Impairment of sperm efficiency in mice following short-term nano-titanium dioxide exposure: An experimental study.

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.

Relation between sperm protamine transcripts with global sperm DNA methylation and sperm DNA methyltransferases mRNA in men with severe sperm abnormalities.

This study aimed to evaluate the relationship between mRNA expression of DNA methyltransferases (DNMTs) such as DNMT1, DNMT3A and DNMT3B mRNA and sperm global DNA methylation with protamine transcripts in the sperm from men with severe sperm abnormalities. Sperm from each semen sample were isolated using a standard gradient isolation procedure by layering 1 mL of 40% (v/v) density gradient medium over 1 mL of 80% (v/v). A total of 30 oligoasthenoteratozoospermic ejaculates (OAT) and 30 normozoospermic ejaculates as controls were compared using real-time quantitative reverse transcriptase polymerase chain reaction for mRNA expression of DNMT1, 3A, 3B, protamine1 (P1) and protamine2 (P2). The enzyme-linked immunosorbent assay was used to detect global DNA methylation in sperm. A p-value of <0.05 was considered statistically significant. In OAT ejaculates, the increased level of DNMT3A, 3B mRNA, sperm global methylation, P1 plus P2 mRNA and decrease of P1-P2 ratio were significantly different. Also the content of protamine transcript was not correlated with sperm parameters. The increased total protamine transcript levels were associated with increased mRNA methyltransferases. The increase of DNMT1 may lead to an increased level of global methylation.

Retinoic acid and/or progesterone differentiate mouse induced pluripotent stem cells into male germ cells in vitro.

Numerous reagents were employed for differentiating induced pluripotent stem cells (iPSCs) into male germ cells; however, the induction procedure was ineffective. The aim of this study was to improve the in vitro differentiation of mice iPSCs (miPSCs) into male germ cells with retinoic acid (RA) and progesterone (P). miPSCs were differentiated to embryoid bodies (EBs) in suspension with RA with or without progesterone for 0, 4, and 7 days. Then, the expression of certain genes at different stages of male germ cell development including Ddx4 (pre meiosis), Stra8 (meiosis), AKAP3 (post meiosis), and Mvh protein was examined in RNA and/or protein levels by real-time polymerase chain reaction or flow cytometry, respectively. The Stra8 gene expression increased in the RA groups on all days. But, expression of this gene declined in RA + P groups. In addition, an increased expression of Ddx4 gene was observed on day 0 in the P group. Also, a significant upregulation was observed in the expression of AKAP3 gene in the RA + P group on days 0 and 4. However, gene expression decreased in P and RA groups on day 7. The expression of Mvh protein significantly increased in the RA group on day 7. The Mvh expression was also enhanced in the P group on day 4, but it decreased on day 7, while this protein upregulated on day 0 and 7 in the RA + P group. The miPSCs have the capacity for in vitro differentiation into male germ cells by RA and/or progesterone. However, the effects of these inducers depend on the type of combination and an effective time.

Evaluation of Sperm Chromatin Integrity Using Aniline Blue and Toluidine Blue Staining in Infertile and Normozoospermic Men.

BACKGROUND: Male infertility is defined as a man lost his ability to fertilize a fertile female naturally. Diagnosis of male infertility cannot be made just according to basic semen analysis. It is necessity to have specific tests for evaluation of chromatin integrity. In this study, an attempt was made to evaluate the sperm chromatin quality in fertile men and infertile subgroup. METHODS: Among 1386 couples, 342 men were categorized into normospermia and 1044 were infertile and they were referred to Yazd Research and Clinical Center for infertility treatment. Standard semen analysis and sperm nuclear maturity tests including aniline blue (AB) and toluidine blue (TB) staining were done. Data were analyzed by SPSS software. The p≤0.05 was considered statistically significant. RESULTS: The mean value of TB staining was significantly higher in infertile group compared to normospermic group (p=0.005). Mean of sperm normal morphology was lower in idiopathic infertile men in comparison with normozoospermic men (p= 0.001). The highest negative correlation was obtained between sperm count and AB staining. Progressive motility was negatively correlated with AB and TB staining in both groups but there was no significant difference between AB staining and progressive motility in men normospermia group. CONCLUSION: Sperm chromatin staining using AB and TB showed a negative association between sperm chromatin condensation with sperm count, normal morphology and progressive motility. It seems that the AB and TB test may be useful for the assessment of male fertility potential.

Vitamin C attenuates negative effects of vitrification on sperm parameters, chromatin quality, apoptosis and acrosome reaction in neat and prepared normozoospermic samples.

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 < 0.05) between different groups, especially in group IV. Data showed sperm chromatin damages and acrosome reaction abnormality increased resulted of vitrification, but, in the groups that added vitamin C (IV, V) rate of damages was decreased and this was notable in the group IV. CONCLUSION: Vitamin C can attenuate the detrimental effects of vitrification on sperm parameters, chromatin quality and rate of apoptosis in both neat semen and prepared spermatozoa of normozoospermic samples.

Sperm chromatin and DNA integrity, methyltransferase mRNA levels, and global DNA methylation in oligoasthenoteratozoospermia.

OBJECTIVE: To investigate sperm chromatin/DNA integrity, global DNA methylation, and DNMT mRNA transcription in men with oligoasthenoteratozoospermia (OAT) compared with normozoospermic men. METHODS: Semen samples from 32 OAT patients who comprised the case group and 32 normozoospermic men who comprised the control group were isolated and purified using a standard gradient isolation procedure according to World Health Organization criteria. DNMT1, DNMT3A, and DNMT3B transcripts were then compared between groups using real-time quantitative reverse-transcription polymerase chain reaction. Global DNA methylation in sperm was determined by an enzyme-linked immunosorbent assay. Protamine deficiency and the proportion of apoptotic spermatozoa were evaluated using chromomycin A3 (CMA3), aniline blue (AB), and toluidine blue (TB) staining, as well as the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The p-values <0.05 were considered to indicate statistical significance. RESULTS: Significantly higher proportions of AB+, TB+, CMA3+, and TUNEL+ spermatozoa, as well as DNMT3A and DNMT3B transcription, were found in the OAT group. Positive correlations were detected between sperm parameters, DNA/chromatin damage, and DNMT3A and DNMT3B transcripts. Global DNA methylation was significantly higher in the OAT patients and had a significant correlation with abnormal results of all sperm chromatin integrity tests, but was not associated with DNMT1, DNMT3A, or DNMT3B expression. CONCLUSION: Oligoasthenoteratozoospermic men showed abnormal sperm parameters, abnormal chromatin/DNA integrity, and a higher global DNA methylation rate, as well as overexpression of DNMT mRNA.

The etiologies of DNA abnormalities in male infertility: An assessment and review.

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 human sperm DNA damage which can affect the potential of fertility. Lifestyle, environment, medical, and iatrogenic factors might be considered to cause dysmetabolism to make distracting interactions and endocrine disrupting compounds. As a result, these may induce chromatin/DNA alteration in germ cells, which may be transmitted across generations with phenotypic consequences. 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 the diagnosis. In chemotherapy cycle 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.

Modern human sperm freezing: Effect on DNA, chromatin and acrosome integrity.

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.

Etiologies of sperm oxidative stress.

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.

Comparison of halogen light and vibroacoustic stimulation on nonreactive fetal heart rate pattern.

BACKGROUND: One of the first-line assessment tools for fetal surveillance is nonstress test (NST), although it is limited by a high rate of false-nonreactive results. This study was performed to investigate if external stimulation from vibroacoustic and halogen light could help in provoking fetal responsiveness and altering NST results. MATERIALS AND METHODS: This is a clinical trial. Sampling was done from April to July 2010. One hundred pregnant women with nonreactive NST for 20 min were allocated in two groups: Vibroacoustic stimulated NST (VNST, n = 50) who received vibration from a standard fetal vibratory stimulator and halogen light stimulated NST (LNST, n = 50) who received a halogen light source for 3 and 10 sec, respectively. Results were compared together and then compared to biophysical profile (BPP) scores as a backup test. We used Mann-Whitney U test, Chi-square test, and Fisher's exact test to compare the variables in the two groups through SPSS version 14. P < 0.05 was considered as statistically significant. RESULTS: Following stimulations, 68% nonreactive subjects in halogen light stimulation group and 62% in vibroacoustic stimulation group changed to reactive patterns. Time to onset of the first acceleration (VNST: 2.17 min; LNST: 2.27 min) and the test duration (VNST: 4.91 min; LNST: 5.26 min) were the same in the two groups. In VNST 89.5% and in LNST 87.5% of nonreactivity followed by score 8 in BPP. There was no significant relation between stimulus NSTs and BPPs. CONCLUSION: Vibroacoustic and light stimulation offer benefits by decreasing the incidence of nonreactive results and reducing the test time. Both halogen light stimulation and vibroacoustic stimulation are safe and efficient in fetal well-being assessment services.