Influence of maternal age on intracytoplasmic sperm injection outcome and global deoxyribonucleic acid methylation in women undergoing intracytoplasmic sperm injection cycle

Background: Intracytoplasmic sperm injection needs sufficient oocytes of high quality in order to increase the rate of fertilization and pregnancy. This study was designed to investigate the influence of maternal age on the ICSI outcomes in women undergoing to first ICSI cycle and to evaluate the influence of maternal age on global DNA methylation.Methods: A total of 242 females were included in this study with a mean age of 30.5±7.3 years. The participants were divided into three groups depending on women's age≤25, N=70; 26-35, N=102 and>35, N=70). The genomic DNA was isolated from the blood samples, then the global DNA methylation was evaluated using ELISA.Results: A significant reduction has been found in the level of anti-Müllerian hormone (AMH), total number of the collected oocyte, mature oocytes, fertilized oocytes and number of embryos transferred in the older females compared to the younger group (p<0.001). While a significant increase has been found in global DNA methylation level in the older females compared to the younger group (p<0.001). A positive significant correlation has been found between global DNA methylation level and maternal age (p<0.001). In contrast, a negative significant correlation has been shown between AMH level, mature oocytes and maternal age (p<0.001).Conclusions: Maternal age has a significant influence on the number of mature oocytes, number of embryos transferred and global DNA methylation. The pregnancy chance is more in the age group less than 35 years

Association between aberrations in DNA methylation patterns of spermatozoa and abnormalities in semen parameters of subfertile males

Infertility affects 10 15% of couples and approximately 50% of cases are linked to male factor infertility. In recent decades, many studies have found that genetic causes, aberrant patterns of DNA methylation and histone modification have been shown to play a role in male infertility or fecundity decline, and have been identified in 15 30% of infertile males. Besides, several studies have illustrated that changes in the DNA methylation of specific genes in germ cells are linked with oligozoospermia, reduced progressive sperm motility, and abnormal sperm morphology. Likewise, a recent study detected aberrant in DNA methylation levels for genes involved in the spermatogenic program and located outside of imprinted regions in poor quality spermatozoa. The present thesis aimed (I) to determine whether sperm DNA methylation level at CpG dinucleotides is different in males suffering from a reduction in fecundity compared to proven fertile males, (II) to determine the relationship between changes in sperm DNA methylation levels in this gene and abnormalities in semen parameters, (III) to identify whether cigarette smoking alters sperm DNA methylation patterns and to determine whether this alteration in sperm DNA methylation is associated with basic semen parameters like sperm count, sperm motility, and morphology, (IV) to evaluate the association between the gene expressions level for the genes included in this study and semen parameters, addition to assessing the correlation between the gene expression and the alteration in spermatozoa DNA methylation levels. The strategy of this research was applied as the following: Infinium 450K BeadChip array was used as screening study, to evaluate the variation in sperm DNA methylation levels between cases and controls groups. Then the differentially methylated CpGs (DMC) underwent to deep bisulfite sequencing to validate on large cohort samples (as validation study). In the end, Quantitative RT-PCR assay was used to evaluate the expression of the gene included in this study. According to the results of the validation study, the bioinformatics analysis found that more than one CpG showed a difference in the methylation level addition to CpGs obtained from the screening study between subfertile males and proven fertile males. Some of this CpGs were located in gene bodies and CpG islands (PRICKLE2, ALS2CR12, ALDH3B2, PTGIR, KCNJ5, MLPH, SMC1b, GAA, PRRC2A, MAPK8/P3, PDE11A, ANXA2, CGß, and TYRO3), while the other CpGs were located in intergenic regions. Besides, a significant correlation was found between the methylation levels at the CpGs in genes related amplicon of subfertile males and semen parameters like sperm count, a percentage of total sperm motility, the percentage of progressive motility, a percentage of nonprogressive motility, a percentage of immotile sperm and sperm vitality. On the other hand, this study showed more than one CpGs have significant differences in methylation levels between current smokers as compared to never smoked males like CpGs namely cg07869343 and cg19169023, which are located in the MAPK8IP3 and TKR genes. Moreover, the results showed a significant correlation between the methylation levels at more than one CpGs in the genes related amplicon of current smoker males and semen parameters such as sperm count, the percentage of total sperm motility, a percentage of sperm immotile, a percentage of progressive motility, and a percentage of sperm normal form. In conclusion, this study identified CpGs related to different genes have an alteration in sperm DNA methylation levels in cases compared to controls groups. In addition, an association between changes in the methylation level for these CpGs and different semen parameters was found. The observed variations may have an influence on sperm phenotype. More studies are needed to clarify the mechanisms relating to these alterations and to discover their significance and functional consequences for male fecundityUnfruchtbarkeit betrifft 10 - 15% der Paare und etwa 50 % der Fälle werden an männliche Faktorunfruchtbarkeit verbunden. In den letzten Jahrzehnten haben viele Studien haben festgestellt, dass die genetische Ursachen, abweichendes Muster der DNA-Methylierung und Histonmodifikation gezeigt haben, eine Rolle in der männlichen Unfruchtbarkeit oder Fruchtbarkeit Rückgang zu spielen, und haben in 15 identifiziert wurde - 30% der infertile Männer. Neben, mehrere Studien haben gezeigt, dass die Veränderungen in der DNA-Methylierung spezifischer Gene in Keimzellen verbunden sind mit oligozoospermie, reduzierte progressive Beweglichkeit der Spermien, die Morphologie der Spermien und abnormal. Ebenso wird eine aktuelle Studie entdeckt aberrante DNA-Methylierung von Genen, die in die spermatogene programm beteiligten und außerhalb der geprägten Regionen in schlechter Qualität der Spermien. Die vorliegende Arbeit soll (I), um zu bestimmen, ob die Spermien-DNA Methylierung bei CpG dinucleotides verschiedenen in Männer leiden unter einer Verringerung der Fruchtbarkeit ist im Vergleich zu den bewährten fruchtbare Männer, (II) die Beziehung zwischen Veränderungen der Spermien-DNA Methylierung Ebenen in diesem Gen und Missbildungen im Samen Parameter zu bestimmen, (iii) um zu ermitteln, ob das Rauchen verändert Spermien-DNA Methylierungsmuster und zu ermitteln, ob diese Änderung der Spermien-DNA Methylierung mit grundlegenden Samen Parameter wie die Zahl der Spermien, die Beweglichkeit der Spermien zugeordnet ist, und die Morphologie, (iv) die Assoziation zwischen den gen Ausdrücke für die Gene, die in dieser Studie und Samen Parameter enthalten, zusätzlich zu der Bewertung der Korrelation zwischen der Genexpression und der Veränderung der Spermien-DNA-Methylierung. Die Strategie dieser Forschung war, wie die Folgenden: Infinium 450K BeadChip Array war als screening Studie verwendet, die Veränderung der Spermien-DNA Methylierung Ebenen zwischen Fällen und Kontrollen Gruppen zu bewerten. Dann die differentiell methylierte CpGs (DMC) wurde zu tief Bisulfite Sequencing auf große Kohorte Proben (als validierungsstudie) bestätigen. Am Ende, Quantitative RT-PCR Assay wurde verwendet, um die Expression des Gens in dieser Studie zu bewerten. Nach den Ergebnissen der Validierungsstudie, die bioinformatik Analyse ergab, dass mehr als ein CpG zeigten einen Unterschied in der methylierung Ebene neben der CpGs vom screening Studie zwischen subfertile Männer und bewährte fruchtbare Männer gewonnen. Einige dieser CpGs wurden in gen Stellen und CpG-Inseln (KRIBBELN 2, ALS 2 CR 12, ALDH3 B2, PTGIR, Kcnj 5, MLPH, SMC 1b, GAA, PRRC2A, MAPK 8 IP3, PDE11A, ANXA2, CGß und TYRO3), während die anderen CpGs in intergenic Regionen entfernt wurden. Außerdem wurde eine signifikante Korrelation zwischen der Methylierung der CpGs in Genen amplikon der Subfertile Männer und Samen Parameter wie die Zahl der Spermien gefunden wurde, einen Prozentsatz der gesamten Beweglichkeit der Spermien, der Prozentsatz der Spermienbeweglichkeit, ein Anteil von Nicht-progressive Motilität, einen Prozentsatz der Immotile Spermien und Spermien Vitalität. Auf der anderen Seite ist diese Studie zeigte mehr als ein CpGs haben signifikante Unterschiede in der Methylierung Ebenen zwischen Rauchern gegenüber nie geraucht Männchen wie CpGs nämlich cg cg 07869343 und 19169023, die in der Mapk 8 IP3 und TKR Gene befinden. Darüber hinaus. Die Ergebnisse zeigten einen signifikanten Zusammenhang zwischen der Methylierung Niveaus an mehr als einem CpGs in den Genen amplikon der aktuellen Raucher Männer und Samen Parameter wie die Zahl der Spermien, der Prozentsatz der gesamten Beweglichkeit der Spermien, einen Prozentsatz der Spermien Immotile, einen Prozentsatz der Spermienbeweglichkeit und ein Prozentsatz der Spermien normale Form an. Im Ergebnis dieser Studie identifizierten CpGs im Zusammenhang mit verschiedenen Genen haben eine Veränderung in der Spermien-DNA Methylierung Ebenen in Fällen verglichen mit Kontrollen Gruppen. Zusätzlich wird eine Verbindung zwischen den Veränderungen in der Methylierung für diese CpGs und verschiedenen Samen Parameter gefunden wurde. Die beobachteten Abweichungen können einen Einfluss auf die spermien Phänotyp. Weitere Studien sind notwendig, um die Mechanismen, die im Zusammenhang mit diesen Veränderungen und deren Bedeutung und funktionelle Konsequenzen für männliche Fruchtbarkeit zu entdecken, zu klären

Impact of Age on Ovarian Response and IVF Outcome during Controlled Ovarian Hyperstimulation in Women from Gaza Strip

Background: Although age is an important factor in female fertility, not much date were focused on the relationship between age and ovarian response and in vitro fertilization (IVF) outcome. However, the female reproductive capacity varies with age. Objective: To assess the impact of age on ovarian response and IVF outcome during controlled ovarian hyperstimulation in women from Gaza Strip. Methods: This prospective cohort study consisted of 75 women attending IVF at Al-Basma Fertility Center in Gaza City. The number of oocytes and embryos were recorded for each female and the occurrence of pregnancy was followed for three months. The obtained data were computer analyzed using SPSS statistical package version 18. Results: The mean age of the study population was 29.2±5.9 years. The total number of oocytes was significantly decreased with increasing age (F= 3.932 and P= 0.024). In this context Pearson correlation test exhibited negative significant correlation between women age and the number of mature oocyte (r=-0.276, P= 0.017). There was an inverse relationship between age and ovarian response (F= 6.773 and P= 0.001), showing good response (9-16 oocytes) at mean age of 26.7±5.0 years. When related to women age, IVF outcome showed that the chance of getting pregnant increased with decreased age (F= 4.278 and p= 0.018). Conclusion: The ovarian response and the chance of getting pregnancy were diminishing with ageing, implying that maternal age should by consider during IVF program

Chromosomal aberrations in males occupationally exposed to chemical pollutants in the gaza strip-palestine

Objective: This study was conducted to evaluate the frequency of chromosomal aberrations in peripheral blood lymphocytes from Palestinian males exposed to various chemical pollutants during their daily work. Subjects and Methods: The study population consisted of 32 males (mean age 35.5 years) distributed as: 14 farmers, 3 plumbers, 5 taxi drivers, 6 paint factory workers, and 4 gas station workers, exposed to pollutants like pesticide, petrol derivatives etc. The control group consisted of 10 healthy Palestinian individuals of the same age and gender, but not exposed directly to pollutants in their jobs. Chromosomes were prepared from peripheral blood lymphocyte cultures using standard methods. The evaluation of chromosomal aberrations was performed following the IPCS (International Program Chemical Safety) guidelines for the monitoring of genotoxicity effects of carcinogens in humans. Results: A significantly higher incidence (2.14%, p< 0.05) of chromosomal aberrations (chromatid breaks, iso-chromatid breaks, chromatid deletions, and acentric fragments) were detected in lymphocyte of the study population. Interestingly, no chromosomal damage at all was recorded in the control group lymphocyte. Conclusion: These results suggest that occupational exposure to chemical pollutants is the cause of the chromosomal aberrations observed in the study population, which could be related to exposure time, since chromosomal aberrations were more frequent in workers exposed for longer times. The increased chromosomal damage detected in the study population can be attributed to the complex mixture of genotoxic compounds to which

Testosterone and gonadotropins in infertile men with Sertoli cell only syndrome from Gaza strip

Aim: To assess serum testosterone and gonadotropins in Sertoli cell only syndrome patients from Gaza Strip. Methods: Based on testicular biopsy, a cross section of 74 Sertoli cell only syndrome patients were enrolled in the study. Age matched 44 fertile men were served as controls. Patients and controls were questioned for their medical history. Blood samples were drawn and serum testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH) were determined by enzyme-linked immunosorbent assay. Data were computer analyzed using SPSS/PC, version 18.0. Results: Varicocele and hormonal problems were significantly more frequent among patients than controls (P< 0.05). Serum testosterone was significantly lower in patients compared to controls (1.7±1.3 versus 5.0±2.2 ng/ml, P= 0.000). In contrast, LH and FSH were significantly higher in patients than controls (12.8±9.7 and 20.8±14.8 mlU/ml versus 6.3±3.1 and 7.7±3.9 mlU/ml, P= 0.000, respectively). Hypergonadotrophic hypogonadism and hypogonadotrophic hypogonadism patients showed lower levels of testosterone compared to the normal reference value (0.9±0.5 and 0.5±0.4 ng/ml versus 2.0-7.0 ng/ml). Higher levels of LH and FSH were recorded in hypergonadotrophic hypogonadism (24.5±2.6 and 37.4±6.7 mlU/ml) compared to the reference values of 2.0-13.0 and 2.5-10.0 mlU/ml, respectively whereas LH and FSH levels were lower in hypogonadotrophic hypogonadism (0.6±0.4 and 0.6±0.5 mlU/ml, respectively). In this context, all hypergonadotrophic hypogonadism and hypogonadotrophic hypogonadism patients showed abnormal levels of testosterone, LH

Smoking‐induced genetic and epigenetic alterations in infertile men

Male fertility rates have shown a progressive decrease in both developing and industrialised countries in the past 50 years. Clinical and epidemiological studies have demonstrated controversial results about the harmful effects of cigarette smoking on seminal parameters. Some studies could not establish a negative effect by tobacco smoking on sperm quality and function, whereas others have found a significant reduction in sperm quality and function. This study reviews the components in cigarette smoke and discusses the effects of smoking on male fertility by focusing extensively on smoking‐induced genetic and epigenetic alterations in infertile men. Chromosomal aneuploidies, sperm DNA fragmentation and gene mutations are discussed in the first section, while changes in DNA methylation, chromatin remodelling and noncoding RNAs are discussed in the second section as part of epigenetic alterations

Antimullerian Hormone as a Predictor of Ovarian Reserve and Ovarian Response in IVF Candidates

Background: Antimullerian hormone (AMH) is expressed only in the gonads. In male, it is secreted by immature sertoli cells and in female by adult granulosa cells of the ovary. AMH is responsible for the regression of mullerian ducts in male fetus. Absence of AMH results in development of female fetus reproductive organs and it is believed to control the formation of primary follicles. Objective: To assess AMH in early follicular phase as a predictor of ovarian reserve among females undergoing in vitro fertilization (IVF) in the Gaza Strip. Methods: In this case-control prospective study, a meeting interview was used for filling in the questionnaire. AMH was determined by enzyme linked immunosorbent assay in 162 women: 81 women undergoing IVF atAl-BasmaFertilityCenter inGazaCity (cases) and 81 healthy women, having at least given birth to one healthy child (controls). The number of oocytes and embryos were recorded for each female in the controls group and the occurrence of pregnancy was followed for the three months. Data were computer analyzed using SPSS statistical package version 13. Results: The AMH mean level in the cases was significantly higher as compared to the controls (3.5±2.3 ng/mL vs. 1.7±0.5 ng/mL; p=0.00). The AMH in cases was significantly decreased with increasing age (3.7±2.0, 3.6±2.4 and 2.1±2.1 ng/mL at ≤25, 26-35 and >35 years, respectively; F=2.327 and p=0.104). The total number of retrieved oocytes was inversely associated with age (12.5±4.5, 11.0±5.4 and 6.9±4.7 at age ≤25, 26-35 and >35 years, F=4.793 and p=0.011). Results showed that the ovarian response to Menotrophin (FSH 75IU, LH 75 IU) stimulation was better with younger age (16 oocytes at mean age of 36.5±5.0, 30.6±5.9, 27.0±4.5 and 26.3±5.2, respectively; F=4.934 and p=0.003). There was a significant positive association between ovarian response in terms of total number of oocytes and AMH levels (16 oocytes at 1.0±0.5, 2.3±1.8, 3.7±1.8 and 5.90±2.9 ng/mL, respectively; F=9.174 and p=0.000), implying that AMH can be used as a good predictor of ovarian reserve and ovarian response. IVF results showed that the chance of pregnancy success increased with decreased age (F=3.077 and p=0.05). Moreover, the maximum level of AMH was observed in females who achieved positive pregnancies (4.5±2.5 ng/mL) followed by negative pregnancies (2.9±1.8 ng/mL) and no cleavage (2.1±1.5 ng/mL) with significant differences (F=6.862 and p=0.002). It is worth-mentioning that AMH levels may be associated with ovarian responsiveness, but not with the probability to achieve a pregnancy. The maximum number of total oocytes was recorded in females who achieved positive pregnancies (13.7±5.1 oocytes). Correlation coefficient revealed that the number of mature oocytes showed strong positive correlation with the AMH levels (r=0.469, p=0.001). Conclusion: AMH can be used in IVF programs as a good predictor of ovarian reserve and ovarian response

Serum Estradiol Level as a Predictor of Ovarian Response and Pregnancy Outcome During Controlled Ovarian Hyperstimulation in Women from Gaza Strip

Background: Currently, controlled ovarian hyperstimulation (COH) is monitored by serum estradiol (E2) levels which are believed to primarily detect functional activity of follicles. Objective: To evaluate estradiol level as a predictor of ovarian response and pregnancy outcome during COH in women from Gaza Strip. Methods: This prospective cohort study consisted of 75 women attending in vitro fertilization (IVF) at Al-Basma Fertility Center in Gaza City. Blood withdrawal for E2 hormone measurement was performed in all the patients and the number of oocytes and embryos were recorded for each female and the occurrence of pregnancy was followed for three months. Obtained data were computer analyzed using SPSS statistical package version 18. Results: The mean age of the study population was 29.2±5.9 years. Questionnaire interview showed that the cause of infertility was mostly referred to husbands. More than half of women seeking IVF had no children and had repeated IVF. The mean level of E2 showed the highest value of 2194.4 (pg/ml) at age group 26-35 years. However, the difference in E2 levels among the age groups was not significant (F= 0.940 and P= 0.395). When related to the number of oocytes retrieved, E2 level showed general increase with increase ovarian response, recording values of 1642.7, 1665.1, 2156.8 and 1798.7 pg/ml with 16 oocytes, respectively, but this change was not significant (F= 0.219 and P= 0.883). The mean level of E2 showed its maximum value of 2143.6 pg/ml in positive pregnancy. However the difference in E2 levels among the different categories of IVF outcome was not

Predictive value of soluble ST2 in adolescent and adult patients with complex congenital heart disease.

BACKGROUND:Soluble suppression of tumorogenicity 2 (sST2) has been shown to be of prognostic value in patients with chronic and acute left heart failure. The present study aims to assess the predictive value of sST2 levels in adult patients with complex congenital heart disease (CHD). METHODS:In 169 consecutive patients with complex CHD and a mean age of 28.2 ± 12.0 years, sST2 levels were compared to 32 healthy controls and associated with clinical status as well as the occurrence of major adverse cardiac events (MACE). Mean follow-up time was 35.6 ± 24.9 months. RESULTS:In CHD patients, median sST2 levels were 29.7 ng/ml compared to 26.4 ng/ml in healthy controls (p = 0.007) and increased with different types of CHD and the severity of MACE. According to ROC analysis, the most important predictors of acute heart/Fontan failure were NYHA class III/IV (AUC 0.804, p<0.001, CI 0.668-0.941), NT-proBNP levels (AUC 0.794, p<0.001, CI 0.640-0.948), γGT levels (AUC 0.793, p<0.001, CI 0.678-0.909) and sST2 levels (AUC 0.742, p = 0.004, CI 0.626-0.858), with NYHA class III/IV as the strongest independent predictor (p<0.001). All-cause mortality was best predicted by sST2 levels (AUC 0.890, p<0.001, CI 0.741-1.000), NT-proBNP levels (AUC 0.875, p = 0.001, CI 0.766-0.984) and NYHA class III/IV (AUC 0.837, p = 0.003, CI 0.655-1.000) with sST2 as the strongest independent predictor (p<0.001). Moreover, AUC increased to 0.918 combining both biomarkers and net reclassification improved with the addition of sST2. CONCLUSION:In patients with complex CHD, sST2 may have additive value to natriuretic peptides for the prediction of all-cause mortality