Therapeutic potential of testosterone on sperm parameters and chromatin status in fresh and thawed normo and asthenozoospermic samples / Potencial terapéutico de la testosterona sobre los parámetros espermáticos y el estado de la cromatina en muestras frescas y descongeladas de normo y astenozoospermia

Background: Hormonal changes alter the physiological level of ROS and cause oxidative stress in the cell. As estimated, hormonal deficiencies, environmental and ideological factors make up about 25% of male infertility. Pathogenic reactive oxygen species (ROS) is a chief cause of unexplained infertility. Limited studies exist on the effects of testosterone on human sperm culture. Therefore, in the current study, the effect of different doses of testosterone on sperm parameters and chromatin quality was investigated. Materials and methods: Semen samples from 15 normospermic and 15 asthenospermic patients were prepared by swim up method, and then were divided into four groups by exposing to different concentrations of testosterone (1, 10, and 100nM) for 45min. Samples without any intervention were considered as control group. All samples were washed twice. Sperm parameters and chromatin protamination were assessed in each group and the remains were frozen. After two weeks, all tests were repeated for sperm thawed. Also, the MSOM technique was used to determine the sperm morphology of class 1. Results: Although sperm parameters were not show any significant differences in normospermic and asthenospermic samples exposed to different concentrations of testosterone before and after freezing, chromatin protamination was significantly decreased in the normospermic samples exposed to 10nM of testosterone before freezing (p<0.006), as well as 1 and 10nM of testosterone after freezing compared to control samples (p=0.001 and p=0.0009, respectively). (AU)

Antecedentes: Los cambios hormonales alteran el nivel fisiológico de las especies reactivas de oxígeno (reactive oxygen species [ROS]) patógenas y provocan estrés oxidativo en la célula. Según estimaciones, las deficiencias hormonales, los factores ambientales y los ideológicos constituyen alrededor del 25% de la infertilidad masculina. Las ROS son una causa principal de infertilidad inexplicable. Existen estudios limitados sobre los efectos de la testosterona en el cultivo de esperma humano. Por lo tanto, en el estudio actual se ha investigado el efecto de diferentes dosis de testosterona sobre los parámetros del esperma y la calidad de la cromatina. Materiales y métodos: Se prepararon muestras de semen de 15 pacientes normospérmicos y 15 astenospérmicos mediante el método swim up, y luego se dividieron en cuatro grupos exponiéndolos a diferentes concentraciones de testosterona (1, 10 y 100nM) durante 45min. Las muestras sin ninguna intervención se consideraron como grupo control. Todas las muestras se lavaron dos veces. En cada grupo se evaluaron los parámetros espermáticos y la protaminación de la cromatina, y los restos se congelaron. Dos semanas después se repitieron todas las pruebas de esperma descongelado. Asimismo, se utilizó la técnica MSOM para determinar la morfología espermática de clase 1. Resultados: Aunque los parámetros espermáticos no mostraron diferencias significativas en las muestras normospérmicas y astenospérmicas expuestas a diferentes concentraciones de testosterona antes y después de la congelación, la protaminación de la cromatina disminuyó significativamente en las muestras normospérmicas expuestas a 10nM de testosterona antes de la congelación (p<0,006), así como a 1 y 10nM de testosterona después de la congelación, en comparación con las muestras de control (p=0,001 y p=0,0009, respectivamente). (AU)

Therapeutic potential of testosterone on sperm parameters and chromatin status in fresh and thawed normo and asthenozoospermic samples.

BACKGROUND: Hormonal changes alter the physiological level of ROS and cause oxidative stress in the cell. As estimated, hormonal deficiencies, environmental and ideological factors make up about 25% of male infertility. Pathogenic reactive oxygen species (ROS) is a chief cause of unexplained infertility. Limited studies exist on the effects of testosterone on human sperm culture. Therefore, in the current study, the effect of different doses of testosterone on sperm parameters and chromatin quality was investigated. MATERIALS AND METHODS: Semen samples from 15 normospermic and 15 asthenospermic patients were prepared by swim up method, and then were divided into four groups by exposing to different concentrations of testosterone (1, 10, and 100nM) for 45min. Samples without any intervention were considered as control group. All samples were washed twice. Sperm parameters and chromatin protamination were assessed in each group and the remains were frozen. After two weeks, all tests were repeated for sperm thawed. Also, the MSOM technique was used to determine the sperm morphology of class 1. RESULTS: Although sperm parameters were not show any significant differences in normospermic and asthenospermic samples exposed to different concentrations of testosterone before and after freezing, chromatin protamination was significantly decreased in the normospermic samples exposed to 10nM of testosterone before freezing (p<0.006), as well as 1 and 10nM of testosterone after freezing compared to control samples (p=0.001 and p=0.0009, respectively). Similarly, chromatin protamination in the asthenospermic samples was significantly decreased at concentration of 1nM of testosterone before and after freezing (p=0.0014 and p=0.0004, respectively), and at concentration of 10nM of testosterone before and after freezing (p=0.0009, p=0.0007) compared to control samples. CONCLUSION: Using a low dose of testosterone in the sperm culture medium, has positive effects on chromatin quality.

Microfluidic devices employing chemo- and thermotaxis for sperm selection can improve sperm parameters and function in patients with high DNA fragmentation.

Conventional sperm processing uses centrifugation has a negative effect on sperm parameters and DNA integrity. We designed and fabricated a novel microfluid device based on chemotaxis and thermotaxis, and compared it with the swim-up method. Twenty normal samples with high DNA fragmentation were included. Each sample was divided into four groups: Group 1, control, Group 2: sperm selection by thermotaxis, Group 3: sperm selection by chemotaxis, and Group 4: sperm selection with thermotaxis and chemotaxis. We used cumulus cells in a microfluid device to create chemotaxis, and, two warm stages to form a temperature gradient for thermotaxis. The spermatozoa were assessed based on the concentration, motility, and fine morphology using Motile Sperm Organelle Morphology Examination, mitochondrial membrane potential (MMP), acrosome reaction (AR), and sperm DNA fragmentation. Concentration (22.40 ± 5.39 vs. 66.50 ± 19.21; p < 0.001) and DNA fragmentation (12.30 ± 3.96% vs. 17.95 ± 2.89%; p < 0.001) after selection in the chemotaxis and thermotaxis microfluid device were significantly lower than control group. The progressive motility (93.75 ± 4.39% vs. 75.55 ± 5.86%, p < 0.001), normal morphology (15.45 ± 2.50% vs. 10.35 ± 3.36, p < 0.001), MMP (97.65 ± 1.81% vs. 94 ± 3.89%, p = 0.02), and AR status (79.20 ± 5.28% vs. 31.20 ± 5.24%, p < 0.001) in the chemotaxis and thermotaxis microfluid device were significantly increased compared to control group. According to these findings, spermatozoa that have penetrated the cumulus oophorus have better morphology and motility, as well as acrosome reactivity and DNA integrity.

Free centrifuge sorting method for high-count sperm preparation improves biological characteristics of human spermatozoa and clinical outcome: A sibling oocytes study.

Sperm processing for assisted reproductive technologies (ART) aims to separate immotile and debris from the motile spermatozoa in the semen. The purpose of this study was to assess the effect of free centrifuge sorting (FCS) approach based on a combination of rheotaxis and swim-up on sperm biological characteristics and ICSI clinical outcomes. Each semen sample was splitted into two equal parts for 67 ICSI cycles with donation oocytes. Parts were processed with the Direct Swim Up (DSU) (control) and with the FCS method (experimental). Sperm quality was assessed in terms of motility, fine morphology, mitochondrial membrane potential, lipid peroxidation and sperm DNA fragmentation. Also Following ICSI, the clinical outcomes were compared between the groups. Sperm progressive motility (93.5 ± 4.1% vs. 78.6 ± 8.2%; p < 0.001), the fraction of Class I (good) morphology (30.2 ± 9.4% vs. 23.7 ± 8.5%; p < 0.0001) and the rate of mitochondrial membrane potential (77.4 ± 7.2% vs. 66.9 ± 5.7%; p < 0.0001) were significantly higher in the FCS compared to DSU groups. The level of lipid peroxidation (0.5 ± 0.05% vs. 0.6 ± 0.06%; p < 0.0001) and concentration of DNA fragmentation (DF) (7.4 ± 1.6% vs. 15.4 ± 2.6%; p < 0.0001) were lower in sperm from the FCS group compared to DSU group. There were higher rates of high-quality embryo formation (p < 0.001), implantation and clinical pregnancy rates (p = 0.03) in the FCS group compared to the control group. The processing of seminal samples using FCS collected spermatozoa with better biological quality and resulted in higher reproductive outcomes in ICSI cycles.

ANXA2, SP17, SERPINA5, PRDX2 genes, and sperm DNA fragmentation differentially represented in male partners of infertile couples with normal and abnormal sperm parameters.

This study aims to evaluate the expression of genes associated with the fertilisation potential and embryo development, sperm DNA fragmentation (SDF), and acrosome reaction in male partners of infertile couples with different sperm parameters compared to fertile men. First, male partners of infertile couples with abnormal (N = 25) and normal sperm parameters (N = 25), and fertile men (N = 10) were included in experimental groups I, II, and controls respectively. The mRNA levels of the Annexin A2 (ANXA2), Sperm protein 17 (SP17), Plasma serine protease inhibitor (SERPINA5), and Peroxiredoxin-2 (PRDX2) genes and SDF were evaluated. To evaluate the maturity of the sperm and oxidative stress, the acrosome reaction, the lipid peroxidation, and total antioxidant were measured. As result, SP17 showed a significantly lower expression in both experimental groups. SERPINA5 was significantly down-regulated in experimental group I that was aligned with the low rate of acrosome reaction. Significant overexpression of PRDX2 was found between experimental group II and controls. Significant higher rates of SDF were seen in both experimental groups compared to the controls. Finally, our data suggest that differentially gene expression of SP17 is a potential diagnostic biomarker in infertile men either with normal or abnormal sperm parameters. SDF is one of the causes of male infertility, independent of the sperm parameters.

Differential apoptotic gene expression in the male partners of infertile couples with normal and abnormal sperm parameters.

OBJECTIVE: The purpose of this study was to investigate the cellular and molecular levels of apoptosis induction in three groups of male partners of infertile couples, one featuring subjects with normal sperm parameters and unexplained male infertility (UMI), one including men with abnormal sperm parameters, and one with fertile men as controls. METHODS: Twenty-five infertile men with abnormal sperm parameters and 25 men with UMI and normal sperm parameters were recruited as experimental group I and experimental group II; 25 fertile men were included as controls. The mRNA levels of Fas, Fas ligand, Caspase 8, Bax, and Bcl2 were measured in the three groups. The cellular rates of early and late apoptosis were assessed using annexin V and propidium iodide staining. RESULTS: The expression of Bax, Bcl2, and the Bax/Bcl2 ratio in experimental group I was significantly higher than that in experimental group II and controls. However, the Bax/Bcl2 ratio was less than 1 among all groups. No significant difference was found among study groups regarding the gene expression of Fas, Fas ligand, and Caspase 8. No significant difference was seen in early apoptotic rates of sperm among study groups. The highest number of necrotic sperm cells was detected in experimental group I. CONCLUSIONS: The findings showed that the external pathways of apoptosis were not activated in the absence of external stimuli of sperm apoptosis in ejaculated sperm. Regardless of fertility status, apoptosis gene induction in the internal pathway was associated with abnormalities in sperm motility and/or morphology in men with abnormal parameters.

Altered methyltransferase gene expression, mitochondrial copy number and 4977-bp common deletion in subfertile men with variable sperm parameters.

Semen parameters have been found to predict reproductive success poorly and are the most prevalent diagnostic tool for male infertility. There are few conflicting reports regarding the correlation of DNMT genes expression, mitochondrial DNA copy number (mtDNAcn) and deletion (mtDNAdel) with different sperm parameters. To investigate DNMT mRNA level, mtDNAcn and deletion in infertile men, with different sperm parameters, compared with fertile men, semen samples from 30 men with unknown male infertility and normal sperm parameters (experimental group I), 30 infertile patients with at least two abnormal sperm parameters (experimental group II) and 30 fertile normozoospermic men (control group) were collected. After semen analysis, total RNA and DNA were extracted. The isolated DNA was used for assessing the respective mtDNAcn and the presence of common 4977 bp deletion in mtDNA by applying real-time quantitative PCR and multiplex PCR, respectively. Synthesized cDNA from total RNAs was used to quantify DNMT1, DNMT3A and DNMT3B transcripts in study groups by using real-time quantitative reverse-transcription PCR. Significantly higher proportions of mtDNAcn were found in experimental group II. DNMT1 was significantly downregulated in both experimental groups and 4977 bp deletion was not detected. Progressive motility and normal morphology were significantly and negatively correlated with mtDNAcn. A significant positive correlation was detected between sperm parameters and DNMT1 mRNA levels. In conclusion, infertile men with different sperm parameter qualities showed elevated mtDNA content. Abnormal sperm parameters associated with DNMT1 gene expression indicate the possibility of changes in some epigenetic aspects of spermatogenesis in subfertile men with different sperm parameters.

The effects of morphine abuse on sperm parameters, chromatin integrity and apoptosis in men.

OBJECTIVE: Morphine is one of the major psychoactive chemicals in opium that can increase the production of free radicals and thus can negatively affect spermatogenesis. The purpose of this study was to demonstrate the effects of morphine consumption on sperm parameters, DNA integrity and apoptosis in men taking morphine. METHODS: In this case-control study, 30 man abusing morphine (cases) and 30 healthy men (controls) were compared in sperm parameters (count, motility and morphology) and sperm chromatin quality, with Aniline Blue (AB), Toluidine blue (TB) and Chromomycin A3 (CMA3) stains. The participants were matched for age, weight, amount and duration of cigarette smoking. RESULTS: In men with morphine dependency, sperm progressive and total motility (p=0.038 and p=0.000, respectively) showed a significant decrease compared to the control group. Concerning morphine abuse, although morphine can decrease the sperm chromatin condensation and increases the rate of sperm apoptosis, these differences were not statistically significant. CONCLUSIONS: According to our results morphine dependence can reduce male fertility by affecting sperm parameters.

The effect of lipopolysaccharide from uropathogenic Escherichia coli on the immune system, testis tissue, and spermatozoa of BALB/c mice.

OBJECTIVE: Uropathogenic Escherichia coli is known to cause urinary tract infections, and the endotoxin (lipopolysaccharide [LPS]) of this bacterium may cause deficiencies of sperm quality and morphology. In the present study, the effects of LPS on mouse sperm were studied, and the levels of interleukin (IL)-17A and possible changes in testis tissue were evaluated. METHODS: LPS of uropathogenic E. coli was extracted using the methanol-chloroform method, followed confirmation using sodium dodecyl sulfate-polyacrylamide electrophoresis. Purified LPS (100 µg/kg) or phosphate-buffered saline was injected intraperitoneally into BALB/c mice for 7 days consecutively in the test and control groups, Mice were sacrificed on days 3, 7, and 42 after the first injection. Blood was tested for levels of IL-17A using the enzyme-linked immunosorbent assay method. Testis tissue and sperm were collected from each mouse and were studied according to standard protocols. RESULTS: The mean sperm count and motility significantly decreased (p=0.03) at 3, 7, and 42 days after the injections. The level of IL-17A in the test groups increased, but not significantly (p=0.8, p=0.11, and p=0.15, respectively). Microscopic studies showed no obvious changes in the morphology of the testis tissue; however, significant changes were observed in the cellular parenchyma on day 42. CONCLUSION: LPS can stimulate the immune system to produce proinflammatory cytokines, resulting in an immune response in the testis and ultimately leading to deficiency in sperm parameters and testis tissue damage. In addition, the presence of LPS could significantly impair sperm parameters, as shown by the finding of decreased motility.

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.

The effects of sesame oil and different doses of estradiol on testicular structure, sperm parameters, and chromatin integrity in old mice.

OBJECTIVE: Studies of the effects of estrogens on the male reproductive system have emphasized the role of these hormones in male fertility. Sesame oil has many phytoestrogenic compounds and may improve male fertility. This study investigated the effects of sesame oil and different concentrations of estrogen on sperm parameters and DNA integrity in male mice. METHODS: Twenty old NMRI (The Naval Medical Research Institute) male mice (40 weeks; weight, 30-35 g) were treated with sesame oil or different concentrations of estrogen (estradiol, 1 and 10 µL/kg/ day) or received no treatment (controls). After 35 days, sperm parameters and DNA integrity were assessed and analyzed. RESULTS: Sperm count, progressive motility, and morphology were decreased in the group that received 10 µL/kg of estradiol. A remarkably lower percentage of DNA fragmentation and protamine deficiency were detected in the group that received 1 µL/kg of estradiol. In the groups that received sesame oil and 1 µL/kg of estradiol, the numbers of spermatogonia and Leydig cells were higher than in controls. The combination of sesame oil and 1 µL/kg of estradiol led to improved sperm parameters and chromatin and testicular structure. CONCLUSION: Based on this study, consumption of sesame oil and a low concentration of estradiol may improve testicular function in older mice.

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.

The effect of testosterone and antioxidants nanoliposomes on gene expressions and sperm parameters in asthenospermic individuals.

BACKGROUND: Antioxidants that used for the infertility treatment cannot have their complete effectiveness, because of their instability in the culture medium. SIGNIFICANCE: One of the most advances, in the drug delivery systems, is nanoliposomes-loaded, as biodegradable and bioavailable carriers. Hormonal and antioxidant agents encapsulating inside the nanoliposomes were used, to increase the effectiveness of antioxidants in the sperm culture medium. MATERIALS: Semen sample from 15 asthenospermia were divided into 10 equal parts. After preparation, the sperms were incubated with free form of drugs and nanocarriers contained resveratrol, catalase, resveratrol-catalase and testosterone for 45 min. All sperm parameters, sperm DNA and gene expressions were evaluated before and after freezing. RESULTS: Before freezing, all nanocarriers and free testosterone showed higher sperm motility compared to free drugs (p=.000). Free Testosterone and free resveratrol-catalase had higher DNA damage compared to nanocarriers (p=.000). Before freezing, the blank nanoliposome and testosterone nanoliposomes had the lowest HSP70 gene expression respectively (p = 0.005) (p = 0.001). After freezing, a significant reduction in sperm motility was observed in the free resveratrol-catalase group (p=.003). Also, a significant increase in sperm viability was observed in the free testosterone and nanoliposomes of blank and testosterone (p > 0.05). The least DNA fragmentation was related to catalase nanoliposomes (p=.000). All nanoliposomes, especially catalase, had the highest percentage of class I morphology compared to the control group (p=.000). CONCLUSIONS: Nanoliposomes could improve the sperm parameters and DNA integrity before and after freezing, by increasing the effectiveness of antioxidants. So, it can be recommended in the ART lab.

L-Carnitine reduces the negative effects of formalin on sperm parameters, chromatin condensation and apoptosis in mice: An experimental study.

BACKGROUND: Formalin is commonly applied as an antiseptic and tissue fixative. It has reactive molecules that lead to its cytotoxic effects. According to recent studies, formalin causes a change in the testicular and sperm structure and L-carnitine (LC) acts as an antioxidant to counteract its effects. OBJECTIVE: This study aimed to investigate the protective effects of LC on the parameters, chromatin condensation and apoptosis of mice sperm exposed to formalin. MATERIALS AND METHODS: In this experimental study, 24 balb/c mice (25-40 gr,10-12 wk) were divided into three groups (n = 8/each): group I without any injections or gavage; group II, received 10 mg/ kg formalin intraperitoneally (I.P); and group III was exposed to formalin and LC, where a dose of 10 mg/kg formalin was injected I.P daily and LC the dose of 100 mg/kg was kept in a solvent solution. After 31 days, the sperm examination was performed as follows: to evaluate chromatin and DNA quality of the sperm, we applied aniline blue (AB), toluidine blue (TB), chromomycin A3 (CMA3), and terminal transferase-mediated deoxy uridine triphosphate biotin end labeling (TUNEL) tests. RESULTS: Sperm parameters such as count, motility, morphology, and viability displayed a significant decrease in the formalin group. While the data exhibited a considerable augment in sperm parameters in the formalin + LC than the formalin and control groups (p < 0.001), significant differences were detected between groups with respect to TB staining, TUNEL test, CMA3 test and AB staining in the formalin and formalin + LC groups. CONCLUSION: LC can reduce the negative effects of formalin on sperm parameters, chromatin stability, and percentage of apoptosis in an animal model.

Does resveratrol affect prepared sperm parameters and chromatin quality in normozoospermic and asthenozoospermic patients before and after freezing? A lab trial study.

BACKGROUND: Previous studies have examined the effect of resveratrol as a potent antioxidant for free radicals in semen. While, the prepared spermatozoa are more affected by ROS factors due to centrifugation and incubation. OBJECTIVE: To evaluate the RSV's effects on the prepared sperm parameters and chromatin quality in both normozoospermic and asthenozoospermic cases before and after freezing. MATERIALS AND METHODS: The sample of 10 normozoospermic and asthenozoospermic men was prepared through the swim-up method. The groups were then divided into two samples of control and experimental (exposure to 30 µmol/l of RSV) to evaluate and compare the sperm parameters and chromatin quality before and after freezing. RESULTS: The motility and viability of spermatozoa were seen to be significantly different before and after freezing separately in the control and treatment samples of the groups (p ≤ 0.001 and p = 0.001, respectively). However, the stated difference between the control and treatment samples of normozoospermic and asthenozoospermic patients were not significant (p > 0.05). In addition, the sperm morphology and chromatin quality were not significantly different between the two samples of each group; nonetheless, chromatin quality of the treated sample was better than that of the control before and after freezing. CONCLUSION: Despite the protective effects of RSV on the semen samples, RSV cannot affect significantly the prepared sperms parameters and chromatin quality in normozoospermic and asthenozoospermic patients.

Association between early embryo morphokinetics plus transcript levels of sperm apoptotic genes and clinical outcomes in IMSI and ICSI cycles of male factor patients.

PURPOSE: The aim was to assess the correlation of sperm apoptotic transcript levels with cleavage stage embryokinetic and pregnancy outcomes of intracytoplasmic morphologically selected sperm injection (IMSI) and ICSI methods in patients with male factor infertility. MATERIAL AND METHODS: Eighty male factor cases were divided into ICSI and IMSI groups. ICSI was done routinely, and for IMSI, sperm was selected at high magnification and injected. On day 3, time-lapse parameters were evaluated, and the best embryos were transferred and followed to delivery. In addition, sperm DNA fragmentation and apoptotic transcript levels were quantified using reverse transcription Q-PCR between the groups. RESULTS: IMSI selected spermatozoa had lower DNA fragmentation and apoptotic transcript levels compared with ICSI (p < 0.0001). Moreover, all cytokinetic variables and cleavage abnormalities were noticeably different between groups (p < 0.0001); the rates of clinical outcomes were higher in the IMSI group. The transcript levels of Caspase 3 showed a moderate negative correlation with s2 and s3 (rs = - 0.57, P = 0.008 and rs = - 0.51, p = 0.021, respectively) in the IMSI group. However, there was no relationship between sperm apoptotic transcript levels and clinical outcomes in two groups. CONCLUSIONS: Sperms selected at high magnification showed lower DNA fragmentation and apoptosis genes transcript. Also, better embryo kinetics and clinical outcomes were confirmed in IMSI than ICSI groups. Some time-lapse parameters may be associated with transcript levels of apoptosis genes. Therefore, these noninvasive techniques may be unique in assisting couples with male factor infertility. TRIAL REGISTRATION: This trial retrospectively registered on 4 July 2020 (IRCT20180130038561N1).

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.

The effect of vitamin C on the gene expression profile of sperm protamines in the male partners of couples with recurrent pregnancy loss: A randomized clinical trial.

OBJECTIVE: Since sperm abnormalities are known to be a major reason for recurrent pregnancy loss (RPL), any defects in DNA structure and chromatin condensation can place embryos at risk in the early stage of development and implantation. As antioxidants such as vitamin C may play a protective role against the destruction of protamine genes in sperm chromatin, this study was conducted to evaluate the effects of vitamin C on chromatin and the expression of protamine genes in the male partners of couples with RPL. METHODS: Twenty male partners of couples with RPL were selected as the intervention group and received vitamin C supplementation (250 mg daily for 3 months). Healthy fertile men (n=20) were included as controls. Sperm chromatin, DNA integrity, and the expression levels of protamine genes were evaluated before and after treatment. RESULTS: Significant differences were found in sperm morphology, protamine deficiency, and apoptosis between the two groups and before and after vitamin C administration. A significant change was found in mRNA levels of PRM1, PRM2, and the PRM1/PRM2 ratio after treatment. CONCLUSION: Daily oral administration of vitamin C may improve human sperm parameters and DNA integrity by increasing protamine gene expression levels in the male partners of couples with RPL. The beneficial effects of vitamin C supplementation as an antioxidant for the male partners of couples with RPL could lead to improved pregnancy outcomes in these cases.

Polymorphisms of sperm protamine genes and CMA3 staining in infertile men with varicocele / Polimorfismos de los genes de la protamina espermática y tinción CMA3 en varones infértiles con varicocele

OBJECTIVES: The aim of this study was to evaluate polymorphisms of sperm protamine genes and their effects on the result of CMA3 staining in varicocele men. MATERIAL AND METHODS: In a case control study, 128 patients with male infertility due to varicocele and 128 controls were recruited. Polymorphisms of PRM1 and PRM2 genes in extracted DNA samples were assessed by PCR-SSCP and sequencing. Protamine deficiency was also indirectly estimated by CMA3 staining. RESULT: Nine different variants including six variants in PRM1 gene and three variants in PRM2 gene were found among varicocele patients. The results showed that sperm count, motility and morphology were significantly different between control group without gene variations and varicocele group who had several variations in their protamine genes (P<0.05). CONCLUSION: Therefore, PRM1 and PRM2 variations in varicocele patients are associated with the production of spermatozoa with more protamine deficiency and this is one of the possible causes of infertility due to varicocele

OBJETIVOS: El objetivo de este estudio fue evaluar los polimorfismos de los genes de la protamina espermática y sus efectos sobre el resultado de la tinción CMA3 en varones con varicocele. MATERIAL Y MÉTODOS: En un estudio de casos y controles, se reclutó a 128 varones con infertilidad por varicocele y 128 controles. Los polimorfismos de los genes PRM1 y PRM2 en muestras de ADN extraídas se evaluaron mediante PCR-SSCP y se realizó su secuenciación. La deficiencia de protamina también se estimó indirectamente mediante tinción CMA3. RESULTADO: Se encontraron 9 variantes diferentes, incluidas 6 variantes en el gen PRM1 y 3 variantes en el gen PRM2 entre los pacientes con varicocele. Los resultados mostraron que el recuento de espermatozoides, la movilidad y la morfología eran considerablemente diferentes entre el grupo de control sin variaciones genéticas y el grupo de varicocele que presentaba algunas variaciones en sus genes de protamina (p < 0,05). CONCLUSIÓN: Por tanto, las variaciones de PRM1 y PRM2 en los pacientes con varicocele están asociadas con la producción de espermatozoides con más deficiencia de protamina y esta es una de las posibles causas de infertilidad debida al varicocele

Polymorphisms of sperm protamine genes and CMA3 staining in infertile men with varicocele.

OBJECTIVES: The aim of this study was to evaluate polymorphisms of sperm protamine genes and their effects on the result of CMA3 staining in varicocele men. MATERIAL AND METHODS: In a case control study, 128 patients with male infertility due to varicocele and 128 controls were recruited. Polymorphisms of PRM1 and PRM2 genes in extracted DNA samples were assessed by PCR-SSCP and sequencing. Protamine deficiency was also indirectly estimated by CMA3 staining. RESULT: Nine different variants including six variants in PRM1 gene and three variants in PRM2 gene were found among varicocele patients. The results showed that sperm count, motility and morphology were significantly different between control group without gene variations and varicocele group who had several variations in their protamine genes (P<0.05). CONCLUSION: Therefore, PRM1 and PRM2 variations in varicocele patients are associated with the production of spermatozoa with more protamine deficiency and this is one of the possible causes of infertility due to varicocele.