 thumbs, good in 21 thumbs, fair in 15 thumbs and poor in 4 thumbs. Three thumbs appeared secondary angular deformity 2 years after operation and one thumb appeared residual osteoepiphysis in the radial side of metacarpophalangeal joint 3 years after operation. The appearance and the function were almost normal after second operation.</p><p><b>CONCLUSIONS</b>The optimal surgical timing of congenital duplication of thumb should be based on the appearance of thumb ossification center. The surgical timing of Wassel type- I and II should be chosen at 1. 5 years old when the ossification center of distal phalanx appears, Wassel type-III and IV should be chosen at 1 year old when the ossification center of proximal phalanx appears, Wassel type-V and VI should be chosen at 2. 5 years old when the ossification center of metacarpal appears, and Wassel type-VII should be chosen at 2. 5 years old. The surgical method should be selected on individualized principle. The key points are reconstruction of collateral ligament, tendon and articular capsule and correction of ulnar deviation (or radial deviation).</p>

Relationship between expression of GYPC and TRIP3 genes and prognosis of acute lymphoblastic leukemia in children / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the relationship of GYPC and TRIP3 gene expression and the prognosis of acute lymphoblastic leukemia (ALL) in children in order to explore the molecular biological mechanisms of recurrence and remission of ALL.</p><p><b>METHODS</b>Thirty-eight newly diagnosed ALL children were enrolled. Of the 38 patients, 31 achieved complete remission (CR) and 12 relapsed. Semi-quantitative RT-PCR was employed to measure blood GYPC and TRIP3 gene expression. Twenty blood samples from normal children were used as controls.</p><p><b>RESULTS</b>Blood GYPC expression in newly diagnosed ALL children was significantly higher than that in the control group (p<0.01) and the CR group (p<0.01). The expression of GYPC gene in the CR group was similar to that in the control group. Other than the control group (p<0.01) and the CR group (p<0.01), the GYPC expression of the relapse group was significantly higher than that in the newly diagnosed group (p<0.01). The CR group showed lower GYPC gene expression than the nonjremission group before treatment (p<0.05). Blood expression of TRIP3 gene in the newly diagnosed and the relapse groups was significantly lower than that in the control group (p<0.05). The CR group had increased TRIP3 gene expression compared with the control group (p<0.01) as well as the newly diagnosed and the relapse groups (p<0.01). Of the 38 newly diagnosed ALL children, the patients with positive TRIP3 expression showed higher remission rate than those with negative TRIP3 (p<0.05). The TRIP3 gene expression before treatment in patients who achieved CR was higher than that in non-remission patients (p<0.05).</p><p><b>CONCLUSIONS</b>A high GYPC gene expression is associated with an unfavorable outcome, in contrast, a high TRIP3 gene expression is associated with a favorable outcome in childhood ALL.</p>