Effect of Formaldehyde and Curcumin on Histomorphological Indices, Gene Expression Associated with Ovarian Follicular Development, and Total Antioxidant to Oxidant Levels in Wistar Rats.

The present experimental study was undertaken to investigate the effect of formaldehyde (FA) and curcumin (CUR) on histomorphological features, antioxidant potential, and messenger ribonucleic acid (mRNA) levels of genes related to follicular development in FA-exposed rats. 24 Wistar female rats were divided into four study groups and given intraperitoneal injections of FA (10 mg/kg) (N = 6), FA (10 mg/kg) + CUR (100 mg/kg) (N = 6), sham (N = 6), and control (N = 6) for 14 days. Ovarian follicular histology, the related gene expression, blood factors, and anti/oxidation potentials were assessed using ovarian tissue and serum, respectively. The klotho was significantly overexpressed in the FA group compared with controls and shams. Contradictory, the factor in germ line alpha was significantly down-regulated in FA and FA + CUR groups compared to shams and controls. A significant decline was seen in the number of ovarian follicles in the FA group, independent of the developmental stage. Regarding the comparison of the FA + CUR group to other groups, a significant change was seen in the number of secondary, graafian, and atretic follicles. The FA group demonstrated significantly lower hemoglobin, red blood cell count, hematocrit, and mean corpuscular hemoglobin concentration than controls. The activity of glutathione peroxidase increased significantly in the FA group than in the controls. Despite the deleterious effects of FA on histological and molecular aspects of rat ovarian follicles, CUR does not appear to have a protective effect against the hazardous effects of this chemical. However, CUR in some cases has positive effects such as reducing follicular destruction and interstitial edema.

Effect of methamphetamine on ultraweak photon emission and level of reactive oxygen species in male rat brain.

All living cells, including neurons, generate ultra-weak photon emission (UPE) during biological activity, and in particular, in the brain, it has been shown that UPE is correlated with neuronal activity and associated metabolic processes. Various intracellular factors, as well as external factors, can reduce or increase the intensity of UPE. In this study, we have used Methamphetamine (METH) as one potentially effective external factor, which is a substance that has the property of stimulating the central nervous system. METH can impair mitochondrial function by causing toxicity via various pathways, including an increase in the number of mitochondria, hyperthermia, the increased metabolic activity of the brain, and the production of glutamate and excess calcium. In addition to mitochondrial dysfunction, METH alters cellular homeostasis, leading to cell damage and the production of excess ROS. The aim of this study is to measure and compare the UPE intensity and reactive oxygen species (ROS) levels of the prefrontal, motor, and visual cortex before and after METH administration. Twenty male rats were randomly assigned to two groups, the control, and METH groups. In the control group, 2 h after injection of normal saline and without any intervention, and in the experimental group 2 h after IP injection of 20 mg/kg METH, sections were prepared from three areas: prefrontal, motor, and V1-V2 cortex, which were used to evaluate the emission of UPE using a photomultiplier tube (PMT) device and to evaluate the amount of ROS. The results showed that the amount of ROS and UPE in the experimental group in all three areas significantly increased compared to the control group. So, METH increases UPE and ROS in the prefrontal, motor, and visual regions, and there is a direct relationship between UPE intensity and ROS production. Therefore, UPE may be used as a dynamic reading tool to monitor oxidative metabolism in physiological processes related to ROS and METH research. Also, the results of this experiment may create a new avenue to test the hypothesis that the excess in UPE generation may lead to the phenomenon of phosphene and visual hallucinations.

Protective effects of curcumin on chromatin quality, sperm parameters, and apoptosis following testicular torsion-detorsion in mice.

OBJECTIVE: The chief outcome of testicular torsion in clinical and experimental contexts is testicular ischemia. Curcumin, a compound with anti-inflammatory and antioxidant properties, has fascinated researchers and clinicians for its promise in the treatment of fertility diseases. METHODS: Thirty-five fully grown male mice were randomly classified into five groups: control, sham, testicular torsion, treatment group 1 (testicular torsion+short-term curcumin), and treatment group 2 (testicular torsion+long-term curcumin). Thirty-five days later, spermatozoa from the right cauda epididymis were analyzed with regard to count and motility. Toluidine blue (TB), aniline blue (AB), and chromomycin A3 (CMA3) staining assays were used to evaluate the sperm chromatin integrity. In addition, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) test was used to assess apoptosis. RESULT: Treatment group 1 exhibited a remarkably elevated sperm count compared to the testicular torsion group. Additionally, notably lower sperm motility was found in the testicular torsion group compared to the control, treatment 1, and treatment 2 groups. Staining (CMA3, AB, and TB) and the TUNEL test indicated significantly greater testicular torsion in the torsion group compared to the control group (p<0.05). The data also revealed notably lower results of all sperm chromatin assays and lower apoptosis in both treatment groups relative to the testicular torsion group (p<0.05). Significantly elevated (p<0.05) AB and TB results were noted in treatment group 1 compared to treatment group 2. CONCLUSION: Curcumin can compensate for the harmful effects of testicular ischemia and improve sperm chromatin quality in mice.

Effects of acrylamide in the presence of vitamin E on sperm parameters, chromatin quality, and testosterone levels in mice.

OBJECTIVE: The present study investigated sperm chromatin quality and testosterone levels in acrylamide-treated mice and the possible protective effects of vitamin E on the fertility potential of spermatozoa. METHODS: Thirty-two adult male mice were divided equally into four groups. Group 1 was the control, group 2 received acrylamide (10 mg/kg, water solution), group 3 received vitamin E (100 mg/kg, intraperitoneal), and group 4 received both acrylamide and vitamin E. After 35 days, spermatozoa from the right cauda epididymis were analyzed in terms of count, motility, morphology, and viability. Sperm DNA integrity and chromatin condensation were assessed by acridine orange (AO), aniline blue (AB), toluidine blue (TB), and chromomycin A3 (CMA3) staining. RESULTS: In acrylamide-treated mice, significantly lower sperm concentration, viability, motility, and testosterone levels were found in comparison with the control and acrylamide+vitamin E groups (p<0.05). In the vitamin E group, significantly more favorable sperm parameters and testosterone levels were found than in the other groups (p<0.05). There were also significantly more spermatozoa with less condensed chromatin in the acrylamide-treated mice than in the other groups. Moreover, significantly more spermatozoa with mature nuclei (assessed by AB, CMA3, AO, and TB staining) were present in the vitamin E group than in the control and acrylamide+vitamin E groups. CONCLUSION: This study revealed the deleterious effects of acrylamide on sperm parameters and sperm chromatin quality. Vitamin E can not only compensate for the toxic effects of acrylamide, but also improve sperm chromatin quality in mice.

Growth differentiation factor 9 and cumulus cell supplementation in in vitro maturation culture media enhances the viability of human blastocysts.

OBJECTIVE: In vitro maturation (IVM) of immature oocytes can be useful for some infertile patients. In IVM programs, the rates of embryo formation and pregnancy are low. Therefore, it is essential to recognize the main factors involved in regulating oocyte maturation in vitro. The purpose of this study was to investigate the effects of growth differentiation factor 9 (GDF9) and cumulus cell (CC) supplementation in IVM medium on the rates of embryo formation and viability of human blastocysts. METHODS: A total of 80 germinal vesicle oocytes from stimulated cycles underwent an IVM program. The oocytes were divided into four groups, where group I consisted of IVM media only and served as the control, group II consisted of IVM+CCs, group III consisted of IVM+GDF9 (200 ng/mL), and group IV consisted of IVM+CCs+GDF9 (200 ng/mL). Intracytoplasmic sperm injection was performed on the IVM oocytes, and the cleavage embryos that were generated were vitrified. Following thawing, the embryos were cultured for 3 additional days, and the viability rates of the developed blastocysts were determined. RESULTS: The maturation rate of the oocytes did not differ significantly across the four groups. The fertilization rate in group II was significantly higher than that in the control group (76.5% vs. 46.2%). Embryo formation was significantly more frequent in all experimental groups than in the control group, while blastocyst formation did not show significant differences in the three experimental groups compared to the control. The mean viability rates in groups II, III, and IV were 58.16%, 55.91%, and 55.95%, respectively, versus 37.78% in the control group (p<0.05). CONCLUSION: Supplementation of IVM culture media with GDF9 and CCs enhanced the fertilization, embryo formation, and viability rates of blastocysts generated from vitrified cleavage embryos.

Effects of different doses of tamoxifen on the sperm parameters and chromatin quality in mice: An experimental model.

BACKGROUND: Tamoxifen (TX) is widely used for the treatment of male factor and idiopathic infertility. It has been shown that TX induces sperm production and so improves male fertility. OBJECTIVE: This study evaluated the effects of different doses of TX on the sperm parameters and chromatin quality in mice. MATERIALS AND METHODS: In this research, 24 male NMRI mice were divided into three groups including group A: control animal receiving vehicle; group B: the group receiving basal diet and TX 0.4 mg/kg/day; and group C: the group receiving basal diet and TX 0.6 mg/kg/day for 35 days. Thereafter, epididymal spermatozoa were analyzed for standard parameters and nuclear chromatin quality using Aniline Blue (AB) and Toluidine Blue (TB) staining. RESULTS: The results indicated that although the TX did not affect the sperm count, motility, and viability parameters, it could elevate the percentage of sperm cells with abnormal morphology and abnormal chromatin at both doses. In addition, in comparison with the control mice, a significant elevation was observed in spermatozoa with residual histones (assessed by AB staining) at high doses of TX. CONCLUSION: Our experimental data in mice suggested that the use of TX for treating male infertility might increase the rates of spermatozoa with abnormal chromatin in a dose-dependent manner.

Effects of Tamoxifen on DNA Integrity in Mice.

BACKGROUND: Tamoxifen (TX) is widely used to treat idiopathic infertility in men. Using TX has been shown to produce sperm in patients with oligospermia and azoospermia and improve male fertility. The aim of this study was to evaluate the effects of TX on DNA and chromatin quality in mice regarding the importance of chromatin quality and sperm DNA at all stages of reproduction. METHODS: 24 male NMRI mice were divided into 3 groups including dose 0.4 mg/kg/day that received basal diet and TX, dose 0.6 mg/kg/day that received basal diet and TX, and group 3 that received vehicle for 35 days as the control. After that, epididymal spermatozoa were analyzed for nuclear DNA quality. One-way ANOVA was performed with a Tukey test to compare sperm DNA fragmentation at different times. The p<0.05 was considered significant. RESULTS: The use of different doses of TX may have detrimental effects on sperm chromatin protamination and DNA integrity in mice. According to Acridine Orange (AO) staining, the rate of increased single-stranded DNA damage was observed at 0/6 mg/kg/day TX dose (p<0.05). CONCLUSION: The use of different studied TX doses in the animal sample was found to increase the amount of protamine deficiency and DNA defect in treated mice.

Do Insulin Replacement and Omega3 Protect the Male Reproductive Function of the Streptozotocin-Induced Diabetic Mice?

Diabetes mellitus (DM), the most common metabolic disease, might affect different organs such as male reproductive system. Experiments have shown that n-3 fatty acids could improve male reproductive function. Present study was performed to examine the effects of omega3 on sperms and testicular parameters in diabetic mice. Adult NMRI male mice were randomly divided into intact and diabetic groups (n = 8). Streptozotocin-induced diabetic animals were divided into 4 groups of diabetic-saline (Dia-Sa), diabetic-insulin (Dia-Ins), diabetic-omega3 (Dia-omg3), and diabetic-insulin-omega3 (Dia-Ins-omg3). Following confirmation of diabetes, different treatments including 3 U/100 g insulin subcutaneously and 400 mg/kg omega3 orally were administered, where applicable according to the treatment groups. Thirty-five days later, the sperm number, motility, progression, and normal morphology were determined. Also, testes diameters and structure including germinal epithelium thickness, seminiferous tubule diameters, Leydig cell number, and testosterone level were assessed. Sperm number, viability, fast motility, testes volume, and serum testosterone level decreased insignificantly in the Dia-Sa group compared with the intact animals. Neither insulin replacement nor omega3 administration could significantly improve the outcome. We might conclude that short periods of diabetes could not significantly affect the male reproductive function. In addition, insulin replacement and/or omega-3 supplementation does not have any profound effects on male reproductive system.

Vitrification of human immature oocytes before and after in vitro maturation: a review.

The use of immature oocytes subjected to in vitro maturation (IVM) opens interesting perspectives for fertility preservation where ovarian reserves are damaged by pathologies or therapies, as in PCO/PCOS and cancer patients. Human oocyte cryopreservation may offer some advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation and postponing childbirth. It also eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In addition, a successful oocyte cryopreservation program could eliminate the need for donor and recipient menstrual cycle synchronization. Recent advances in vitrification technology have markedly improved the oocyte survival rate after warming, with fertilization and implantation rates comparable with those of fresh oocytes. Healthy live births can be achieved from the combination of IVM and vitrification, even if vitrification of in vivo matured oocytes is still more effective. Recently, attention is given to highlight whether vitrification procedures are more successful when performed before or after IVM, on immature GV-stage oocytes, or on in vitro matured MII-stage oocytes. In this review, we emphasize that, even if there are no differences in survival rates between oocytes vitrified prior to or post-IVM, reduced maturation rates of immature oocytes vitrified prior to IVM can be, at least in part, explained by underlying ultrastructural and biomolecular alterations.

Mesenchymal Stem Cell-Conditioned Medium Modulates Apoptotic and Stress-Related Gene Expression, Ameliorates Maturation and Allows for the Development of Immature Human Oocytes after Artificial Activation.

The aim of the present study was to determine whether mesenchymal stem cell-conditioned medium (MSC-CM) modulates apoptotic and stress-related gene expression, and ameliorates maturation and developmental potential of immature human oocytes after artificial activation. A total of 247 surplus immature germinal vesicle (GV) oocytes obtained from infertile women were allocated into two in vitro maturation (IVM) groups: 1: GV oocytes (n = 116) matured in vitro (fIVM), and 2: GV oocytes (n = 131) that were vitrified, then in vitro matured (vIVM). Also, two maturation media were used: Alpha-minimum essential medium (α-MEM) and human umbilical cord-derived MSCs (hUCM). After 36 h of incubation, the IVM oocytes were examined for nuclear maturation. In IVM-matured oocytes, cytoplasmic maturation was evaluated after artificial activation through Ionomycin. Moreover, the quantitative expressions of B-cell CLL/lymphoma 2 (BCL2), BCL2-associated X protein (BAX), superoxide dismutase (SOD), and Heat shock proteins (HSP70) in matured oocytes were assessed by quantitative Real-time polymerase chain reaction (qRT-PCR) and compared with fresh and vitrified in vivo matured oocytes, which were used as fIVM and vIVM controls, respectively. The highest maturation rate was found in hUCM in fIVM, and the lowest maturation rate was found using α-MEM in vIVM (85.18% and 71.42%, respectively). The cleavage rate in fIVM was higher than that in vIVM (83.4% vs. 72.0%). In addition, the cleavage rate in α-MEM was lower than that in the hUCM (66.0% vs. 89.4%). Furthermore, the difference between parthenote embryo arrested in 4-8 cells (p < 0.04) and the quality of embryo arrested in 8-cell (p < 0.007) were significant. The developmental stages of parthenote embryos in hUCM versus α-MEM were as follows: 2-4 cell (89.45% vs. 66.00%, respectively), 4-8 cell (44.31% vs. 29.11%, respectively), morula (12.27% vs. 2.63%, respectively), and blastocysts (2.5% vs. 0%, respectively). The messenger RNA (mRNA) expression levels of BCL2, BAX and SOD were significantly different (p < 0.05) between the matured IVM oocytes. Overall, hUCM showed potential efficacy in terms of ameliorating oocyte maturation and in promoting the development and mRNA expression of BAX, BCL2, and SOD.

Ultrastructure of in vitro Matured Human Oocytes.

BACKGROUND: Approximately 20% of recovered oocytes are immature and discarded in intracytoplasmic sperm injection (ICSI) procedures. These oocytes represent a potential resource for both clinical and basic science application. OBJECTIVE: The aim of this study was to evaluate the ultrastructure architecture of in vitro matured human oocytes using transmission electron microscopy (TEM). MATERIALS AND METHODS: A total of 204 immature oocytes from infertile patients who underwent ICSI cycles were included in this prospective study. Immature oocytes were divided into two groups: (i) GV oocytes (n = 101); and (ii) MI oocytes (n = 103). Supernumerary fresh in vivo matured oocytes (n = 10) were used as control. RESULTS: The rates of maturations were 61.38% for GV and 73.78% for MI oocytes in IVM medium (P = 0.07). However, the rate of oocyte arrest was significant between groups (P <0 .05). Ultrastructurally; in vitro and in vivo matured oocytes appeared round, with a homogeneous cytoplasm, an intact oolemma and an intact zona pellucida. However, immature oocytes indicated numerous large mitochondria-vesicle complexes (M-VC). CONCLUSIONS: Ultrastructural changes of M-VC in IVM groups emphasize the need for further research in order to refine culture conditions and improve the implantation rate of in-vitro matured oocytes.

Contribution of human oocyte architecture to success of in vitro maturation technology.

 The use of ovarian stimulation for infertility treatment is associated with side effects of ovarian hyperstimulation syndrome (OHSS) and potential cancer risk. This is also true in high risk women such as those polycystic with ovary (PCO) and polycystic ovarian syndrome (PCOS). In vitro maturation (IVM) of oocytes was primarily developed to make IVF safe for women with PCO and at high risk of OHSS. The application of IVM of oocytes to assist clinical infertility treatment remains poor because of the reduced developmental competence of oocytes after IVM, despite several decades of research. Reduced meiotic maturation and fertilization rates, as well as low blastocyst production reveal short-term developmental insufficiency of oocytes when compared with in vivo-matured counterparts. In this review, the structural role of human oocytes, revealed by different technical approaches, to the success of IVM technology is highlighted.

The effect of vitrification on ultrastructure of human in vitro matured germinal vesicle oocytes.

OBJECTIVE: To describe the possible effects of cryotop vitrification on maturation rate and ultrastructural morphology of human in vitro matured germinal vesicle (GV) oocytes. STUDY DESIGN: A total of 301surplus immature GV oocytes obtained from infertile patients were allocated into two groups: (i) GV oocytes (n=150) matured in vitro (fIVM), and (ii) GV oocytes (n=151) that were first vitrified, then matured in vitro (vIVM). Supernumerary fresh in vivo matured oocytes (n=10) were used as controls. The maturation media was Ham's F10 supplemented with FSH+LH and human follicular fluid. After 36h of incubation, the oocytes were investigated for nuclear maturation and ultrastructural changes using transmission electron microscopy (TEM). RESULTS: Oocyte maturation rates were reduced (P<0.001) in vIVM (45.92%) in comparison with fIVM oocytes (75.33%). The rate of degeneration was also significantly higher in vIVM than in the fIVM group (44.4% vs. 6.0%). Large and numerous mitochondria and minute vesicles of smooth endoplasmic reticulum (SER) complexes (MV complexes) were observed in both fIVM and vIVM groups. In addition, TEM revealed a drastic reduction in amount of cortical granules (CGs) at the cortex of vitrified-warmed GV oocytes, as well as appearance of vacuoles and small mitochondria-SER aggregates in the ooplasm. CONCLUSION: The vitrification procedure is associated with ultrastructural alterations in specific oocyte microdomains, presumably related to the reduced competence of cryopreserved oocytes for maturation. This information emphasizes the need for further work on advancing the cryotechnology of human oocytes.