Prm1 Genes Research Articles

Polymorphism detection and characterization of sperm cells chromatin remodeling associated genes in Murrah buffalo.

Seasonal variations significantly impact buffalo bull semen production and quality, particularly during the summer months. Understanding the genetic basis of these changes is important for managing bull fertility and improving sperm quality. The present study focused on characterizing and identifying polymorphisms in chromatin remodeling genes, protamines (PRMs) and Transition Nuclear Proteins (TNPs) in Murrah buffalo bulls with varying semen quality due to seasonal effects. Our findings revealed none of the coding region variation in PRM1, PRM2, TNP1, and TNP2, these genes are highly conserved in buffalo. Two intronic variants were identified, including G16C in PRM1 intron 1 and intronic SNP in PRM2 intron 1(G96A). The complete CDS of consensus sequence of bubaline PRM1 was 86.3% identical and 94.1% similar to the bovine PRM1. Whereas the complete CDS of consensus sequence of bubaline TNP2 was 78.2% identical and 91.0% similar to bovine TNP2. Further, no statistically significant differences in the fold change of TNP1, TNP2, PRM1, and PRM2 levels between the hot summer SNA and SA groups and the winter SNA and SA groups This study represents the first comprehensive report on the characterization of bubaline PRM1 (complete CDS), PRM2 (partial CDS), TNP1 (partial CDS), and TNP2 (complete CDS) genes in buffalo sperm cells. Results of the study, clearly indicate that the genes associated with protamine (PRM1 and TNP2) are highly conserved in Bubalus bubalis. Understanding these genetic underpinnings can have implications for improving buffalo bull fertility and semen quality.

Sperm RNA quantity and PRM1, PRM2, and TH2B transcript levels reflect sperm characteristics and early embryonic development.

Spermatozoa have a highly complex RNA profile. Several of these transcripts are suggested as biomarkers for male infertility and contribute to early development. To analyze the differences between sperm RNA quantity and expression of protamine (PRM1 and PRM2) and testis-specific histone 2B (TH2B) genes, spermatozoa from 33 patients who enrolled in assisted reproduction treatment (ART) program were analyzed. Sperm RNA of teratozoospermic (T), oligoteratozoospermic (OT), and normozoospermic (N) samples was extracted, and the differences in transcript levels among the study groups were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The correlations of total RNA per spermatozoon and the expression of the transcripts were evaluated in relation to sperm characteristics and preimplantation embryo development. The mean (±standard deviation) RNA amount per spermatozoon was 28.48 (±23.03) femtogram in the overall group and was significantly higher in the OT group than that in N and T groups. Total sperm RNA and gene expression of PRM1 and PRM2 genes were related to preimplantation embryo development and developmental arrest. Specific sperm characteristics were correlated with the expressions of PRM1, PRM2, or TH2B genes. We conclude that the sperm RNA amount and composition are important factors and might influence early embryonic development and also differ in different cases of male infertility.

Effect of naringenin on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo (Bubalus bubalis) bull sperm

Our objectives were to explore the effect of naringenin addition in the semen extender on the post-thaw 1) sperm quality, 2) fertility-associated gene expression, and 3) fertilization potential of buffalo bull sperm. In experiment 1, semen samples (n = 32) from four Nili-Ravi buffalo bulls were pooled (n = 8) and diluted with the tris-citric acid (TCF-EY) extender containing different concentrations of naringenin, i.e., placebo (DMSO), 0 (control), 50, 100, 150 and 200 μM naringenin. After dilution, semen samples were packed in 0.5 mL French straws, cryopreserved and analyzed for post-thawed sperm quality and gene expression. Computer-assisted Semen Analysis, Hypo-osmotic Swelling test, Normal Apical Ridge assay, Rhodamine 123, Acridine orange, Propidium iodide staining and Thiobarbituric Acid Reactive Substances assay were performed to assess sperm motility parameters, plasma membrane functionality, acrosome integrity, mitochondrial membrane potential, DNA integrity, viability and lipid peroxidation, respectively. Expression levels of sperm acrosome-associated SPACA3, DNA condensation-related PRM1, anti-apoptotic BCL2, pro-apoptotic BAX, and oxidative stress-associated ROMO1 genes were evaluated through qPCR. Results revealed that total and progressive motility, plasma membrane functionality, acrosome integrity, mitochondrial membrane potential, DNA integrity and viability were higher (P < 0.05) with 50, 100 and 150 μM naringenin compared to 200 μM naringenin, placebo and control groups. Moreover, all naringenin-treated groups improved catalase activity, and reduced lipid peroxidation compared to placebo and control groups (P < 0.05). Relative expression levels of SPACA3 and PRM1 genes were higher (P < 0.05) with 150 μM naringenin compared to all groups except 100 μM (P > 0.05). No difference (P > 0.05) in the expression level of BCL2 gene was observed among all groups. Furthermore, BAX gene was expressed higher (P < 0.05) in the 200 μM naringenin group, whereas no difference (P > 0.05) in expression was noticed among the remaining groups. In addition, ROMO1 gene was expressed lower (P < 0.05) in all naringenin-treated groups compared to the control. In experiment 2, the in vivo fertility of semen doses (n = 400; 200/group) containing optimum concentration of naringenin (150 μM; depicted better in vitro sperm quality in experiment 1) was compared with control during the breeding season. Buffaloes were inseminated 24 h after the onset of natural estrus and palpated transrectal for pregnancy at least 60 days post-insemination. The fertility rate of 150 μM naringenin group was higher (P = 0.0366) compared to the control [57.00 ± 0.03 % (114/200) vs. 46.50 ± 0.04 % (93/200), respectively]. Taken together, it is concluded that naringenin supplementation in semen extender improves post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm, more apparently at 150 μM concentration.

In vitro proliferation and differentiation of mouse spermatogonial stem cells in decellularized human placenta matrix.

Utilizing natural scaffold production derived from extracellular matrix components presents a promising strategy for advancing in vitro spermatogenesis. In this study, we employed decellularized human placental tissue as a scaffold, upon which neonatal mouse spermatogonial cells (SCs) were cultured three-dimensional (3D) configuration. To assess cellular proliferation, we examined the expression of key markers (Id4 and Gfrα1) at both 1 and 14 days into the culture. Our quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed a notable increase in Gfrα1 gene expression, with the 3D culture group exhibiting the highest levels. Furthermore, the relative frequency of Gfrα1-positive cells significantly rose from 38.1% in isolated SCs to 46.13% and 76.93% in the two-dimensional (2D) and 3D culture systems, respectively. Moving forward to days 14 and 35 of the culture period, we evaluated the expression of differentiating markers (Sycp3, acrosin, and Protamine 1). Sycp3 and Prm1 gene expression levels were upregulated in both 2D and 3D cultures, with the 3D group displaying the highest expression. Additionally, acrosin gene expression increased notably within the 3D culture. Notably, at the 35-day mark, the percentage of Prm1-positive cells in the 3D group (36.4%) significantly surpassed that in the 2D group (10.96%). This study suggests that the utilization of placental scaffolds holds significant promise as a bio-scaffold for enhancing mouse in vitro spermatogenesis.

(249) Association of Single Nucleotide Polymorphisms (rs2301365 &amp; Rs737008) in PRM1 Gene and Male Infertility: A Meta-Analysis

Abstract Introduction In the sperm nucleus, protamine is a major DNA-binding protein that replaces histone. It is a basic arginine-rich protein responsible for DNA compaction. Protamin I (PRM1) is one of the important genes that produces protamine. Mutations and polymorphisms in PRM1 may increase the odds of male infertility. Objective The present meta-analysis investigated the association of single nucleotide polymorphisms (SNPs) (rs2301365 &amp; rs737008) in the PRM1 gene and male infertility. Methods The original research articles, published till July 2023 in the English language, on the association of rs2301365 and rs737008 in the PRM1 gene and male infertility, were retrieved from Science Direct, PubMed, NCBI and Google Scholar. Results A total of 129 studies were retrieved by searching in the different databases, after the removal of duplicates, meta-analysis, and abstracts, 14 were finally selected. In 11 case-control studies, the association of the rs2301365 (-190C &amp;gt;A) polymorphism was presented as the meta-analysis of different models, Allelic model (C vs. A), Dominant model (CC + CA vs. AA), Recessive model (CC vs. CA + AA), Additive model (CC vs. AA), Heterozygous model (AC vs. AA). Allelic, additive, dominant and heterozygous models reported that the -190C&amp;gt;A (rs2301365) increased the odds of male infertility. The ethnic subgroup analysis showed the same pattern of association for the Caucasian infertile men. However, none of these overall and subgroup analyses based on ethnicity showed an association between rs2301365 (-190C &amp;gt;A) and infertility in Asian men. For rs737008 (139C &amp;gt; A) in the meta-analysis of 14 studies, none of these overall and subgroup analyses based on ethnicity showed an association with male infertility. Trial sequential analysis (TSA) for the allelic model of rs2301365 (-190C &amp;gt;A) showed the required sample size was 2252 at 90% power and 5% type-I error, whereas the cumulative z-curve crossed the trial sequential monitoring boundaries of harm. However, the sample size was greater than required therefore further studies will not likely change results. For rs737008 (139C &amp;gt; A), TSA recommended more studies to be conducted. Conclusions The PRM1 rs2301365 (-190C &amp;gt;A) was associated with male infertility in overall analysis and Caucasian men. Further studies on this SNP may not change results. However, rs737008 (139C &amp;gt; A) was not associated with male infertility. However, further studies are recommended. Disclosure No.

Analysis of Correlation between PRM1, STK35, and IFT27 Gene Expression Levels and Holstein Bull Semen Quality Parameters

Analysis of Correlation between PRM1, STK35, and IFT27 Gene Expression Levels and Holstein Bull Semen Quality Parameters

Ameliorative effect of crocin on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo (Bubalus bubalis) bull sperm.

Buffalo bull sperm suffer more cryoinjuries due to lipid peroxidation of high structural polyunsaturated fatty acid contents than cattle sperm. Consequently, the post-thaw fertilization potential of buffalo bull sperm is compromised. Crocin is a carotenoid known for its antioxidant potential through scavenging reactive oxygen species. Objectives of the current study were to investigate the effect of crocin addition in the semen extender on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm. Semen samples (n = 32) from four Nili-Ravi buffalo bulls were extended with tris-citric acid extender containing different concentrations of crocin (0 mM; control, 0.5, 1, 1.5 and 2 mM). The extended semen was packed in 0.5 mL French straws (25 × 106 sperm/straw) and cryopreserved in liquid nitrogen. Computer-assisted semen analysis, hypo-osmotic swelling test, normal apical ridge assay, Rhodamine 123, acridine orange, propidium iodide staining, and thiobarbituric acid reactive substances assay were performed to assess sperm motility parameters, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential, DNA integrity, viability, and lipid peroxidation, respectively. Expression levels of sperm acrosome-associated SPACA3, DNA condensation-related PRM1, anti-apoptotic BCL2, pro-apoptotic BAX, and oxidative stress-associated ROMO1 genes were evaluated through qPCR. The fertility of semen doses containing the most potent concentration of crocin (based on optimum post-thaw semen quality) was compared with control during the breeding season. Buffaloes (n = 400; 200/group) were inseminated 24 h after the onset of oestrus and transrectally palpated for pregnancy at least 60 days post-insemination. Results revealed that 0.5 and 1 mM crocin improved sperm post-thaw total motility, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential and viability, and 1 and 1.5 mM crocin enhanced catalase activity and reduced lipid peroxidation compared to control (p < .05). Moreover, 1 mM crocin improved sperm post-thaw progressive motility, kinematics, and DNA integrity, and 1.5 mM crocin enhanced plasma membrane integrity than control (p < .05). Expression levels of SPACA3, PRM1 and BCL2 genes were higher (p < .05) with 1 mM crocin compared to other groups. In contrast, no difference (p > .05) was noticed in expressions of BAX and ROMO1 genes among all groups. The fertility rate of semen doses containing the most potent concentration (1 mM) of crocin was higher (p = .0465) compared to control (56 ± 0.03% vs. 46 ± 0.04%, respectively). In conclusion, 1 mM crocin in the semen extender improves post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm.

Correlation analysis of the PRM1, STK35 and IFT27 level of expression genes with the quality of native and deposited seed of holsting bulls

The research aim is a correlation analysis of the PRM1, STK35 and IFT27 genes mRNA expression with the native and decryoconserved sperm quality indicators of Holstein bulls to search for effective transcriptomic biomarkers of bull semen. In the study course, native and decryoconserved sperm of seven Holstein bulls were used. To solve the study problems, eight indicators of sperm quality were studied, the studied genes in native and decryoconserved spermatozoa expression was analyzed in real time PCR. Nonparametric probabilistic and statistical methods were used, the analysis of rank correlation was carried out using Spearman’s criterion. Higher expression of the studied genes was mainly noted in frozen--thawed sperm compared to native. The mRNA expression level of the protamine gene (PRM1) did not give a reliable correlation with sperm parameters: The level ITF27 gene mRNA expression was significantly positively correlated with the content of defective cells from frozen--thawed sperm (0.714, p=0.05) and dead cells (0.714, p=0.0545) from native sperm. A negative correlation was noted with the content of normal cells in frozen--thawed sperm (-0.750, p=0.038) and live cells (-0.714, p=0.0545) in native sperm. The ITF27 gene transcript (mRNA) showed a negative correlation (-0.703, p=0.0545) in terms of the acrosome defect of frozen--thawed spermatozoa. In terms of the content of reactive oxygen species (ROS), the mRNA of the ITF27 gene had a significant positive correlation (0.786, p=0.0251). The STK35 gene transcript (mRNA) was the only one of all the studied mRNAs that had an average negative correlation with sperm motility in native (-0.692, p=0.052) and frozen--thawed sperm (-0.876, p=0.035). These studies can be used to create a system of non-invasive transcriptional markers of bull sperm quality.

FTHL17, PRM2, CABYR, CPXCR1, ADAM29, and CABS1 are highly expressed in colon cancer patients and are regulated in vitro by epigenetic alterations

BackgroundColon cancer is a serious public health issue and a major cause of cancer-related mortality worldwide, including Saudi Arabia. Knowledge of genes associated with colon cancer development and progression is essential for identifying new cancer-specific biomarkers to improve the diagnosis of colon cancer. MethodsThe expression levels of FTHL17, PRM2, CABYR, CPXCR1, ADAM29, and CABS1 in 15 adjacent colon cancer and normal colon tissue samples from male patients were investigated using reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR) assays. qRT-PCR analysis was also used to determine whether reducing DNA methyltransferase (via 5-aza-2′-deoxycytidine treatment) or histone deacetylation (via trichostatin treatment) increased the expression levels of the tested genes. ResultsThe analysis of the 15 colon cancer and adjacent normal colon tissue samples revealed that all six genes were expressed in both groups, but their expression levels were significantly higher in the colon cancer group. Furthermore, the mRNA expression levels of the FTHL17, PRM2, CABYR, CPXCR1, and ADAM29 genes were considerably upregulated after treatment of HCT116 and Caco-2 cells with 5-aza-2′-deoxycytidine and trichostatin. However, the CABS1 gene was activated only with trichostatin treatment. ConclusionsThe findings of this study suggest that FTHL17, PRM2, CABYR, CPXCR1, ADAM29, and CABS1 are suitable candidate biomarkers of colon cancer and their expressions are regulated by hypomethylation and hyperacetylation.

The anti-spermatogenic activity of nanoliposomes loaded with Heracleum persicum phenolic compounds in Balb/C mice.

There are various types of bioactivities that have been reported for Heracleum persicum species, such as antioxidant, anti-inflammatory, and cytotoxicity properties. In the current study, the bio-accessibility of H. persicum bioactive compounds was improved by purifying its phenolic-enriched fractions (PEF) and encapsulating them into nanoliposomes to analyze its cytotoxic impacts on mice testicular tissue and their fertility status. Nano liposomal H. persicum PEF (NL-HPEF) was prepared by ultrasound-based encapsulation of HPEF and L-agranular lecithin mixture. The size, morphology, and stability of NL-HPEF were characterized by dynamic light scattering, field emission scanning electron microscopy, and zeta potential analysis. The 18 white male Balb/c mice (20-25g) at 3 treatment groups were provided to study the NL-HPPF cytotoxicity by measuring the mice liver enzyme including aspartate aminotransferase (AST), ALP and alanine aminotransferase (ALT), testis lipid peroxidation, and testicular tissue destruction levels. Moreover, the mice's fertility was evaluated by studying the Adam3, Prm1, Spata19, and Tnp2 gene expression in the testicular tissues. The obtained results manifested that the synthesized NL-HPEF was stable (193.7nm) and exhibited a notable cytotoxic impact on the mice's liver (ALT and AST enhancement levels) and testicular tissues. Moreover, their increasing treatment doses impaired the male mice's fertility by decreasing the sperm count, viability, and motility. In addition, fertility suppression was verified by decreasing serum testosterone and downregulating the Adam3, Prm1, Spata19, and Tnp2 gene expression in their testicular tissues. The male mice's fertility was significantly (p<0.05) suppressed by increasing treatment doses of NL-HPEF. Hence, the NL-HPEF could be considered a promising alternative to replace the male chemical contraceptives drugs.

Sperm Head Morphology Alterations Associated with Chromatin Instability and Lack of Protamine Abundance in Frozen-Thawed Sperm of Indonesian Local Bulls.

This study aimed to analyze various alterations in the morphology of the sperm head and its association with nucleus instability and insufficient sperm protamine. Frozen-thawed semen from twenty local Indonesian bulls was used for all stages in this study. The results of sperm head defect assessments are used for bull grouping, high (HD) and low (LD). Sperm DNA damage was assessed using Acridine Orange and Halomax. The PRM1 protein abundance was carried out using an enzyme immunoassay, while PRM1 gene expression was carried out using the RT-qPCR. PRM deficiency was performed using CMA3. Several kinds of sperm head defects in the HD were significantly higher (p < 0.05) than in the LD bulls. Sperm DNA damage showed a significant (p < 0.05) difference between the HD and LD bulls. PRM1 abundance was significantly (p < 0.05) decreased in HD bulls. PRM deficiency was significantly (p < 0.05) higher in HD bulls than in LD bulls. PRM deficiency in bulls correlated significantly (p < 0.01) with sperm head defects, DNA damage, and PRM1 abundance. The lack of sperm protamine might affect the sperm nucleus's stability and induce morphological alterations in the sperm head.

Susceptibility to azoospermia by haplotype analysis of protamine 1 and protamine 2 variants

Infertility remains a major global problem, affecting 10–15% of couples worldwide, with half of the cases due to male factors. Protamines, found only in the testes, are essential for sperm chromatin condensation and paternal genome imprinting during spermatogenesis. Seventy testicular samples from infertile men were analyzed for the expression level of PRM1 and PRM2. 448 infertile males were additionally examined for PRM1 and PRM2 polymorphism. There was a significant association of rs2070923 polymorphism of PRM2 in both additive and recessive models (OR (95% CI) 1.32[0.92; 1.88, p = 0.023] and 2.26[1.12; 4.57, p = 0.017], respectively). The TT genotype of rs2070923 PRM2 was also correlated with lower gene expression of PRM2 in the infertile group (p = 0.03). Moreover, our result showed that the frequency of GTCT haplotype was higher than average in azoospermic patients (OR = 2.67; 95%CI = 1.17–2.53; p = 0.041) and that gene-gene interaction played a role in male infertility. The linkage disequilibrium plot showed that rs2301365 was strongly associated with rs2070923. For the first time, we reported a significant haplotype of PRM1 and PRM2 genes associated with male infertility in Iranian population. These results are likely to contribute to a better understanding of the causes of male infertility.

Impact of waterpipe and tobacco cigarette smoking on global DNA methylation and nuclear proteins genes transcription in spermatozoa: a comparative investigation

Background Waterpipe smoking is harmful and dangerous, and it is a growing threat to public health. Objectives This study was performed to evaluate the influence of waterpipe smoking on global DNA methylation, DNA fragmentation, and protamine deficiency in spermatozoa compared to cigarette heavy smokers and nonsmokers, and to determine whether the transcription levels of spermatozoa nuclear proteins genes ‘PRM1, PRM2, and H2BFWT’ in waterpipe smokers are different compared to cigarette heavy smokers and nonsmokers. Methods A total of 900 semen samples were collected from males with a mean age of 32.5 ± 6.3 years (300 waterpipe smokers, 300 cigarette heavy smokers, and 300 nonsmokers). The nucleic acids were isolated from purified spermatozoa, and then the global DNA methylation and transcription levels of the PRM1, PRM2, and H2BFWT genes were assessed using ELISA and qPCR, respectively. Results A significant increase was found in the level of global DNA methylation (8.6 ± 0.6 ng/μl vs. 7.1 ± 0.6 ng/μl and 4.7 ± 0.6 ng/μl, p < 0.001), protamine deficiency (72.8 ± 15.3 vs. 51.7 ± 19.2 and 15.3 ± 5.9%, p < 0.001), and DNA fragmentation (73.4 ± 13.4 vs. 50.5 ± 18.9 and 9.3 ± 4.3%, p < 0.001) in waterpipe smokers compared to cigarette heavy smokers and nonsmokers. A significant increase was shown in the transcription levels of PRM1, PRM2, and H2BFWT genes in waterpipe smokers compared to cigarette heavy smokers and nonsmokers (p < 0.001). A down-regulation was found in the transcription level of these genes in different smoker groups compared to nonsmokers (<0.001). Conclusion This study suggests that waterpipe smoking is more harmful than cigarette smoking on semen parameters, global DNA methylation, and transcription of nuclear protein genes.

Gene Polymorphism, Microdeletion, and Gene Expression of PRM1, PRM2, AZFc in Infertile Males.

Background: Male infertility contributes to roughly 15% of all infertility cases in couples. The most common cause of male infertility is azoospermia, which is caused by genetic mutations. The connection between various single nucleotide polymorphisms in the PRM genes and AZF region microdeletions with male infertility has not been reported. In this case-control study, 100 infertile males (33 with azoospermia, 48 with oligozoospermia, and 19 with severe oligozoospermia) were chosen as the study subjects, and 100 fertile males were selected. Total DNA from peripheral blood was used to amplify two sequence-tagged site markers through multiplex PCR to detect AZFc partial deletions, and SNPs in PRM1 and PRM2 were determined through PCR-RFLP. Furthermore, quantitative real-time PCR was conducted to evaluate PRM1, PRM2, and DAZ1 (found in the AZFc region) expression levels in testis tissue. The frequency of the rs779337774 SNP in the PRM2 gene in the study population had no significant differences. However, a significant association was observed between the rs737008CA genotype (P= 0.013) and the C allele (P= 0.025) as a risk factor for male infant mortality. The deletion of sY254 and sY255 was discovered in azoospermia and severe oligozoospermia patients. Furthermore, all of these genes showed considerably low expression levels. However, only DAZ1 was identified with diagnostic biomarker potential (AUC=0.742). When these genes expression levels are reduced, the likelihood of spermatozoa retrieval in azoospermic individuals is elevated. Furthermore, no significant association was observed between PRM2 polymorphism and azoospermia; however, the CA genotype of PRM1 polymorphism is significantly associated with azoospermia incidence.

Protective effect of selenium and vitamin C on the fertility of male rats given penconazole.

Penconazole is used in agriculture and human and veterinary medicine applications. It has been included in the acute toxicity hazard category by the WHO. This study examines the protective effect of selenium and vitamin C on the fertility of male rats given penconazole. Nine groups of rats were given penconazole at concentrations of 50 and 75 mg/ml and selenium and vitamin C at concentrations of 0.5 and 100 mg/ml, respectively. Serum levels of LH and FSH were measured with ELISA kits; β-actin, GPX4, AQP7, PRM2, and BAX gene expression was evaluated with real-time PCR performed on the left testis of each rat. LH, FSH, and testosterone levels were lower in the groups given penconazole (50 and 75 mg/kg). Histopathology showed that the groups given penconazole had the lowest number of spermatogonia and primary spermatocytes; these numbers were greater in the groups receiving penconazole together with selenium or vitamin C; and the highest counts were observed in separate groups given Se and vitamin C. GPX4, AQP7, PRM2 and BAX gene expression in the groups receiving penconazole was different from controls and was modulated by treatment with selenium or vitamin C. This study showed that antioxidant compounds have a strengthening effect on the reproductive system and can mitigate the destructive effects of chemical fungicides.

Impact of tobacco smoking in association with H2BFWT, PRM1 and PRM2 genes variants on male infertility.

Tobacco's genotoxic components can cause a wide range of gene defects in spermatozoa such as single- or double-strand DNA breaks, cross-links, DNA-adducts, higher frequencies of aneuploidy and chromosomal abnormalities. The aim in this study was to determine the correlation between sperm quality determined by standard parameters, sperm DNA maturity tested by Chromomycin A3 (CMA3) staining, sperm DNA fragmentation tested by TUNEL assay and tobacco smoking in association with the single nucleotides polymorphisms (SNP) of three nuclear protein genes in spermatozoa (H2BFWT, PRM1 and PRM2). In this study, semen samples of 167 male patients were collected and divided into 54 non-smokers and 113 smokers. The target sequences in the extracted sperm DNA were amplified by PCR followed by Sanger sequencing. The results showed the presence of three variants: rs7885967, rs553509 and rs578953 in H2BFWT gene in the study population. Only one variant rs737008 was detected in PRM1 gene, and three variants were detected in the PRM2 gene: rs2070923, rs1646022 and rs424908. No significant association was observed between the concentration, progressive motility, morphology and the occurrence of H2BFWT, PRM1 and PRM2 SNPs. However, sperm parameters were significantly lower in heavy smokers compared to controls (p < 0.01) (sperm count: 46.00 vs. 78.50 mill/ml, progressive motility: 15.00% vs. 22.00%, and morphology 4.00% vs. 5.00%, respectively). Moreover, the heavy smoker individuals exhibited a considerable increase in CMA3 positivity and sDF compared to non-smokers (p < 0.01) (29.50% vs. 20.50% and 24.50% vs. 12.00%, respectively). In conclusion, smoking altered sperm parameters and sperm DNA integrity, but did not show a linkage with genetic variants in H2BFWT, and protamine genes (PRM1 and PRM2).

Correlation of Single Nucleotide Polymorphisms of PRM1, PRM2, PYGO2, and DAZL Genes with Male Infertility in North West of Iran.

Almost half of infertility is related to male factors. Although the effect of genetic factors on male infertility is identified, about 30%-50% still has no proven cause and is classified as idiopathic infertility. This study was performed to investigate the correlation of some single nucleotide polymorphisms of PYGO2, DAZL, PRM1, and PRM2 genes with male infertility in idiopathic cases among the Iranian population. In this case-control study, 120 idiopathic azoospermia or severe oligospermia patients in the range of 25-45 years and 120 fertile men in the same age range were recruited as case and control groups, respectively. Eight different single nucleotide polymorphisms including PRM1 rs737008, PRM1 rs423668, PRM2 rs1646022, PRM2 rs11645592, PYGO2 rs141722381, PYGO2rs61758741, DAZL rs75931701, and DAZL rs188506466 were genotyped by using ampli ficat ion-r efrac tory mutation system polymerase chain reaction methods. Hardy-Weinberg was calculated by using online website. Statistical Package for Social Sciences software was applied for statistical analysis. P value <.05 was considered significant. Thirty percent of the samples were regenotyped to confirm the obtained results. The obtained results showed a significant correlation between PYGO2 rs141722381 in the heterozygote form (odds ratio: 2.803, 95% CI: 1.397-5.626). Heterozygote over-dominance was also observed in this variant (odds ratio: 2.637, 95%CI: 1.321-5.264). There was no significant association between other studied single nucleotide polymorphisms and male infertility. This study proposed a novel single nucleotide polymorphism as a predisposition of male infertility among the Iranian population, but more studies in larger populations are needed to confirm the results.

Dynamic Expression and Chromatin Incorporation of ACT and CREM Transcription Factors in Testis Tissues of Infertile Men.

ObjectiveActivator of CREM in the testis (ACT) is a tissue specific transcription factor which activates cAMP responsive element modulator (CREM), a key transcription factor in differentiation of round spermatids into mature spermatozoa. They bind to CRE region in the promoters of transition protein genes (TNP1, TNP2) and protamine genes (PRM1 and PRM2), which are essential for sperm chromatin compaction, and regulates their transcription. This study was conducted to consider the expression of ACT and CREM and their regulatory roles on the expression of PRM1, PRM2, TNP1 and TNP2 genes in testis tissues of infertile men.Materials and MethodsIn this case-control study, testicular biopsies were collected from 40 infertile men and classified into three groups: obstructive azoospermia (OA, n=10, positive control), round spermatid maturation arrest (SMA, n=20), Sertoli cell-only syndrome (SCOS, n=10, negative control group). Using quantitative real-time polymerase chain reaction (PCR), the expression profile of ACT, CREM, TNP1, TNP2, PRM1 and PRM2 genes were assessed in testicular samples and incorporation of ACT and CREM proteins on the promoters of PRM1, PRM2, TNP1 and TNP2 genes were also evaluated by ChIP-real time PCR. ResultsOur results demonstrated significant decrease in the expression levels of ACT, CREM and in their incorporations on their target genes in SMA group in comparison to control groups (P<0.05).ConclusionThese data confirm that there is low expression and incorporation of ACT and CREM and of their target genes in infertilities which are associated with post-meiotic arrest.

Comparison of polymorphism 139C > A ( (rs737008) of protamine 1 gene in infertile men with diagnosis of oligospermia and asthenospermia

The male infertility accounts for about half of the infertility in couples. The idiotypic asthenospermia and oligospermia, which mostly occur as a result of genetic mutations, are among the main causes of male infertility. Until now, the relationship between different SNPs in the PRM1 gene and male infertility have been reported. In this study, we evaluated the possible correlation between 139C > A (rs737008) SNP in the PRM1 gene and asthenospermia/oligospermia in patients who referred to the Gerash Infertility Center. To aim this, three groups were considered in this study including healthy fertile males, asthenospermia patients and the patients suffering from oligospermia. After DNA extraction from their blood samples, the PCR was carried out to amplify a 558 bp PRM1 gene fragment. Then, the RFLP technique was performed to identified the SNP in the PCR products. Our results showed that the frequency of the 139C > A (rs737008) SNP in the population study was 41%. We found no significant differences between the SNP and asthenospermia/oligospermia in the current study. According to the demographic data, no significant differences were also found between smoking or alcohol consumption and male infertility in this study.

P–076 Probability of sperm retrieval in azoospermic patients and mRNA expression profile of JMJD1A, TNP2 and PRM2 : in a subset of karachi population

Abstract Study question To access successfulness of sperm retrieval by evaluating the mRNA expression profile of JMJD1A, TNP1, TNP2, PRM1 and PRM2 in patients undergoing surgical sperm retrieval procedure. Summary answer Probability of sperm retrieval in azoospermia is decreased when mRNA expression profile of JMJD1A TNP2 and PRM2 in testicular tissue is decreased. What is known already Studies have been done on expression of JMJD1A in non-obstructive azoospermic patients in other part of the world with smaller sample size but this is the first study in Pakistan with larger number of patients. Study design, size, duration: Crossectional study, 100 azoospermic patients coming for purpose of sperm retrieval by TESE or micro-TESE in Australian Concept Infertility Medical Center, Karachi,from March, 2018 to December, 2019 Participants/materials, setting, methods All recruited azoospermic patients were evaluated by history, physical examination, and hormonal assessment. RNA was extracted by pureLink RNA Micro kit and mRNA expression of the JMJD1A, TNP1, TNP2, PRM1 and PRM2 genes was determined using innu-SCRIPT One Step RT_qPCR SyGreen kit. For quantitative variables independent t test and for categorical variables chi-square/ Fisher Exact test was used. Unadjusted and adjusted prevalence ratio were reported by using cox regression algorithm. Main results and the role of chance: The patients were categorized into (i) Group-I: Patients with successful sperm retrieval n = 42, (ii) Group-II: Patients with unsuccessful sperm retrieval n = 58. The patients were categorized into (i) Group-I: Patients with successful sperm retrieval n = 42, (ii) Group-II: Patients with unsuccessful sperm retrieval n = 58. Azoospermic men in the successful sperm retrieval group had significantly decreased expression of JMJD1A (P &amp;lt; 0.001), TNP2(P &amp;lt; 0.001), and PRM2 (P 0.008). In addition to this regarding hormonal parameters: FSH (P 0.004), LH(P &amp;lt; 0.001), TSH(P&amp;lt;.011) were significantly different in azoospermic men with successful and unsuccessful sperm retrieval. In multivariate analysis, after adjusting for the other covariates, a significant association was found between JMJD1A, TNP2, PRM2 and successful sperm retrieval (p-value &amp;lt;0.05). Limitations, reasons for caution It is unicentric and outcomes for fertilization were not assessed. Azoospermic patients from multi-centeres were difficult because of lack of facility of sperm retrieval procedures at these centers and it was difficult to follow the fertrilization outcome. Wider implications of the findings: This will be useful for making the decision in azoospermic men to proceed for ICSI or not. In addition to this, the repetition of unnecessary surgical procedures can be avoided, as the azoospermic men often undergo number of rounds of ICSI, with the hope of becoming biological father. Trial registration number non-clinical trials