Epigenotoxic effect of dimethyl sulfoxide on buffalo somatic cells and buffalo-bovine interspecies somatic cell nuclear transfer embryos

Objective: In the present study, we investigated the possible epigenotoxic effect of dimethyl sulfoxide [DMSO] on buffalo fibroblast cells and on reconstructed oocytes during buffalo-bovine interspecies somatic cell nuclear transfer [iSCNT] procedure and its effect on rate and quality of blastocyst which derived from these reconstructed oocytes

Materials and Methods: In this experimental study, cell viability of buffalo fibroblasts was assessed after exposure to various concentration [0.5, 1, 2 and 4%] of DMSO using MTS assay. The epigenetic effect of DMSO was also assessed in terms of DNA methylation in treated cells by flowcytometry. Reconstructed oocytes of buffalo-bovine iSCNT exposed for 16 hours after activation to non-toxic concentration of DMSO [0.5%] to investigate the respective level of 5-methylcytosine, cleavage and blastocyst rates and gene expression [pluripotent genes: OCT4, NANOG, SOX2, and trophectodermal genes: CDX2 and TEAD4] of produced blastocysts

Results: Supplementation of culture medium with 4% DMSO had substantial adverse effect on the cell viability after 24 hours. DMSO, at 2% concentration, affected cell viability after 48 hours and increased DNA methylation and mRNA expression of DNMT3A in fibroblast cells. Exposure of reconstructed oocytes to 0.5% DMSO for 16 hours post activation did not have significant effect on DNA methylation, nor on the developmental competency of reconstructed oocyte, however, it decreased the mRNA expression of NANOG in iSCNT blastocysts

Conclusion: Depending on the dose, DMSO might have epigenotoxic effect on buffalo fibroblast cells and reconstructed oocytes and perturb the mRNA expression of NANOG in iSCNT blastocysts

Effect of vitamin D supplementation on intracytoplasmic sperm injection outcomes: a randomized double-blind placebo-controlled trial

Background: Despite numerous studies indicating an imperative role for reproduction, however, the role of Vitamin D supplementation on outcomes of assisted reproductive techniques remains controversial. This clinical trial was per- formed to evaluate the effect of Vitamin D supplementation 6 weeks prior to intracytoplasmic sperm injection [ICSI] on fertility indices

Materials and Methods: The present study was a double-blind clinical trial conducted on infertile women was randomly allocated into two groups: Vitamin D supplementation [42 participants] and placebo [43 participants]. Serum Vitamin D was measured before and six to eight weeks after treatment, on the day of ovum pick up. Results were analyzed using SPSS16 and fertility indices were compared between the two groups

Results: No significant difference was observed between the intervention and control groups regarding the mean number of oocytes retrieved, percentage mature oocyte, fertilization rate and the rate of good quality embryos [all P>0.05]. But, percentages of the individual with suitable endometrium [7-14 mm thickness] were significantly higher in the Vitamin D compared to control group [P=0.011]. The rate of chemical [47.6 vs. 25.5%, P=0.013] and clinical pregnancy rate [38.1 vs. 20.9%, P=0.019] were also significantly higher in the Vitamin D compared to control group

Conclusion: The present study reveals that consuming Vitamin D for 6 weeks prior to ICSI improves quality of endo- metrium, rate of chemical and clinical pregnancy

Hypothesis: a challenge of overexpression Zfp521 in neural tendency of derived dental pulp stem cells

Neurodegenerative diseases have now become a major challenge, especially in aged societies. Most of the traditional strategies used for treatment of these diseases are untargeted and have little efficiency. Developments in stem cell investigations have given much attention to cell therapy as an alternative concept in the regeneration of neural tissues. Dental pulp stem cells [DPSCs] can be readily obtained by noninvasive procedures and have been shown to possess properties similar to well-known mesenchymal stem cells. Furthermore, based on their neural crest origin, DPSCs are considered to have a good potential to differentiate into neural cells. Zfp521 is a transcription factor that regulates expression of many genes, including ones involved in the neural differentiation process. Therefor based on neural crest origin of the cell and high expression of neural progenitor markers, we speculate that sole overexpression of Zfp521 protein can facilitate differentiation of dental stem cells to neural cells and researchers may find these cells suitable for therapeutic treatment of neurodegenerative diseases

Assessment of PGC1 Alpha -FNDC5 axis in granulosa cells of PCOS mouse model

Background: Polycystic ovarian syndrome [PCOS] is a metabolic and endocrine disorder which is characterized by hyperandrogenism, anovulation or oligomenor-rhea and polycystic ovarian morphology. It is believed that modulation in metabo-lism of granulosa cells of PCOS patients may lead to infertility. One of the metabolic modulators is FNDC5 and its cleaved form, irisin. The axis of PGC1 Alpha - FNDC5 pathway is one of the main factors affecting cellular energy balance the purpose of this study was to evaluate this pathway in granulosa cells derived from PCOS mice model in comparison with control group

Methods: In the present study, PCOS mouse model was developed by injection of dehydroepiandrosterone [DHEA] hormone in 20 mice for a period of 20 days. Also, 20 uninjected mice were used as the control. Meanwhile, a vehicle group consisted of mice which received daily subcutaneous sesame oil injection [n=20]. Relative ex-pressions of PGC1á and FNDC5 in granulosa cells were evaluated by RT-qPCR. Analysis of gene expressions was calculated by the Delta Delta CT method and the relative levels of mRNA were normalized to GAPDH transcript levels. Differences in genes expression among three groups were assessed using one-way ANOVA, Tukey's Post Hoc test

Results: Our results showed that expression of FNDC5 was significantly reduced in granulosa cells of DHEA-induced PCOS mice compared with control and vehicle groups [p<0.05], while there was no significant differences in PGC1 Alpha expression among different groups

Conclusion: Down regulation of FNDC5 transcript level may contribute in metabol-ic disturbance of granulosa cells derived from PCOS ovary apart from PGC1 Alpha levels which remained unchanged

role of local anesthesia instillation in pain alleviation post laparoscopy

Background: gynecological laparoscopy is now becoming a more popular technique for diagnosis and treatment of infertility cases; however gynecological laparoscopy is usually followed by Post-operative pain which may be attributed to small incision in abdominal wall or as a result of diaphragmatic irritation by inflating gases

Aim of the Study: was to assess the role of local anesthesia instillation in pain relief after laparoscopy

Patients and methods: the present study was a randomized trial study performed on 146 women aged 20-35 years, admitted to Ain Shams University hospitals. Patients were split into 2 equal groups, Group[A]: further subdivided into A1 and A2 whom undergone diagnostic and operative laparoscopy respectively- patients administrated 20ml of 0.25% of Bupivacaine instilled intraperitoneal-and Group [B]: further subdivided into B1 and B2 -whom undergone diagnostic and operative laparoscopy respectively-Patients administrated 20ml of 0.75% of Ropivacaine instilled intra-peritoneal. Vital signs and Pain scores were measured preoperatively, immediate postoperative, 1 hours, 2 hours, 4 hours, 8 hours, 12 hours postoperatively. Data was collected, tabulated, then analyzed using IBM[copyright sign] SPSS[copyright sign] Statistics version 22 [IBM[copyright sign] Corp., Armonk, NY]

Results: administration of 20mL of Ropivacaine 0.75% at the end of technique gave pain relief for four hours in group B1 and pain relief for three hours in group B2, associated with an increase in heart rate and blood pressure for two hours in group B1 and B2 for three hours

Conclusion: instillation of local anesthetic after laparoscopy promotes pain relief post operatively and further minimizes the postoperative hospital stay

Evidence of oocyte polarity in bovine; implications for intracytoplasmic sperm injection and somatic cell nuclear transfer

Objective: We recently demonstrated spatial regionalization of maternal transcripts and proteins within unfertilized ovine oocyte. Here, we investigated the likelihood of oocyte polarity for the first time in bovine

Materials and Methods: In this experimental study, in vitro matured bovine oocytes were used for manual bisection [into oocyte halve that were near-to [HNS] and far-from [FS] spindle] or trisection [into MII-spindle [S], the spindle-side half [NS], and the distal half unassociated with the spindle [FS]]. Prepared pools of oocyte substructures were used for comparative quantitative real-time polymerase chain reaction [RT-qPCR]. To map the possible preferential sperm entry point [SEP], the spatial relationship between SEP and MII-spindle was measured 5 hours post-fertilization

Results: The proportional amount of maternal mRNA in S oocyte fragment was estimated to be 6 to 11-fold higher than NS and FS counterparts. The relative abundances of Nanog, Oct4, Fgf4 and Tead4 were significantly higher in HNS oocyte fragment compared t0 FS. The relative abundances of Ctnb, Carm1, Rex1, Sox2 and Cdx2 were comparable between HNS and NS oocyte fragments. FS oocyte fragment possessed significantly higher transcripts of Gata4 compared to HNS. The distribution of certain transcripts related to pluripotency and lineage commitment were different depending upon the region of the oocyte; either enriched at S [Tead4, Nanog, Ctnb and Sox2], NS [Oct4], or FS [Gata6]. The SEP in almost [90%] fertilized oocytes was located in MII-hemisphere

Conclusion: The observation of spatial restriction of mRNAs and SEP within MII-oocyte may indicate that the principal forces of oocyte polarity are evolutionary conserved. This may in turn highlight the need for refinements in the methodology of intracytoplasmic sperm injection [where a sperm is injected far from the MII-spindle] and somatic cell nuclear transfer [where a major amount of regulative mRNAs that are associated with MIIspindle is removed during enucleation]

Methylation status of H19/IGF2 differentially methylated region in in vitro human blastocysts donated by healthy couples

Backgrund: Imprinted genes are a unique subset of few genes, which have been differentially methylated region [DMR] in a parental origin-dependent manner during gametogenesis, and these genes are highly protected during pre-implantation epigenetic reprogramming. Several studies have shown that the particular vulnerability of imprinting genes during suboptimal pre- and peri-conception micro-environments often is occurred by assisted reproduction techniques [ART]. This study investigated the methylation status of H19/IGF2 DMR at high-quality expanding/expanded human blastocysts donated by healthy individuals to evaluate the risks linked to ART

Method: Methylation levels of H19/IGF2 DMR were analyzed by bisulfite conversion and sequencing at 18 CpG sites [CpGs] located in this region

Result: The overall percentage of methylated CpGs and the proportion of hyper-methylated clones of H19/IGF2 DMR in analyzed blastocysts were 37.85 +/- 4.87% and 43.75 +/- 5.1%, respectively. For validation of our technique, the corresponding methylation levels of peripheral human lymphocytes were defined [49.52 +/- 1.86% and 50%, respectively]

Conclusion: Considering the absence of in vivoproduced human embryos, it is not possible to conclude that the methylation found in H19/IGF2 DMR is actually normal or abnormal. Regarding the possible risks associated with ART, the procedures should be optimized in order to at least reduce some of the epigenetic risks

Rational development of a polycistronic plasmid with a CpG-free bacterial backbone as a potential tool for direct reprogramming

Objective: Induced pluripotent stem cells are generated from somatic cells by direct reprogramming. These reprogrammed pluripotent cells have different applications in biomedical fields such as regenerative medicine. Although viral vectors are widely used for efficient reprogramming, they have limited applications in the clinic due to the risk for immunogenicity and insertional mutagenesis. Accordingly, we designed and developed a small, non-integrating plasmid named pLENSO/Zeo as a 2A-mediated polycistronic expression vector

Materials and Methods: In this experimental study, we developed a single plasmid which includes a single expression cassette containing open reading frames of human LIN28, NANOG, SOX2 and OCT4 along with an EGFP reporter gene. Each reprogramming factor is separated by an intervening sequence that encodes a 2A self-processing peptide. The reprogramming cassette is located downstream of a CMV promoter. The vector is easily propagated in the E. coli GT115 strain through a CpG-depleted vector backbone. We evaluated the stability of the constructed vector bioinformatically, and its ability to stoichiometric expression of the reprogramming factors using quantitative molecular methods analysis after transient transfection into HEK293 cells

Results: In the present study, we developed a nonviral episomal vector named pLENSO/Zeo. Our results demonstrated the general structural stability of the plasmid DNA. This relatively small vector showed concomitant, high-level expression of the four reprogramming factors with similar titers, which are considered as the critical parameters for efficient and consistent reprogramming

Conclusion: According to our experimental results, this stable extrachromosomal plasmid expresses reliable amounts of four reprogramming factors simultaneously. Consequently, these promising results encouraged us to evaluate the capability of pLENSO/Zeo as a simple and feasible tool for generation of induced pluripotent stem cells from primary cells in the future

Relationship between potential sperm factors involved in oocyte activation and sperm DNA fragmentation with intra-cytoplasmic sperm injection clinical outcomes

Objective: The present study aimed to simultaneously evaluate the association between expression of three potential factors [post-acrosomal sheath WW domain-binding protein [PAWP], phospholipase Czeta [PLCzeta], and truncated form of the kit receptor [TR-KIT]] as candidates of oocyte activation with fertilization rate and early embryonic development

Materials and Methods: In this experimental study, semen samples were collected from 35 intra-cytoplasmic sperm injection [ICSI] candidates and analyzed according to World Health Organization criteria [2010]. Each sample was divided into two parts. The first part was processed for insemination by density-gradient centrifugation [DGC] and the second part was prepared for assessment of sperm morphology [Papanicolaou staining], DNA fragmentation [transferase dUTP nick end labeling [TUNEL]], and three Sperm-borne oocyte-activating factor [s] [SOAFs]-PLCzeta, PAWP, and TR-KIT

Results: Significant positive correlations existed between the percentages of PLCzeta, PAWP, and TR-KIT with fertilization rate. In addition, significant negative correlations existed between the percentage of DNA fragmentation with the percentages of PLCzeta and PAWP. We did not find a relationship between percentages of PLCzeta, PAWP, and TR-KIT with embryo quality and pregnancy rate [P>0.05]. There was a significant negative correlation between percentage of DNA fragmentation with fertilization and embryo quality

Conclusion: Oocyte activation was associated with the studied sperm factors [PAWP, PLCzeta, and TR-KIT]. These factors might hold the potential to be considered as diagnostic factors in the assessment of semen samples to evaluate their potential to induce oocyte activation. In addition, we observed a significant association between DNA fragmentation with fertilization, as well as embryo quality and expression of PAWP and PLCzeta, which indicated that men with high degrees of DNA fragmentation might require artificial oocyte activation. Whether such action should take place, and its cost and benefits should be evaluated in the future

Chances to have a boy after gender selection by pre-implantation genetic screening are reduced in couples with only girls and without a boy sired by the male partner

Background: Gender selection and family planning have their roots in human history. Despite great interest in these fields, very few scientific propositions exist which could explain why some family do not attain the desired sex. Therefore, the aim of this study was to evaluate whether sex of previous child or children could affect the outcomes of pre-implantation genetic screening [PGS]

Materials and Methods: This historical cohort study including 218 PGS cases referring to Isfahan Fertility and Infertility Center [IFIC]. Couples were grouped as those who their male child passed away or her husbands' has a son[s] from their previous marriage [n=70] and couples who just have daughter [n=148]. Male normal blastocysts were transferred for both groups. The outcomes of PGS including pregnancy, implantation and abortion rates, along with possible confounding factors were compared between the two groups

Results: Significant differences in pregnancy, implantation and abortion rates were observed between couples whose their male partner had/has one boy [n=70] compared to those who have just girl[s] [n=148] despite similar number and quality of male normal blastocyst transferred in the two groups. Confounding factors were also considered

Conclusion: The Y- bearing spermatozoa in male partners with no history of previous boy have lower ability to support a normal development to term, compared to male partners with previous history of boy requesting family balancing

Analysis of promyelocytic leukemia in human embryonic carcinoma stem cells during retinoic acid-induced neural differentiation

Background: Promyelocytic leukemia protein [PML] is a tumor suppressor protein that is involved in myeloid cell differentiation in response to retinoic acid [RA]. In addition, RA acts as a natural morphogen in neural development

Objectives: This study aimed to examine PML gene expression in different stages of in vitro neural differentiation of NT2 cells, and to investigate the possible role of PML in pluripotency and/or neural development

Materials and Methods: RA was used as a neural inducer for in vitro neural differentiation of NT2 cells. During this process PML mRNA and protein levels were assessed by quantitative real time RT-PCR [QRT-PCR] and Immunoblotting, respectively. Furthermore bisulfite sequencing PCR [BSP] was used to assess PML promoter methylation in NT2 cells and NT2 derived neuronal precursor cells [NT2.NPCs]

Results: QRT-PCR results showed that, PML had maximum expression with significant differences in NT2 derived neuronal precursor cells relative to NT2 cells and NT2 derived neural cells [NT2.NCs]. Numerous isoforms of PML with different intensities appeared in immunoblots of pluripotent NT2 cells, NT2.NPCs, and NT2.NCs. Furthermore, the methylation of the PML promoter in NT2.NCs was 2.6 percent lower than NT2 cell

Conclusions: The observed differences in PML expression in different cellular stages possibly could be attributed to the fact that PML in each developmental state might be involved in different cell signaling machinery and different functions. The appearance of different PML isoforms with more intensity in neural progenitor cells; may suggest apossible role for this protein in neural development

Epidemiological, clinical and bacteriological studies on bacterial lamb enteritis at behera province, Egypt

Lamb enteritis is economically important problem because several agents may be involved in the etiology of it. This study was conducted to throw light on bacterial enteritis in some sheep flocks in Behera province. From 1200 examined lambs, 650 showed enteritis [54.16%]. Enteritis rate were higher in closed system [83.70%0 than in open system [46.80%]. Mortality rate was [4.16%] and case fatality was [7.69%]. Bacteriological examination of fecal samples revealed that 190 lambs [29%] were positive for pathogenic bacterial culture and 460 lambs [71%] were negative. The isolated bacteria were E. coli pathogenic form from 65 cases [34.20%] which was the most predominant bacterial isolate. Other bacteria isolated were salmonella from 10 cases [5.26%], clostridia isolated from 15 cases [7.89%], proteus species isolated from 25 cases [13.10%], shigella isolated from 20 cases [10.52%], klel'siclla isolated from 15 cases [7.89%] and mixed infection was reported in 40 cases [21%]. Serotyping of E coli revealed that O55/K50 [35] [2 isolates], while O78/K80 [B-], O125.K70 [B15], O101/K99 and O22/K11 [L] and each of them [l isolates], while the untypable isolates was [4 isolates]. Salmonella typing revealed that S. typhimurium serotype B [2 isolates], S. enteritidis serotype Dl [1 isolate] and the un typable salmonella isolates [2 isolates]. The antimicrobials susceptibility of isolated bacteria was done to determine its susceptibility

[Relationship between occurrence of guillan-barre syndrome and mass campaign of measles and rubella immunization in Iranian 5-14 years old children]

Several case reports and epidemiologic studies have reported a relation between different vaccines including measles, rubella, and mumps and Guillain-Barre syndrome [GBS]. In this study we investigated relation between receiving measles and/or rubella vaccines and occurrence of Guillain-Barre syndrome after national immunization campaign in 2003 in Iran. In this study we used National Surveillance system for acute flaccid paralysis data from the beginning of 2002 to the end of 2004. We studied 5-14 year-old children. Three-year time span of the study was divided into fifteen 10 weeks periods, and number of reported and approved cases of Guillain-Barre syndrome in each time period has been analyzed supposing their distribution was according to Poisson distribution. One of time periods has been chosen in a manner that its first 4 weeks included embedded National Immunization and following 6 weeks were designated for covering incubation period of Guillin-Barre syndrome. Of total 553 reported flaccid paralysis cases in 5-15 year-old children 370 were approved to be Guillain-Barre [67%]. Estimated average incidence of Guillain-Barre syndrome in<15 year-olds is 1/100,000/year [95% CI: 0.88-13], and in 5-15 year-old children is 0.6/100,000/year [95% CI: 0.58-083]. Thus, number of expected cases in 25 cases each 10 weeks time period was 23.8 of GBS that were reported in the time period which coincided with immunization campaign. Probability of occurring>/=25 cases of GBS in that time period according to Poison distribution with expected case numbers of 23.8 is equal to 0.43 [P=0.43]. Yearly incidence rate of GBS in this study was similar to other studies. According to our results, there was no increase in GBS incidence in the 4 weeks national Immunization Campaign and 6 weeks after it in comparison to other 10 weeks periods before or after this time period

role of sperm chromatin anomalies on the outcome of assisted reproductive techniques

Sperm DNA is known to contribute one half of the genomic material to the offspring. The integrity of sperm DNA is important in fertilization, embryonic and fetal development, and postnatal child well being. The nature has created multiple barriers that allow only the fittest sperm to reach and fertilize an oocyte. However, assisted reproductive techniques [ART], like IVF and ICSI, may allow sperms with abnormal genomic material to enter the oocyte with minimal effort. This article describes structure of sperm DNA and different mechanism involved in sperm chromatin anomalies and DNA damage. Furthermore, this study elaborates possible sperm selection methods that may improve the outcome of ART

Basic fibroblast growth factor: a marker predicting the severity of joint destruction in juvenile rheumatoid arthritis

To investigate whether serum basic fibroblast growth factor [bFGF], as a representative of angiogenesis, is to be considered a useful marker for predicting joint destruction in juvenile rheumatoid arthritis [JRA]. bFGF was measured by ELISA in 30 serum samples and 8 joint fluid samples obtained from JRA patients. Patients were evaluated at baseline and 12 months later. A] Clinically with the overall articular severity score. B] Laboratory tests including ESR, complete blood count and rheumatoid factor. C] Radiologically using modified Larsen index, mal-alignment and van Rossum's scores. A group of 20 healthy children and another of 10 patients with other collagen vascular diseases were also studied for comparison. Serum bFGF was significantly higher in JRA patients than those with other collagen vascular diseases and the controls with 90% sensitivity and 100% specificity at a cut-off level of 0.203 pg/ml [control mean + 3SD]. Polyarticular JRA patients had a significant higher rate of positive bFGF than those with systemic onset or pauciarticular types. Synovial fluid bFGF levels were significantly higher than the corresponding serum values. Serum bFGF correlated positively with disease activity markers and the articular severity score. Radiological evaluation revealed higher rate of seropositive bFGF in patients with severe joint destruction than in those with mild form of joint affection. Moreover, patients whose serum bFGF levels were >0.231 pg/ml showed deterioration of the clinical and radiological scores of joints when reevaluated after one year. Serum bFGF concentration helps to assess the degree of joint involvement and is a useful prognostic marker for predicting the severity of joint destruction in JRA patients