Expression of miR-302 in human embryo derived from in-vitro matured oocyte

Background: The expression of miR-302 over the period of early embryogenesis could possibly regulate the maternal transcript clearance. Zygotic transcription activation is mostly related to maternal messages degradation. Objective: In this study, the effects of in-vitro maturation technique (IVM) on the expression of miR-302 in human embryo produced from immature and mature human oocytes (matured in vitro and in vivo, before sperm exposure) obtained from females under gonadotrophin therapy were evaluated for assisted reproduction. Materials and Methods: Immature oocytes were cultured in vitro. The injection of oocytes-producing polar bodies was given using fresh sperm. Then, the embryo quality score was assessed in the IVM group compared with the control group. In both the groups, embryos with normal morphology were included in the molecular study. Only one blastomere was removed from three-day embryos and then the embryos were frozen. The expression of miR-302 in embryos was measured through quantitative realtime polymerase chain reaction. Results: Our data showed a significant reduction of miR-302 expression in the IVM group vs. the control group (p = 0.02). The embryo quality score showed a significant difference between the two groups (p = 0.01). Conclusion: The present study demonstrated that the IVM process had a negative effect on the expression level of miR-302 in human pre-implantation embryos. Considering the major role of expression miR-302, a reduced potential in miR-302 expression could be related to a decrease in the early embryonic development

Expression of Endothelial Nitric Oxide Synthase in Testicular Cells of Men with Obstructive Azoospermia; a Case-Control Study

Introduction: Obstructive azoospermia is one of the causes of post-testicular infertility in men and previous studies have reported inconsistent levels of endothelial nitric oxide synthase (eNOS) enzyme in these patients. Accordingly, the present study aimed to provide further evidence on the expression of eNOS enzyme in patients with azoospermia. Materials and Methods: In this case-control study, 10 patients, who were diagnosed with azoospermia and were referred to the infertility center for treatment or diagnosis, and 7 healthy fertile men were recruited. An informed written consent was obtained from included subjects and they underwent testicular biopsies. Samples were assessed via immunohistochemical methods to determine their levels of eNOS expression. Results: Both leydig and sertoli cells were found to express eNOS, while this enzyme was not expressed in normal germinal cells. The only significant difference between the two groups was the level of eNOS expression in sertoli cells which was found to be higher in patients with obstructive azoospermia compared to the control group (P<0.001). Conclusion: According to the results of this study, sertoli cells and their interactions with germinal cells of seminiferous tubule might play an important role in sperm quality and a subsequent successful fertilization

Aberrant Wnt/β-Catenin Signaling Pathway in Testis of Azoospermic Men​

Purpose: The Importance and key role of Wnt/β-catenin signaling pathway in spermatogenesis is known. Abnormalities of this pathway in Sertoli and germ cells leads to infertility. Leydig cells play an important role in spermatogenesis and male reproduction. As of now, exact position of the Wnt/β-catenin signaling pathway disorders in the tissue and possible involvement of Leydig cells has not been investigated. Methods: Samples of our previous study were used for common Y chromosome microdeletions screening and common CFTR gene mutations.1 β-catenin gene expression were evaluated and compared between testicular tissue obtained by testicular sperm extraction (TESE) in two groups of obstructive (n=10) and non-obstructive (n=10) azoospermic infertile men. Location of β-catenin accumulation was detected by immunofluorescence technic and quantitatively compared in the tissue followed by counterstaining with anti-vimentin antibody. It was used as specific marker of leydig cells to determine and confirm the cells in which this gathering was occurred. Results: β-catenin gene expression does not have a significant difference between the obstructive azoospermia (0.998) and non-obstructive azoospermia group (0.891). β-catenin was abnormally aggregated in leydig cell of non-obstructive azoospermic men. Conclusion: Gathering β-catenin in cytoplasm of leydig cells can disrupt spermatogenesis and cause infertility in men

Male obesity is associated with sperm telomere shortening and aberrant mRNA expression of autophagy-related genes

Abstract Background Obesity is regarded a global public health crisis. It has been implicated in a variety of health problems, but when it comes to male fertility, how and to what extent obesity affects it are poorly understood. Accordingly, semen samples from 32 individuals with obesity (body mass index (BMI) ≥ 30 kg/m2) and 32 individuals with normal weight (BMI: 18.5-25 kg/m2) were obtained. Here, for the first time, we examined the association between obesity, relative sperm telomere length (STL) and autophagy-related mRNA levels such as Beclin1, AMPKa1, ULK1, BAX, and BCL2. Each group was also evaluated for conventional semen parameters, sperm apoptotic changes, DNA fragmentation index (DFI), sperm chromatin maturation, and reactive oxygen species (ROS) levels. Results Based on our findings, there was a marked reduction in relative STL in individuals with obesity as compared to the normal-weight group. We also found a significant negative correlation between relative STL and age, BMI, DFI, percentage of sperm with immature chromatin, and intracellular ROS levels in patients with obesity. In the normal-weight group, relative STL was only negatively correlated with DFI and intracellular ROS levels. Regarding mRNA expression, there was considerable upregulation of Beclin1, ULK1, and BCL2 in the group with obesity compared to the normal-weight group. Obesity was also found to be associated with a considerable decline in semen volume, total sperm count, progressive motility, and viability in comparison to normal-weight individuals. Furthermore, obesity was associated with considerably higher percentages of DFI, sperm with immature chromatin, late-stage apoptosis, and elevated ROS levels. Conclusion According to our findings, obesity is associated with sperm telomere shortening and aberrant autophagy-related mRNA expression. It should be emphasized that telomere shortening in sperm may be an indirect consequence of obesity due to the oxidative stress associated with the condition. Nevertheless, further investigation is required for a more comprehensive understanding

Vitrification induced apoptosis in spermatozoa from fertile and subfertile men

Background: Phospholipids are distributed asymmetrically between inner and outer leaflets of the plasma membrane of live cells. Early during apoptosis, this asymmetry is disrupted and phosphatidylserine becomes exposed on the outside surface of the plasma membrane. There is little information about the effects of vitrification on apoptosis. Objective: The aim of the present study was to evaluate the effect of vitrification on apoptosis of subfertile and fertile men. Materials and Methods: In this study, semen samples were collected from subfertile (n=20) and fertile men (n=10) after 48h abstinence of intercourse. After semen analysis according to WHO criterias, each semen sample was divided into two portions. First portion was assessed by Annexin V-flous staining kit for showing apoptosis in subfertile and fertile men and second portion was assessed after vitrification-thawing. Results were analyzed by Paired t-test and Independent t-test. Results: After vitrification-thawing, mean percentage of apoptotic spermatozoa has increased 6 and 3 times in subfertile and fertile men respectively. This difference is significant. Conclusion: Vitrification-thawing could disrupted membrane asymmetry and caused apoptosis. Therefore, it will cause reduction of functional spermatozoa in access of Assisted Reproduction Technologies (ART)

In vitro application of Matrigel enhances human blastocyst formation and hatching

Background: Matrigel (extracellular matrix) can improve the growth of many cell types in vitro. Objective: The aim of the present study was to determine the effect of Matrigel on the development of 2-4 cells human embryos in culture. Material and Methods: Surplus 2-4 cells human embryos, resulting from ICSI, were divided into two groups (control and test). Quality of embryos in both groups was morphologically similar. The test group (n=140) was cultured in Hams' F10 supplemented with 10% human serum albumin and 150 µl liquid Matrigel. The control group (n=140) was cultured in the same medium devoid of Matrigel. Embryos were cultured for an additional 4 days and their morphology was assessed every 24 hours. Both groups were then statistically compared. Results: The percentage of the human embryos that reached the morula stage in the control and test groups were 79.2% and 80%, respectively (p>0.05). However, 36.4% of embryos reached the blastocyst stage in the test group as compared to 5.7% in the control group after 144 hours in culture. This difference was statically significant (p <0.01). In addition, culture of embryos on Matrigel and medium versus medium alone significantly improved in vitro hatching (25.7% versus 3.5%; p <0.01). Conclusion: Matrigel at low concentration enhances human blastocyst formation and hatching in vitro

Adverse effects of ciprofloxacin on testis apoptosis and sperm parameters in rats

Background: Ciprofloxacin is a commonly prescribed antibiotic in the treatment of genitourinary tract infection. Objective: The aim of this study was to investigate the effects of ciprofloxacin on testis apoptosis and sperm parameters in rat. Materials and Methods: Twenty male Wistar rats were selected and randomly divided into two groups; control (n=10) and experimental (n=10). The experimental group was orally received 12.5 mg/kg ciprofloxacin daily for 60 days and the control group just received water and food. Rats were then killed and sperm removed from cauda epididymis and analyzed for sperm motility, morphology, and viability. Testis tissues were also removed and prepared for TUNEL assay to detect apoptosis. Results: Results showed that ciprofloxacin significantly decreased the sperm concentration, motility (p<0.05) and viability (p<0.001). In addition, ciprofloxacin treatment resulted in a significant decrease in the number of spermatogenic cells (spermatogonia, spermatocyte, spermatid and sperm) in the seminiferous tubules when compared with the control group. The apoptotic germ cells per seminiferous tubular cross section was significantly increased in the experimental group (15.11±3.523) as compared with the control group (7.3±0.762) (p<0.05). Conclusion: It is concluded that ciprofloxacin has the toxicological effects on reproductive system in male rats

Vitamins C, E and lipid peroxidation levels in sperm and seminal plasma of asthenoteratozoospermic and normozoospermic men

Background: It has been shown that reactive oxygen species (ROS) can lead to deleterious effects on a range of sperm parameters. Vitamins E and C are capable of reducing ROS levels and lipid peroxidation. Objective: The aim of study was to determine the level of lipid peroxidation as indicated by Malondialdehyde (MDA) and vitamins E and C in sperm and seminal plasma of asthenoteratozoospermic and normozoospermic men and their relationships with semen parameters. Materials and Methods: Forty men with normozoospermic and 60 infertile men with asthenoteratozoospermic semen profiles were randomly selected. Semen analysis was done according to the WHO standard. Sex hormonal profiles were measured by ELISA methods. The level of vitamins (C and E) and MDA were measured by HPLC and tiobarbiotic acid, respectively. Results: MDA concentration in the spermatozoa (0.1±0.06 nmol/ml) and seminal plasma (1.9±0.35 nmol/ml) of asthenoteratozoospermic were significantly higher than in normozoospermic males (p<0.001).The level of vitamins E and C in seminal plasma of normozoospermic were significantly higher than in asthenoteratozoospermic males (p<0.01). However, the amount of vitamin C in the spermatozoa of normozoospermic was significantly less than in asthenoteratozoospermic males (p<0.01). Sensitivity, specificity, positive and negative prognostic value of MDA of seminal plasma and spermatozoa were more than vitamins C and E. The level of vitamin C of spermatozoa had more diagnostic value when compare to vitamins C and E in seminal plasma. Conclusion: The level of MDA in seminal plasma and spermatozoa and vitamin C in spermatozoa may be a diagnostic tool for etiology of infertility in the asthenoteratozoospermic patients

Exposure of neonatal mice to Sevoflurane may induce male germ cell apoptosis in testicular tissue after puberty

Background: common use of sevoflurane in congenital defects during repeated surgeries may have detrimental effects on spermatogenesis after puberty. Objective: This study investigated sevoflurane effects on spermatogenesis process in male mature mice after exposure in prepubertal time. Materials and Methods: 24 neonatal NMRI male mice were randomly classified in three groups. Experimental 1 and 2 groups (exposure to 1 minimum alveolar concentration (MAC) and 2 MAC sevoflurane, respectively in 2 lit/min oxygen (O2) for 7 days (30 min, daily) and control. All groups were sacrificed after 2 months. Histological assessment, immunohistochemistry and apoptosis process was done. Bax and Bcl2 expression was evaluated in the testicular tissue by real time Poly Chain Reaction. Results: Our results showed that the integrity of testicular tissue was preserved in both experimental groups. Count of spermatogonial cells had significant decrease in group 2 compared to others. The rate of apoptosis in spermatogonial cells was 15&plusmn;3% and 9&plusmn;2% in the group 2 and 1, respectively. Also, Bax/Bcl2 ratio was 0.2615, 1.0070 and 9.3657 in control, experimental group 1 and 2, respectively. This result was significant (p&le;0.002) between groups 2 with other groups. Conclusion: Continuous exposure of 2 MAC sevoflurane in 2 lit/min O2 simultaneous during prepubertal may create more testicular tissue damage in terms of cellular and molecular function compared to continuous exposure to lower level of sevoflurane by increase in ratio of Bax/Bcl2 and apoptosis in germ cells after puberty