The effect of vitamin C on the gene expression profile of sperm protamines in the male partners of couples with recurrent pregnancy loss: A randomized clinical trial / 대한생식의학회지

Objective@#Since sperm abnormalities are known to be a major reason for recurrent pregnancy loss (RPL), any defects in DNA structure and chromatin condensation can place embryos at risk in the early stage of development and implantation. As antioxidants such as vitamin C may play a protective role against the destruction of protamine genes in sperm chromatin, this study was conducted to evaluate the effects of vitamin C on chromatin and the expression of protamine genes in the male partners of couples with RPL. @*Methods@#Twenty male partners of couples with RPL were selected as the intervention group and received vitamin C supplementation (250 mg daily for 3 months). Healthy fertile men (n=20) were included as controls. Sperm chromatin, DNA integrity, and the expression levels of protamine genes were evaluated before and after treatment. @*Results@#Significant differences were found in sperm morphology, protamine deficiency, and apoptosis between the two groups and before and after vitamin C administration. A significant change was found in mRNA levels of PRM1, PRM2, and the PRM1/PRM2 ratio after treatment. @*Conclusion@#Daily oral administration of vitamin C may improve human sperm parameters and DNA integrity by increasing protamine gene expression levels in the male partners of couples with RPL. The beneficial effects of vitamin C supplementation as an antioxidant for the male partners of couples with RPL could lead to improved pregnancy outcomes in these cases.

Analysis of PRM1 and PRM2 polymorphisms in Iranian infertile men with idiopathic teratozoospermia

Single nucleotide polymorphisms [SNPs] in a number of genes involved in sperm maturation are considered as one of the main factors for male infertility. The aim of the present case-control study was to examine the association of SNPs in protamine1 [PRM1] and protamine2 [PRM2] genes with idiopathic teratozoospermia. In this case-control study, some SNPs in PRM1 [c.49 C>T, c.102 G>T and c.230A>C] and PRM2 [rs545828790, rs115686767, rs201933708, rs2070923 and rs1646022] were investigated in 30 idiopathic infertile men with teratozoospermia [case group] in comparison with 35 fertile men [controls]. Genotyping of SNPs was undertaken using polymerase chain reaction [PCR]-direct sequencing. For PRM1, c.230A>C, as a synonymous polymorphism, was detected in both teratozoo- spermic men [heterozygous n=26, homozygous minor n=1] allele frequency C[48] A[52] and controls [heterozygous n=15, homozygous minor n=4]. All cases and controls were genotyped for rs545828790 in PRM2, a missense poly- morphism, as well as rs115686767 and rs201933708, both of which synonymous variants. The findings showed an intronic variant in PRM2 [rs2070923] was also present in both groups. Also, rs1646022, a missense polymorphism, occurred in teratozoospermic men [heterozygous n=10, homozygous minor n=5] and controls [heterozygous n=13, homozygous minor n=2]. However, there were no significant differences in SNPs of PRM1 and PRM2 between the two groups, however, for c.230A>C, the frequency of the CA genotype was significantly higher in infertile men with teratozoospermia [P=0.001]. We demonstrate that PRM2 G398C and A473C polymorphisms were associated with the teratozoospermia and its genetic variation was in relation to semen quality, sperm apoptosis, and morphology in the Iranian population. This study is a preliminary study and presenting data as part of a future comprehensive study to clinically establish whether these gene polymorphisms are biomarkers for susceptibility to teratozoospermia

Sperm chromatin and DNA integrity, methyltransferase mRNA levels, and global DNA methylation in oligoasthenoteratozoospermia / 대한생식의학회지

OBJECTIVE: To investigate sperm chromatin/DNA integrity, global DNA methylation, and DNMT mRNA transcription in men with oligoasthenoteratozoospermia (OAT) compared with normozoospermic men. METHODS: Semen samples from 32 OAT patients who comprised the case group and 32 normozoospermic men who comprised the control group were isolated and purified using a standard gradient isolation procedure according to World Health Organization criteria. DNMT1, DNMT3A, and DNMT3B transcripts were then compared between groups using real-time quantitative reverse-transcription polymerase chain reaction. Global DNA methylation in sperm was determined by an enzyme-linked immunosorbent assay. Protamine deficiency and the proportion of apoptotic spermatozoa were evaluated using chromomycin A3 (CMA3), aniline blue (AB), and toluidine blue (TB) staining, as well as the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The p-values < 0.05 were considered to indicate statistical significance. RESULTS: Significantly higher proportions of AB+, TB+, CMA3+, and TUNEL+ spermatozoa, as well as DNMT3A and DNMT3B transcription, were found in the OAT group. Positive correlations were detected between sperm parameters, DNA/chromatin damage, and DNMT3A and DNMT3B transcripts. Global DNA methylation was significantly higher in the OAT patients and had a significant correlation with abnormal results of all sperm chromatin integrity tests, but was not associated with DNMT1, DNMT3A, or DNMT3B expression. CONCLUSION: Oligoasthenoteratozoospermic men showed abnormal sperm parameters, abnormal chromatin/DNA integrity, and a higher global DNA methylation rate, as well as overexpression of DNMT mRNA.

relationship between the expression levels of miR-135a and HOXA10 gene in the eutopic and ectopic endometrium

Background: the study of microRNA expression can be effective in the diagnosing and treating different diseases. miR-135a is one of the most important micro-ribonucleic acids involved in endometriosis. Among the genes that become the target of the miR-135a and are subjected to changes in the endometrium of patients with endometriosis is HOXA10 gene which is expressed in the endometrium in response to steroid hormones

Objective: the aim of this study was to evaluate the expression of miR-135a and its relationship with the level of HOXA10 gene expression in both endometrial ectopic and eutopic tissues in patients with endometriosis compared to the control samples

Materials and Methods: in this prospective case-control study, both case-eutopic and case-ectopic tissue samples were obtained from 17 women with endometriosis and the eutopic endometrial tissue was sampled from 17 women with normal endometrium as the control group. The gene's expression of miR-135a and HOXA10 were investigated using quantitative reverse transcription PCR [q-RT PCR]

Results: a significant decrease in the expression of HOXA10 gene was detected in case-eutopic during the luteal phase compared to the control samples [p=0.001], while in the case-ectopic, the expression of this gene was increased [p=0.681] compared to the control samples. In addition, the expression miR-135a in the luteal phase showed a remarkable increase in the case-eutopic endometrial tissue [p=0.026] as well as a significant decrease in the case-ectopic endometrial tissue compared to the control samples [p=0.008]

Conclusion: considering the inverse relations between the over-expression of miR-135a and the reduction of HOXA10, it seems that miR-135a may be applied as an endometrial diagnostic and therapeutic biomarker

Frequency of chromosomal aneuploidy in high quality embryos from young couples using preimplantation genetic screening

Background: Selection of the best embryo for transfer is very important in assisted reproductive technology [ART]. Using morphological assessment for this selection demonstrated that the correlation between embryo morphology and implantation potential is relatively weak. On the other hand, aneuploidy is a key genetic factor that can influence human reproductive success in ART

Objective: The aim of this lab trial study was to evaluate the incidence of aneuploidies in five chromosomes in the morphologically high-quality embryos from young patients undergoing ART for sex selection

Materials and Methods: A total of 97 high quality embryos from 23 women at the age of 37or younger years that had previously undergone preimplantation genetic screening for sex selection were included in this study. After washing, the slides of blastomeres from embryos of patients were reanalyzed by fluorescence in-situ hybridization for chromosomes 13, 18 and 21

Results: There was a significant rate of aneuploidy determination in the embryos using preimplantation genetic screening for both sex and three evaluated autosomal chromosomes compared to preimplantation genetic screening for only sex chromosomes [62.9% vs. 24.7%, p=0.000]. The most frequent detected chromosomal aneuploidy was trisomy or monosomy of chromosome 13

Conclusion: There is considerable numbers of chromosomal abnormalities in embryos generated in vitro which cause in vitro fertilization failure and it seems that morphological characterization of embryos is not a suitable method for choosing the embryos without these abnormalities

Evaluation of sperm protamine deficiency and apoptosis in infertile men with idiopathic teratozoospermia / 대한생식의학회지

OBJECTIVE: Sperm morphology plays an important role in infertility, especially in cases of defects in the heads of spermatozoa. Tapered-head or elongated-head spermatozoa are examples of morphological abnormalities. The aim of this study was to compare the semen parameters, levels of protamine deficiency, and frequency of apoptosis between patients with normozoospermia and those with teratozoospermia with tapered-head spermatozoa. METHODS: Fifty-two semen samples (27 patients with tapered-head sperm and 25 fertile men) were collected and semen analysis was performed according to the World Health Organization criteria for each sample. Protamine deficiency and the percentage of apoptotic spermatozoa were evaluated using chromomycin A3 (CMA3) staining and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assays, respectively. RESULTS: Sperm concentration, motility, and normal morphology in the tapered-head spermatozoa (cases) were significantly lower than in the normozoospermic samples (controls). CMA3-reactive spermatozoa (CMA3+) in the case group were more common than in the controls. Apoptotic spermatozoa (TUNEL-positive) were significantly more common in the cases than in the controls. CONCLUSION: This analysis showed that tapered-head spermatozoa contained abnormal chromatin packaging and exhibited a high rate of apoptosis, which can be considered to be an important reason for the impaired fertility potential in teratozoospermic patients with tapered-head spermatozoa.

The effects of different types of media on in vitro maturation outcomes of human germinal vesicle oocytes retrieved in intracytoplasmic sperm injection cycles / 대한생식의학회지

OBJECTIVE: Optimizing in vitro maturation (IVM) media to achieve better outcomes has been a matter of interest in recent years. The aim of this prospective clinical trial was to investigate the effects of different media on the IVM outcomes of immature oocytes at the germinal vesicle (GV) stage. METHODS: A total of 400 immature oocytes at the GV stage with normal morphology were retrieved from 320 infertile women aged 31±4.63 years during stimulated intracytoplasmic sperm injection (ICSI) cycles. They were divided into groups of homemade IVM medium (I, n=100), cleavage medium (II, n=100), blastocyst medium (III, n=100), and Sage IVM medium (IV, n=100) and cultured for 24 to 48 hours at 37℃. ICSI was performed, and the rates of fertilization and embryo formation were compared across the four groups. RESULTS: In the 400 retrieved GV oocytes, the total maturation rates showed significant differences in groups I to IV (55%, 53%, 78%, and 68%, respectively, p<0.001). However, there were no significant differences in the fertilization, embryo formation, or arrest rates of metaphase II oocytes across these groups. In all groups, GV maturation was mostly completed after 24 hours, with fewer oocytes requiring 48 hours to mature (p<0.01). Moreover, the rate of high-quality embryos was higher in group IV than in the other groups (p=0.01). CONCLUSION: The quality of the IVM medium was found to affect clinical IVM outcomes. Additionally, blastocyst medium may be a good choice in IVM/ICSI cycles as an alternative IVM medium.

Relationship between sperm protamine deficiency and apoptosis in couples with unexplained repeated spontaneous abortions

Background: Etiology of more than half of Recurrent Spontaneous Abortion. The etiology of more than 50 percent of Recurrent Spontaneous Abortions [RSA] cases has been remained unexplained. It is supposed that RSA may have "paternal effect" due to supply 50% of embryonic genomic content by male gamete

Objective: The aim of present study was to evaluate the role of sperm apoptosis and protamine deficiency at same time in RSA cases

Materials and Methods: Forty fertile [control] and 40 unfertile men with RSA [case] were enrolled in this case-control study. Semen analysis was performed in accordance with WHO criteria and sperm apoptosis and protamine deficiency were evaluated by cell apoptosis detection kit and chromomycin A3, respectively

Results: Results showed significant different between normal morphology and total motility in two groups. Case group had higher percentage of spermatozoa with protamine deficiency and apoptosis compared to controls significantly

Conclusion: Our results showed that in cases of RSA, in addition to abnormal sperm parameters, we have a high percentage of spermatozoa with protamine deficiency and apoptosis and these two anomalies may consider as important causes of idiopathic recurrent abortions. It should be advised that sperm chromatin and DNA examinations are useful tools in the process of RSA treatments

Sperm parameters, protamine deficiency, and apoptosis in total globozoospermia

Globozoospermia is a severe form of teratozoospermia [incidence < 0.1%] in infertile men that is characterized by round headed sperm and acrosomeless in semen. To compare the semen parameters, protamine deficiency, and apoptosis in ejaculated spermatozoa between globozoospermic and normozoospermic men. Thirty six semen samples were divided into two groups including 15 infertile men with total globozoospermic [> 90% round-headed sperm] and 21 healthy donors with normal spermograms as controls. Semen analysis was performed according to World Health Organization criteria [2010]. Sperm protamine deficiency was assessed using Chromomycin A3 [CMA3] staining and the rate of apoptotic spermatozoa was evaluated with TUNEL assay. Sperm concentration, motility, and normal morphology in globozoospermic men were significantly decreased compared with controls [p<0.05]. The rate of CMA3-reacted spermatozoa [CMA3+] in globozoospermic men was higher than controls [65.93 +/- 11.77 vs. 21.24 +/- 7.37, respectively, p<0.0001]. The rate of apoptotic spermatozoa [TUNEL positive] were significantly increased in globozoospermic cases with respect to the controls [17.60 +/- 10.72 and 5.95 +/- 3.02, respectively, p<0.0001]. There was no significant correlation between sperm protamine deficiency and apoptosis in globozoospermic men. Globozoospermic samples contain a higher proportion of spermatozoa with abnormal chromatin packaging and DNA fragmentation than normozoospermic samples. Therefore, in addition to absence of acrosome in the spermatozoa of globozoospermic patients, the high percentage of spermatozoa with immature chromatin and apoptotic marker may be considered as the other etiologies of infertility in these patients

Cytotoxicity of some 1-[2,4-dihydroxyphenyl]-3-[4-phenylpiperidin-1-yl] prop-2-en-1-one derivatives using MTT assay

Some N-substituted piperidine structures were synthesized and evaluated for cytotoxic activity against four different cell lines using the standard MTT assay method and Doxorubicin was used as the reference drug. The result of cytotoxic activity as a measurement of IC50 values revealed that three of the synthesized compounds were active against breast cancer cell line. Compound 6a bearing hydroxyl at para position of piperidine ring was the most active compound within this series. The N-subtituted piperidine with propene substructure could be considered as a lead structure for further studies of structure activity relationship to develop more potent compounds in future. The compounds were evaluated against four different cell lines using the standard MTT assay method and doxorubicin was used as the reference drug. The IC50 values were determined by constructing dose-response curves and revealed that three of the synthesized compounds were active against breast cancer cell lines. Compound 6a bearing hydroxyl at para position of piperidine ring was the most active compound within this series

comparison of anxiety and depression rate between medical staff of infertility centers and obstetrics and gynecology centers of Yazd, Shiraz, Isfahan and Kerman hospitals

Regarding the close and continuous interaction of infertility staff with hopeless infertile couples and in the contrary the atmosphere of happiness especially in obstetric wards make a sense that considering anxiety and depression it would be a difference between these two wards. The objective of this study is the comparison of the rate of depression and anxiety between the two wards of infertility and obstetrics and gynecology. This study is a descriptive-correlation study based on cross-sectional method. 199 individuals who were the staff of infertility and obstetrics and gynecology wards in four provinces enrolled in this study through stratified sampling. Data collection was done by demographic questionnaire, Spiel Berger and Beck depression inventory tests. Data were analyzed by SPSS software using ANOVA test. The result showed the rate of anxiety in obstetrics and gynecology staff of Isfahan center [54.69 +/- 13.58] and depression rate had increased level in infertility staff of Shiraz center [14.94 +/- 10.87]. Overall, there was significant correlation between anxiety, depression and work place [p=0.047, 0.008 respectively]. According to ANOVA test, the mean value of anxiety level was higher in the staff of four obstetrics and gynecology centers and one infertility center. As long as we know that infertile couples have little chance for success rate and obstetrics and gynecology wards patients have little risk of failure in treatment, it could be mentioned that the anxiety and depression in the staff are not correlated with the client illness

investigation on mitochondrial DNA deletions and telomere shortening during multiple passages of adult stem cells

Limited resources for adult stem cells necessitate their in vitro culture prior to Clinical use. Investigating mitochondrial DNA [mtDNA] and telomere shortening has proved to be important indications of stem cell validity. This study was designed to investigate these indicators in multiple passages of three adult stem cell lines which were produced in our stem cell laboratory. In this study, Dental Pulp Stem Cells [DPSCs], Periapical Follicle Stem Cells [PAFSCs] and Human Foreskin Fibroblast [HFF] cell lines were expanded for 20 passages. After 1, 5, 10, 15 and 20 passages, expanded cells were harvested and DNA was extracted for further studies. Common mtDNA mutation was detected by multiplex PCR and telomere shortening was tested by Southern blot analysis. The common deletion was not detected in any of the stem cells or cell lines after several passages. In addition, Southern blot analysis indicated that the mean difference of telomere length between first and last passage was 0.25 kb in DPSC, 0.1 kb in PAFSC and 0.32 kb in HFF which indicates that the mean telomere length in various passages of the samples showed insignificant changes. Absence of mtDNA mutations in adult stem cell lines indicates good mitochondrial function even after 20 passages. In addition, absence of telomere shortening indicates stem cells validity after multiple passages. It is hoped this information could pave the way for using in vitro expansion of adult stem cells for future Clinical applications

Mesenchymal Stem Cells Improved the Ultrastructural Morphology of Cerebral Tissues after Subarachnoid Hemorrhage in Rats

Subarachnoid hemorrhage (SAH) causes widespread disruption in the cerebral architecture.The process of SAH is complicated and many people lose their lives or become disabled after injury. Mesenchymal stem cells (MSCs) are considered as good candidate for repair of cerebral damage. The aim was to assess the ultrastructural changes in the rat cerebral tissue after intravenous transplantation of MSCs. Female Wistar rats (8 per group) weighing 275~300 g were assigned to control (SAH+PBS) and experimental groups (SAH+MSCs).The samples from middle cerebral arterial wall and parietal cerebral tissue were prepared for transmission electron microscopy (TEM) according to standard protocol. Fine architectures of the vessel wall, including the contraction of the inner layer, smooth muscle layer,as well as neural cells were observed after SAH. Cerebral arterial wall and cortex, including neuronal and glial cells were injured post SAH. But, administration of MSCs improved the structural integrity of cerebral tissues. Changes were much more balanced with their relative improvement in some areas. The role of MSCs for repairing the injured cerebral tissues post experimental SAH was approved by electron microscopy.

Evaluating the role of silver nanoparticles on acrosomal reaction and spermatogenic cells in rat

Nanoparticles have wide range of application while there are some reports regarding their probable effects on male reproductive system and spermatozoa. The aim of this study was to evaluate the effect of different doses of silver nanoparticles [AgNPs] [70nm] on acrosome of rat spermatozoa and number of spermatogenic cells. In this experimental study, in experimental group, 32 male wistar rats [8 rats/group] received oral feeding AgNPs every 12 hr in one spermatogenesis period [48 days] by means of gavages in 25, 50, 100 and 200 mg/kg concentration [experimental groups 1-4, respectively]. The control group [8 rats] was treated on schedule with distilled water. Spermatozoa were stained by triple staining protocol for acrosome reaction. Histological evaluation on testis sections was performed using tissue processing and hematoxylin-eosin [H and E] staining. There was significant difference between the control group and the experimental group 1 for acrosome reaction [11.00 +/- 0.00 and 24.25 +/- 3.68, respectively, p=0.01]. There was only significant reduction in spermatogonia cells in experimental group 4. Experimental groups 2, 3 and 4 showed a significant reduction in the number of primary spermatocytes and spermatids as well as spermatozoa. But there were no significant differences between different groups for Sertoli cell number and seminiferous tubule diameter. It seems that Ag NPs have acute and significant effects on spermatogenesis and number of spermatogenic cells and also on acrosome reaction in sperm cells. More experimental investigations are necessary to elucidate better conclusion regarding the safety of nanoparticles on male reproduction system.

Characterization of mesenchymal stem cells from human dental pulp, preapical follicle and periodontal ligament

Human dental stem cells have high proliferative potential for self-renewal that is important to the regenerative capacity of the tissue. The aim was to isolate human dental pulp stem cells [DPSC], periodontal ligament stem cells [PDLSC] and periapical follicle stem cells [PAFSC] for their potential role in tissue regeneration. In this experimental study, the postnatal stem cells were isolated from dental pulp, preapical follicle and periodontal ligament .The cells were stained for different stem cell markers by immunocytochemistry. To investigate the mesenchymal nature of cells, differentiation potential along osteoblastic and adipogenic lineages and gene expression profile were performed. For proliferation potential assay, Brdu staining and growth curve tests were performed. Finally, all three cell types were compared together regarding their proliferation, differentiation and displaying phenotype. The isolated cell populations have similar fibroblastic like morphology and expressed all examined cell surface molecule markers. These cells were capable of differentiating into osteocyte with different capability and adipocyte with the same rate. PAFSCs showed more significant proliferation rate than others. Reverse transcriptase PCR [RT-PCR] for nanog, oct4, Alkaline phosphatase [ALP] and glyceraldehydes-3-phosphate dehydrogenease [GADPH] as control gene showed strong positive expression of these genes in all three isolated cell types. PDLSCs, DPSCs and PAFSCs exist in various tissues of the teeth and can use as a source of mesenchymal stem cells for developing bioengineered organs and also in craniomaxillofacial reconstruction with varying efficiency in differentiation and proliferation