Evaluation of mouse oocyte in vitro maturation developmental competency in dynamic culture systems by design and construction of a lab on a chip device and its comparison with conventional culture system

Objective: In conventional assisted reproductive technology [ART], oocytes are cultured in static microdrops within Petri dishes that contain vast amounts of media. However, the in vivo environment is dynamic. This study assesses in vitro oocyte maturation through the use of a new microfluidic device. We evaluate oocyte fertilization to the blastocyct stage and their glutathione [GSH] contents in each experimental group

Materials and Methods: In this experimental study, we established a dynamic culture condition. Immature oocytes were harvested from ovaries of Naval Medical Research Institute [NMRI] mice. Oocytes were randomly placed in static [passive] and dynamic [active] in vitro maturation [IVM] culture medium for 24 hours. In vitro matured oocytes underwent fertilization, after which we placed the pronucleus [PN] stage embryos in microdrops and followed their developmental stages to blastocyst formation after 3 days. GSH content of the in vitro matured oocytes was assessed by monochlorobimane [MCB] staining

Results: We observed significantly higher percentages of mature metaphase II oocytes [MII] in the passive and active dynamic culture systems [DCS] compared to the static group [P<0.01]. There were significantly less mean numbers of germinal vesicle [GV] and degenerated oocytes in the passive and active dynamic groups compared to the static group [P<0.01]. Fertilization and blastocyst formation rate in the dynamic systems were statistically significant compared to the static cultures [P<0.01]. There was significantly higher GSH content in dynamically matured oocytes compared to statically matured oocytes [P<0.01]

Conclusion: Dynamic culture for in vitro oocyte maturation improves their developmental competency in comparison with static culture conditions

DNA methylation and histone acetylation patterns in cultured bovine adipose tissue-derived stem cells [BADSCs]

Many studies have focused on the epigenetic characteristics of donor cells to improve somatic cell nuclear transfer [SCNT]. We hypothesized that the epigenetic status and chromatin structure of undifferentiated bovine adipose tissue-derived stem cells [BADSCs] would not remain constant during different passages. The objective of this study was to determine the mRNA expression patterns of DNA methyltransferases [DNMT1, DNMT3a, DNMT3b] and histone deacetyltransferses [HDAC1, HDAC2, HDAC3] in BADSCs. In addition, we compared the measured levels of octamer binding protein-4 expression [OCT4] and acetylation of H3K9 [H3K9ac] in BADSCs cultures and different passages in vitro. In this experimental study, subcutaneous fat was obtained from adult cows immediately post-mortem. Relative level of DNMTs and HDACs was examined using quantitative real time polymerase chain reaction [q-PCR], and the level of OCT4 and H3K9ac was analyzed by flow cytometry at passages 3 [P3], 5 [P5] and 7 [P7]. The OCT4 protein level was similar at P3 and P5 but a significant decrease in its level was seen at P7. The highest and lowest levels of H3K9ac were observed at P5 and P7, respectively. At P5, the expression of HDACs and DNMTs was significantly decreased. In contrast, a remarkable increase in the expression of DNMTs was observed at P7. Our data demonstrated that the epigenetic status of BADSCs was variable during culture. The P5 cells showed the highest level of stemness and multipotency and the lowest level of chromatin compaction. Therefore, we suggest that P5 cells may be more efficient for SCNT compared with other passages

Association between increased expression of endothelial isoform of nitric oxide synthase in the human fallopian tube and tubal ectopic pregnancy

Tubal ectopic pregnancy [tEP] is the most common type of extra-uterine pregnancy and the most common cause of maternal mortality. Nitric oxide [NO] is a molecule that incorporates in many physiological processes of female reproductive system. Recent studies have demonstrated the possible role of endothelial isoform of nitric oxide synthase [eNOS] enzyme in the regulation of many reproductive events that occur in the fallopian tube [FT]. The aim of this study was to evaluate the expression of eNOS in the FTs of women with tEP. In this case-control study, a total number of 30FTs samples were obtained from three groups including: 10 FTs of women that bearing an EP, 10 FTs from the non-pregnant women at luteal phase of the menstrual cycle, and 10 FTs of healthy pregnant women [n=10]. Samples were fixed in 10% buffered formalin and then were evaluated by immunohistochemistry. Localization of eNOS was seen in secretory and ciliated luminal epithelium and vascular endothelium of all groups. However, we did not observed the expression of eNOS in smooth muscle cells of all groups. Expression of eNOS in luminal epithelium of women with EP compared to non-pregnant women at luteal phase of menstrual cycle and healthy pregnant group showed statistically significant increase [p=0.00]. Significant difference in expression of eNOS was not observed in luminal epithelium of FTs of women at luteal phase compared to healthy pregnant groups [p=0.78]. This study indicates that changes in expression of eNOS in luminal epithelium of FT may lead to development of EP.

Immuno-histochemical localization of endothelial nitric oxide synthase in testicular cells of men with non-obstructive azoospermia

Non obstructive azoospermia [NOA] is one of the causes of male infertility in which spermatogenesis process is disturbed. Recent studies suggested the possible role of endothelial nitric oxide synthase [eNOS] in spermatogenesis process. The aim of the present study is to evaluate the expression of eNOS in human testicular tissue in men with NOA and men with normal spermatogenesis by using immunocytochemistry. In this case-control study, testicular biopsies were obtained from 10 men with NOA and 7 men with normospermia who were attended to infertility center for diagnosis or infertility treatment. Immunohistochemistry was used to localize the isoform of eNOS in these tissues and the intensity of staining was semi quantitively assessed. In addition, the histopathological evaluation was examined in both groups. The isoform of eNOS enzyme activity was detected in the cytoplasm of sertoli and leydig cells in both groups. There was, however, a considerable variability in the intensity of staining between two groups. The expression of eNOS in Leydig cells in control group was significantly [p<0.05] higher than those in the NOA group. In contrast, expression of eNOS in Sertoli cells in NOA was more than those in the control group. eNO Simmune staining was absent in the normal germ cells but was intense in the abnormal germ cells with piknotic neucleous. The most histopathological finding were hypospermatogenesis [27.2%], Sertoli cell only syndrome [18.1%] and tubular fibrotic [13.6%]. These results suggested that increase level of eNOS may play an important role in the apoptosis process in the abnormal germ cells and disturbance of spermatogenesis process