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Summary: DNA methylation plays important roles in genome stability and regulation of gene expression. This study was designed to determine the influence of cigarette smoking on sperm DNA methylation. From a genome‐wide survey on sperm samples, differentially methylated target CpGs should be selected and subjected to local deep bisulphite sequencing. Obtained methylation data are compared to sperm parameters and (ICSI) outcome. Similar to pilot study, samples were subjected to Infinium 450K BeadChip arrays to identify alterations in sperm DNA methylation between smokers and nonsmokers males. Routine testing on a significantly altered CpG site was performed on more samples using local deep bisulphite sequencing. Of approximately 485,000 CpG sites analysed, only seven CpGs were found to show a significant DNA methylation difference of >20% with the top six CpGs overlapping common SNP sites. The remaining CpG site (cg19455396) is located in intron 12 of the TAP2 gene. The results of deep bisulphite sequencing showed only a tendency towards hypomethylation in the smoking group. This study could not detect biologically relevant CpG positions that are altered in sperm DNA methylation on the influence of cigarette smoking beyond individual‐specific effects that may be caused by other environmental factors. [ABSTRACT FROM AUTHOR]

Background: Protamine content is necessary for proper sperm chromatin condensation and subsequent male fertility. The exact effect of smoking on male fertility remains controversial. The objective of this study was to evaluate the effect of smoking on protamine content of sperm in smoker and non-smoker patients.Methods: Protamines 1 (P1) and 2 (P2) were quantified by gel electrophoresis in the sperm of 53 smokers and 63 non-smokers. Sperm DNA fragmentation was analyzed employing the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) assay and non-condensed chromatin was evaluated using chromomycin A(3) (CMA(3)). Levels of smoking and oxidative stress markers were determined in seminal plasma using an enzyme linked immunosorbant assay (ELISA) and chemical reactions.Results: Protamine 2 concentrations were significantly lower (P < 0.050) in smokers than in non-smokers. In contrast P1/P2 ratios were significantly higher (P < 0.010) in smokers (1.34 ± 0.46 ng/10(6) sperm) than in non-smokers (1.11 ± 0.20 ng/10(6) sperm). The oxidative stress and smoking markers, reactive oxygen species (ROS), malondialdehyde, 8-Hydroxyguanosine (8-OHdG) and cotinine were significantly higher (P < 0.010) in smokers than in non-smokers, and correlated significantly (P < 0.050) with P1/P2 ratios. P2 showed significant negative (P < 0.050) correlations with ROS, 8-OHdG and cotinine. CMA(3) and TUNEL were also significantly higher (P < 0.010) in smokers (36.4 ± 8.1 and 17.4 ± 5.3%) than in non-smokers (29.8 ± 7.1 and 11.3 ± 4.2%).Conclusions: This is the first study to evaluate the effect of smoking on protamines. Abnormal elevation of the P1/P2 ratio appears to be associated with aberrant P2 expression in smokers. These results suggest that induced oxidative stress by cigarette smoking may have significant inverse effect on the protamination process by disrupting P2. [ABSTRACT FROM AUTHOR]

The aims of this study were (i) to determine and compare the concentration of reactive oxygen species (ROS) and total antioxidant status (TAS) in seminal plasma and sperm parameters of the male partners of patients undergoing IVF or intracytoplasmic sperm injection (ICSI) treatment and (ii) to establish the relationship between ROS and TAS concentrations and sperm quality and their effect on fertilization and pregnancy rate of patients who achieved a pregnancy and those who were unsuccessful. Twenty-six IVF and 22 ICSI patients were included in this study. The ROS concentration in seminal plasma and sperm concentration, vitality (eosin test), motility, morphology, membrane integrity (HOS test), maturity (chromomycin, CMA3) and DNA fragmentation (TUNEL) results and their relationship to fertilization and pregnancy were analysed. ROS concentrations were similar regarding the seminal plasma of male partners of patients who achieved a pregnancy and those who were unsuccessful. The other semen parameters, concentration, motility, vitality, membrane and DNA integrity, were comparable in both groups. However, both groups demonstrated a negative correlation between ROS concentration and sperm vitality, membrane integrity and morphology. Moreover, an inverse correlation was found between TUNEL, vitality, and membrane integrity. In conclusion, ROS concentration in seminal plasma affects the quality of spermatozoa. A negative correlation between the ROS concentration in seminal plasma and fertilization rate in both IVF/ICSI programmes was shown. [ABSTRACT FROM AUTHOR]

Semen analysis constitutes the most important investigation of male infertility. However, the true anomalies present in defective sperm cells have been only partially characterized. The integrity of the sperm chromatin may play the most important role, particularly in ICSI, where most of the natural selection mechanisms are bypassed. This study was carried out to characterize sperm morphology (strict criteria), to evaluate chromatin condensation and sperm count in native semen as well as after semen preparation by the swim-up technique, and to eventually evaluate any correlation between these parameters. Semen from 90 men was analyzed for the above parameters in both the fresh and processed semen. Whereas the sperm count decreased after sperm preparation by the swim-up technique in comparison to the value in the fresh semen (p<.001), there was an increase in the percentage of morphologically normal (p<.001) and chromatin-condensed sperm (p=.99). However, there was no correlation between sperm morphology, chromatin condensation, and sperm count either in the fresh or in the processed semen samples. These results suggest that sperm morphology, sperm count, and chromatin condensation are independent parameters that should be evaluated separately in the assessment of male fertility in an assisted reproduction program. [ABSTRACT FROM AUTHOR]

Anaesthetic management for oocyte retrieval may interfere with the results and success rate of an in vitro fertilization (IVF) program. This study was conducted to compare the effects of two different anaesthetic techniques (general anaesthesia versus sedation) used for oocyte retrieval on IVF outcome. For general anaesthesia we used a combination of remifentanil (UltivaTM) with either propofol or isoflurane in hypnotic concentrations. For sedation the protocol included midazolam, diazepam or propofol according to clinical needs. In total, 202 women were enrolled in the study. 96 women opted for sedation and 106 for general anaesthesia. The number of collected oocytes was significantly higher with general anaesthesia (10.54±5.43 [mean±SD]) than with sedation (6.25±3.65, p<0.0001), whereas the number of fertilized oocytes was not different (4.70±3.57 vs. 4.23±2.90). There were no significant differences in cleavage and pregnancy rates. We therefore believe that remifentanil-based general anaesthesia without nitrous oxide is a suitable alternative to sedation and may be recommended for IVF oocyte retrieval if general anaesthesia is requested. [ABSTRACT FROM AUTHOR]

Introduction: Transvaginal puncture for oocyte retrieval is a short-lasting but painful procedure. We hypothesized that a sole infusion of the ultra-short acting mu-agonist remifentanil may be a suitable and well-controllable single-agent analgesic technique that can dose-dependently be applied to spontaneously breathing patients.Methods: Fifty consenting adult women were enrolled in this prospective trial. A sedative premedication was omitted, all patients received 3 L/min of inhaled oxygen, and a sole remifentanil infusion was started with 0.25 microg/kg/min. Remifentanil was adjusted as needed for pain relief (in steps of 0.05 microg/kg/min) and finished after the last puncture. Dosage requirements, vital functions, oxygen saturation (as achieved by pulse oximetry, psO(2)), adverse drug effects and the level of sedation (LOS 1-5; 1 = asleep/unarousable, 4 = calm/awake) were recorded. Remifentanil plasma concentrations were achieved by STANPUMP pharmacokinetic simulation. Data are presented as mean +/- SD.Results: A total of 50 women (31.8 +/- 5.1 yr, 67.3 +/- 14. ASA I or II ) were investigated. Follicular aspiration lasted 10.8+/-5.2 min, and remifentanil was infused for 19.7+/-8.3 min. Dosage requirements were 0.25 microg/kg/min in 70% of all patients, 0.3 microg/kg/min in 22%, 0.2 microg/kg/min in 6%, and 0.4 microg/kg/min in 2% of all cases. Vital signs (baseline, after 1(st) puncture, end of surgery) nearly remained unchanged: heart frequency = 85 +/- 15, 87 +/- 17, 90 +/- 17 bpm, systolic blood pressure = 129 +/- 12, 132 +/- 13, 131 +/- 14 mmHg; respiratory rate = 116 +/- 4, 15 +/- 4 breaths/min; psO(2) = 99 +/- 1, 99 +/- 1, 99 +/- 2%. LOS was 4.0 (all), 3.9 +/- 0.3, 3.9 +/- 0.3. Remifentanil plasma concentrations were 5.0 +/- 1.3 ng/mL at the start, 6.6 +/- 1.3 at the end of surgery and 1.2 +/- 0.5 at PACU arrival. Adverse drug effects: 54% itching, no muscle rigidity. 94% of all women would choose this technique again.Conclusions: The sole infusion of remifentanil is a suitable and satisfying single-agent monitored anaesthesia care technique for oocyte retrieval. However, close anaesthetic observation - especially of the respiratory function - is mandatory. [ABSTRACT FROM AUTHOR]

A significant association between male subfertility, imperfect spermiation and abnormal nuclear condensation has been suggested. The DNA content of spermatozoa might be responsible for inducing alterations in sperm morphology. The final nuclear shape, which is species-specific, depends on chromatin condensation during spermatogenesis as well as a precise organization of DNA within the nucleus. Many reports have described the association between disturbances in sperm chromatin condensation, morphology and male infertility. Chromatin condensation is achieved by gradual substitution of lysine-rich somatic histones by testis-specific histone and finally by protamine. In this study two groups of patients were compared: the first consisted of 63 patients who had undergone intracytoplasmic sperm injection (ICSI) with freshly ejaculated spermatozoa whereas the second included 47 patients assigned to ICSI with testes biopsy-extracted spermatozoa. In both groups chromatin condensation was assessed by aniline blue staining and morphology evaluated according to strict criteria. The condensed chromatin and morphology of spermatozoa were significantly (P < 0.0001) less in the second group compared to the first. However the fertilization, cleavage, implantation and pregnancy rates were almost the same in both investigated groups. There was no significant difference between the two groups with respect to ICSI outcome. The percentage of chromatin condensation (nuclear maturity) and morphologically-normal spermatozoa were significantly higher (P < 0.0001) in the ejaculated spermatozoa than in those from testis biopsy but the ICSI outcome (fertilization, cleavage, implantation and pregnancy rates) was the same. In view of these results the fertilization capability and the embryo quality obtained using testis biopsy extracted spermatozoa is not influenced by chromatin condensation and sperm morphology in testicular sperm extraction (TESE)-ICSI programmes. Therefore, it could be said that neither chromatin condensation nor morphology of testis extracted sperm could predict the fertilization, implantation and pregnancy rate in TESE-ICSI programmes. [ABSTRACT FROM PUBLISHER]

The main purpose of this study was to determine the possible relationship between chromatin condensation (Aniline Blue staining), the morphology of spermatozoa according to strict criteria, and the fertilization, cleavage and pregnancy rate in an intracytoplasmic sperm injection (ICSI) programme. A total of 60 patients were divided into two groups (27 versus 34) according to sperm stainability by Aniline Blue. The first group involved patients having a positive Aniline Blue staining test with 0-29% stained. The fertilization rate in this group was 60.8%, cleavage rate 54.4% and pregnancy rate 18.5%. In the second group in which > 29% spermatozoa were positively stained, the fertilization rate was 62.1%, cleavage rate 62.0% and pregnancy rate 35.3%. There was no statistically significant difference between the two groups. Furthermore, the influence of morphology according to strict criteria after Papanicolaou staining on successful fertilization, cleavage and pregnancy was studied in 85 patients who were divided into two groups according to the percentage of morphologically normal sperm. The fertilization, cleavage and pregnancy rates were 44.21, 63.37, and 39.47% respectively in the first group (< 4%), the corresponding values for the second group (> 4%) were 56.50, 46.04 and 21.21%. There was no significant correlation between the fertilization (P = 0.722), cleavage (P = 0.519) and pregnancy (P = 0.096) rates in either group. This study demonstrates that neither chromatin condensation (Aniline Blue staining) nor morphology could assess the fertilization potential, cleavage and pregnancy rate in an ICSI programme. [ABSTRACT FROM AUTHOR]