[ role and effect of HSPA2 in male infertility]

The heat shock proteins [HSPs] are a group of homologous proteins encoded with a multigene family. They can be divided into five groups based on both size and function; including HSP27, HSP60, HSP70, HSP90 and HSP100. Expression of HSPs can be constitutive and can increased when cells undergo metabolic and/or heat stress. HSPA2/Hsp70-2 is a kind of HSP70 and constitutively expressed in testis. This protein is essential for sperm maturation and is associated with infertility. HSPA2 expression has been shown to occur in two phases in human testis; 1] meiosis as an element of the synaptonemal complex and 2] spermiogenesis durinat sperm maturation and is considesered as an index for matured sperm. Several functions have been proposed for HSPA2 protein in human testis including; protein folding, formation and desynapsis of synaptonemal complex, repair of DNA strand breaks, plasma membrane remodeling, and the removal of excess cytoplasm. Background literature suggests that HSPA2 play a key role in spermatogenesis. Therefore, the aim of this biological- medicine study was the evaluation of the role and effect of HSPA2 in male infertility

Selection of the most appropriate medium for assessing motility and DNA uptake of bovine spermatozoa

The aim of this study was to select the best medium to maintain sperm motility during sperm-DNA incubation and assess the DNA uptake by spermatozoa of Iranian Holstein bulls and its effects on sperm motility. Frozen sperms from an Iranian Holstein bull were thawed and centrifuged. Motile sperms were separated through Puresperm gradient [40/80%] followed by two times washing in SP-TALP medium. Then, sperms were washed once [PBS, Opti-MEM and SP-TALP] and incubated with DNA in each media followed by sperm motility estimation. The plasmid pEGFP-C1 was linearized and incubated with sperms at 37°C for 1 hour. Sperm-DNA mixture was treated with DNase I and the sperm pellet was washed with PBS. DNA extraction from sperms and supernatants from the last washing were used as template for PCR. Data was analyzed using SAS package and mean comparisons between sperm motility in different media were performed. Sperm motility after incubation in PBS, Opti-MEM and SP-TALP were 40[ +/- 2.89], 2[ +/- 1.53] and 54[ +/- 4.41] percent, respectively. PCR results from transfected sperms indicated that EGFP transgene internalized into the bovine sperms and DNaseI treatment could not eliminate it. In conclusion the best medium for sperm and DNA incubation was SPTALP. The DNA not only could attach to the post acrosomal region of spermatozoa but also could integrate into it. So bovine spermatozoa can be used as transgene carrier into oocyte

[ Effect of chick Embryonic Somites on Neural Rosette Formation in Mouse Embryonic Stem Cells]

The aim of the present study is to understand if EBs can generate neural rosette upon co-culture with chick embryo sornites. The mouse ES cells, line Royan Bi, were cultured in hanging drops to induce embryoid bodies [EBs] formation. Somites were isolated from the chick embryos and then embedded in alginate solution. Finally, alginate beads containing somites were co-cultured with EBs. RA was added to some EBs according to 2-12+12+ protocol Mean percentage of EBs containing early and late rosettes in somite, control and RA was 1456%, 2.6% and 0.0%, respectively and what is important to rosette formation in EBs was the preseice of neural inducing components as well as the time course of neural differentiation of EBs Chick embryonic somites can induce ES cells -derived EBs to generate rosttte structures with neuron formation capacity

[In vitro development of cattle embryo as affected by glucose, serum and EDTA]

The aim of this study was to evaluate the effect of different modifications of sequential synthetic oviductal_fluid [SOF] culture system on developmental competence of in vitro matured/fertilized cattle embryos. Bovine oocytes were matured and fertilized in vitro and then presumptive zygotes were randomly cultured for up to 9 days in different modifications of SOF culture system to consider the effects of glucose, serum and EDTA on embryo development. All the embryo culture systems were efficient to support bovine embryo development till blastocyst stage. There was no significant difference in the ratios of embryos; however, the ratios of blastocyst and also hatchability of embryos cultured in SOF C [51.3%, 43.0% and 83.8%, respectively] were significantly higher than those of all the other SOF groups. Furthermore, while glucose had a partial improving effect on embryo development, a significant decrease in embryo development beyond the morula stage was observed in embryos cultured in SOF system with initial supplementation of EDTA compared with all the other groups. It was concluded that appropriate modifications of SOF culture systems can result in significantly great in vitro embryo development

[Origins and evaluation of DNA damage in infertile individual]

Assisted reproductive techniques [ART] are considered as the first line of treatment in overcoming infertility. The scope of success of these methods depend on several factors including integrity of sperm and oocyte. Obviously, paternal genomic content has an important effect on fertilization potential in infertile couples. Thus, sperm DNA damage is a genomic disorder that exists in different degree in infertile men. Since the first reports on sperm DNA integrity, this subject has become the focus of numerous studies. It has been reported that individuals with high percentage of sperm DNA damage have lower sperm parameters and, fertilization rate. Therefore, this review is based on our current understanding of mechanisms involved in sperm DNA damage and also will describe different procedures for evaluation of sperm DNA damage

[Effect of demecolicne treatment on the developmental competency of in vitro matured bovine oocytes]

The aim of this study was to investigate whether demecolicne treatment of matured bovine oocytes adversely affects the process of in vitro fertilization and embryo development. Bovine Cumulus Oocyte Complexes [COC's] were matured in vitro and then were randomly allocated to two treatment groups of common concentrations of demecolicne [0.05 and 0.4 micro g/ml for 30 min] and a control group. COC's were then fertilized and cultured in vitro for up to 9 days when the ratios of in vitro embryo development and the viability of the hatched blastocysts were assessed and compared with the control group [p<0.05]. The ratios of the cleavage and blastocyst formation of demecolicne treated groups [0.4 and 0.05 micro g/ml] were 68.6, 63.5% and 23.3, 32.8%, which were not significantly different from the control group [73.3, 29.0%], respectively. The results of cell-viability were also not significantly different between the control vs. treatment groups. Since the overall indices of in vitro embryo development revealed no significant difference between the demecolicne treated compared to control bovine oocytes, it seems that demecolicne treatment of matured bovine oocytes may not compromise their potency for further in vitro development

Bone marrow-mesenchymal stem cells as a suitable model for assessment of environmental pollution

Stem cells are considered as ideal model for assessment of environmental toxins on proliferation, multipotency and differentiation. The aim of this study was to investigate the effect of lead as harmaful environmental pollutant on proliferation and neural differentiation of murine bone marrow-MSCs. In this experimental study, MSC cells were exposed to different concentrations of lead [0 to 100 micro M] for 24h, and the level of cell proliferation was determined by 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-dipheny-2H-tetrazolium bromide [MTT] reduction assay. In addition, DNA fragmentation was evaluated with comet assay at a single cell level. To induce the neural phenotype, MSCs were cultured for 2 days in the presence of 50 micro Pb[2+] for 48 h. At the end of this period, the medium was replaced by fresh medium supplemented with 1 mM beta-ME for 24 hr and then fresh medium supplemented with 7 mM beta-ME for 4 hours respectively. The expression of neural marker such as nestin, MAP2, and tau was assessed by immunocytochemistry, while the expression of neuronal specific genes such as Neur-1, Nestin, and beta-tubulin III was determined by RT-PCR analysis. Exposure to lead reduced the level of cell proliferation in a dose-dependent manner. The comet assay of cells exposed to lead showed varying degrees of DNA damaged. Change in cell morphology was observed 1 to 4 hr post neural exposure. The percentage of the MAP2 positive cells was reduced significantly at greater than 40 micro M lead concentration. This observation was further verified by assessment of the expression of neural markers. This study clearly indicated lead is highly cytotoxic to MSCs and these cells appear to be an excellent choice for establishment of guidelines for environmental hazards and drugs on cell proliferation and differentiation

[ efficiency of Zeta method in separation of sperm with normal morphology and chromatin structure]

Selection of mature sperm with normal morphology and chromatin structure is needed for assisted reproduction technology [ART] procedures. At present, various sperm separation methods exist to select mature sperm. The selection of mature sperm based on electrical charge of membrane is one of these methods. Therefore, the aim of this study was to evaluate the efficiency of the Zeta potential for selection of normal sperm in term of morphology and chromatin structure integrity. In this descriptive-analytical study, semen samples from 70 infertile couples referring to Isfahan Fertility and Infertility center were evaluated. Semen analysis was carried out according to WHO criteria. The remaining of semen samples were used for Zeta method. Then Sperm morphology, protamine deficiency and DNA fragmentation were assessed using papanicolaou staining, chromomycin A3 [CMA3] staining, and sperm chromatin dispersion [SCD] test, respectively. In addition, we compared the result of Zeta group with control group. Coefficients of correlation and paired-samples t-test were carried out using SPSS 11.5, to compare results among between two groups. The results of this study show that the mean of abnormal morphology, protamine deficiency and DNA fragmentation were 88.19 +/- 7.37, 67.17 +/- 17.34, 32.87 +/- 8.65 in control group, and 80.17 +/- 10.26, 58.40 +/- 18.20, 18.19 +/- 8.64, in the Zeta group, respectively. Zeta group can decrease significantly the percentage of abnormal morphology, protamine deficiency and DNA fragmentation compared to control group [P<0.05]. Zeta method can be used in separation of mature sperm with normal morphology, normal protamine contained and intact DNA

Isolation of mesenchymal stem cells from dental pulp of exfoliated human deciduous teeth

The exfoliated human deciduous tooth [SHED] contain multipotent stem cells that identified to be a population of highly proliferative and clonogenic .These cells are capable of differentiating into a variety of cell types including neural cells, adipocytes, and odontoblasts. Normal exfoliated human deciduous incisors collected from six- to nine-years-old children. The pulp was separated from the crown and digested with collagenase .Single cell solutions were cultivated in alpha-MEM supplemented with ES-FCS. After two to three days, the cells reached confluency and were trypsinized and cultured for further passages. The passage-4 cells were analyzed with CD34, CD45, CD105, CD166, CD31, CD90 and CD146 markers that indicated these cells had a mesenchymal stem cell [MSC] identity. We examined the cells for Alkaline Phosphatase activity to investigate the mesenchymal [stromal] nature.Finally, the cells were differentiated into the osteoblastic and adipocytic lineages in different subcultures and analysed by RT-PCR and different staining protocols. Viable cells growing out of the explants showed elongated shapes in clusters. These cells showed alkaline phosphatase activity. Flow cytometry results revealed high expression of pluripotent stem cell markers .In some area of the osteoinductive cultures nodule-like structures were observed that showed red mineralizing area upon staining with Alizarin Red.In adipogenic cultures lipid vesicles appeared after five weeks of induction with Oil Red. This study show that pulp contains cells with high plasticity and proliferation capacity and can be easily isolated without any serious intervention

Detection of antisperm antibodies by indirect flow cytometry: Comparison with direct mixed agglutination reaction test

The immunobead binding test [IBT] and the mixed agglutination reaction [MAR] are the most commonly used methods for detection of antisperm antibodies [ASA]. The detection of ASA by flow cytometry [FCM] was first described by Haas and Cunningham. Both assays can be performed as direct or indirect methods. In this study, indirect FCM was compared with the direct MAR for detection of ASA. Semen samples were obtained from 80 men [infertile couples] in Isfahan Fertility and Infertility Center. Seminal plasma samples were incubated with ASA-negative donor sperm. Then, surface-bound antibody was detected with FITC-labeled antihuman immunoglobulin directed against IgA and IgG in the indirect FCM assay. ASAs bound to the surface of patients' sperm were detected by direct MAR test. The indirect FCM correlates with direct MAR for detection of IgA antisperm antibodies [r=0.55 and P=0.006]. The indirect FCM, however, does not correlate with direct MAR for the detection of IgG antisperm antibodies [r=0.25 and P=0.25]. Some of the ASAs in seminal fluid bind to spermatozoa. Therefore, indirect tests to detect ASAs in seminal plasma are likely to miss the presence of IgG antisperm antibodies while they effectively detect IgA antisperm antibodies

In vitro biocompatibility of eight orthodontic materials in human oral fibroblasts

In orthodontic therapy, different materials are used and subjected to a damp oral environment, which can modify their properties. The purpose of this study was to evaluate the biocompatibility of eight metallic and non metallic orthodontic materials in human oral fibroblast culture. The specimens consisted of eight clinically used materials, including two coil springs, two glass ionomer cements, two O-rings and two orthodontic acrylic resins. Teflon was used as a negative control. After a release period of each material in the culture medium for 20 days, the viability of oral cultivated fibroblasts was compared to the negative control by MTT assay. Biocompatibility was also assessed using the direct method in which cell viability was evaluated by an invert phase contrast microscope, after direct exposure to the tested materials. MTT assay showed stainless steel [P.V=0.665] and Bayer acrylic resin [P.V=0.l79] to be biocompatible with gingival fibroblasts. Significant differences were observed between the biocompatibility of all study groups [Nickel titanium [PV=0.037], Acropars acrylic resin [PV=0.014] and both studied O-rings [PV=0.001], [PV=0.016]] and the control group. According to the direct method, stainless steel and Nickel titanium showed grade 1, glass ionomer cements grade 4 and other materials revealed grade 0 cytotoxicity. The two studied glass ionomer cements were highly cytotoxic. Bayer acrylic resin was more biocompatible than Acropars acrylic resin

Evaluating semen parameters and time to pregnancy [TTP] in 234 Couples in Isfahan

Defining the lowest normal values of semen parameters, which are required for fertility, is of utmost importance in the diagnosis and management of infertile couples. These values are defined periodically by W.H.O. However, it has been emphasized that semen parameters should be determined regionally or nationally. The objective of this study was to evaluate semen parameters in fertile couples in Isfahan. Semen samples were obtained from partners of 234 pregnant women referring to gynecologists throughout Isfahan. Questionnaires, including time to pregnancy [TTP], were filled out. Semen samples were analyzed according to WHO guidelines. Results were analyzed and odds ratios were calculated by the use of SPSS statistical software and the level of significance was considered <.05%. The 10% cut-off point for the values such as volume, density, total count, motility and normal morphology, being considered as the minimum requirement for fertility, were 1ml, 45x10[6] per ml, 75x10[6] per ejaculate, 57% and 28% respectively. Semen parameters with TTP of less than 6 months were grouped according to the mentioned cut-off points and the pregnancy ratio and relative risks of pregnancy were calculated for each group. The results showed no significant differrence between the relative risks for pregnancy with respect to the cut-off points. Due to lack of information on sperm parameters in different parts of the world, regional and national evaluations of these parameters is of great value for demographic studies. Genetic characteristics and regional climate as environment, may affect sperm parameters. Isfahan, for example, is situated in a region with warm and dry climate and this may justify the low mean volume and higher concentration of sperm in the obtained samples from the subjects

Effects of acrosomal activity and morphology on fertilization rates following ICSI

Fertilization failures after ICSI may be due to different factors related to oocyte, sperm or both of them. Considering the importance of sperm morphology, acrosomal activity in oocyte activation and fertilization rates, this study was done to evaluate the relationship between a series of events occurring during spermiogenesis such as sperm morphology and acrosomal activity as an index for acrosomal integrity and the relationship between sperm ability to induce oocyte activation with fertilization rates following ICSI. Semen samples from 68 infertile couples undergoing ICSI at Isfahan Fertility and Infertility Center were assessed. Some of each semen sample was analyzed for semen parameters according to WHO criteria, most of it was used for ICSI and the rest for Papanicolaou staining and gelatinolysis test to evaluate sperm morphology based on the strict criteria and acrosin activity respectively. The results were analyzed by SPSS software [Version 11.5] and correlation coefficient was determined. P-values less than 0.05 were considered statistically significant. Results from gelatinolysis showed that the mean halo diameter had a significant positive correlation with sperm concentration [r=0.343, p=0.004], motility [r=0.282, p=0.020], sperm morphology according to the WHO criteria [r=-0.314, p=0.009], fertilization rate [r=0.270, p=0.026] and the percentage of halo formation [r=0.853, p=0.001]. The results of the study revealed that during ICSI, spermatozoa with small acrosome, which are likely to have reduced gelatinolysis test parameters [Smaller percentages of halos and smaller mean halo diameters], have lower fertilizing potential. As gelatinolysis test is considered as an index for acrosomal and perinuclear theca integrity, lower fertilizing ability in these spermatozoa could be likely due to their reduced levels of sperm associated oocyte activating factors [SAOAFs]. Therefore, studies for the identification and measurement of SAOAF levels in these kinds of spermatozoa are proposed

relationship between sperm protamine aberrations with protamine P1/ protamine P2 ratio

The aim of this study was to evaluate the relation between protamine deficiency assessed by CMA3 and protamine 1/ protamine 2 [P1/P2] ratio. This study was carried out on 71 patients referring to Isfahan Fertility and Infertility center. Semen analysis was assessed according to WHO criteria. CMA3 staining was use to determine protamine deficiency. P1/P2 ratio was evaluated by nuclear protein extraction, acetic acid urea poly acrylamide gel-electrophoresis and analysis of protein bands with related software. Western blot was carried out with primary anti P1 and anti P2 antibody to determine protamine 1 and protamine 2. Of the 71 patients 45 patients underwent Intracytoplasmic Sperm Injection [ICSI]. A negative significant correlation was observed between fertilization rate with protamine deficiency and P1/P2 ratio. However, no significant correlation was observed between protamine deficiencies with P1/P2 ratio. The results of this study showed that protamine deficiency can be assessed by CMA3; however this procedure does not indicate type of protamine deficiency

[Effect of seminal vesicles and dithiotritol [DTT] on stability of sperm chromatin in IVF and ICSI patients]

Different studies have shown that there is no relation between sperm chromatin stability andfertilization rate in both IVF and ICSI patients. However, the relation between SDS tests, as a detergent, alongwith DTT as reducer of disulphide bridges has not been studied so far in ICSI patients. Since differentconcentrations of DTT can induce different degrees of sperm chromatin decondensation, the aim of this studywas to evaluate the effect of different concentrations of DTT on sperm chromatin decondensation in IVF andICSI cases.During this study, 85 patients were divided into two groups according to their treatment procedure[IVF or ICSI].Semen samples of each patient was evaluated for sperm chromatin tests including SDS,SDS+EDTA and SDS+DTT for assessment of free thiole groups level [-SH], amount of non covalent bondbetween Zn and thioles[-SH Zn SH-] and levels of disulfide bond [-S-S-] in sperm chromatin, respectively. Inthis study, seminal fructose concentration, corrected seminal fructose level and true corrected fructose level asindicators of seminal vesicle function on sperm chromatin stability were assessed.No correlation was observed between any of the above tests and rate of fertilization, both in IVF andICSI cases. However, in IVF patients, a significant correlation was observed between SDS, SDS+DTT test andseminal fructose level, while in ICSI patients, only a significant correlation was observed between SDS+DTTand corrected or true fructose concentration.Since no correlation was observed between sperm chromatin test and fertilization rate, it issuggested that the chromatin status of these samples are adequate for fertilization to take place and extent ofdisulphide bridges has no effect on fertilization rate. However, the amount of disulphide bound present in spermsof ICSI and IVF patients are different, and this difference is related to seminal vesicle performance in thesepatients

Effect of sperm protamine deficiency on fertilization and embryo development post-ICSI

The aim of this study was to evaluate the effect of sperm protamine deficiency on fertilization and embryo development post ICSI. Semen samples from 27 patients candidate for ICSI were assessed for semen parameters. Sperm processing was carried out using pure sperm and percentage of protamine deficient sperm was assessed by Chromomycin A3 post processing. Correlation between fertilization rate, embryo quality and cleavage score with protamine deficiency was assessed using SPSS statistical programme. A significant negative correlation was observed between fertilization rate and embryo quality score on day 3 with percentage of protamine deficient sperms. Semen samples with high percentage of protamine deficient sperms have lower fertilization rate and lower potential to develop good quality embryos

[Correlation between in vitro fertilization with the level of antisperm antibody in seminal plasma measured by flow cytometry]

Antisperm antibodies [ASA] are present in%8-21 of infertile men. In vitro Fertilization [IVF] has been recommended as an effective procedure in couples with immunological male factor. Although this procedure has been found to bypass the inhibitory effect of antisperm antibodies on fertilizing ability of spermatozoa but the fertilization rate is reduced about%40 for ASA positive samples. The goal of present study was to investigate the correlation between anti-sperm antibodies measured by indirect flow cytometry and fertilization rate in infertile couples undergoing in vitro Fertilization [IVF]. Semen samples were collected from 80 infertile men undergoing IVF cycle in Isfahan fertility and infertility center. Couples were classified based on fertilization rate into high and low groups. 52 couples had high [>50%] and 28 couples had low fertilization rate [

Effect of human sperm MTT viability assay on outcome of intraycytoplasmic sperm injection

Sperm MTT Viability Assay has been shown to be a suitable test for differentiation of viable from non-viable sperms. In this procedure MTT is converted to observable purple MTT Formazan by mitochondrial dehydrogenase in the midpiece region and therefore viable sperms can be distinguished which makes this test suitable for ICSI. Therefore, in order to study the effect of MTT positive sperms on fertilization, cleavage and blastocyst formation, 109 fresh human oocytes [metaphase II] were divided in to two groups; one group was injected with MTT positive sperms and the other one was taken as control. The results of study showed that there is not significant difference with respect to fertilization, cleavege and blastocyst formation between these two groups. Therefore, if MTT proves to be nether mitogenic nor teratogenic, sperm MTT viability assay might be useful for ICSI in patients with absolute or severe asthenospermia, especially in cases with tail abnormality

Effect of human follicular fluid on implantation of mouse embryo

Prevention of implantation is considered as one of the suitable methods for contraception or interception. Therefore during this study the effect of human follicular fluid [FF] on mouse implantation was evaluated. The results obtained during this study show that FF contains enzymes and upon incubation with mouse uterine tissue, results in delimitation of epithelium. This effect was inhibited by heat inactivation or addition of EDTA to FF. In vivo uterine wash with follicular fluid on days 3,4,5 and 6-post mating prevents implantation and therefore significantly reduces implantation and pregnancy rate. However uterine wash on day 7 post mating with FF had no effect on implantation and pregnancy rate. On day 3,4,5,6 and 7 with Ham's 10 as control had no effect on implantation or pregnancy rate. Addition of EDTA also prevented the in vivo effect of FF, suggesting that the active agent present in FF is likely to be a metalloprotoinase which inactivates with heat and addition of EDTA. Taking into consideration the fact that FF does not affect the pregnancy of the next cycles, thus the FF or its active agent can be considered as good interceptive agent for prevention of pregnancy rate