Association of Single Nucleotide Polymorphisms in the PYGO2 and PRDM9 Genes with Idiopathic Azoospermia in Iranian Infertile Male Patients.

Background: Azoospermia is a risk factor for infertility affecting approximately 1% of the male population. Genetic factors are associated with non-obstructive azoospermia (NOA). Pygo2 and PRDM9 genes are involved in the spermatogenesis process. The present study aimed to assess the association of single nucleotide polymorphism (SNP) in the Pygo2 (rs61758740 and rs61758741) and PRDM9 (rs2973631 and rs1874165) genes with idiopathic azoospermia (IA). Methods: A cross-sectional study was conducted from October 2018 to August 2019 at Rooya Infertility Centre (Qom, Iran). A total of 100 infertile patients with NOA and 100 men with normal fertility were enrolled in the study. Tetra-primer amplification refractory mutation system-PCR method was used to detect SNPs rs61758740, rs61758741, and rs2973631. The restriction fragment length polymorphism method was used for SNP rs1874165. In addition, luteinizing, follicle-stimulating, and testosterone hormone levels were measured. Results: The results showed a significant increase in luteinizing and follicle-stimulating hormone levels in the patient group (P<0.001), but a non-significant difference in testosterone levels in both groups. SNP rs61758740 (T>C) was associated with the increased risk of azoospermia (OR: 2.359, 95% Cl: 1.192-4.666, P=0.012). SNP rs2973631 showed a significant difference in genotype frequency between the patient and control groups in the dominant, recessive, and codominant models. However, in the case of SNP rs1874165, the difference was significant in the dominant, codominant, and overdominant models. Conclusion: There is an association between azoospermia and SNPs in Pygo2 and PRDM9 genes in Iranian infertile male patients with IA. SNPs can be considered a risk factor for male infertility. It should be noted that this article was published in preprint form on the website of europepmc (https://europepmc.org/article/ppr/ppr416800).

Effect of Lactobacillus plantarum on folliculogenesis in deep frying oil-fed rats.

Today, the tendency towards Western World diet characterized by a high consumption of Deep Frying Oil (DFO), as well as female infertility has increased. On the other hand, probiotics are living microorganisms that can benefit human health. Therefore, this study aimed to investigate the effect of a probiotic treatment (Lactobacillus plantarum) on the process of follicular growth in rats fed with DFO. Twenty adult female Wistar rats were divided into four groups: control, DFO treatment, DFO treatment group receiving probiotic and the healthy group receiving probiotic for one month. After blood sampling and dissection, ovarian tissue was examined for the number of ovarian follicles at different stages. In addition, the expression of Bmp15 (Gdf-9b) and Gdf9 genes was assessed by the real-time PCR method. The ELISA test was also used to measure hormonal changes (LH and FSH). Data showed that rats treated with DFO had a significant decrease in follicle numbers, hormone levels and Bmp15 and Gdf9 gene expression. Moreover, the number of atretic follicles was increased. Treatment of rats with the probiotic reduced the observed side effects of DFO. Thus, treatments of rats with the probiotic mitigated some of the observed side effects of DFO. An increase in primordial follicles and a reduction of atretic follicles was indicated compared to the DFO group (P ≤ 0.001). Lactobacillus plantarum could reduce the detrimental effects of DFO on folliculogenesis through its beneficial effects.

USP7 and SET9 Expression in The Oligospermic Human Semen: A Case-Control Study.

BACKGROUND: Oligospermia is defined as a less than 15 million per milliliter sperm in each ejaculation of semen. Proper and complete spermatogenesis requires the expression of a large number of genes. As a result, stopping the expression of any of them may lead to disrupt the process of spermatogenesis. In order to understand the disorders of spermatogenesis, it is necessary to study expression of effective genes in the spermatogenesis process. Therefore, in the present study, USP7 and SET9 (SETD7) gene expression was examined in the healthy and oligospermic men. MATERIALS AND METHODS: In this case-control study, semen samples of individuals with normal sperm and oligospermia were collected from men who referred to the Roya Clinic (Qom, Iran) according to World Health Organization (WHO) parameters after obtaining consent. Then the expression of USP7 and SET9 genes in two groups was analyzed using quantitative polymerase chain reaction (qPCR). RESULTS: There was no difference forage between the healthy and oligospermic individuals (P=0.889). The data showed that, USP7 gene expression in the patients was 3.99 times higher than the control group (P<0.001). The expression of SET9 gene in the patient was 1.28 times less than the control group, which was not significant (P=0.231). The results indicated that USP7 gene expression was increased in the 84% of oligospermic individuals. CONCLUSION: The USP7 gene can be considered as one of the molecular markers in the development of oligospermia.

The effect of acrylamide on sperm oxidative stress, total antioxidant levels, tyrosine phosphorylation, and carboxymethyl-lysine expression: A laboratory study.

BACKGROUND: Acrylamide (AA) is a reactive molecule produced during food processing at temperatures above 120∘C. OBJECTIVE: To evaluate the impact of different concentrations of AA on human sperm parameters, oxidative stress and total antioxidant capacity (TAC). MATERIALS AND METHODS: In this laboratory study, semen samples were obtained from healthy donors referred to the Taleghani Hospital, Tehran, Iran between June and July 2019. Samples were divided into four groups (n = 10/each): one control and three treatment groups (0.5, 1, and 2 mM of AA). After 2 hr of exposure to AA, the superoxide dismutase and malondialdehyde levels were measured based on colorimetric methods. The TAC was determined by the ferric-reducing antioxidant power assay. Flow cytometry was performed to measure the intracellular reactive oxygen species generation. Also, immunohistochemistry was done to determine the effect of AA on tyrosine phosphorylation and carboxymethyl-lysine expression. RESULTS: Results of the study demonstrated that the motility and viability of spermatozoa were significantly decreased after AA exposure (p < 0.001). This decrease was also seen in the TAC and superoxide dismutase activity as well as in the phosphotyrosine percentage compared with the control (p < 0.01). However, the carboxymethyl-lysine and prooxidant activity including reactive oxygen species generation and lipid peroxidation level increased (p < 0.001). CONCLUSION: Overall, the results confirmed the detrimental effect of AA on human spermatozoa which may be due to oxidative stress and decreased total antioxidant levels. AA may reduce fertility by reducing sperm capacitation and motility.

The effect of acrylamide on mitochondrial membrane potential and glutathione extraction in human spermatozoa: A laboratory study.

BACKGROUND: Acrylamide (AA) is a compound used in the industrial production of polyacrylamide. AAs affects by creating oxidative stress. It produces reactive oxygen species and leads to lipid peroxide. Lipid peroxidation in the cell membrane is one of the most important oxidations in the sperm, which can disrupt the fluidity and permeability of cell membranes and damage all cells. OBJECTIVE: To investigate the different concentrations of AA on human sperm parameters based on the World Health Organization standard and its impact on mitochondrial membrane potential and sperm glutathione levels. MATERIALS AND METHODS: In this laboratory study, we examined the different concentrations of AA on human sperm parameters based on the World Health Organization standard and its impact on mitochondrial membrane potential by flow cytometry and sperm glutathione levels by ELISA assay. RESULTS: The results were reported as the mean fluorescence intensity of JC and the index was observed to decrease following the effect of AA in mitochondrial membrane potential (Δ Ψ m). The results of ELISA test to study the level of intracellular glutathione showed that with the increase in the concentration of AA exposed to sperms, there was a significant reduction in the level of intracellular glutathione. CONCLUSION: AA destroys the sperm membrane integrity under apoptotic and oxidative inductions with a negative impact on mitochondrial function and antioxidative enzyme in sperm such as glutathione.

Identification of Nrf2/STAT3 axis in induction of apoptosis through sub-G 1 cell cycle arrest mechanism in HT-29 colon cancer cells.

We investigated the role of stattic as an adjuvant molecule to increase the cytotoxicity of 5-fluorouracil (5-FU) through specific inhibition of molecular targets, signal transducer and activator of transcription 3 (STAT3) and nuclear factor erythroid 2-related factor 2 (Nrf2) in HT-29 colon cancer cells. Cytotoxicity and apoptotic effects were investigated by methylthiazolyldiphenyl-​tetrazolium bromide assay and flow cytometry analysis, respectively. Real-time polymerase chain reaction was applied to assess the messenger RNA (mRNA) level of STAT3, Nrf2, and apoptotic genes including Bax, Bcl-xl, and Bcl-2. The antitumor effect of 5-FU in combination with stattic induced synergistic effect in HT-29 cells with combination indexes (CIs) 0.49. Flow cytometric results related to apoptotic confirmed that there was up to 40% increase in the population of apoptotic cells in HT-29 colon cancer cells incubated with 5-FU and stattic compared with control groups. Our data from gene expression determined a substantial diminish in the mRNA levels of the Nrf2 and antiapoptotic gene Bcl-2 along with a noticeable increase in the level of the proapoptotic Bax in HT-29 colon cells that underwent cotreatment with 5-FU and stattic (P < 0.05). Moreover, the results exhibited that stattic can be used as adjuvant chemotherapy besides the 5-FU. This therapeutic approach in colon cancer could mediate 5-FU chemoresistance via modulating therapeutic targets (ie, STAT3 and Nrf2 pathways) and decreased 5-FU-related adverse effects.

Neuroprotective Effect of Total and Sequential Extract of Scrophularia striata Boiss. in Rat Cerebellar Granule Neurons Following Glutamate- Induced Neurotoxicity: An In-vitro Study.

Neuroprotective effect of the extract from aerial parts of Scrophularia striata Boiss (Scrophulariaceae) was investigated against glutamate-induced neurotoxicity on cultured rat pups Cerebellar Granule Neurons (CGNs). CGNs from 8 days old Sprague-Dawley rat were prepared and cultured. The experiments were performed after 8 days in culture. The plant was collected from the northeastern part (Ruin region) of Iran and air-dried at room temperature. The total extract was prepared with maceration of prepared powder in ethanol 80% for three times. SEQUENTIAL EXTRACTS WERE OBTAINED USING DRIED AND POWDERED AERIAL PARTS WITH INCREASINGLY POLAR SOLVENTS: petroleum ether, chloroform, ethyl acetate and methanol 80% solution. Cultured cells were exposed to 125 µM of glutamate for 12 h following a 24 h of incubation with test fractions at concentration of 10 mcg/mL. Morphological assay was performed using invert light microscope after fixation and staining with haematoxylin. Neuronal viability was measured using MTT assay. Statistical analysis was done using SPSS software. One way analysis of variance (ANOVA) was performed by Tukey post-hoc test. Values were considered statistically significant when p-value ≤ 0.05. Results of this study showed a significant neuroprotective activity of high polarity methanolic fraction of aerial parts of Scrophularia striata against glutamate-induced neurotoxicity in a dosedependent manner. Treatment with 10 mcg/mL of the fractions showed the best result.

Melatonin effect during different maturation stages of oocyte and subsequent embryo development in mice.

BACKGROUND: It is important to protect oocytes and embryos from oxidative stress in the culture medium. Melatonin has been shown to be a direct free radical scavenger. OBJECTIVE: Effect of melatonin during in vitro oocyte maturation, fertilization and embryo development of mouse oocytes was evaluated. MATERIALS AND METHODS: Oocytes from supper-ovulated mouse were divided to two groups: cumulus oocyte complexes (COCs, group I) and denuded COC (d-COCs, group II). The oocytes were cultured in maturation medium with different doses of melatonin (1×10(1)-10(5) nM). The cumulus expansion and nuclear status were evaluated after 24 h of in-vitro maturation. The oocytes were used for in-vitro fertilization. The fertilized oocytes were cultured in medium supplemented with different doses of melatonin. RESULTS: The expansion (86.79%) and maturation (80.55%) rate of COCs increased in supplemented medium with 10 nM of melatonin vs. control group (73.33%), p=0.006 and p=0.026 respectively), but oocytes without cumulus cells indicated higher maturation rate at higher melatonin doses (10 and 100 M, 84.34% and 79.5% respectively( vs. 69.33% in control group (p=0.002). Fertilization rate was higher in treated medium with 1 µM of melatonin (93.75%, p=0.007). The rate of cleavage and blastocyst formation was promoted in medium supplemented with 10 and 100 nM of melatonin (92.37% and 89.36% vs. 81.25% in control group, p=0.002). We observed a dose dependent response to melatonin treatment in this experiment. CONCLUSION: Exogenous melatonin can promote cumulus cell expansion, in vitro oocyte maturation, and embryo development. However we investigated a dose-dependent response in different stages of maturation and development. It may reflect sensitive rate of oocytes and embryos to culture conditions.

The effect of melatonin on the developmental potential and implantation rate of mouse embryos.

OBJECTIVE: Melatonin is a scavenger agent that has been used to promote in vitro embryo development. This study was designed to show the effects of melatonin on the quality and quantity rate of preimplantation mouse embryo development and pregnancy. MATERIALS AND METHODS: In this experimental study, super ovulated, mated mice were killed by cervical dislocation to collect two-cell zygotes from the oviduct of pregnant 1 day NMRI mice. Zygotes were cultured to the hatching blastocyst stage and the numbers of embryos at different stages were recorded under an inverted microscope. The cleavage rates of two-cell zygotes were assayed until the blastocyst and hatching blastocyst stage in drops of T6 medium that contained either melatonin (1, 10, and 100×10(6), 10 and 100×10(9) M) or no melatonin. The cell numbers of blastocysts were determined by differential staining, implantation outcomes were studied, and development and pregnancy rate were compared by the Chi-square (development) and Fisher's exact (pregnancy rate) tests. RESULTS: The addition of 10 and 100 nM melatonin to the embryo culture media promoted the development of the two-cell stage embryos to blastocyst and hatching blastocysts (p<0.01) and caused a significant increase in total cell number (TCN), trophoectoderm (TE), and inner cell mass (ICM) of the blastocysts (p<0.01). A difference was observed in the percentage of transferred embryos that were successfully implanted between the control and treatment groups (p<0.05). CONCLUSION: The data indicate that 10 and 100 nM of melatonin positively impact mouse embryo cleavage rates, blastocyst TCN, and their implantation. Therefore, melatonin at low concentrations promotes an embryonic culture system in mice.

Evaluation of Enamel Matrix Derivative (EMD) Teratogenicity on the Rat Embryo Neural Crest Culture.

Enamel matrix derivative Emdogain (EMD) is widely used in periodontal treatment in spite of the fact that its effect on the developing embryo has not been elucidated. The aim of this study was to investigate the teratogenic effect of EMD on the rat embryo neural crest cells. The neural crest is a unique population of cells that migrates from the dorsal neural tube along defined pathways and produces various cell types including the melanocytes, neuronal and glial cells of the sensory, autonomic and enteric nervous system as well as the chromaffin cells of the adrenal gland. These cells have been used extensively for in-vitro studies of neurogenesis. Cultured cells by micromass culture method derived from midbrain of six embryos (13 day postcoitum; 34-36 smites) and exposed to various concentrations of EMD for 5 days at 37°C and differentiated foci were counted. Retinoic Acid (20 µg/mL) was used as standard positive control. These cells were stained using Mayer's hematoxylin which is specific for staining differentiated cell nucleus. Neutral red staining determines cell viability rather than related cell differentiation but is used for normalization of Mayer's hematoxylin results. At the concentration as low as 8 µg/mL of EMD, no toxic effect on fetal cells was observed and it is suggested that EMD has no teratogenic effect at studied concentrations.

Effect of three vitrification methods on development of two-cell mouse embryos.

The purpose of this study was to investigate the effect of three vitrification procedures [conventional straw (CS), open pulled straw (OPS) and closed pulled straw (CPS)] on the development of two-cell mouse embryos. Two-cell mouse embryos were randomly divided into vitrified and non-vitrified control groups. Embryos in the vitrified group were cryopreserved within a combination of 5.5 M ethylene glycol and 1M sucrose as cryoprotectants, loaded within three different straws (CS, OPS and CPS) and warmed in stepwise sucrose solutions. The survived embryos from each procedure were cultured in human tubal fluid (HTF). The non-vitrified control embryos were also cultured in the same manner. The rates of the development in all the groups were daily determined and statistically compared. On day 4 of the cultivation period, several expanded blastocysts from each group were randomly selected and stained either with propidium iodide (PI) and bisbenzimide or with terminal transferase- mediate DNA end labeling (TUNEL) Technique. The mean number of the inner cell mass (ICM), trophoectoderm (TE), necrotic and apoptotic cells were counted and statistically compared. The survival rate of embryos in CPS was significantly higher than that in OPS and CS. The rate of hatched blastocysts did not differ in the three vitrification procedures, but in comparison with that of the control, CS and OPS showed a significant reduction. The mean number of ICM and TE decreased in CS and OPS, whereas in CPS it was almost identical to that of the control. The incidence of apoptosis and necrosis appeared to be almost similar in all the groups. In conclusion, CPS seems to be an effective, easy and rapid method for the cryopreservation of two-cell mouse embryos.