فهرست مطالب mansoureh movahedin

Decellularizing testis tissue and recellularizing with spermatogonial stem cells (SSCs) seems to be a promising approach to restore fertility in prepubertal boys who undergoes cytotoxic therapies.

Testis tissue decellularization was performed by adding 1% SDS and confirmed by histological analysis and DNA quantification. The MTT assay was performed for biocompatibility analyses. SSCs were derived from male mice and cultured in αMEM medium for two weeks. Expanded SSCs were seeded onto the DTM scaffold. The recellularized DTM scaffold disc was cultured in a static cultivation system for one week, then transferred in a dynamic mini-perfusion bioreactor for two weeks. The expression of Id4, Plzf, Gfrα, Prm, Sycp3, ABP, Ki67, Bax, and Bcl2 genes were assessed in SSCs and recellularized DTM after static and dynamic cultivations.

DNA qualification indicated that approximately 99% of the DNA components were removed from DTMs. Hematoxylin-eosin, Masson's trichrome, and DAPI staining confirmed the effective recellularization. Dynamic cultivation of recellularized DTMs at the flow rate of 10 ml/h provided optimum conditions. The expression of SSCs-specific genes of Id4, Plzf, and Gfrα-1 and post-meiosis genes of Scp3, prm1, and ABP was insignificantly higher in the DTMs group than in the control group. Ki67 expression was shown no difference between groups. An insignificant lower expression of the Bax and higher expression of Bcl2 genes was detected in the DTMs group compared to the control.

Our results indicated that SSCs could successfully be attached to the DTMs and effectively proliferate in the mini-perfusion bioreactor.

Due to myelin and axonal insults in multiple sclerosis individuals, motor coordination problems and endocrine imbalance may develop.

This study aims to evaluate the role of chronic demyelination on the hypothalamic-pituitary-gonadal axis in the mouse model of multiple sclerosis.

20 adult C57/BL6 male mice were divided into 2 groups (n = 10/each) as follows: the control group (CONT) received a regular diet for 17 wk; and the experimental group (cuprizone [CPZ]) was fed with 0.2% CPZ for 12 wk and, then CPZ was withdrawn for 5 wk. Serum testosterone, histopathology of the brain and testis, and sperm analysis were evaluated.

The hypothalamic myelin content was significantly decreased in the arcuate nucleus following the 12 wk of CPZ consumption compared to the CONT group, and the statistical difference remained until 17 wk. Testosterone levels declined significantly in the CPZ group compared to the CONT group in the 12th and 17th wk. A significant decrease was observed in the height of the seminiferous epithelium and the interstitial tissue area, and the number of seminiferous epithelial cells in the CPZ group compared to the CONT group in the 12th and 17th wk. The sperm count, motility, and viability in the CPZ group significantly decreased compared to the CONT group in the 12th and 17th wk of the study.

Chronic demyelination induced by CPZ intoxication, maybe through damage to the hypothalamus arcuate nucleus, leads to the hypothalamic-pituitary-gonadal axis disturbance and damage to the testis and spermatogenesis subsequently.

For patients who had testicular tissue cryopreserved before receiving gonadotoxic therapies, transplantation of testiculartissues and cells has been recommended as a potential therapeutic option. There are no studies that indicate thegeneration of sperm after human immature testicular tissue (ITT) or spermatogonial stem cells (SSCs) transplantation.The use of releasing scaffolds and localized drug delivery systems as well as the optimizing transplantation site canplay an effective role in increasing the efficiency and improving the quality of testicular tissue and cell transplantationin animal models. Current research is focused on optimizing ITT and cell transplantation, the use of releasingscaffolds, and the selection of the right transplantation site that might restore sperm production or male infertilitytreatment. By searching the PubMed and Google Scholar databases, original and review papers were collected. Searchterms were relevant for SSCs and tissue transplantation. In this review, we'll focus on the potential advantages of usingscaffolds and choosing the right transplantation site to improve transplantation outcomes.

The aim of this research was to assess the effect of melatonin on the diameter of colonies, apoptosis status and apoptosis related genes expression in sheep’s spermatogonial stem cells (SSCs). SSCs at the basal membrane of seminiferous tubules were isolated from testes of Afshari sheep using enzymatic digestion steps. The samples assigned into four groups. The control group received basic medium and the other groups contained H2O2 (30 µM), melatonin (1 nmol) and melatonin+H2O2 (30 µM H2O2 along with 1 nmol melatonin), respectively. The cells were cultured for 3 weeks and the colonies’ diameter were evaluated on the 5th, 14th and 21th days of culture. At the end of culturing period, apoptosis status and apoptosis related genes expression (BAX and BCL2) were evaluated. On the 5th and 14th days of culture, the diameter of colonies were higher (P≤0.05) in the control and melatonin groups compared to the other groups. On the 21th day of culture, the highest and the least (P≤0.05) diameter of colonies were observed in melatonin and H2O2 groups, respectively. The least and the highest (P≤0.05) rate of apoptotic SSCs, BAX expression and BAX/BCL2 ratio were observed in melatonin and H2O2 groups, respectively. Moreover, the highest (P≤0.05) expression of BCL2 gene was found in melatonin group. In conclusion, using melatonin in culture medium could be an effective way to improve the quality and decrease apoptosis status in sheep’s SSCs.

Spermatogonial stem cells (SSCs) because of its ability to be reprogrammed into embryonic-like stem cells (ELSCs) can be a new source of pluripotent stem cells which can play a promising role in regenerative medicine. In this study, SSCs were transdifferentiated into neuron-like cells (NLCs) using two-step differentiation protocol. pluripotency and germ cells markers were analyzed in SSCs and ELSCs. Also neural markers were analyzed in ELSCs and NLCs.

Neonatal rat testes were mechanically dissected and digested then was cultured in DMEM supplemented with 15% FBS. The medium was replaced with DMEM containing LIF, mercaptoethanol, EGF, bFGF, and GDNF. After 5 weeks, ELSCs colonies appeared. SSCs and ELSCs were evaluated by Stra8, plzf (germ cells markers) Oct4, and sox2 (pluripotency markers) using qRT-PCR. The ELSCs colonies were isolated and cultured in DMEM containing 0.5 mM lithium chloride. In day 5, ELSCs transdifferentiated to NLC. They were evaluated using neural marker including Neurofilament 200 (NF-200), choline acetyltransferase (CAT), synaptophysin (Syp), Nestin (Nes), Neurogenin1 (NG1), Neurod1 (Nd1), and Neurofilament 68 (NF-68)gene expression.

Result showed increasing expression of Oct4 and sox2 genes and low level of Stra8 and plzf expression in ELSCs than SSCs. After neural transdifferentiation by lithium chloride induction, neural markers were examined by RT-PCR in ELSCs and NLCs. The result showed expression of NF-200, CAT, Syp, Nes, NG1, Nd1 and NF-68 in NLCs opposed to ELSCs.

This study indicates lithium chloride can promote ELSCs to transdifferentiate into NLCs.

Epigenetic and genetic changes have important roles in stem cell achievements. Accordingly, the aim of thisstudy is the evaluation of the epigenetic and genetic alterations of different culture systems, considering their efficacy inpropagating human spermatogonial stem cells isolated by magnetic-activated cell sorting (MACS). In this experimental study, obstructive azoospermia (OA) patient-derived spermatogonial cells were divided into two groups. The MACS enriched and non-enriched spermatogonial stem cells (SSCs) were cultured in the control and treated groups; co-culture of SSCs with Sertoli cells of men with OA, co-culture of SSCs with healthy Sertoli cells of fertile men, the culture of SSCs on PLA nanofiber and culture of testicular cell suspension. Gene-specific methylation by MSP, expression of pluripotency (NANOG, C-MYC and OCT-4), and germ cells specific genes (Integrin α6, Integrin β1, PLZF) evaluated. Cultured SSCs from the optimized group were transplanted into the recipient azoospermic mouse. The use of MACS for the purification of human stem cells was effective at about 69% with the culture of the testicular suspension, being the best culture system. Upon purification, the germ-specific gene expression was significantly higher in testicular cell suspension and treated groups (P≤0.05). During the culture time, gene-specific methylation patterns of the examined genes did not show any changes. Our data from transplantation indicated the homing of the donor-derived cells and the presence of human functional sperm. Our in vivo and in vitro results confirmed that culture of testicular cell suspension and selection ofspermatogonial cells could be effective ways for purification and enrichment of the functional human spermatogonial cells. The epigenetic patterns showed that the specific methylation of the evaluated genes at this stage remained constant with no alteration throughout the entire culture systems over time.

Sperm cryopreservation results in damage to membrane integrity, sperm viability, sperm motility, and DNAstructure. We aimed to evaluate the effect of plasma rich in growth factors (PRGF) on sperm parameters during the freeze-thaw process. In the first phase of this prospective study, after sperm preparation, 10 normozoospermic specimens were cryopreserved by rapid freezing with different concentrations of PRGF including 0, 1, 5, and 10% to find the optimum dose. Sperm motility and viability were assessed in this phase. In the second phase of the study, based on the results of first phase, 25 normal sperm samples were frozen with 1% PRGF. All sperm parameters including motility, viability, acrosome reaction, and DNA integrity were assessed before freezing and after thawing. The rates of progressive and total sperm motility and viability were significantly higher in 1% PRGF compared to control, 5%, and 10% PRGF in the first phase (P<0.05). Supplementation of freezing medium with 1% PRGF could significantly improve all sperm parameters including sperm motility, viability, normal morphology, acrosome integrity, chromatin structure, chromatin integrity, DNA denaturation, and DNA fragmentation in comparison with the control group. It appears that the supplementation of freezing medium with 1% PRGF could protect human sperm parameters during cryopreservation.

It was in the early 20th century when the quest for in vitro spermatogenesis started. In vitro spermatogenesis is critical for male cancer patients undergoing gonadotoxic treatment. Dynamic culture system creates in vivo-like conditions. In this study, it was intended to evaluate the progression of spermatogenesis after testicular tissue culture in mini-perfusion bioreactor. In this experimental study, 12 six-day postpartum neonatal mouse testes were removed and fragmented, placed on an agarose gel in parallel to bioreactor culture, and incubated for 8 weeks. Histological, molecular and immunohistochemical evaluations were carried out after 8 weeks. Histological analysis suggested successful maintenance of spermatogenesis in tissues grown in the bioreactor but not on agarose gel, possibly because the central region did not receive sufficient oxygen and nutrients, which led to necrotic or degenerative changes. Molecular analysis indicated that Plzf, Tekt1 and Tnp1 were expressed and that their expression did not differ significantly between the bioreactor and agarose gel. Immunohistochemical evaluation of testis fragments showed that PLZF, SCP3 and ACRBP proteins were expressed in spermatogonial cells, spermatocytes and spermatozoa. PLZF expression after 8 weeks was significantly lower (P<0.05) in tissues incubated on agarose gel than in the bioreactor, but there was no significant difference between SCP3 and ACRBP expression among the bioreactor and agarose gel culture systems. This three-dimensional (3D) dynamic culture system can provide somewhat similar conditions to the physiological environment of the testis. Our findings suggest that the perfusion bioreactor supports induction of spermatogenesis for generation of haploid cells. Further studies will be needed to address the fertility of the sperm generated in the bioreactor system.

The octamer-binding transcription factor-4 (OCT4) is known as an established important regulator of pluripotency, as well as a genetic “master switch” in the self-renewal of embryonic stem and germ cells. OCT4B1, one of the three spliced variants of human OCT4, plays crucial roles in the regulation of pluripotency and stemness. The present study developed a transgenic mouse model containing an OCT4B1-expressing construct under the transcriptional direction of mouse mammary tumor virus promoter (pMMTV) to evaluate the role of OCT4B1 in the function of male germ cells in terms of fertility potential. Additionally, the effect of ectopic OCT4B1 overexpression on endogenous OCT4 expression was examined in mouse embryonic stem cells (mESCs). The pMMTV-OCT4B1cDNA construct was injected into the pronuclei of 0.5-day NMRI embryos. Transgenic mice were identified based on the PCR analysis of tail DNA. Further, Diff-Quik staining was applied to assess sperm morphology, while the other sperm parameters were analyzed through a conventional light microscopic evaluation according to World Health Organization (WHO) criteria. The fertility rate was scored by using in vitro frtilization (IVF) method. Furthermore, mESCs was electroporated with the OCT4B1cDNA-containing constructs, followed by analyzing through employing semi-quantitative RT-PCR and western blotting. The results demonstrated the changes in sperm morphology, as well as a statistically significant decrease in the other sperm parameters (count, viability, and motility) and fertility rate (p<0.05) in the transgenic mice compared with the control group. The assessment of the cause of the embryonic stem cell (ESC) death following transfection revealed a significant reduction in the endogenous OCT4 expression at both mRNA and protein levels in the transfected mESCs compared to the control ones. In general, the in vivo results suggested a potential role of OCT4B1 in the spermatogenesis process. These results represented that the overexpression of OCT4B1 may induce its role in spermatogenesis and fertility rate by interfering endogenous OCT4 expression. However, further studies are required to clarify the mechanisms underlying OCT4B1 function.

Conventional methods for the skin aging are based mainly on physiological or biochemical observations. The aim of this study is to provide a non-invasive method based on the extraction of biomechanical parameters of the skin resulting from the processing of sequential high-frequency ultrasound images in order to investigate the process of skin lesions.

Consecutive ultrasound images of the epidermal and dermal layers were recorded and saved with a 40 MHz imaging system. In order to evaluate the process of skin damage, 25 C57BL6 mice were exposed to UVB radiation. The mechanical parameters of the skin derived from the processing of sequential ultrasound images were also estimated weekly with the motion estimation algorithm (gradient and block matching) during the injury generation process and results were reported as a mean and standard deviation. Validation of the method was performed by invasive tensiometric test. Correlation between the two methods was performed by Pearson correlation analysis. Statistical  repeated measures analysis of  variance was used to compare the statistical trend of changes over 5 weeks.

Significant correlation was obtained between elasticity extracted from non-invasive ultrasonic image processing method and invasive tensiometric method with a correlation coefficient of more than 0.79. By processing the sequential ultrasound images, the axial elastic and the shear modulus of the skin layers were significantly increased, which indicate the increased skin firmness during  the ultraviolet radiation (p <0.05). Also, the ratio of changes in the elastic modulus on the thirty-fifth day was 11 times more than zero-day. The shear modulus on the 35th day of ultraviolet radiation was 2.2 times more than zero-day. The results of repeated measures analysis of  variance showed that during irradiation with ultraviolet waves, the axial elastic modulus and the shear modulus of the skin layers obtained by processing sequential ultrasound images also increased significantly, indicating an increase in skin firmness (p <0.05).

Based on the findings of the present study a non-invasive method according to the extraction of local biomechanical parameters of skin based on the processing of sequential high-frequency ultrasound images for detecting the skin damage caused by ultraviolet radiation is proposed.

Mice are the most commonly used animal in reproductive research and following the urgent need for these type of studies and also due to the increased interest in the ethical principle of animal rights, four various inbred and outbred strains of the laboratory mouse were evaluated to select the more efficient one for reproductive research.

60 female and 16 male of strains (C57, CD1, NMRI, and Balb/c) weighing 25 to 30g and aged 6 to 8 weeks were evaluated under same conditions at different stages of mature oocyte collection, fertilization and in vitro embryo development up to the blastocyst stage. The data were analyzed using a chi-square test, and the selected significance level was p<0.05.

Among the four strains, the highest to lowest fetal survival rates were for the CD1, NMRI, Balb /C and C57 mice, respectively and  their values ​​were 38.9, 14.4, 9.1 and 3.1%, individually.

Considering the results, we conclude that it is not possible to obtain optimal results for some strains due to using same instructions. The results showed that the highest rate of fertilization and embryo development up to the 8-cell stage was observed in the outbred CD1 mice. It seems that this strain is more applicable than others for reproductive research. In addition, we believe that using different medium during fertilization and embryo development as well as laboratory conditions, probably assist in improving the embryo production while minimized the required number of animals and allowed the achievement of the desired result.

Spermatogenesis is a programmed route for germ cell proliferation and differentiation that can produce abundant numbers of spermatozoa. The antioxidants play a vital role in decreasing oxidative stress production in cells; therefore, the extraction of plants with antioxidant property can prevent cell damage. In the present study, antioxidant effects of Calligonum extract on proliferation and colonization rate of spermatogonial cells were assessed.

After isolation and culturing of spermatogonial stem cells (SSCs) on neonatal mice (4-5 days old) and identification by PLZF and Oct4 markers, the therapeutic effect ofCalligonum comosum extract on cells treated with H2O2 was measured. The cultured cells were divided into four groups: Control, Calligonum, H2O2 and Calligonum + H2O2 groups. Induced oxidative stress cells were treated with 10 μg/ml extract for 3 weeks. Reactive oxygen species (ROS) levels were assessed by the flow cytometry, and proliferation and total antioxidant capacity (TAC) were evaluated by cell count and ferric reducing ability of plasma (FRAP) assay, respectively. Also, the apoptosis rate was measured with P53 and Bax genes by the real- time PCR method.

After three-week treatment, ROS level was significantly lower in the Calligonum group than in the H2O2 group. Antioxidants levels were significantly higher in Calligonum group than in the H2O2 group (P≤0.05). There was also a strong inverse relationship between the two groups. Proliferation and colonization rate were significantly higher in Calligonum + H2O2 group than in H2O2 group (P≤0.05). Finally, the results suggested that P53 and Bax expression decreased in Calligonum + H2O2 group compared to H2O2 group.

The results of present study revealed that 30 μM doses of H2O2 increased oxidative stress and apoptosis on the one hand and decreased proliferation of SSCs on the other hand. As a plant with antioxidant effect, Calligonum could reduce the level of ROS and apoptosis, and increase proliferation, colonization rate and TAC.

In testis, the extracellular matrix (ECM) in addition to the supportive role for cells in the seminiferous epithelium, is also essential for the accurate functioning of these cells. Thus, using a decellularized testicular ECM (DTECM), as a scaffold for three-dimensional (3D) culture of testicular cells can mimic native ECM for studying in vitro spermatogenesis.  The rat testis was decellularized via perfusion of 0.5% sodium dodecyl sulfate (SDS) for 48 hr, followed by 1% Triton X-100 for 6 hr, and then 1% DNase I for 1 hr. The efficiency of decellularization was evaluated by histology, immunohistochemistry (IHC), scanning electron microscopy (SEM), and MTT test. The prepared scaffolds were recellularized with testicular cells and cultured and assessed with hematoxylin-eosin (H&E) staining after two weeks.  Based on the H&E image, no trace of cell components could be observed in DTECM. IHC images demonstrated collagen types I and IV, laminin, and fibronectin were preserved. Masson’s trichrome and alcian blue staining revealed that collagen and glycosaminoglycans (GAGs) were retained, and the SEM image indicated that 3D testicular architecture remained after the decellularization process. Based on the results of the MTT test, DTECM was cytocompatible, and H&E images represented that DTECM supports testicular cell arrangements in seminiferous tubule-like structures (STLSs) and organoid-like structures (OLSs).   The results showed that the applied protocol successfully decellularized the testis tissue of the rat. Therefore, these scaffolds may provide an appropriate vehicle for in vitro reconstruction of the seminiferous tubule.

Generating functional gametes for patients with male infertility is of great interest. We investigated dif-ferent cultural systems for proliferation of SSCs derived from obstructive azoospermic patients.

Testicular cells were obtained from men with obstructive azoospermia. After enzymatic digestion process, cells were assigned to various groups: culture of SSCs in the dish without cover (control group), co-culture of SSCs with infertile Sertoli cells (I), co-culture of SSCs with fertile Sertoli cells (II), culture of SSCs on nanofiber (covered with laminin) (III), culture of testicular cell suspension (IV). Then cells were cultured and colony formation, gene-specific methylation (by MSP), quantitative genes expression of pluripotency (Nanog, C-Myc, Oct-4) and specific germ cell (Integrin α6, Integrin β1, PLZF) genes were evaluated in five different culture systems.

Our findings indicate a significant increase in the number and diameter of colonies in IV group in com-pare to control group and other groups. Expression of germ specific genes in IV group were significantly increased (P≤ 0.05) and levels of expression of pluripotency genes were significantly decreased in this group (P≤ 0.05) compared with other groups. Gene-specific pattern of methylation of examined genes showed no changes in cul-ture systems during the culture era.

A microenvironment capable of controlling the proliferation of cell colonies can be restored by tes-ticular cell suspension.

Biological scaffolds are derived by the decellularization of tissues or organs. Various biological scaffolds, such as scaffolds for the liver, lung, esophagus, dermis, and human testicles, have been produced. Their application in tissue engineering has created the need for cryopreservation processes to store these scaffolds.

The aim was to compare the two methods for prolong storage testicular scaffolds.

In this experimental study, 20 male NMRI mice (8 wk) were sacrificed and their testes were removed and treated with 0.5% sodium dodecyl sulfate followed by Triton X-100 0.5%. The efficiency of decellularization was determined by histology and DNA quantification. Testicular scaffolds were stored in phosphate-buffered saline solution at 4ºC or cryopreserved by programmed slow freezing followed by storage in liquid nitrogen. Massonchr('39')s trichrome staining, Alcian blue staining and immunohistochemistry, collagen assay, and glycosaminoglycan assay were done prior to and after six months of storage under each condition.

Hematoxylin-eosin staining showed no remnant cells after the completion of decellularization. DNA content analysis indicated that approximately 98% of the DNA was removed from the tissue (p = 0.02). Histological evaluation confirmed the preservation of extracellular matrix components in the fresh and frozen-thawed scaffolds. Extracellular matrix components were decreased by 4°C-stored scaffolds. Cytotoxicity tests with mouse embryonic fibroblast showed that the scaffolds were biocompatible and did not have a harmful effect on the proliferation of mouse embryonic fibroblast cells.

Our results demonstrated the superiority of the slow freezing method for prolong storage of testicular scaffolds.

In the present study, a numerical simulation was conducted to investigate the separation of blood cells using an integrated dielectrophoretic-photophoretic method in a new microfluidic device. In this simulation, the migration behavior of human blood cells under laser radiation with a wavelength of 522 nm and in the presence of fluid flow has been investigated. Studies show that the photophoretic migration of red cells under the irradiation of laser beam is higher than platelets and other blood cells, so that the magnitude of the applied photoelectric force on the red blood cells has been calculated about 9 times that of the white blood cells under the irradiation of laser beam of 50 μm. In this separation using photophoretic forces, red cells were first separated from the platelets and white cells. Subsequently, using the hydrodynamic forces induced by the fluid on the particles and the dielectrophoretic forces, the separation of the platelets from the white blood cells was carried out in different branches of the microchannel. In this study, the dielectrophoretic forces were created using electrode arrays, located on the one side of the microchannel of the microfluidic device. The combination of dielectrophoretic and photophoretic methods has led to a reduction in the magnitude of the required peak to peak voltage for electrodes to a remarkable magnitude of 3v. The proposed design, in addition to high separation efficiency, has a negligible cell loss, so that it can be used as an effective method in many diagnostic processes and medical applications.

Recreational use of illicit drugs is one of the main factors affecting male fertility. However, the mech-anisms of heroin smoke-associated damage to mature spermatozoa are still completely unknown. The aim of this study was to concomitantly examine the levels of protamine-2 gene and protein concentrations, the amount of miRNA-122 in seminal plasma and semen analysis findings in heroin-addicted men.

In a case control study, twenty-four fertile men that lacked any recreational drug abuse were considered as the healthy group, and 24 addicted men who used only heroin for at least four months were selected as the addicted group. Semen samples were gathered by masturbation after 2 - 5 days of sexual abstinence. Following the preparation of a semen analysis by computer-assisted sperm analysis according to WHO (2010), the level of protamine-2 gene expression in sperm and miRNA-122 in seminal plasma was measured using real-time sqPCR. Also, protamine-2 protein concentrations were quantified by nuclear protein extraction, SDS-Page and western blotting.

Among the studied variables, body mass index (27.75 ± 0.88 vs. 22.30 ± 0.36, p = 0.001), seminal pH (7.79 ± 0.06 vs. 7.58 ± 0.06, p = 0.003), white blood cell count in semen (1.69 ± 0.41 vs. 8.61 ± 1.73, p = 0.001), motility (65.51 ± 2.57 vs. 41.96 ± 3.58, p = 0.001) and survival rate (87.41 ± 1.00 vs. 71.50 ± 4.59, p = 0.002) of sperm cells was significantly different between the healthy and addicted groups. In addition, the levels of protamine-2 gene and protein expression in the addicted group (0.05 ± 0.02 and 0.10 ± 0.02, respectively) were significantly lower than the healthy group (3.59 ± 0.94 and 0.27 ± 0.06, respectively) (p = 0.002 and p = 0.017, respectively). Seminal miRNA-122 levels in addicted men (3.51 ± 0.73) were statistically higher than in healthy men (1.52 ± 0.54) (p = 0.034).

This is one study on human infertility that evaluates the effects of heroin on protamine deficiency and seminal small RNAs expression levels. Heroin abuse may lead to male infertility by causing leukocytospermia, asthenozoospermia, protamine deficiency, and seminal plasma miRNA profile alteration.

Induction of in vitro spermatogenesis can be useful for infertility treatment in azoospermic patients and those undergoing chemotherapy. Different culture systems have been used to achieve this goal. This review study was performed with the aim to evaluate the application of 3D culture and testicular scaffolds in the establishment of in vitro spermatogenesis. In this review study, the information on the application of 3D culture and testicular scaffolds in induction of in vitro spermatogenesis was searched in databases such as SID, Magiran, PubMed, Irandoc, Iranmedx Scopus, Google Scholar, Web of Science using the keywords of three dimensional culture, testicular scaffold, spermatogenesis, spermatogonial stem cells without time limitation. Data analysis was carried out qualitatively. Finally, 35 papers in English and Persian were used to compile the article. In order to induce of in vitro spermatogenesis, three-dimensional culture methods such as testicular tissue culture, soft agar culture system, natural biomaterial scaffolds such as collagen, and scaffolds derived from decellularized testis have been used.

Three-dimensional culture using spermatogonial stem cells and scaffolds can be used in vitro for induction of spermatogenesis, but there are further technical and ethical challenges in the path of fertile sperm production for the treatment of infertility.

Cryopreservation of immature testicular tissue before chemo/ radiotherapy is the only option to preserve fertility of cancer-affected prepubertal boys. To avoid reintroduction of malignant cell, induction of in vitro spermatogenesis could be considered. Induction of in vitro spermatogenesis using spermatogonial cells requires a suitable platform for cell growth and proliferation. The extracellular matrix of the testis could be used for adhesion, proliferation, migration and differentiation of spermatogonial cells. The extracellular matrix of the testis consists of glycosaminoglycans (GAGs), fibronectin, collagen and laminin. It can mimic specific microenvironment of testis. The extracellular matrix as a biological scaffold provided an appropriate platform for proliferation and differentiation of spermatogonial cells. Biological scaffolds were developed using decellularization of tissues and organs.  Decellularization is a process that removes the cells, their nuclei and debris from tissues and organs without sever damage to structure and biochemical component of the tissues. The aim of our study was decellularization of whole testis for preparation of scaffold and evaluation of spermatogonia cells homing after injection into the scaffold

In order to prepare the scaffolds, adult mouse testes and different concentrations of detergents were used.  Initially, the adult mice were scarified using chloroform and their testes were removed and washed with PBS, then decellularization was performed using different concentrations of detergents according following protocols. Protocol 1: The testes were immersed in 0.1% SDS solution for 24 hours Protocol 2:  The testes were immersed in 0.5% SDS solution for 24 hours. Protocol 3: The testes were immersed in 1% SDS solution for 24 hours. Protocol 4: The testes were immersed in 0.5% SDS solution for 18 hours, then washed with PBS and immersed in 0.5% Triton solution for 18 hours. In order to remove detergents, scaffolds were washed using PBS and disinfected by 70% ethanol. All protocols of decellularization and washing were done at room temperature on orbital shaker with 50 rpm speed. The efficiency of the decellularization process was determined by hematoxylin-eosin staining and DNA quantification. To evaluate the preservation of collagen and GAGs, Massonchr('39')s trichrome staining and alcian blue staining were done respectively. Confirmation of fibronectin, collagen 4 and laminin presence in decellularized scaffolds was done using immunohistochemistry (IHC). The quantity of totlal collagen and GAGs in scaffolds was evaluated using Sicrol assay kit and Blyscan assy kit respectively. The biocompatibility of testicular scaffolds was evaluated using MTT test.  Initially, mouse embryonic fibroblast cells were cultured on testicular scaffold for 24 hours and 72 hours. Then, the culture medium was removed and 200 μl of MTT reagent with a concentration of 0.5 mg / ml was added to the cells and incubated at 37 ° C for 4 h. Finally, 200 micrometers of DMSO was added and the samples were transferred to the 96 well plates and located in ELISA reader. In order to evaluation of spermatogonial cells support by scaffolds, the isolated cells from neonatal testes were injected to scaffolds via efferent ductile and then cultured on agarose gel for two weeks. Histological studies were carried out at the end of culture.

The results of hematoxylin-eosin staining showed that immersion testis in 0.1% SDS solution and 0.5% SDS solution couldn’t   decellularize the testes.  On the other hand, immersion testis in 1% SDS solution led to destruction of seminiferous base membrane. Immersion testis in 0.5% SDS and 0.5% Triton resulted in complete decellularization of the testes without severe damage to seminiferous base membrane. In order to further evaluation of methods efficiency, the amount of DNA residue in the scaffolds was extracted using kit and examined by nanodrop.Spectrophotometric analysis showed 50%  and 70% of DNA were removed  in first and second  methods respectively, while more than 98% of DNA was removed  in  third and  forth  methods. The first and second methods were discarded due to inefficiency in DNA removal from the testes and third method due to destruction of the basement membrane of the tubes. So, the scaffolds that prepared by forth method were selected for further evaluation. The result of alcian blue staining indicated the good preservation of the GAGs in decellularized testes scaffolds .The result of thrichrom staining confirmed the preservation of collagen decellularized testes scaffolds. Presence of blue fibers in the scaffold (representing collagen fibers) and the lack of red dots (representing the cell nuclei) indicate that the prepared scaffolds are cell-free and Collagen strands are well preserved. Examination of fluorescent microscopic images showed that extracellular testicular matrix proteins including fibronectin, collagen type 4 and laminin were expressed in testicular scaffolds, indicating preservation of these proteins in scaffolds. Quantified evaluation of GAGs and collagen content of decellularized scaffolds showed that there was no significant reduction in GAGs and collagen level in scaffolds compared to testes. In order to evaluation of the cytotoxicity of testicular scaffolds, MTT test was done. The results of the MTT test showed that the survival rate of mouse embryo fibroblastic cells didn’t show significant difference after 24 and 72 hours of culture in the presence of testicular scaffolds compared to culture without scaffolds, so the scaffolds were biocompatible and did not negative effect on cell survival. Mouse embryo fibroblastic cells could metabolize MTT in the presence of scaffolds, so mitochondria of the cells were active in the presence of scaffolds and led to the survival and proliferation of cells.  Examination of hematoxylin-eosin images showed that the injected cells were located on basement membrane of seminiferous tubules and in the interstitial space and created colonies that resemble organoid structures. The tubes were completely collapsed in control group, and no cells were seen in the scaffolds.

Immersion of adult mouse testes in 0.5% SDS solution and 0.5% triton solution was an effective method for decellularization of whole testes without severs damage to seminiferous tubules. Our decellularization method could preserve important proteins of extra cellular matrix including fibronectin, collagen type 4 and laminin in testicular scaffolds. Decellularized testicular scaffolds were biocompatible and did not have a harmful effect on MEF and spermatogonial cells viability. Also prepared scaffolds could support the proliferation of spermatogonial cells during two weeks culture

The study aimed to evaluate the impact of Calligonum extract and US radiation on sperm parameters of cryopreserved human semen samples.

In this experimental study, twenty-five semen specimens were obtained from healthy semen donors and incubated in human tubal fluid (HTF) medium supplemented with 10% human serum albumin (HSA) for 45 minutes. Samples were treated with Calligonum extract (10 μg/ml) alone (CGM group) and US radiation (LIPUSexposed group) alone or a combination of both treatments (CGM+LIPUS). The US group received US stimulation (in both continuous and pulsed wave modes) at a frequency of 1 MHZ and intensity of 200 mW/cm2 for 200 seconds. Sperm morphology was assessed by Diff-Quik staining. The DNA fragmentation was evaluated the Halo sperm kit. Sperm parameters was analyzed by a computer-assisted semen analysis system. Reactive oxygen species (ROS) was assessed by flow cytometry.

The results showed that the treatment with Calligonum extract significantly (P<0.05) increased the progressive motility of spermatozoa in the CGM group as compared with the control group. The application of low-intensity US significantly (P<0.05) decreased the motility and viability of spermatozoa in the US group when compared with the control group. Our findings also indicated that the use of both low-intensity US in continuous mode and Calligonum extract slightly increased progressive motility; however, such an increase was not statistically significant. The rate of DNA fragmentation was considerably higher (P<0.05) in control and LIPUS-exposed groups than the other groups.

Treatment of spermatozoa with Calligonum extract slightly improved the sperm parameters due to its antioxidant activity, on the other hand, according to our results, US radiation did not improve sperm parameters which may be due to interference with the motility of sperm, as well as its physical effects on spermatozoa.

Infertility is one of the global health problems. The bacterial infection of semen and genital tract is one of the causes of the decline in male fertility. Defects in spermatogenesis and sperm function may be due to cellular reactions against microbial agents, as well as the direct effect of bacteria on germ cells. The recognition of the interaction between bacteria and reproductive system plays an important role in treating infertile men. The aim of this study was to examine the in vitro direct effect of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) on the motility, morphology, and viability of human spermatozoa, and compare the effect of these bacteria on the quality of sperm.

The study was carried out on swim-up-separated spermatozoa from 33 men referred to the Nikan Infertility Treatment Center in Tehran, Iran, in 2018. After the removal of seminal plasma, the sperm suspension of each sample was divided into three parts. The first and second parts (i.e., experimental groups) were coincubated with E. coliand S. aureus, respectively, for 90 min at 37°C, and the third part (i.e., control group) was incubated at the same condition but without bacteria. Sperm parameters were assessed after the incubation. Statistical analysis was done to find the significant differences between the experimental groups and control group.

The results showed that E. coliand S. aureus infection caused a significant decline in the morphology, motility, and viability of two experimental groups in comparison to that reported for the control group (P<0.05).

The results of this study revealed that the contact of bacteria with ejaculated spermatozoa can decrease sperm motility, morphology, and viability, with potential consequences for male fertility.

Adipose-derived stem cells (ADSCs), with suitable and easy access, are multipotential cells that have the ability for differentiation into other mesodermal and transdifferentiate into neural phenotype cells. In this study, Lithium chloride (LiCl) was used for in vitro transdifferentiation of rat ADSCs into neuron-like cells (NLCs). ADSCs were isolated from the rats’ perinephric region using Dulbecco΄s Modified Eagle΄s Medium (DMEM) with Fetal Bovine Serum (FBS), cultured for 3 passages, characterized by flowcytometry and differentiation into adipogenic and osteogenic phenotypes. The ADSCs were exposed to 0.1, 0.5, 1, 1.5, 2, 5, and 10 millimolar (mM) LiCl without serum for 24 hr. The optimum dose of LiCl was selected according the maximum viability of cells. The expression of neurofilament light chain (NfL), neurofilament high chain (NfH), and nestin was evaluated by immunocytochemistry. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to evaluate the amount of synaptophysin, neurogenin-1, neuroD1, NfL, NfH, and nestin genes’ expression in ADSCs and NLCs. The optimum dose of LiCl was 1 mM in 24 hr. The transdifferentiated ADSCs showed cytoplasmic extension with synapse-like formation. Synaptophysin, neurogenin-1, neuroD1, NfL, NfH, and nestin genes were significantly expressed more in NLCs than in ADSCs. LiCl can induce ADSCs into neural phenotype cells with higher expression of neural and neuronal genes.

The aim of this study was to investigate two enkephalin-degrading enzymes, aminopeptidase N (APN/CD13) and endopeptidase (NEP/CD10), gene and protein expression levels in sperm samples of fertile and heroin-addicted men, and the correlation between their expressions and semen quality.

This case-control study, semen collected from 24 normozoospermic healthy (as a control group) and 24 heroin-addicted men donors (as case or addiction group). Sperm cells isolated by Cook Medical gradient (40%–80%) and followed up by swim-up techniques were used for real-time qPCR and flow cytometry techniques to assess APN/CD13 and NEP/CD10 genes and proteins subsequently. Semen parameters were analyzed by computer-assisted sperm analysis.

The findings revealed that sperm total motility (41.07 ±3.63 vs. 63.03 ± 3.31 %, p = 0.0001) and progressive motility (35.21 ± 2.64 vs. 20.93 ± 3.22%, p =0.001), and viability (69.9 ± 4.69 vs. 86.81 ± 1.26 %, p =0.002) were significant differences in addicted group vs. control ones. APN and NEP gene expression levels in the addicted group decreased compared with the control ones (1.00 ± 0.67 vs. 0.36 ± 0.13, p = 0.008 and 1.07 ± 0.11 vs. 0.52 ± 0.12 0.002, respectively). Flow cytometry analysis showed that the average percent of APN/CD13 in heroin consumers significantly decreased compared with the healthy ones, while NEP/CD10 rate between two groups was similar. We also observed that duration of drug dependence is correlated with sperm viability (r =-0.627, p =0.016) and motility (r= -0.410, p= 0.05), NEP (r= -0.434, p= 0.049), and APN (r -0.641, p= 0.002) gene expression levels.

We concluded that semen quality and enkephalin-degrading enzymes were altered in heroin-addicted men.

We present a four-branch model of the dielectrophoresis (DEP) method that takes into consideration the inherent properties of particles, including size, electrical conductivity, and permittivity coefficient. By using this model, bioparticles can be continuously separated by the application of only a one-stage separation process.

In this numerical study, we based the separation process on the differences in the particle sizes. We used the various negative DEP forces on the particles caused by the electrodes to separate them with a high efficiency. The particle separator could separate blood cells because of their different sizes.

Blood cells greater than 12 μm were guided to a special branch, which improved separation efficiency because it prevented the deposition of particles in other branches. The designed device had the capability to separate blood cells with diameters of 2.0 μm, 6.2 μm, 10.0 μm, and greater than 12.0 μm. The applied voltage to the electrodes was 50 V with a frequency of 100 kHz.

The proposed device is a simple, efficient DEP-based continuous cell separator. This capability makes it ideal for use in various biomedical applications, including cell therapy and cell separation, and results in a throughput increment of microfluidics devices.

  To investigate the effects of heroin on sperm parameters, histone-to-protamine transition ratios in mature sperm, and serum reproductive hormone levels in active heroin users. Semen and blood samples were collected from 25 men who used only heroin for at least 12 months and the same number healthy men who did not use any drugs and did not suffer from infertility problems. Computer-based analysis, Aniline blue staining, and hormonal assessment were performed to provide valuable new information on the relationship between addiction and semen profile and serum reproductive hor mone levels. Our finding showed that semen pH (7.8 vs. 7.75), sperm motility (42.93 ± 3.89% vs. 68.9 ± 2.68%), and viability (73.27 ± 3.85% vs. 86.48 ± 1.05%), and sperm histone replacement abnormalities (32.33 ± 10.89% vs. 5.56 ± 0.85%) were significant differences in addicted group vs. non-exposed ones (P ? .05). In addition, serum sex hormone levels were not significantly differed between groups. There was a correlation between the amount of daily heroin consumption and LH level. We also observed that duration of drug dependence is correlated with sperm abnormalities. We concluded that heroin consumption affect sperm maturities such as histone-to-protamine ratio and impair semen profile in general and particularly sperm morphology and motility. Heroin may be considered as one of the idiopathic male infertility reason.