A new cryotop vial device system provides an aseptic cryoprotectant-free and centrifuge-free cryopreservation of human spermatozoa (a closed system).

Single sperm cryopreservation is a new method to preserve small numbers of spermatozoa in small droplets. So far, several devices have been introduced for this technique, but more studies are needed for its optimization. The aim of this study was optimizing the previous device for low number of spermatozoa and low volume semen, which led to design of Cryotop Vial device. Normal semen samples from 25 patients were prepared by swim-up method and divided into four groups: Fresh (F), Rapid freezing (R) and ultra-rapid freezing with Cryotop Device (CD) and Cryotop Vial Device (CVD). In R group, the diluted sperm suspension with sperm freezing medium was cooled in the vapor phase and then immersed in liquid nitrogen. Ultra rapid freezing was performed with sucrose in small volume using the Cryotop Device (CD) or Cryotop Vial Device (CVD). Sperm viability, motility, fine morphology, mitochondrial activity and DNA fragmentation were assessed in all samples. All sperm parameters decreased significantly in all the cryo groups compared to the fresh group. The comparison between the cryo groups showed that progressive motility (69.28 ± 6.82 vs. 55.68 ± 9.04, and 54.76 ± 5.34, p < 0.001) and viability (77.36 ± 5.48 vs.68.84 ± 8.51, p < 0.001, and 70.04 ± 7.44, P = 0.002) were significantly higher in the CVD compared to the CD and R groups respectively. DNA fragmentation was also significantly lower in both ultra-rapid freezing groups (CD and CVD) compared to the R group. Fine morphology and mitochondrial activity were not different between the cryo groups. The CVD as a cryoprotectant and centrifuge-free technique preserved sperm motility, viability and DNA integrity after cryopreservation better than other groups.

The effect of pentoxifylline and calcium ionophore treatment on sperm cell biology in oligoasthenoteratozoospermia samples.

The objective of this study was to assess the effects of pentoxifylline (PTX) and Ca2+ ionophore (CI) A12387 treatment on some biological characteristics of sperm cells in oligoasthenoteratozoospermia (OAT) patients. After processing, each sample was divided into four groups: 1, control; 2, exposed to 3.6 mM PTX; 3, exposed to 5 µm calcium ionophore (CI); and 4, exposed to both PTX and CI; 30 min at 37°C. Sperm motility was measured before and after preparation. Acrosome reaction (AR), status of sperm vacuoles, mitochondrial membrane potential (MMP) and DNA fragmentation were assessed using PSA-FITC staining, motile sperm organelle morphology examination (MSOME), JC-1 staining and sperm chromatin dispersion (CSD) test, respectively. Treatment with PTX and CI led to increased and decreased sperm motility, respectively (P < 0.05). Furthermore, vacuole status and rates of sperm DNA fragmentation were not significantly different among groups (P > 0.05). Moreover, the data showed that the rates of AR and disrupted MMP were significantly different between groups (P < 0.05). In conclusion, in vitro application of PTX not only did not have any adverse effects on sperm cell biology characteristics, but also can rectify the harmful effect of CI.

Pentoxifylline treatment had no detrimental effect on sperm DNA integrity and clinical characteristics in cases with non-obstructive azoospermia.

The aim of this study was to assess the consequences of treatment with pentoxifylline (PTX), an inducer of sperm motility, on sperm DNA fragmentation (SDF) and clinical characteristics in non-obstructive azoospermia (NOA) patients. The pilot study included 15 NOA patients. Half of each sperm sample before and after rapid freezing, was treated with PTX (3.6 mM /l, 30 min) as the PTX group and the remaining samples were considered as the control. SDF and sperm motility were assessed in each group. The clinical study comprised 30 fresh testicular sperm extractions (TESE) and 22 post-thawed TESE intracytoplasmic sperm injection cycles. Half of the mature oocytes from each patient were injected with PTX-treated spermatozoa and the remaining oocytes were injected with non-treated spermatozoa. Fertilization was assessed at 16 h post injection. Embryo transfer was carried out on day 2 after fertilization. Chemical pregnancy was assessed 2 weeks after transfer. PTX was found to significantly increase (P < 0.05) sperm motility. There was an insignificant difference in SDF rates between the groups (P > 0.05). In patient ovaries given fresh TESE, there was not any significant difference in clinical characteristics (P > 0.05). In patient ovaries given post-thawed TESE, there was a significant difference in the number of 2PN and in embryo formation (P < 0.05). Differences in the results of chemical pregnancy were insignificant (P > 0.05) between the groups. In addition, there was not any correlation between DNA fragmentation index and sperm motility and laboratory outcomes. Therefore, obtaining viable spermatozoa using PTX was more effective in post-thawed TESE regime patients in terms of 2PN and in embryo formation, deprived of damaging effects on sperm DNA integrity.

Efficacy of Ficus carica leaf extract on morphological and molecular behavior of mice germ stem cells.

Infertility is one of the most prevalent health disorders in reproductive-age males and females. Ficus carica (Fc), an herbal plant, has been used traditionally for the treatment of different diseases such as infertility especially in Iranian folk medicine. This study examined the effects of Fc leaf extract on the proliferation of mice spermatogonial stem cells (SSCs). Phenolic, flavonoid content, major polyphenolic compounds and antioxidant activity of the extract was evaluated respectively by Folin-Ciocateu, aluminum chloride, HPLC and the FRAP and DPPH methods. Testicular cells of neonate mice were extracted and their identity was confirmed using cytokeratin for Sertoli and Oct-4, CDHI and PLZF for SSCs. Effects of Fc (0.0875, 0.175, 0.35, 0.71 and 1.42 mg/ml) was evaluated at third, 7th, 9th and 14th days of culture by colony assay. The expression of the Mvh, GFRα1 and Oct-4 genes and the viability and proliferation of cultured cells was assessed at the end of the culture period. The extract has a rich phenolic and flavonoid content such as Rutin, Psoralen, Bergapten and Caffeoylmalic acid using HPLC analysis. It also had a potent reducing and radical scavenging activity. Morphology of colonies was similar in all groups. Higher viability, proliferation, colony number and diameter of SSCs was seen in the presence of Fc leaf extract in a dose-dependent manner so that higher number and diameter of colonies were observed in two higher doses of 0.71 and 1.42 mg/ml, separately for each time point relative to other groups. The Mvh, Oct-4 and GFRα1 genes expression had no significant differences between groups. It seems that Fc leaf extract not only had no any cytotoxic effects on the viability and proliferation of SSCs but also support their stemness state. So, this culture system can be employed for enrichment of germ stem cells for use in clinical applications.

Fertility preservation strategies for cancerous women: An updated review.

Due to the increase in cancer among young women, the risk of premature ovarian insufficiency with subsequent infertility has been raised. Fertility preservation restores reproductive potential along with increasing life expectancy in these patients. Given the articles on new options for treating cancerous women, we searched the keywords, including fertility preservation, in vitro maturation (IVM), and ovarian cryopreservation. This review focuses on the currently available procedures, including in (IVM) of retrieved immature oocytes, oocyte, embryo, and ovarian tissue cryopreservation (OTC). OTC is a helpful procedure that restores ovarian function and natural pregnancy. Also, we summarized the literature that reported the qualification of using the abovementioned procedures, comparing the cryopreservation methods including vitrification and slow freezing. Due to the impressive clinical development of OTC in cancerous patients, it is recommended as a standard treatment in cryopreservation strategies.

Challenges on the effect of cell phone radiation on mammalian embryos and fetuses: a review of the literature.

Cell phones operate with a wide range of frequency bands and emit radiofrequency-electromagnetic radiation (RF-EMR). Concern on the possible health hazards of RF-EMR has been growing in many countries because these RF-EMR pulses may be absorbed into the body cells, directly affecting them. There are some in vitro and in vivo animal studies related to the consequences of RF-EMR exposure from cell phones on embryo development and offspring. In addition, some studies have revealed that RF-EMR from cellular phone may lead to decrease in the rates of fertilization and embryo development, as well as the risk of the developmental anomalies, other studies have reported that it does not interfere with in vitro fertilization or intracytoplasmic sperm injection success rates, or the chromosomal aberration rate. Of course, it is unethical to study the effect of waves generated from cell phones on the forming human embryos. Conversely, other mammals have many similarities to humans in terms of anatomy, physiology and genetics. Therefore, in this review we focused on the existing literature evaluating the potential effects of RF-EMR on mammalian embryonic and fetal development.

Does in vitro application of pentoxifylline have beneficial effects in assisted male reproduction?

Application of nonspecific phosphodiesterases inhibitors, such as pentoxifylline (PTX), is a strategy utilised to aid sperm selection from immotile sperm samples prior to ICSI. No extensive studies have yet been performed to verify the safety of the clinical outcomes of ICSI after PTX administration. In this article, we summarise the data reported in the literature that assess the implication of in vitro usage of PTX on sperm parameters, as well as clinical outcomes during assisted male reproduction programme.

In vitro effects of date palm (Phoenix dactylifera L.) pollen on colonization of neonate mouse spermatogonial stem cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Date palm (Phoenix dactylifera L.) pollen (DPP) is widely used as a folk remedy for male infertility treatment, and has well known medicinal effects. AIM OF THE STUDY: This study aimed to determine the in vitro effects of DPP on the efficiency of neonate mouse spermatogonial stem cells (SSCs) proliferation. MATERIAL AND METHODS: Sertoli and SSCs were isolated from 6 to 10-days-old mouse testes, and their identity was confirmed using immunocytochemistry against cytokeratin for sertoli cells and PLZF, Oct-4 and CDH-1 for SSCs. Isolated testicular cells were cultured in the absence or presence of 0.06, 0.25 and 0.62mg/ml concentrations of DPP aqueous extract for 2 weeks. The number and diameter of SSC colonies were assessed during third, 7th, 9th and 14th day of culture, and the expression of the Mvh, GFRα-1 and Oct-4 was evaluated using quantitative PCR at the end of the culture period. The significance of the data was analyzed using ANOVA and paired samples t-test and Tukey and Bonferroni test as post hoc tests at the level of p≤0.05. RESULTS: Pattern assay of colony formation showed that SSCs numbers increased in the present of 0.62mg/ml concentration of DPP extract with higher slop relative to other groups (P <0.05). Colony diameters had no significant difference between groups in 3th, 7th, 9th and 14th days after culture. The Mvh and Oct-4 genes expression had no significant difference between groups, while GFRα1 expression was increased significantly in cells treated with 0.06mg/ml concentration relative to other groups (P<0.05). CONCLUSION: It seems that co-culture of SSCs with sertoli sells in the presence of low doses of DPP can increase SSCs proliferation and keep their stemness state, while higher concentrations can differentiate the treated cells.

Effects of Common Fig (Ficus carica) Leaf Extracts on Sperm Parameters and Testis of Mice Intoxicated with Formaldehyde.

Formaldehyde (FA) is the leading cause of cellular injury and oxidative damage in testis that is one of the main infertility causes. There has been an increasing evidence of herbal remedies use in male infertility treatment. This assay examines the role of Ficus carica (Fc) leaf extracts in sperm parameters and testis of mice intoxicated with FA. Twenty-five adult male mice were randomly divided into control; sham; FA-treated (10 mg/kg twice per day); Fc-treated (200 mg/kg); and FA + Fc-treated groups. Cauda epididymal spermatozoa were analyzed for viability, count, and motility. Testes were weighed and gonadosomatic index (GSI) was calculated. Also, histoarchitecture of seminiferous tubules was assessed in the Haematoxylin and Eosin stained paraffin sections. The findings showed that FA significantly decreased GSI and increased percentage of immotile sperm compared with control group. Disorganized and vacuolated seminiferous epithelium, spermatogenic arrest, and lumen filled with immature germ cells were also observed in the testes. However, Fc leaf extracts improved sperm count, nonprogressive motility of spermatozoa, and GSI in FA-treated testes. Moreover, seminiferous tubule with spermatogenic arrest was rarely seen, indicating that Fc has the positive effects on testis and epididymal sperm parameters exposed with FA.

In vitro cytotoxicity effects of date palm (Phoenix dactylifera L.) pollen on neonate mouse spermatogonial stem cells.

There is a fast growing tendency in the use of herbal remedies in developing countries. One of the traditional medicines used for male infertility treatment is date palm (Phoenix dactylifera) pollen (DPP). Isolated spermatogonial stem cells and sertoli cells using enzymatic digestion were grown in Dulbecco's modified Eagle's medium supplemented with 4% foetal bovine serum in the absence or presence of 0.06, 0.25 and 0.62 mg/mL concentrations of aqueous extract of DPP for 2 weeks. The assessment of mean number of the whole cells and the living cells showed that there were no significant differences between the mean viability percentage and proliferation rate between control and experimental groups (P>0.05). As there are no cytotoxicity effects of DPP in our cultural system, this system can be utilised for the enrichment or differentiation of these cells in clinical applications, cell replacement therapy, tissue regeneration and tissue engineering applications.