Correlation between sperm mitochondrial ND5 and ND6 gene variations and total fertilisation failure.

INTRODUCTION: The purpose of this study was to investigate the correlation between sperm mitochondrial NADH dehydrogenase subunit 5 (ND5) and NADH dehydrogenase subunit 6 (ND6) gene variations and total fertilisation failure (TFF). MATERIAL AND METHODS: A total of 232 sperm samples at the fresh in vitro fertilisation (IVF) cycle or the half-intracytoplasmic sperm injection (ICSI) cycle were collected for this retrospective controlled study on Han Chinese people between July 2011 and April 2014. Of the 232 total samples, 45 were from the IVF-TFF group and 187 were from couples with normal fertilisation (fertilisation rate > 50%). The mitochondrial ND5 and ND6 gene variations and sperm haplotypes were confirmed using nested PCR and DNA sequencing. RESULTS: Ten homozygous variations were newly discovered, namely C12417T, T12441A, C12543A, C13650A, C13765A, T13769C, C13775T, A13776G, C13785A and C13845T. The gene variation rates of six sites, C12417T, C13650A, C13765A, T13769C, C13785A and C13845T, in the TFF group were significantly higher than those in the control group (p < 0.05). There were 231 heterozygous variations discovered; however, only nine heterozygous sites (12441, 12561, 12735, 13164, 13743, 13812, 13928, 14172 and 14368) had significantly higher gene variation rates than those in the control group (p < 0.05). In addition, the results showed that haplogroup C did not affect TFF (p > 0.05), and the fertilisation failure rates of haplogroup R and haplogroup D4a were both higher than those in the control group (p < 0.05). CONCLUSIONS: Our results suggested that the ND5 and ND6 gene variations are correlated with TFF. Furthermore, this study indicated that haplogroup R and haplogroup D4a might be risk factors for TFF.

The effectiveness of different down-regulating protocols on in vitro fertilization-embryo transfer in endometriosis: a meta-analysis.

BACKGROUND: To investigate the effectiveness of the GnRH-a ultra-long protocol, GnRH-a long protocol, and GnRH-a short protocol used in in vitro fertilization-embryo transfer (IVF-ET) in infertile women with endometriosis. METHODS: We searched PubMed, Embase, Web of Science, Cochrane Library, Elsevier Science Direct, OA Library, Google Scholar, China National Knowledge Infrastructure (CNKI), Wanfang Data Knowledge Service Platform, China Science and Technology Journal database, and the China Biology Medicine disc for randomized controlled trials (RCTs) and observational studies (non-RCTs) to evaluate the efficacy of the GnRH-a ultra-long protocol, GnRH-a long protocol, and GnRH-a short protocol in IVF-ET in infertile patients with endometriosis. RESULTS: A total of 21 studies in compliance with the standard literature were included, and RCT and non-RCT studies were analyzed separately. This meta-analysis showed that the GnRH-a ultra-long protocol could improve the clinical pregnancy rate of infertile patients in RCT studies, especially in patients with stages III-IV endometriosis (RR = 2.04, 95% CI: 1.37~3.04, P < 0.05). However, subgroup analysis found the different down-regulation protocols provided no significant difference in improving clinical outcomes in patients with endometriosis in the non-RCT studies. CONCLUSION: This study suggests that the GnRH-a ultra-long protocol can improve the clinical pregnancy rate of the patients with stages III-IV endometriosis in RCT studies. Although it is generally believed that the results of RCT are more reliable, the conclusions of the non-RCT studies cannot be easily neglect, which let us draw conclusions more cautious.

Association between sperm mitochondrial ND2 gene variants and total fertilization failure.

The objective of this study was to explore the association of sperm mitochondrial ND2 (MT-ND2) gene variants with total fertilization failure (TFF). A retrospective comparative study of 246 cases of fresh in vitro fertilization (IVF) cycles or half-intracytoplasmic sperm injection cycles in the Han Chinese population was performed from July 2011 to May 2017. A total of 59 cases undergoing TFF, and 187 control cases with normal fertilization (fertilization rates >50%) were included. The sperm mitochondrial genovariation was determined using nested sequencing. A total of 32 homoplasmic variants and 47 heteroplasmic variants of MT-ND2 gene were observed in this study. There were no significant differences in the frequencies of the 32 homoplasmic variants of MT-ND2 gene between the TFF and control groups. A total of 53 pair-wise comparisons were performed, and the general characteristics of the IVF failure and control subjects were adjusted in logistic models. Data suggested that there were no significant differences in the frequencies of point 4914, 5320, and 5426 heteroplasmic variants of MT-ND2 gene between the TFF and control groups. In addition, no significant difference was observed in the frequency of mtDNA haplogroup D or haplogroup G between the IVF failure group and the normal fertilization group. This study suggests that the MT-ND2 gene variants might not be associated with TFF. ABBREVIATIONS: ATP: adenosine triphosphate; dNTP: deoxy-ribonucleoside triphosphate; FADH2: flavin adenine dinucleotide; FDR: false discovery rate; FSH: follicle-stimulating hormone; IVF: in vitro fertilization; LH: luteinizing hormone; MTATP6: mitochondrially encoded ATP synthase 6; MTCYB: mitochondrially encoded cytochrome b; mtDNA: mitochondrial DNA; MT-ND2: mitochondrial ND2; NADH: nicotinamide adenine dinucleotide; ND2: NADH dehydrogenase subunit 2; OXPHOS: oxidative phosphorylation; PCR: single nucleotide polymorphisms; SNPs: single nucleotide polymorphisms; TFF: total fertilization failure.

MicroRNA-150 regulates steroidogenesis of mouse testicular Leydig cells by targeting STAR.

Leydig cells are essential for male reproductive development throughout life. Production of androgens as well as intermediate steroids is tightly regulated. Although microRNAs (miRNAs) are suggested to play important roles in spermatogenesis, little is currently known regarding the regulation of steroidogenesis by miRNAs in Leydig cells. Here, we found that miR-150 was predominantly expressed in Leydig cells within mouse testis. Therefore, we determined steroidogenesis of the Leydig cells in which miR-150 was knocked down or overexpressed using miR-150 antagomir and agomir, respectively. Compared with negative control group, a significant increase of STAR expression was observed in miR-150 antagomir-treated Leydig cells. Conversely, STAR expression was significantly reduced in miR-150 agomir-transfected Leydig cells. Production of sex-steroid precursors and testosterone of Leydig cells was also negatively controlled by miR-150. We further identified Star as a target of miR-150 using luciferase reporter assay. Finally, we confirmed that miR-150 was necessary for steroidogenesis and spermatogenesis in vivo via intratesticular injection of miR-150 antagomir or agomir. Taken together, our studies suggest that miR-150 negatively regulates the expression of STAR and steroidogenesis of Leydig cells in mice.

Role of PI3K p110ß in the differentiation of human embryonic stem cells into islet-like cells.

To investigate the effects of the PI3K inhibitors on the differentiation of insulin-producing cells derived from human embryonic stem cells. Here, we report that human embryonic stem cells induced by phosphatidylinositol-3-kinase (PI3K) p110ß inhibitors could produce more mature islet-like cells. Findings were validated by immunofluorescence analysis, quantitative real-time PCR, insulin secretion in vitro and cell transplantation for the diabetic SCID mice. Immunofluorescence analysis revealed that unihormonal insulin-positive cells were predominant in cultures with rare polyhormonal cells. Real-time PCR data showed that islet-like cells expressed key markers of pancreatic endocrine hormones and mature pancreatic ß cells including MAFA. Furthermore, this study showed that the expression of most pancreatic endocrine hormones was similar between groups treated with the LY294002 (nonselective PI3K inhibitor) and TGX-221 (PI3K isoform selective inhibitors of class 1ß) derivatives. However, the level of insulin mRNA in TGX-221-treated cells was significantly higher than that in LY294002-treated cells. In addition, islet-like cells displayed glucose-stimulated insulin secretion in vitro. After transplantation, islet-like cells improved glycaemic control and ameliorated the survival outcome in diabetic mice. This study demonstrated an important role for PI3K p110ß in regulating the differentiation and maturation of islet-like cells derived from human embryonic stem cells.

Polymorphisms in the MT-ATP6 and MT-CYB genes in in vitro fertilization failure.

The purpose of this study was to investigate whether fertilization failure after in vitro fertilization could be explained by polymorphisms in MT-ATP6 and MT-CYB genes. We performed a prospective comparative study of 111 fresh IVF cycles in Han Chinese between July 2011 and February 2013. Human sperm mitochondrial DNA (mtDNA) variants in the MT-CYB and MT-ATP6 genes were screened by polymerase chain reaction (PCR) and direct sequencing. Forty-six couples had low fertilization rates (< or =30%) or total fertilization failure, and 65 controls with normal fertilization. One unreported point mutation (A15472G) was found in this study. There were 7 and 3 polymorphic sites in the MT-ATP6 and MT-CYB, respectively. Interestingly, the frequencies of points 8701 and 15301 homozygous variants in study group were significantly higher than those in control group. However, the frequencies of the points 8701, 9075 and 15,301 heterozygous variants in study group were significantly lower than those in control group (4.35% versus 16.92%, 15.22% versus 32.31% and 6.52% versus 33.84%, respectively, p < 0.05). In addition, the frequency in subjects harboring A8701G and G15301A variants in study group was significantly higher than that in control group (63.04% versus 33.85%, p < 0.05). This study suggests that, in part, polymorphisms in the MT-ATP6 and MT-CYB genes may contribute to the unexpected fertilization failure.

Comparisons of the effects of long-acting and short-acting GnRH agonists on embryo quality, endometrial thickness and pregnancy rate in human in vitro fertilization.

INTRODUCTION: The aim was to compare the efficacy of long-acting and short-acting gonadotropin-releasing hormone (GnRH) agonists by long protocol on embryo quality, endometrial thickness and pregnancy rate in in vitro fertilization. MATERIAL AND METHODS: In this retrospective study, long-term pituitary downregulation, achieved with long- and short-acting GnRH agonists (GnRHa), was performed for patients undergoing in vitro fertilization (n = 175). RESULTS: There were no significant differences between the long and short-acting GnRH group (63.16% vs. 66.26%, p > 0.05), and the secondary and primary infertility group (63.47% vs. 66.86%, p > 0.05) in embryo quality. Logistic regression analysis showed that type of infertility and endometrial thickness were significantly associated with pregnancy outcome. Patients in the long-acting GnRHa group had a thicker endometrium on the day of human chorionic gonadotrophin (hCG) administration (10.79 ±2.62 mm vs. 9.64 ±1.97 mm, p < 0.01), lower serum luteinizing hormone (LH) concentration (1.21 ±1.13 vs. 2.53 ±3.39) and a higher pregnancy rate (59.60% vs. 43.42%, p < 0.05) than those of patients in the short-acting GnRHa group. CONCLUSIONS: This work suggests that types of agonist protocol and infertility may not affect embryo quality. Type of infertility and endometrial thickness may be positive predictors for clinical pregnancy, but the key finding is that the long-acting GnRHa protocol may be an effective method of improving endometrial thickness, endometrial receptivity and pregnancy rate in in vitro fertilization.

Three step derivation of cartilage like tissue from human embryonic stem cells by 2D-3D sequential culture in vitro and further implantation in vivo on alginate/PLGA scaffolds.

In this study a three step culture system, 2D-3D sequential culture in vitro and further implantation in vivo was developed to induce human embryonic stem cells (hESCs) into cartilage like tissues. Five-day-old embryoid bodies were plated for chondrogenic induction for 27 days (step1), then the cells were suspended in alginate and seeded onto polylactic-co-glycolic acid (PLGA) scaffolds for 3D cultivation for 7 days (step 2) and the cells/alginate/PLGA complexes were further transplanted into nude mice for 8 weeks (step 3). At same time, some of complexes were cultured in vitro up to 8 weeks. At the end of step 1, cells exhibited fibroblast-like morphology and expressed chondrocyte-specific markers, Sox 9 and collagen II. During the following 8 weeks of 3D cultivation in vitro, cells displayed spherical morphology, decreased immunoreactivity to Sox-9 and increased one to collagen II, demonstrated further differentiation to mature chondrocyte. In implanted grafts, not only cells appeared typical chondrocytes shape and markers but also cartilage like tissues were formed. These results indicate that 2D-3D sequential culture in vitro is an efficient protocol to induce hESCs differentiates into chondrocytes, while the three step culture system may be an appropriate procedure to derive cartilage like tissues from hESCs.

The reversal of hyperglycaemia in diabetic mice using PLGA scaffolds seeded with islet-like cells derived from human embryonic stem cells.

Islet-like cells derived from embryonic stem (ES) cells may be a promising therapeutic option for future diabetes treatment. Here, we demonstrated a five-stage protocol with adding exendin-4 instead of nicotinamide finally could generate islet-like cells from human embryonic stem (ES) cells. Immunofluorescence analysis revealed a high percentage of c-peptide positive cells in the derivation. However, in addition to insulin/c-peptide, most cells also coexpressed PDX-1 (pancreas duodenum homeobox-1), glucagon, somatostatin or pancreatic polypeptide. Insulin and other pancreatic beta-cell-specific genes were all present in the differentiated cells. Insulin secretion could be detected and increased significantly by adding KCL in high glucose concentration in vitro. Furthermore, subcutaneous transplantation of scaffolds seeded with the islet-like cells or cell transplantation under kidney capsules for further differentiation in vivo could improve 6h fasted blood glucose levels and diabetic phenotypes in streptozotocin-induced diabetic SCID mice. More interestingly, blood vessels of host origin, characterized by mouse CD31 immunostaining, invaded the cell-scaffold complexes. This work reveals a five-stage protocol with adding exendin-4 may be an effective protocol on the differentiation of human ES cells into islet-like cells, and suggests scaffolds can serve as vehicles for islet-like cell transplantation.