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Abstract Background: Titanium dioxide nanoparticles (TiO 2 NPs) are widely used in many compounds. Recent evidence has displayed some cytotoxic effects of TiO 2 NPs on male reproduction. Objective: The effects of TiO 2 NP administration on sperm parameters and chromatin and seminiferous histopathology of male mice were investigated. Materials and Methods: In this experimental study, 32 NMRI male mice (35 ± 3 gr, 8-12-week-old) were divided into four groups (n = 8/each): treated groups were fed orally with 2.5 (group I), 5 (group II) and 10 (group III) mg/kg/day TiO 2 NPs for 40 days and the control group received phosphate buffered saline. Sperm parameters, DNA integrity and chromatin quality were assessed using chromomycin A3, aniline blue, toluidine blue staining and TUNEL. Hematoxylin eosin staining was performed to measure spermatogenic cells and the total diameter of seminiferous tubules. Also, sex hormone and malondyaldehyde levels were measured. Results: Abnormal sperm tails rose in group III (28.87 ± 4.91) in comparison with the control group (12.75 ± 3.95). However, chromomycin A3 staining and TUNEL showed higher levels in group III in comparison with the control group, whereas aniline blue and toluidine blue staining showed no differences. A significantly lower spermatogenesis index and lumen parameters were observed in group III. Leydig cell numbers, cellular diameters and the area of the seminiferous tubules were lower in the treated groups. The testosterone level was also lower in these groups and the percentage of malondyaldehyde in the seminal fluid was higher. Conclusion: Exact mechanisms of TiO 2 NPs are not clear; however, cytotoxic and genotoxic effects of TiO 2 NPs may relate to oxidative stress. Given their widespread use, TiO 2 NPs should be a public health focus of attention.

ABSTRACT Background and Aim: All studies on mammals and rodents have revealed the presence of protamine 1 (P1) and protamine 2 (P2) in the sperm which is indicative of the expression of these two genes at different molecular levels. The aim of this study was to investigate the effect of protamine and its disorders on male fertility potential. Materials and Methods: Using keywords of sperm, protamine, male infertility, and chromatin we searched PubMed and Google Scholar databases between 1980 and 2020. Results: Sperm concentration, motility, and morphology in the patients with variable P1/P2 ratios were significantly reduced compared to those in the individuals with normal P1/P2 ratios which were directly associated with reduced fertility rate. The most common protamine abnormality in the infertile men was increased P1/P2 ratio which was frequently associated with a decreased level of P2 and increased level of P2 precursors. Increased levels of histone B2 (H2B) in sperm and lower levels of protamine have been reported. Any disturbances in the histone expression process cause inconvenient early chromatin condensation, transcription arrest, as well as spermatogenesis failures. Conclusion: The results of this study showed that the protamine transcripts ratio can be used as a marker for male fertility. Histones/protamines mRNAs ratios are important for sperm quality and therefore can be used as predictors for male infertility. Altered levels of protamines may result in an increased susceptibility to injury in the sperm DNA causing infertility or poor outcome in assisted reproduction.

Objective: Aim of this study was to evaluate the effects of vitamin C on sperm parameters, sperm chromatin quality and apoptosis resulted of vitrification in neat semen and prepared spermatozoa of normozoospermic samples. Material and methods: Forty semen samples from normozoospermic men were included in this prospective study. Each sample was divided into five groups. Group I: control or fresh semen, group II: semen prepared by swim-up method and then vitrified, group III: neat semen was vitrified, group IV: vitamin C (600 μM) was added to prepared spermatozoa and then vitrified and group V: vitamin C (600 μM) was added to neat semen and then vitrified. After warming, sperm analysis was done accordingly. For evaluating the sperm chromatin/DNA integrity status and acrosome reaction, we used toluidine blue (TB), acridine orange (AO), terminal transferase mediated deoxyuridine triphosphate biotin end labeling (TUNEL) and double staining tests. Results: All of the sperm parameters (count, motility, morphology and viability) had significant differences (P

Objective: Presence of vitrification method in sperm freezing and the introduction of solid surface vitrification beside rapid freezing in vapour, opens an easy and safe way to help infertility centres. While the effects of cryopreservation on motility, morphology and viability of sperm are documented, the question of the probable alteration of sperm DNA, chromatin and acrosome integrity after freezing and thawing procedures in different methods is still controversial. Materials and methods: Normal sample were collected according to WHO strict criteria. Sperm suspensions were mixed 1:1 with 0.5 M sucrose and divided into four equal aliquots for freezing: fresh, nitrogen direct immersion vitrification (Vit), solid surface vitrification (SSV) and in vapour (Vapour). Sperm suspensions were transferred into a 0.25 ml sterile plastic. Then straw was inserted inside the 0.5 ml straw. For thawing, the straws were immersed in a 42 °C water bath. Beside the sperm parameters, we assessed the acrosome reaction by double staining, chromatin integrity by toluidine blue (Tb) and chromomycin A3 (CMA3) and DNA integrity by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) respectively. Results: In progressive motility, the highest rate occurred in Vit (39.9 ± 13.3). Moreover, the lowest rate of immotile sperm was in Vit (32.7 ± 16.3). In normal morphology, the group Vit was similar to the fresh, while SSV and Vapour were significantly different from the fresh. The percentage of acrosome-reacted sperms was more in Vit (81.3 ± 10.2) than the fresh group. TUNEL+ results showed that DNA fragmentation was significantly increased in Vit (p-value = 0.025). While in SSV and Vapour results were comparable to fresh. There was a significant correlation between TUNEL+ and normal morphology, TB, CMA3 and presence of intact acrosome. Conclusion: Sperm in Vapour was healthier in terms of DNA, chromatin and acrosome integrity. In contrast of higher motility and normal morphology; DNA, chromatin and acrosome integrity were decreased in Vit. However, these findings were more acceptable in SSV or Vapour.

The sperm DNA damage may occur in testis, genital ducts, and also after ejaculation. Mechanisms altering chromatin remodeling are abortive apoptosis and oxidative stress resulting from reactive oxygen species. Three classifications of intratesticular, post-testicular, and external factors have been correlated with increased levels of sperm DNA damage which can affect the potential of fertility. Alcohol consumption may not increase the rate of sperm residual histones and protamine deficiency; however, it causes an increase in the percentage of spermatozoa with DNA fragmentation and apoptosis. In a medical problem as spinal cord injury, poor semen parameters and sperm DNA damage were reported. Infection induces reactive oxygen species production, decreases the total antioxidant capacity and sperm DNA fragmentation or antigen production that lead to sperm dysfunctions and DNA fragmentation. While reactive oxygen species generation increases with age, oxidative stress may be responsible for the age-dependent sperm DNA damage. The exposing of reproductive organs in older men to oxidative stress for a long time may produce more DNA-damaged spermatozoa than youngers. Examining the sperm chromatin quality in testicular cancer and Hodgkin’s lymphoma patients prior to chemotherapy demonstrated the high incidence of DNA damage and low compaction in spermatozoa at the time of diagnosis. In chemotherapy cycles with genotoxic agents in cancer patients, an increase in sperm DNA damage was shown after treatment. In overall, those factors occurring during the prenatal or the adult life alter the distribution of proteins associated with sperm chromatin induce changes in germ cells which can be detected in infertile patients.

Objective: To examine the effect of co-culture system with CCs on the in vitro maturation (IVM) of immature oocytes retrieved from mice. Design: This is an experimental study. Setting: Research and Clinical Center for Infertility of Yazd. Sample: A total of 144 oocytes from 5 to 6 weeks mice. Material: After dissecting ovaries, denuded GVs (DGVs) were considered as control group and put in IVM medium. In cumulus oocyte complexes (COCs) as exp I, intact COCs were put in IVM medium and observed for maturation after 24 h and 48 h. In exp II, GVs were co-cultured with mature CCs. For exp II, COCs were incubated for 30 min before removing oocytes. Then, denuded mature oocytes were removed and the remaining CCs were used to culture denuded GVs recovered from ovaries. Main outcome measures: Maturation rates of oocytes after 24 h and 48 h. Results: The rate of oocyte maturation was 77.36 ± 14.4 in control group. In exp I and exp II, the rates of maturation were 91.32 ± 22.5 and 63.33 ± 7.4, respectively (P = 0.04). However, maturation rate did not increase after 24 h. The maturation rate in metaphase I (MI) oocytes was as low as 15% in different groups at 24 h and did not increase by 48 h. Degeneration rate was increased from time 0 to 48 h, but not significant in different times. Conclusion: Presence of intact COCs significantly improved the oocyte maturation rates in animal model. However, co-culture of GVs with mature CCs did not improve IVM program in mice.

Sperm is particularly susceptible to reactive oxygen species (ROS) during critical phases of spermiogenesis. However, the level of seminal ROS is restricted by seminal antioxidants which have beneficial effects on sperm parameters and developmental potentials. Mitochondria and sperm plasma membrane are two major sites of ROS generation in sperm cells. Besides, leukocytes including polymer phonuclear (PMN) leukocytes and macrophages produce broad category of molecules including oxygen free radicals, non-radical species and reactive nitrogen species. Physiological role of ROS increase the intracellular cAMP which then activate protein kinase in male reproductive system. This indicates that spermatozoa need small amounts of ROS to acquire the ability of nuclear maturation regulation and condensation to fertilize the oocyte. There is a long list of intrinsic and extrinsic factors which can induce oxidative stress to interact with lipids, proteins and DNA molecules. As a result, we have lipid peroxidation, DNA fragmentation, axonemal damage, denaturation of the enzymes, over generation of superoxide in the mitochondria, lower antioxidant activity and finally abnormal spermatogenesis. If oxidative stress is considered as one of the main cause of DNA damage in the germ cells, then there should be good reason for antioxidant therapy in these conditions