Role of Binder of SPerm homolog 1 (BSPH1) protein in mouse sperm-egg interaction and fertilization.

In mice, the Binder of Sperm Homolog 1 protein is exclusively expressed in the epididymis. BSP proteins play a role in the membrane modification events that occur during sperm capacitation. In the current study, we investigated the role of mouse recombinant BSP homolog 1 (rec-BSPH1) in sperm-egg interaction. Mouse oocytes were co-incubated with different concentrations of rec-BSPH1 or control proteins and then inseminated with sperm. To establish whether rec-BSPH1 interfered with in vitro fertilization of mouse oocytes, rec-BSPH1 binding to egg and sperm was first tested using an immunodetection assay. In separate experiments, sperm were immuno-neutralized by anti-rec-BSPH1 antibodies to indirectly verify the implication of BSPH1 in sperm-egg interaction and fertilization. The study revealed a dose-dependent inhibition of fertilization when oocytes were pre-incubated with rec-BSPH1. Moreover, sperm immuno-neutralization with anti-rec-BSPH1 antibodies led to dramatic motility changes, followed by compromised fertilization. In view of these results, we conclude that BSPH1 could be a marker of sperm fertility and thus an eventual target for male contraceptive development.

Efficient Wound Healing Using a Synthetic Nanofibrous Bilayer Skin Substitute in Murine Model.

Treatment of full-thickness skin wounds with minimal scarring and complete restoration of native tissue properties still exists as a clinical challenge. A bilayer skin substitute was fabricated by coating human amniotic membrane (AM) with electrospun silk fibroin nanofibers, and its in vivo biological behavior was studied using murine full-thickness skin wound model. Donut-shaped silicon splints were utilized to prevent wound contraction in mouse skin and simulate re-epithelialization, which is the normal path of human wound healing. Skin regeneration using the bilayer scaffold was compared with AM and untreated defect after 30 d. Tissue samples were taken from healed wound areas and investigated through histopathological and immunohistochemical staining to visualize involucrin (IVL), P63, collagen I, CD31, and vascular endothelial growth factor. In addition, mRNA expression of IVL, P63, interleukin-6, and cyclooxygenase-2 was studied. The application of bilayer scaffold resulted in the best epidermal and dermal regeneration, demonstrated by histopathological examination and molecular analysis. In regenerated wounds of the bilayer scaffold group, the mRNA expression levels of inflammatory markers (interleukin-6 and cyclooxygenase-2) were downregulated, and the expression pattern of keratinocyte markers (IVL and P63) at both mRNA and protein levels was more similar to native tissue in comparison with AM and no-treatment groups. There was no significant difference in the expression level of collagen I, CD31, and vascular endothelial growth factor among different groups. Conclusively, these promising results serve as a supporting evidence for proceeding to clinical phase to examine the capacity of this bilayer scaffold for human skin regeneration.

Correction to: "Comparative repair capacity of knee osteochondral defects using regenerated silk fiber scaffolds and fibrin glue with/without autologous chondrocyes during 36 weeks in rabbit model.

The published online of the original version contains mistakes.

Polymorphisms in the Estrogen Receptor Beta Gene and the Risk of Unexplained Recurrent Spontaneous Abortion.

BACKGROUND: Recurrent Spontaneous Abortion (RSA) is caused by multiple genetic and non-genetic factors. Around 50% of the RSA cases have no known etiology and are considered as Unexplained RSA (URSA). Estrogens, via binding to their receptors, play an important role in female reproduction. This study aimed to investigate whether single nucleotide polymorphisms (SNPs; +1082G/A, +1730G/A and rs1256030 C/T) in the estrogen receptor beta (ESR2) gene are associated with susceptibility to URSA in a population of Iranian women. METHODS: In this case-control study, the study groups consisted of 240 subjects with a history of URSA and 102 fertile women as controls. Serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) were measured on day 2-3 of menstrual cycle. Two functional SNPs, +1082G/A (a silent mutation in exon 5) and +1730G/A (3' untranslated region of the exon 8), and one intron, rs1256030C/T, in the ESR2 gene were genotyped, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. RESULTS: Serum levels of LH were significantly increased in URSA women. No significant differences in distribution of +1082G/A, +1730G/A and rs1256030C/T between URSA and control groups were observed. CONCLUSION: Our findings suggest that the studied SNPs on ESR2 gene may not be associated with URSA.

Comparative repair capacity of knee osteochondral defects using regenerated silk fiber scaffolds and fibrin glue with/without autologous chondrocytes during 36 weeks in rabbit model.

The reconstruction capability of osteochondral (OCD) defects using silk-based scaffolds has been demonstrated in a few studies. However, improvement in the mechanical properties of natural scaffolds is still challengeable. Here, we investigate the in vivo repair capacity of OCD defects using a novel Bombyx mori silk-based composite scaffold with great mechanical properties and porosity during 36 weeks. After evaluation of the in vivo biocompatibility and degradation rate of these scaffolds, we examined the effectiveness of these fabricated scaffolds accompanied with/without autologous chondrocytes in the repair of OCD lesions of rabbit knees after 12 and 36 weeks. Moreover, the efficiency of these scaffolds was compared with fibrin glue (FG) as a natural carrier of chondrocytes using parallel clinical, histopathological and mechanical examinations. The data on subcutaneous implantation in mice showed that the designed scaffolds have a suitable in vivo degradation rate and regenerative capacity. The repair ability of chondrocyte-seeded scaffolds was typically higher than the scaffolds alone. After 36 weeks of implantation, most parts of the defects reconstructed by chondrocytes-seeded silk scaffolds (SFC) were hyaline-like cartilage. However, spontaneous healing and filling with a scaffold alone did not eventuate in typical repair. We could not find significant differences between quantitative histopathological and mechanical data of SFC and FGC. The fabricated constructs consisting of regenerated silk fiber scaffolds and chondrocytes are safe and suitable for in vivo repair of OCD defects and promising for future clinical trial studies.

Fabrication and characterization of nano-fibrous bilayer composite for skin regeneration application.

Full thickness wound healing with minimal scarring and complete restoration of normal skin properties still remains as a clinical challenge. In this study, a bilayer skin substitute has been fabricated to biomimic the microstructure of natural extracellular matrix of the skin. Human amniotic membrane (HAM) and silk fibroin nano-fibers were combined to produce bilayer construct, which was further treated and characterized. HAM was obtained from healthy mothers and de-epithelized by means of fine enzymatic method to preserve the extracellular structure. Fibroin protein was extracted from fresh Bombyx mori cocoons and transformed to uniform nano-fiberous structure, which was used as a coating layer on the de-epithelized membrane. Surface modification through oxygen plasma treatment was attempted to further induce hydrophilicity. Subsequently, scaffolds were fully characterized in terms of morphology, mechanical properties, hydrophilicity and cell culture response. Histological and immunohistological staining demonstrated localization of fibronectin, cell denudation and structural integrity of HAM after de-epithelization. Scanning electron microscopy images showed bead-free silk fibroin nano-fibers with the average diameter of 250nm. Water contact angle of bilayer scaffolds reduced dramatically to 26.34° after oxygen plasma treatment, which is correlated with more hydrophilic surface. Due to fibroin nano-fiber coating, mechanical properties of HAM improved significantly. Tensile Young's modulus and tensile strength increased from 16.14MPa and 68.46MPa to 25.69MPa and 108.03MPa, respectively. 14days in vitro cultivation of mouse embryonic fibroblasts on the scaffolds revealed that bilayer scaffolds are able to support cell attachment and proliferation. Plasma-etched scaffolds provided the best niche for cell-matrix crosstalk by allowing cells to penetrate beneath the pores and to integrate in fibers direction. The obtained results suggest that the presented nano-fibrous bilayer composite based on HAM is a potential substitute for skin regeneration application.

Comparative evaluation of in vivo biocompatibility and biodegradability of regenerated silk scaffolds reinforced with/without natural silk fibers.

Nowadays, exceptional advantages of silk fibroin over synthetic and natural polymers have impelled the scientists to application of this biomaterial for tissue engineering purposes. Recently, we showed that embedding natural degummed silk fibers in regenerated Bombyx mori silk-based scaffold significantly increases the mechanical stiffness, while the porosity of the scaffolds remains the same. In the present study, we evaluated degradation rate, biocompatibility and regenerative properties of the regenerated 2% and 4% wt silk-based composite scaffolds with or without embedded natural degummed silk fibers within 90 days in both athymic nude and wild-type C57BL/6 mice through subcutaneous implantation. In all scaffolds, a suitable interconnected porous structure for cell penetration was seen under scanning electron microscopy. Compressive tests revealed a functional relationship between fiber reinforcement and compressive modulus. In addition, the fiber/fibroin composite scaffolds support cell attachment and proliferation. On days 30 to 90 after subcutaneous implantation, the retrieved tissues were examined via gross morphology, histopathology, immunofluorescence staining and reverse transcription-polymerase chain reaction as shown in Figure 1. Results showed that embedding the silk fibers within the matrix enhances the biodegradability of the matrix resulting in replacement of the composite scaffolds with the fresh connective tissue. Fortification of the composites with degummed fibers not only regulates the degradation profile but also increases the mechanical performance of the scaffolds. This report also confirmed that pore size and structure play an important role in the degradation rate. In conclusion, the findings of the present study narrate key role of additional surface area in improving in vitro and in vivo biological properties of the scaffolds and suggest the potential ability of these fabricated composite scaffolds for connective tissue regeneration. spjba;30/6/793/FIG10885328215601925F1fig1-0885328215601925Figure 1.Illustrative summary of the main methods and findings.RS: regenerated silk; RSF: regenerated fibroin/ silk fiber composite scaffolds; H&E: Hematoxylin and eosin; COX-1: Cyclooxygenase.

Tissue Engineering and Regenerative Medicine in Iran: Current State of Research and Future Outlook.

During two decades ago, Iran has exhibited remarkable increase in scientific publication in different aspects including tissue engineering and regenerative medicine (TERM). The field of TERM in Iran dates comes back to the early part of the 1990 and the advent of stem cell researches. Nowadays, Iran is one of the privileged countries in stem cell therapy in the Middle East. The next major milestone in TERM was application and fabrication of scaffolds for tissue engineering in the early 2000s with a focus on engineering bone and cartilage tissue. A good amount of thoughtful works has also yielded prototypes of other tissue substitutes such as nerve conduits, liver, and even heart. However, forward movement to clinical application of these products is still far from offering clinically acceptable solutions. In this study, we have presented a comprehensive review on the efforts of Iranian scientists in different issues of tissue engineering and regenerative medicine field.

Quantification of sperm specific mRNA transcripts (PRM1, PRM2, and TNP2) in teratozoospermia and normozoospermia: New correlations between mRNA content and morphology of sperm.

Sperm mRNAs could be used as a predictor of fertilization capacity since the transcriptional profile of a gamete is critical for the production of viable human sperm. The aim of this study was to determine if PRM1, PRM2, and TNP2 transcripts in spermatozoa from normozoospermic and teratozoospermic men correlate with sperm morphology and/or assisted-reproduction outcomes. Human ejaculates were collected from 138 men referred to an infertility clinic, and were separated in two groups, teratozoospermic (n =72) and normozoospermic (n =66), based on World Health Organization criteria (2010). Chromomycin A3 and analine blue staining were used to evaluate protamination and chromatin integrity, respectively. Quantitative reverse-transcriptase PCR was performed for PRM1, PRM2, and TNP2. This analysis revealed significantly higher PRM1 and PRM2 mRNA copy numbers in normozoospermic versus teratozoospermic samples (P < 0.001). In contrast, TNP2 transcript abundance was significantly higher in teratozoospermic versus normozoospermic samples (P < 0.001) and positively correlated with sperm-head defects (P < 0.05). Sperm-tail defects negatively correlated (P < 0.05) with both PRM1 and PRM2 transcripts in normozoospermic samples. No significant differences were observed between the two groups when comparing transcript levels to the outcome of intracytoplasmic sperm injection cycles (P > 0.05), and a normal PRM1/PRM2 mRNA ratio (∼1) was observed in more than 70% of successful cycles. Thus, the quantity of PRM1, PRM2, and TNP2 transcripts and the PRM1/PRM2 mRNA ratio affect spermiogenesis, sperm morphology, and the function of mature human sperm. These mRNAs could therefore be used as biomarkers for the diagnosis of male infertility.

Molecular and serologic diagnostic approaches; the prevalence of herpes simplex in idiopathic men infertile.

BACKGROUND: Human pathogens that can cause infertility may also affect sperm count and quality. Viral infections can be considered as direct and/or indirect cause of male factor infertility. OBJECTIVE: Our goal was to investigate the prevalence of herpes simplex virus in the semen of infertile men attending the Avicenna Infertility Clinic, and to compare it with the herpes virus serology results. MATERIALS AND METHODS: This cross sectional study was conducted during 2009-2010. Infertile men participating without any clinical signs of infection with herpes simplex virus, and no obvious cause for their infertility were included. Semen and blood samples were used for Polymerase Chain Reaction (PCR) and serologic testing for these people. Two samples were collected: one ml semen sample to verify the existence of genital herpes simplex virus in infertile men, and blood samples of 217 individuals tested for antibodies to herpes simplex virus. Data were analyzed by SPSS 16. RESULTS: According to the PCR results of semen samples the prevalence of herpes simplex in semen was 12% and serologic test showed 3.2% prevalence within blood. Nine to 10% of IgM negative were PCR positive and only 2-3% of IgM positive were PCR positive. Between herpes serologic studies with positive controls and negative controls by using both tests, there was a significant positive relationship (r=0.718 and p<0.001). The relationship between semen PCR test results and serological survey of herpes patients with a negative control in both Pearson and Spearman tests was positive and significant (r=0.229 and p=0.001). Correlation between the PCR results of semen samples with two positive control subjects and a positive IgM test was statistically confirmed (r=0.235 and p<0.001). CONCLUSION: We recommend that if there is suspicion to herpes simplex as a microorganism that theoretically could impact semen parameters and cause infertility it is prudent to use PCR technique on semen sample rather than ELISA on serum.

Evaluation of an Aqueous-Ethanolic Extract from Rosmarinus officinalis (Rosemary) for its Activity on the Hormonal and Cellular Function of Testes in Adult Male Rat.

Rosmarinus officinalis has been used in traditional medicine extensively. This study evaluated the hormonal and cellular effects of Rosmarinus officinalis extract on testes of adult rats. Thirty male Wistar rats (in three groups) received 50 or 100 mg/Kg b.w of Rosmarinus officinalis extract (made from the plant's leaves, flower and stem) (treatment groups) and 10 mL/Kg b.w normal saline (control group) respectively, on a daily bases by gavage route for 60 days. Then, spermatological properties, histometric parameters and sperm dynamics, testis and body weight, testicular cell population and serum testosterone level were analyzed by an acceptable method. Results showed that the mean serum testosterone level was decreased significantly in both treatment groups (50 and 100 mg/Kg b.w) during the experiment time, compared with control group (p < 0.05). However, Rosmarinus officinalis did not change the total count, motility and viability of sperm. In addition, Rosmarinus officinalis at both doses did not change body and testes weight and their ratio. Furthermore, Rosmarinus officinalis increased the number of Spermatogonia at both doses, Spermatocyte at doses of 50 mg/Kg b.w, Leydig cell and Spermatid at dose of 100 mg/Kg b.w significantly (p < 0.05). Rosmarinus officinalis did not significantly affect the number of Spermatozoid and Sertoli cells. In conclusion, it seems that Rosmarinus officinalis may have some hormonal and cellular effects on the testes which can contribute the spermatogenesis process in rat. Rosmarinus officinalis may have antiandrogenic effect potentially indicating the possibility of developing herbal male contraceptive.

A survey on the prevalence of Chlamydia trachomatis and Mycoplasma genitalium infections in symptomatic and asymptomatic men referring to urology clinic of labbafinejad hospital, tehran, iran.

BACKGROUND: Chlamydia trachomatis and Mycoplasma genitalium infections are the most prevalent sexually transmitted bacterial infections in the world that cause urogenital infections in both men and women. It appears that infertility is a complication of these infections. OBJECTIVE: This study was designed to estimate the prevalence of Chlamydia trachomatis and Mycoplasma genitalium in symptomatic and asymptomatic men and to assess risk factors associated with infection. PATIENTS AND METHODS: Urine specimens were collected from 200 men; 100 of them were symptomatic and 100 asymptomatic. Samples were examined by PCR to detect the infections. RESULTS: C. trachomatis was detected in 20% of symptomatic and in 4% of asymptomatic men (P < 0.001). The prevalence of M. genitalium was revealed to be 12% and 2% in symptomatic and asymptomatic men, respectively (P < 0.01). Four of 100 men in the symptomatic group were infected with both organisms. C. trachomatis infection was associated with dysuria, urethral discharge, testicular swelling, and genital ulcer (P < 0.05). M. genitalium infection was related with dysuria, testis inflammation, pelvic pain and low educational level (P < 0.05). Furthermore, the prevalence of infections at ages 30-39 years was more than other ages. CONCLUSIONS: Considering the role of these bacteria in urogenital infections, a screening test is recommended. Since the PCR assay is a highly sensitive and specific assay for the detection of these bacteria in male urine specimens, it provides a noninvasive technique for routine screening.

The association of seminal plasma antioxidant levels and sperm chromatin status with genetic variants of GSTM1 and GSTP1 (Ile105Val and Ala114Val) in infertile men with oligoasthenoteratozoospermia.

In this study we aimed to examine the effects of genetic variants of GSTM1 and GSTP1 (Ile105Val and Ala114Val) on GST activity, seminal oxidative stress and sperm chromatin status in infertile men with oligoasthenoteratozoospermia (OAT). The study population (n=121) consisted of 95 infertile men with OAT and 26 controls with normozoospermia. Multiplex polymerase chain reaction (PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods were utilized to detect the aforesaid genetic variants. We measured GST activity and total antioxidant capacity (TAC) of seminal plasma by spectrophotometry. Sperm chromatin integrity and maturity were assessed using toluidine blue and chromomycin A3 (CMA3-positive sperm) staining, respectively. The analysis showed that subgroups of GSTM1 null and GSTP1 C/T+T/T genotypes in comparison with GSTM1 present and GSTP1 wild type (C/C) genotypes did not have statistically significant differences in both OAT or normozoospermic men considering sperm concentration and motility, percentage of CMA3-positive sperm, seminal plasma TAC, sperm chromatin integrity and GST activity. Thus, the findings of our study suggest that there are no significant associations between GSTM1 and GSTP1 polymorphisms and sperm parameters at conventional or at molecular levels including OS status, sperm chromatin integrity or maturity in Iranian infertile men with OAT and normozoospermia. However, these polymorphisms could be related to the fertility status of the studied population but not evaluated in this study.

Effects of very rapid versus vapor phase freezing on human sperm parameters.

The aim of the present study was to compare the effects of two freezing methods, vapor phase and very rapid freezing, with and without cryoprotectant on semen parameters in men with normal semen criteria. Cryopreservation was done on semen samples from 31 men by two methods of vapor phase freezing and very rapid freezing, with and without Test Yolk buffered glycerol (TYBG) as cryoprotectant. The motility, viability, acrosome and DNA integrity were evaluated on fresh and post-thaw samples. Post-thaw sperm progressive motility was significantly higher in the presence of TYBG in the vapor phase cryopreservation (%6.30 ± 3.74) compared with the very rapid freezing method (%2.2 ± 1.97 and %4.00 ± 2.42 in the presence and absence of TYBG, respectively). There was no significant difference in viability, acrosome status and DNA integrity between two methods in presence or absence of TYBG. The very rapid freezing method in the absence of TYBG showed better sperm motility but viability, acrosome and DNA integrity were similar to the presence of TYBG. The results show that cryopreservation of human spermatozoa together with seminal plasma by using vapor phase method is better than very rapid freezing method to preserve sperm progressive motility; however very rapid freezing method is quick and simple and do not require special cryoprotectant. It can be used for cryopreservation of small number of spermatozoa in IVF centers.

The inductive effects of Centella asiatica on rat spermatogenic cell apoptosis in vivo.

Centella asiatica (L.) Urban has been traditionally used for the treatment of various disease and as a food for thousands of years in various parts of the world including eastern Asia, China and India. The goal of this study was to investigate the effects of Centella asiatica aqueous leaf extract on the induction of spermatogenic cell apoptosis in male rats. After lethal dose (LD(50)) assessment of plant extract, rats were divided in five groups. The experimental groups received orally 10, 50, 80 and 100 mg/kg aqueous leaf extract daily for 60 days and the control group received just water. After 60 days, body and testis weight were measured and blood samples were taken from the heart. To evaluate apoptosis and histological changes, tissue samples obtained from rat testes were stained by TUNEL assay and hematoxylin and eosin stain. Results showed that the sperm count, motility, and viability and the number of spermatogenic cells in the seminiferous tubules were significantly decreased compared with the control group. The number of apoptotic germ cells per seminiferous tubule cross-section was significantly increased in the experimental group (18.11 ± 3.5) compared with the control group (8.7 ± 0.81) (P < 0.05). Serum testosterone, follicle-stimulating hormone, and luteinizing hormone levels also showed significant decreases in the experimental groups (P < 0.05). There was also a significant decrease in testis weight in experimental groups compared with the control group (P < 0.05). It is concluded that Centella asiatica has toxicological effects on the reproductive system in male rats and, therefore, it is suggested that leaf extracts of Centella asiatica possess antifertility effects in the male rat.

Differentiation of bovine spermatogonial stem cells into osteoblasts.

Spermatogonial Stem Cell (SSC) technologies provide multiple opportunities for research in the field of biotechnology and regenerative medicine. The therapeutic use of Embryonic Stem Cells (ESCs) is restricted due to severe ethical and immunological concerns. Therefore, we need a new pluripotent cell type. Despite well-known role of germ cells in the gametogenesis, some facts apparently show their multipotentiality. In the present study, bovine SSCs were co-cultured with Sertoli cell for 7 days. Sertoli cells and SSCs were identified by Vimentin and Oct-4 immunocytochemical staining method, respectively. In order to differentiate SSCs into osteoblasts, we used consecutive inducer media without separation of the colonies. We characterized osteoblasts using Alizarin red staining.