Cytogenetic Evaluation of Couples With Spontaneous Abortion, Still Birth and Recurrent Miscarriage in Qazvin: Report and Review

Background: Chromosomal abnormality plays an important role in different types of miscarriages. Objectives: The present study was designed to investigation chromosomal anomalies in three groups of couples with recurrent abortion (RA), spontaneous abortion (SA) and still birth (SB). PatientsandMethods: In this retrospective study, the frequency of chromosomal aberrations was investigatedamong 260 couples with miscarriage, which had referred to the cytogenetic section of a reference laboratory in Buali hospilal, Qazvin, Iran from 2009 to 2014. Metaphase spreads were analyzed using G-banding. Results: In this study, 7.6% of couples had chromosomal aberrations including, balanced reciprocal translocations, robertsonian translocations, inversions and sex chromosome aneuploidy. Frequency of balanced translocations was higher, specifically in couples with SA. Conclusions: In this investigation we showed that chromosomal abnormalities could be one of the important causes of miscarriages. Cytogenetic evaluation of couples, which experienced different types of miscarriage, may prevent unnecessary treatments. Keywords: Recurrent Abortion, Spontaneous Abortion, Still Birth, Chromosome Abnormalit

Underexpression of hsa-miR-449 family and their promoter hypermethylation in infertile men: A case-control study

Background: Post-transcriptional microRNAs (miRNAs) have an important pattern in the spermatogenesis process. Objective: Study of the expression and methylation of hsa-miR-449 family in sperm samples of infertile men. Materials and Methods: In this case-control study, we recruited 74 infertile men (with asthenozoospermia, teratozoospermia, asthenoteratozoospermia, and oligoasthenoteratozoospermia) and 30 control samples. Methylation-specific PCR (MSP) method was used for methylation evaluation of hsa-miR-449 a, b, c promoter. By Real time PCR (qRT-PCR) method,we showed downregulation of hsa-miR-449 a, b, c in the sperm samples of infertile men and compared it to their fertile counterparts. Results: There was significant underexperssion, in hsa-miR-449-b in oligoasthenoteratospermic samples (p = 0.0001, F = 2.9). About the methylation pattern, infertile men showed high frequency of methylation in the promoter of hsa-miR-449 a, b, c in comparison to controls (60.8% vs 23.3%), the highest amount of methylation was observed in oligoasthenoteratospermia samples (81.2%). Conclusion: In this study, low expression and high methylation of hsa-miR-449-b were observed in infertile men in compared to control samples, which can be one of the causes of defective spermatogenesis. Key words: Spermatogenesis, miR-449, Expression, Epigenetic

Cell-free seminal mRNA of DDX4 and TNP1 Genes as Potential Biomarkers of the Presence of Sperm in the Testicular Tissue

Introduction: Non-obstructive azoospermia (NOA) is one of the reasons for infertility in men, and different factors including genetic factors are involved in its development. Since taking biopsies of the testicular tissue for assisted reproductive technologies (ARTs) is invasive and time-consuming, and the testicular tissue is heterogeneous, introducing a biomarker for predicting the possibility of the presence of sperm in the testicle can increase the ART efficiency. Accordingly, Cell-free seminal mRNA (CFs-mRNA), which is found in many fluids of the body including the seminal fluid of NOA individuals, can be employed as a biomarker for this purpose. Materials and methods: This study was conducted on 15 men suffering NOA, candidates for testicular sperm extraction (TESE), along with 15 healthy men as control. The testicular tissue of 10 patients was examined using hematoxylin and eosin staining and then classified according to Johnsen scoring. RNA was extracted from the cell-free plasma of semen samples and cDNA was synthesized. The Expression level of TNP1 and DDX4 genes was investigated using real-time polymerase chain reaction (PCR). Results: The expression of CFS-mRNA of the DDX4 gene was observed in only one sample of NOA individuals (10%), showing a score of 8. Further, the expression of CFS-mRNA of the TNP1 gene was observed only in two samples (20%) of NOA patients whose scores were 3 and 8. Conclusion: Insufficiency or lack of expression of CFS-mRNA of TNP1 and DDX4 genes may be helpful in predicting the absence of sperm in the testicular tissue of NOA patients in terms of sperm retrieval for ART. Yet, further studies with more specific and sensitive techniques are required to achieve a more solid and precise conclusion

Modulation of the Expression of the GABAA Receptor β1 and β3 Subunits by Pretreatment with Quercetin in the KA Model of Epilepsy in Mice -The Effect of Quercetin on GABAA Receptor Beta Subunits-

Objectives: Quercetin is a flavonoid and an important dietary constituent of fruits and vegetables. In recent years, several pharmacological activities of quercetin, such as its neuroprotective activity and, more specifically, its anti-convulsant effects in animal models of epilepsy, have been reported. This study evaluated the role of quercetin pretreatment on gene expression of γ-amino butyric acid type A (GABAA) receptor beta subunits in kainic acid (KA)-induced seizures in mice. Methods: The animals were divided into four groups: one saline group, one group in which seizures were induced by using KA (10 mg/kg) without quercetin pretreatment and two groups pretreated with quercetin (50 and 100 mg/kg) prior to seizures being induced by using KA. Next, the messenger ribonucleic acid (mRNA) levels of the GABAA receptor β subunits in the hippocampus of each animal were assessed at 2 hours and 7 days after KA administration. Quantitative real-time polymerase chain reaction (RT-PCR) assay was used to detect mRNA content in hippocampal tissues. Results: Pretreatments with quercetin at doses of 50 and 100 mg/kg prevented significant increases in the mRNA levels of the β1 and the β3 subunits of the GABAA receptor at 2 hours after KA injection. Pretreatment with quercetin (100 mg/kg) significantly inhibited β1 and β3 gene expression in the hippocampus at 7 days after KA injection. But, this inhibitory effect of quercetin at 50 mg/kg on the mRNA levels of the β3 subunit of the GABAA receptor was not observed at 7 days after KA administration. Conclusion: These results suggest that quercetin (100 mg/kg) modulates the expression of the GABAA receptor β1 and β3 subunits in the KA model of epilepsy, most likely to prevent compensatory responses. This may be related to the narrow therapeutic dose range for the anticonvulsant activities of quercetin

Bioinformatics design of CRISPR guide RNA for genomic knockout of ABCB1 gene

Abstract Background: Over-expression of P-Glycoprotein (Pgp) induces acquired drug resistance. Therefore, targeting Pgp as a dominant efflux transporter involved in emergence of multidrug resistance (MDR) has become a major strategy for reversibility of sensitivity to chemotherapy. Objectives: The aim of this study was to design sgRNAs targeting ABCB1 in order to knockout and inhibit the expression of Pgp in Adriamycin resistant (A2780/ADR) ovarian cancer cell line. Methods: This study was performed as a bioinformatics and computational research in Qazvin University of Medical Sciences in collaboration with the Isfahan University of Medical Sciences during 2015-2016. All the 28 exons of the ABCB1 gene were separately investigated in terms of single guide RNA (sgRNA) target sites with regards to the highest on-target and lowest off-target activities, using www.deskgen.com website. Three sgRNA sequences were chosen and synthesized by the GeneCopoeia company. All the plasmids were validated after extraction using BamH1 and EcoR1 restriction enzymes. Results: Sequences of the three sgRNAs were selected close to the start codon (ATG) in order to maximize the possibility of exons 4 and 5 knockout. Digested pCRISPR-CG01, using BamH1 and EcoR1, was electrophorized on 1.5% agarose gel. Detection of the two 330bp and 10100bp fragments on the gel confirmed the integrity of the plasmid and success of the restriction enzyme digestion. Conclusions: The vectors containing the designed sgRNA sequences and CRISPR associated protein (Cas9) can inhibit Pgp gene expression in cell lines over-expressing this gene, including A2780/ADR. Keywords: Drug Resistance, P-Glycoprotein, Ovarian Cancer, CRISP

Increasing viability, numbers, and motility of sperm in men with normal spermatogenesis exposed to saffron extract after freezing- thawing process

Background: Sperm freezing method is used frequently in assisted reproductive techniques, on the other hand in different studies negative effect of freezing have been shown on different sperm parameters. Objective: The aim of this study was to determine the effect of saffron extract as an antioxidant, on the different sperm parameters in men with normal spermatogenesis after freezing-thawing process. Methods: In this case-control study, collecting of samples was done in 2015 year from the Infertility Treatment Center, ACECR Branch of Qazvin, Qazvin, Iran. These men had normal spermatogenesis and their spouse had infertility problem. Semen samples was divided in two groups, control without saffron extract, and case with 50 mg/ml saffron extract. Then, samples freezed with snap freezing method. After two weeks, they were thawed and different sperm parameters were assessed. Data were analyzed by two-tail T test. Findings: Our results showed, mean percent of viability (72 ± 0.99), motility (87 ± 0.43), and the number of sperm cells (62.5 ± 3.8) in treaded group was elevated significantly (P<0.01) compared to the control group (46.6±1.1), (62.3 ± 0.33), and (45 ± 4.3) respectively. However, about the morphology we don’t observed significant difference (P>0.05). Conclusion: Our results showed that possibly antioxidant agents of saffron extract could scavenge free radicals and thus, optimize different sperm parameters (viability, motility, and number) after freezing and thawing. Keywords: Saffron extract, Freezing, Thawing, Sperm parameter

Comparison of Protamine 1 to Protamine 2 mRNA Ratio and YBX2 gene mRNA Content in Testicular Tissue of Fertile and Azoospermic Men

Background: Although aberrant protamine (PRM) ratios have been observed in infertile men, the mechanisms that implicit the uncoupling of PRM1 and PRM2 expression remain unclear. To uncover these mechanisms, in this observational study we have compared the PRM1/PRM2 mRNA ratio and mRNA contents of two regulatory factors of these genes. Materials and Methods: In this experimental study, sampling was performed by a multi- step method from 50 non-obstructive azoospermic and 12 normal men. After RNA extraction and cDNA synthesis, real-time quantitative polymerase chain reaction (RTQPCR) was used to analyze the PRM1, PRM2, Y box binding protein 2 (YBX2) and JmjC-containing histone demethylase 2a (JHDM2A) genes in testicular biopsies of the studied samples. Results: The PRM1/PRM2 mRNA ratio differed significantly among studied groups, namely 0.21 ± 0.13 in azoospermic samples and -0.8 ± 0.22 in fertile samples. The amount of PRM2 mRNA, significantly reduced in azoospermic patients. Azoospermic men exhibited significant under expression of YBX2 gene compared to controls (P<0.001). mRNA content of this gene showed a positive correlation with PRM mRNA ratio (R=0.6, P=0.007). JHDM2A gene expression ratio did not show any significant difference between the studied groups (P=0.3). We also observed no correlation between JHDM2A mRNA content and the PRM mRNA ratio (R=0.2, P=0.3). Conclusion: We found significant correlation between the aberrant PRM ratio (PRM2 under expression) and lower YBX2 mRNA content in testicular biopsies of azoospermic men compared to controls, which suggested that downregulation of the YBX2 gene might be involved in PRM2 under expression. These molecules could be useful biomarkers for predicting male infertility

Pathological Variants of Aminoacyl-tRNA-synthetase-Interacting Multifunctional Protein 1 Gene in an Iranian Consanguineous Family With Autosomal Recessive Intellectual Disability

Background Intellectual disability (ID) is one of the most common neurodevelopment disorders that caused by both environment and genetic factors. Genetic diseases account for 50% of ID incidents and have important role in its development. One of the most important risk factors of ID in most countries is consanguineous marriage. In consanguineous families, the risk of developing autosomal recessive ID is 3.6-fold higher. There is high prevalence of consanguineous marriage in Iran (about 40 %). Objective In this study, we aimed to investigate the pathological variants of aminoacyl-trna-synthetaseinteracting multifunctional protein 1 (AIMP1) in an Iranian consanguineous family with multiple-ID affected members. Methods this analytical epidemiological study, whole exome sequencing method was used to examine the molecular etiology in two female ID patients of a consanguineous family living in Qazvin, Iran. Sanger sequencing was carried out for validating potential causative variants in patients, and co-segregation analysis for other family members. Findings A stop-gain variant (p. Arg158*) in the AIMP1 gene was identified as pathological variant in the study family according to American College of Medical Genetics and Genomics guidelines. Conclusion The found variant in the AIMP1 gene caused truncated protein and clinical manifestations such as developmental delay, ID, spastic paraplegia, thin corpus callosum, and speech impairment in the two patients