Impact of Tobacco smoking on sperm nuclear proteins genes : H2BFWT, TNP1, TNP2, PRM1, and PRM2 and its influence on male infertility

During fertilization, the spermatozoon task is not limited to just deliver the male genome to the oocyte, it is beyond that. During the last stages of spermatogenesis, the spermatozoa develop to have a highly organized genome. The sperm DNA is compacted by nearly 85% of protamines and 15 % of histones carrying epigenetic signals, together with different kind of RNA molecules and proteins participate in post-fertilization events and mainly in embryo development. About 50% of infertility cases are referred to as idiopathic infertility, around 15% of it is due to genetic factors and 35% due to environmental factors. Tobacco smoke is one of the lifestyle factors that present a big threat for human health and it is believed to have an influence on male fertility and sperm quality by inducing epigenetic and/or genetic modulations on sperm genome. Thus, this study comes to fulfil a part in the remained gap in the comprehensive understanding of the expression and regulation of the genes in the human spermatozoa. The purposes of this study were first, to find out the effect of Tobacco smoke on sperm quality determined by standard parameters (WHO, 2010), sperm DNA maturity tested by Chromomycin A3 (CMA3) staining, sperm DNA fragmentation tested by TUNEL assay, and clinic outcomes after ICSI therapy. Second, to quantify the transcript levels of five nuclear proteins genes: H2BFWT, TNP1, TNP2, PRM1, and PRM2 by RT-PCR, correlate them with the previous parameters and determine the smoking effect on this gene expression and regulation. Finally, to determine the single nucleotides polymorphisms in three genes: H2BFWT, PRM1, and PRM2 by Sanger sequencing and their association to smoking and previous parameters. The study population (n=167) were male partners, randomly collected, of couples undergoing intracytoplasmic sperm injection (ICSI) therapy, in reproductive age (25-49 years). The patients were divided into two groups: heavy-smokers group and non-smokers group. In heavy-smokers group a significant decrease (p<0.01) in standard semen parameters in comparison to non-smokers has been shown: sperm concentration (62.17 ± 51.68 mill/ml vs. 88.09 ± 63.42 mill/ml), progressive motility (PR) (14.86 ± 10.95% vs. 27.31 ± 21.78%), and sperm normal morphology (4.01 ± 2.88% vs. 10.87 ± 12.11%). Besides, the mean percentage of protamine deficiency (CMA3 positivity) and sperm DNA fragmentation (sDF) were significantly higher (p<0.01) in heavy-smokers than in nonsmokers (33.30 ± 23.33% vs. 20.35 ± 13.43% and 26.86 ± 19.77% vs.14.23 ± 13.07% respectively). A significant positive correlation has been found between CMA3 positivity and sDF (r=0.484, p=0.0001) in the group of heavy-smokers, and no correlation (r=0.256, p=0.098) between these two parameters in the non-smokers group. In the present study, by comparing the ICSI results between the heavy-smokers and nonsmokers, the pregnancy rate was significantly higher in the group of non-smokers than the heavy-smokers group (0.60 ± 0.49% vs. 0.38 ± 0.48%, p=0.013), other parameters showed no significant differences. Furthermore, the transcript level of each studied gene mRNA (mean delta Ct) was differentially expressed between the heavy-smokers and non-smokers groups and this difference was highly significant (p<0.01). H2BFWT, TNP1, TNP2, PRM1 and PRM2 genes were down-regulated in spermatozoa of heavy-smoker compared to non-smoker (Fold change<0.5) and were significantly correlated between each other (p<0.01). Moreover, the protamine mRNA ratio, in the current study, was significantly higher in the heavy-smokers group in comparison to the non-smokers group (0.60 ± 1.08 vs. 0.11 ± 0.84, p=0.001). In the non-smokers group, protamine ratio correlated positively with the expression levels of TNP2 (r=0.349, p=0.032) and PRM2 (r=0.488, p=0.001). However, in the heavysmokers group, it correlated positively to TNP2 transcript (r=0.307, p=0.004), PRM2 transcript (r=0.445, p=0.0001), and H2BFWT transcript (r=0.342, p=0.001). Only in the group of heavy-smokers, the protamine ratio significantly correlates with CMA3 positivity (r=0.413, p=0.0001) and sDF (r=0.302, p=0.003). Moreover, the present study demonstrated the absence of a connection between genetic variations founded in H2BFWT gene (rs7885967, rs553509 and rs578953), protamines genes PRM1 (rs737008) and PRM2 (rs2070923 and rs1646022) and fertility alteration in heavy-smoker and non-smoker males. In conclusion, the results of this study demonstrated that smoking has inverse effects on sperm quality, sperm DNA integrity, mRNA expression levels of H2BFWT, TNP1, TNP2, PRM1and PRM2 genes and protamine mRNA ratio, but has no effect on the nucleotides sequences of these genes. This suggests that the RNA of the studied genes and protamine mRNA ratio in the sperm of male partners of ICSI patients are good predictive factors to evaluate the sperm quality and its fertilizing capacity.Das Spermium hat während der Befruchtung mehrere Aufgaben. Zum einen muss das männliche Genom zur Eizelle. Des Weiteren entwickeln sich die Spermien in den letzten Stadien der Spermatogenese zu einem hoch organisierten Genom. Die Spermien-DNA wird aus Protaminen (ca. 85%) und Histonen (ca. 15%) gebildet, zusammen mit verschiedenen Arten von RNA-Molekülen und Proteinen. Diese sind vor allem an Prozessen nach der Fertilisation beteiligt und nehmen eine wichtige Rolle in der Embryonalentwicklung ein. Etwa 50% der Fälle von Unfruchtbarkeit werden als idiopathische Unfruchtbarkeit bezeichnet, davon sind 15% auf genetische Faktoren und 35% auf Umweltfaktoren zurückzuführen. Tabakrauch ist einer der Lebensstilfaktoren, die eine große Bedrohung für die menschliche Gesundheit darstellen. Es wird angenommen, dass Rauchen Einfluss auf die männliche Fruchtbarkeit und Spermienqualität hat, indem es epigenetische und/oder genetische Modulationen auf das Spermiengenom induziert. Ziel dieser Studie war es, zunächst die Wirkung von Tabakrauch auf die Spermienqualität zu ermitteln.Die Spermienqualität wurde bestimmt durch Standardparameter (WHO, 2010), die DNA-Integrität (gemessen mit Chromomycin A3 Färbung, CMA3) und die DNAFragmentierung (gemessen durch den TUNEL-Test). Des Weiteren wurde das klinische Outcome nach ICSI Therapie dokumentiert. Transkriptionswerte von fünf atomaren Proteingenen wurden ebenfalls quantifiziert: H2BFWT, TNP1, TNP2, PRM1 und PRM2 von RT-PCR.Es wurde untersucht, ob die Gene mit den bisherigen Parametern korrelieren und ob es einen Effekt des Rauchens auf die Genexpression, bzw. Genregulation gibt. Um die einzelne Nukleotid-Polymorphismen zu bestimmen, wurden 3 Gene (H2BFWT, PRM1 und PRM2) durch Sanger-Sequenzierung untersucht.Ihre Assoziation mit dem Rauchen, sowie mit den anderen Spermienparametern wurde ebenfalls gemessen. Die Studienpopulation (n=167) waren männliche Probanden, die sich einer intrazytoplasmatischen Spermieninjektion (ICSI) unterzogen hatten. Das Alter betrug 25-49 Jahre. Die Patienten wurden in zwei Gruppen eingeteilt: Die Gruppe der schweren Raucher und die Nichtraucher-Gruppe. Bei der Gruppe der Raucher wurde eine signifikante Abnahme (p<0,01) bei den Standardparametern im Vergleich zu Nichtrauchern gezeigt: Spermienkonzentration (62,17 ± 51,68 mill/ml vs. 88,09 ± 63,42 mill/ml), progressive Beweglichkeit (PR) (14,86 ± 10,95% vs. 27,31 ± 21,78%) und Spermienmit normaler Morphologie (4,01 ± 2,88% vs. 10,87 ± 12,11%) waren bei Rauchern im Vergleich zu den Nichtrauchern reduziert. Außerdem war der Mittelanteil von Protamin-Mangel (CMA3-Positivität) und der DNAFragmentierung von Spermien (sDF) bei Rauchern signifikant höher (p<0,01) als bei Nichtrauchern (33,30 ± 23,33% vs. 20,35 ± 13,43% und 26,86 ± 19,77% vs. 14,23 ± 13,07%). Es wurde eine signifikante, positive Korrelation zwischen die CMA3-Positivität und sDF (r=0,484; p=0,0001) in der Gruppe der Raucher festgestellt. Dagegen gab es keine Korrelation (r=0,256; p=0,098) zwischen diesen beiden Parametern in der Nichtraucher- Gruppe. In der vorliegenden Studie war die Schwangerschaftsrate nach ICSI in der Gruppe der Nichtraucher deutlich höher als die der Raucher-Gruppe (0,60 ± 0,49% vs 0,38 ± 0,48%; p=0,013). Darüber hinaus wurde die Transkriptionsstufe jedes untersuchten Gens mRNA (mean delta ct) zwischen den Rauchern und Nichtrauchern differenziert ausgedrückt.Dieser Unterschied war signifikant (p<0,01). Die Gene H2BFWT, TNP1, TNP2, PRM1 und PRM2 wurden in Spermien der Raucher im Vergleich zum Nichtraucher (Foldchange<0,5) herunterreguliert und signifikant miteinander korreliert (p<0,01). Zudem lag das Protamin-MRNAVerhältnis in der Raucher-Gruppe deutlich höher (0,11 ± 0,84 vs. 0,60 ± 1,08, p=0,001). In der Gruppe der Nichtraucher korrelierte das Protaminsverhältnis positiv mit den Ausdruckstufen TNP2 (r=0,349; p=0,032) und PRM2 (r=0,488; p=0,001). In der Gruppe der Raucher korrelierte es jedoch positiv mit TNP2-Transkript (r=0,307; p=0,004), PRM2- Transkript (r=0,445; p=0,0001) und H2BFWT-Transkript (r=0,342; p=0,001). Nur in der Gruppe der Raucher korreliert das Protaminsverhältnis signifikant mit CMA3-Positivität (r=0,413; p=0,0001) und sDF (r=0,302; p=0,003). Darüber hinaus hat die vorliegende Studie gezeigt, dass es keinen Zusammenhang zwischen genetischen Variationen gibt, die im H2BFWT-Gen (rs7885967, rs553509 und rs578953), Protaminen-Gen PRM1 (rs737008) und PRM2 (rs2070923 und rs1646022) vorkommen. Zusammenfassend haben die Ergebnisse dieser Studie gezeigt, dass Rauchen einen negativen Einfluss auf die Spermienqualität hat.Die Spermienqualität, die mRNAExpressionsstufen der H2BFWT, TNP1, TNP2, PRM1-und PRM2-Gene und das ProtaminmRNA- Verhältnis waren bei Nichtrauchern reduziert, hatten aber keine Auswirkungen auf die Nukleotid-Sequenzen dieser Gene. Dies deutet darauf hin, dass die RNA der untersuchten Gene, sowie das Protamin-mRNA-Verhältnis in den Spermien männlicher Partner von ICSI-Patienten gute Vorhersage-Faktoren sind, um die Spermienqualität und ihre Fertilität zu bewerten

A Systematic Review of the Impact of Mitochondrial Variations on Male Infertility

According to current estimates, infertility affects one in four couples trying to conceive. Primary or secondary infertility can be due either to both partners or only to the man or the woman. Up to 15% of infertility cases in men can be attributed to genetic factors that can lead to irreversible partial or complete spermatogenic arrest. The increased use of assisted reproductive technology (ART) has provided not only insights into the causes of male infertility but also afforded a diagnostic tool to detect and manage this condition among couples. Genes control a variety of physiological attributes, such as the hypothalamic–pituitary–gonadal axis, development, and germ cell differentiation. In the era of ART, it is important to understand the genetic basis of infertility so as to provide the most tailored therapy and counseling to couples. Genetic factors involved in male infertility can be chromosome abnormalities or single-gene disorders, mitochondrial DNA (mtDNA) mutations, Y-chromosome deletions, multifactorial disorders, imprinting disorders, or endocrine disorders of genetic origin. In this review, we discuss the role of mitochondria and the mitochondrial genome as an indicator of sperm quality and fertility

Tobacco smoking and its impact on the expression level of sperm nuclear protein genes: H2BFWT, TNP1, TNP2, PRM1 and PRM2

The aim of this current study was to investigate the influence of tobacco smoke on sperm quality determined by standard parameters, on sperm DNA maturity tested by chromomycin A3 (CMA3) staining, on sperm DNA fragmentation tested by TUNEL assay and on the transcript level of sperm nuclear proteins H2BFWT, PRM1, PRM2, TNP1 and TNP2 genes quantified by RT‐PCR. One hundred forty‐one (141) sperm samples (43 nonsmokers (G.1) and 98 heavy smokers (G.2)) of couples undergoing ICSI were enrolled in this study. In G2, a significant decrease in standard semen parameters in comparison with nonsmokers was shown (p < .01). In contrast, protamine deficiency (CMA3 positivity) and sperm DNA fragmentation (sDF) were significantly higher in G2 than in G1 (p < .01). Furthermore, the studied genes were differentially expressed (p < .01), down‐regulated in the spermatozoa of G.2 compared to that of G.1 (fold change <0.5) and were significantly correlated between each other (p < .01). Moreover, in comparison with G1, the protamine mRNA ratio in G2 was significantly higher (p < .01). It can therefore be concluded that smoking alters mRNA expression levels of H2BFWT, TNP1, TNP2, PRM1 and PRM2 genes and the protamine mRNA ratio and consequently alters normal sperm function

Hydrochemistry and stable isotopes (δ 18 O and δ 2 H) tools applied to the study of karst aquifers in Southern Mediterranean basin (Teboursouk area, NW Tunisia)

Karst aquifers receive increasing attention in Mediterranean countries as they provide large supplies water used for drinkable and irrigation purposes as well as for electricity production. In Teboursouk basin, Northwestern Tunisia, characterized by a typical karst landscape, the water hosted in the carbonates aquifers provides large parts of water supply for drinkable water and agriculture purposes. Groundwater circulation in karst aquifers is characterized by short residence time and low water-rock interaction caused by high karstification processes in the study area. Ion exchange process, rock dissolution and rainfall infiltration are the principal factors of water mineralization and spatial distribution of groundwater chemistry. The present work attempted to study karstic groundwater in Teboursouk region using hydrochemistry and stable isotopes (δ18O and δ2H) tools. Karst aquifers have good water quality with low salinity levels expressed by TDS values largely below 1.5 g/l with Ca-SO4-Cl water type prevailing in the study area. The aquifers have been recharged by rainfall originating from a mixture of Atlantic and Mediterranean vapor masses

Impact of heavy alcohol consumption and cigarette smoking on sperm DNA integrity

The purposes of the presents study were to investigate the impact of alcohol consumption and cigarette smoking on semen parameters and sperm DNA quality, as well as to determine whether tobacco smoking, or alcohol consumption causes more deterioration of sperm quality. Two hundred and eleven semen samples of men were included in this study. Four groups were studied: heavy smokers (N = 48), heavy drinkers (N = 52), non-smokers (n = 70), and non-drinkers (n = 41). Semen parameters were determined according to WHO guidelines, protamine deficiency assessed by chromomycin (CMA3) staining, and sperm DNA fragmentation (sDF) evaluated by TUNEL assay. Sperm parameters were significantly higher in non-smokers versus smokers and in non-drinkers versus drinkers (p < 0.005). However, protamine deficiency and sDF were significantly lower in non-smokers versus smokers and in non-drinkers versus drinkers (p < 0.0001). No significant difference in the semen analysis parameters was observed between heavy smokers and heavy drinkers (semen volume: 3.20 ± 1.43 vs. 2.81 ± 1.56 ml, semen count: 65.75 ± 31.32 vs. 53.51 ± 32.67 mill/ml, total motility: 24.27 ± 8.18 vs. 23.75 ± 1.75%, sperm vitality: 36.15 ± 18.57 vs. 34.62 ± 16.65%, functional integrity: 41.56 ± 18.57 vs. 45.96 ± 17.98% and the morphologically normal spermatozoa: 28.77 ± 11.82 vs. 27.06 ± 13.13%, respectively). However, protamine deficiency was significantly higher among drinkers than smokers (37.03 ± 9.75 vs. 33.27 ± 8.56%, p = 0.020). The sDF was also significantly higher among drinkers than smokers (22.37 ± 7.60 vs. 15.55 ± 3.33%, p < 0.0001). Thus, cigarette smoking, and heavy alcohol intake can deteriorate sperm quality. However, alcohol consumption deteriorates sperm maturity and damages DNA integrity at significantly higher rates than cigarette smoking

An additional marker for sperm DNA quality evaluation in spermatozoa of male partners of couples undergoing assisted reproduction technique (IVF/ICSI): Protamine ratio

The aim of this study was to evaluate the relationship between the protamine ratio (P1/P2), DNA fragmentation of spermatozoa and protamine deficiency. Patients were grouped into fertile (G1; n = 151) and sub‐fertile (G2; n = 121). DNA fragmentation in spermatozoa was analysed by a TUNEL assay (terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labelling), and the protamination was determined by CMA3 staining, while Western blot was used to measure protamine P1 and P2. While sperm DNA fragmentation (SDF) and protamine ratio were significantly elevated in G2 compared with G1 (12.31 ± 7.01% vs. 17.5 ± 9.5%; p = .001) and (0.91 ± 0.43 vs. 0.75 ± 0.42; p = .003); respectively, the CMA3 positive showed no difference at all between G1 and G2. In G1, the CMA3 positive correlated negatively with the P1/P2 ratio and SDF (r = −.586, r = −.297; p = .001 respectively). In contrast, the protamine ratio correlated positively with SDF (r = .356; p = .001). In G2, no correlation was observed between CMA3 positive, SDF and the P1/P2 ratio but the P1/P2 ratio showed a positive correlation with SDF (r = .479; p = .001). In conclusion, the spermatozoa DNA deterioration was closely associated with abnormal protamination but showed an association with the protamine ratio, more than with CMA3 positive. Therefore, for the evaluation of DNA damage in spermatozoa, the P1/P2 ratio might act as an additional biomarker

The Impact of Heavy Smoking on Male Infertility and Its Correlation with the Expression Levels of the PTPRN2 and PGAM5 Genes

Smoking has been linked to male infertility by affecting the sperm epigenome and genome. In this study, we aimed to determine possible changes in the transcript levels of PGAM5 (the phosphoglycerate mutase family member 5), PTPRN2 (protein tyrosine phosphatase, N2-type receptor), and TYRO3 (tyrosine protein kinase receptor) in heavy smokers compared to non-smokers, and to investigate their association with the fundamental sperm parameters. In total, 118 sperm samples (63 heavy-smokers (G1) and 55 non-smokers (G2)) were included in this study. A semen analysis was performed according to the WHO guidelines. After a total RNA extraction, RT-PCR was used to quantify the transcript levels of the studied genes. In G1, a significant decrease in the standard semen parameters in comparison to the non-smokers was shown (p < 0.05). Moreover, PGAM5 and PTPRN2 were differentially expressed (p ≤ 0.03 and p ≤ 0.01, respectively) and downregulated in the spermatozoa of G1 compared to G2. In contrast, no difference was observed for TYRO3 (p ≤ 0.3). In G1, the mRNA expression level of the studied genes was correlated negatively with motility, sperm count, normal form, vitality, and sperm membrane integrity (p < 0.05). Therefore, smoking may affect gene expression and male fertility by altering the DNA methylation patterns in the genes associated with fertility and sperm quality, including PGAM5, PTPRN2, and TYRO3

Groundwater mixing and geochemical assessment of low-enthalpy resources in the geothermal field of southwestern Tunisia

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Association between the single nucleotide variants of the mitochondrial cytochrome B gene (MT-CYB) and the male infertility

Background Idiopathic male infertility can be attributed to genetic predispositions that afect sperm performance and function. Genetic alterations in the mitochondrial DNA (mtDNA) have been linked to certain types of male infertility and abnormal sperm function. Mutations in the mitochondrial cytochrome B (MT-CYB) gene might lead to some defciencies in mitochondrial function. Thus, in the current study, we aimed to investigate the efect of mutations in the MT-CYB gene on sperm motility and male infertility. Methods and results Semen specimens were collected from 111 men where 67 men were subfertile and 44 were fertile. QIAamp DNA Mini Kit and REPLI-g Mitochondrial DNA Kit from QIAGEN were used to isolate and amplify the mito chondrial DNA. Followed by PCR and Sanger sequencing for the target sequence in the MT-CYP gene. Sequencing of the MT-CYB gene revealed a total of thirteen single nucleotide polymorphisms (SNPs). Eight SNPs were non-synonymous vari ant (missense variant) including: rs2853508, rs28357685, rs41518645, rs2853507, rs28357376, rs35070048, rs2853506, and rs28660155. While fve SNPs were Synonymous variant: rs527236194, rs28357373, rs28357369, rs41504845, and rs2854124. Among these SNPs, three variants showed a signifcant diference in the frequency of the genotypes between subfertile and fertile groups: rs527236194 (T15784C) (P=0.0005), rs28357373 (T15629C) (P=0.0439), and rs41504845 (C15833T) (P=0.0038). Moreover, two SNPs showed a signifcant association between allelic frequencies of rs527236194 (T15784C) (P=0.0014) and rs41504845 (C15833T) (P=0.0147) and male subfertility. Conclusion The current study showed a signifcant association between the MT-CYB gene polymorphisms and the develop ment of male infertility. In particular, rs527236194, rs28357373 and rs41504845 variants were found to be the most related to the subfertility group. Further studies on larger and other populations are required to reveal the exact role of this gene in the development of male infertility. In addition, functional studies will be helpful to elucidate the molecular impact of the MT-CYP polymorphisms on mitochondrial function

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)