Label-Free Evaluation of Chromatin Condensation in Human Normal Morphology Sperm Using Raman Spectroscopy.

Chromatin condensation is one of the main factors essential for sperm function. Evaluation of chromatin condensation by current methods render the assessed sperm unsuitable for assisted reproduction. We examined the Raman spectra of normal morphology sperm to determine whether a non-invasive confocal Raman spectroscopy can detect spectral differences between groups having different levels of chromatin condensation. Semen samples from 85 donors who underwent ICSI were obtained. Chromomycin A3, aniline blue and acridine orange staining were performed to evaluate the protamine deficiency, histone retention and DNA fragmentation respectively. Raman spectra were obtained from 50 normal morphology sperm for each donor. Spectral analysis was performed using home written programs in LabVIEW software and samples were grouped based on chromomycin A3 staining. Raman peaks intensities at 670 cm-1, 731 cm-1, 785 cm-1, 858 cm-1, 1062 cm-1, 1098 cm-1, 1185 cm-1, 1372 cm-1, 1424 cm-1, 1450 cm-1, 1532 cm-1, 1618 cm-1 and 1673 cm-1 were significantly correlated with at least one of the sperm staining methods. The median intensity of the Raman peaks at 670 cm-1, 731 cm-1, 785 cm-1, 1062 cm-1, 1098 cm-1, 1185 cm-1, 1372 cm-1, 1424 cm-1, 1450 cm-1, 1532 cm-1, 1618 cm-1 and 1673 cm-1 show a significant difference between the CMA3≤41 and CMA3>41groups. The Raman spectroscopic measurements represent a promising diagnostic tool that has the ability to label-free detect sperm with chromatin abnormalities, such as improper chromatin condensation and DNA fragmentation to a certain degree similar to that of the existing staining techniques at the individual cell level.

DNA methylation level of spermatozoa from subfertile and proven fertile and its relation to standard sperm parameters.

The epigenetic mechanism plays an important role in spermatogenesis such as DNA methylation where this episode is represented by either switching genes on or off. Twenty-eight samples (14 case and 14 controls) were subjected to Infinium 450K BeadChip arrays to identify genomic regions that differ in sperm DNA methylation patterns in the subfertile compared to the proven fertile group. Then two CpGs were validated by deep bisulphite sequencing on 82 sperm samples. The results screening study revealed eight CpGs were significantly different in their sperm DNA methylation levels between cases and control group. The results of the validation study for the two CpGs (cg19779893 and cg19406113) showed that a significant variation in the methylation level at 2 CpGs of 3 CpGs related to cg19779893 site amplicon in cases compared to the controls. Moreover, six CpGs related to the cg19406113 site amplicon showed significant differences in sperm DNA methylation between the cases and the control group. Furthermore, there was a significant decrease in the sperm parameters in the cases compared to the control group. This study found two CpGs altered in their sperm DNA methylation levels. In addition, a strong association was found between changes in the sperm DNA methylation levels in these CpGs sites and sperm parameters.

Relationship between nuclear DNA fragmentation, mitochondrial DNA damage and standard sperm parameters in spermatozoa of fertile and sub-fertile men before and after freeze-thawing procedure.

The aim of this study was to assess the stability of nuclear and mitochondrial DNA (n-DNA and mt-DNA) of spermatozoa under freeze-thawing and to find out the correlation between them and their association with standard sperm parameters. Forty-three semen samples were collected from fertile (G.1; n = 29) and sub-fertile (G.2; n = 14). N-DNA fragmentation was determined by TUNEL assay and mt-DNA using caspase 3 staining. Each semen sample was frozen at -196°C by the programmed freezer. Freeze-thawing decrease vitality, total motility and membrane integrity from (43.02 ± 22.74%; 31.63 ± 18.15%; 51.5 ± 24.82%) to (22.71 ± 17.3%; 9.21 ± 6.61%; 34.64 ± 19.92% respectively [p < .001]). G.1 native spermatozoa stained positive with TUNEL and caspase 3 were (14.85 ± 17.6% and 5.8 ± 11.59%) and increased after freeze-thawing to 27.54 ± 19.74% (p = .004) and 7.3 ± 6.13% (p = .01) respectively. In G.2, TUNEL and caspase 3 were (19.84 ± 17.52% and 7.53 ± 8.56%) and increased to (29.48 ± 16.97% [p = .03] and 10.21 ± 11.73%). In conclusion, freeze-thawing process affects not only semen parameters but also n-DNA and mt-DNA. Therefore, n-DNA and mt-DNA could be used as sensitive parameters for assessment of the cryodamage of human spermatozoa.

Influence of extended incubation time on Human sperm chromatin condensation, sperm DNA strand breaks and their effect on fertilisation rate.

The purpose of this study was to determine influence of extended incubation time on sperm chromatin condensation and DNA strand breaks and their effect on fertilisation rate. Forty couples undergoing ICSI therapy were included. Semen was prepared by PureSperm gradient centrifugation and divided into two parts. The first part (G1) was used immediately for ICSI, whereas the second part (G2) was kept in the incubator at 37°C, 5% and 90% Humidity for 5 hr, and thereafter, the capacitated spermatozoa were used for ICSI. The TUNEL test and chromomycin CMA3 were used to evaluate the DNA strand breaks and chromatin condensation respectively. The percentage of condensed chromatin was 73.92 ± 12.70 in the group 1 and 81.13 ± 10.31% in group 2 (p = .001). However, the double-strand breaks were 11.15 ± 8.67% in G.1 and 16.30 ± 11.12% in G.2. (p = .001). Fertilisation rate in the (Group 1) was 62.45% and 69.17% in (Group 2). There was a positive correlation between condensed chromatin and fertilisation rate (r = 0.846, p = .001) and a negative correlation with DNA double-strand breaks (r = -0.802; p = .001). In conclusion, the prolonged sperm incubation (5 hr) leads to a higher chromatin condensation and to a significantly increased number of DNA strands double breaks with no influence on fertilisation rates.

Impact of cigarette-smoking on sperm DNA methylation and its effect on sperm parameters.

DNA methylation is an epigenetic modification of the genome. The purpose of this study was to determine the influence of cigarette-smoking on sperm DNA methylation from a genomewide survey of sperm samples and to ascertain its effect on sperm parameters. Twenty-eight sperm DNA samples (from 14 fertile smokers as a case study and 14 proven fertile nonsmokers as controls) were subjected to Infinium 450K BeadChip arrays to identify the changes in the DNA methylation level between the two groups. Then, deep bisulphite sequencing was used to validate five CpGs on 78 samples. The results from the Infinium 450K found that only 11 CpGs showed a significant difference in DNA methylation between the case and the control groups. Five CpGs of the eleven (cg00648582, cg0932376, cg19169023, cg23841288 and cg27391564) underwent deep bisulphite sequencing where cg00648582, related to the PGAM5 gene, and the cg23841288 CpGs, related to the PTPRN2 gene amplicons, showed a significant increase in their DNA methylation level in more than one CpG in the case group. In contrast, a significant decrease was found at cg19169023 and at its various neighbouring CpGs in the TYRO3 gene-related amplicons. Furthermore, this study demonstrated a significant correlation between the variation in sperm DNA methylation level and standard sperm parameters in the case group.

The effects of bacterial infection on human sperm nuclear protamine P1/P2 ratio and DNA integrity.

The purpose of this study was to detect the effects of bacterial infection on human sperm nuclear protamines and DNA fragmentation. In this study, 120 semen samples were collected from unselected male partners of couples consulting for infertility in infertility and obstetrics clinic. All the samples were screened bacteriologically according to World Health Organization guidelines, and also sperm parameters and DNA fragmentation were evaluated. The concentrations of protamines P1 and P2 were quantified using acid urea acrylamide gel electrophoresis. Of a total number of 120 sample, 36 (30%) of them were infected with bacteria. Nine species of bacteria belonging to five genera, Staphylococcus, Escherichia, Streptococcus, Enterococcus and Klebsiella, were identified. The comparison between infected (36) and noninfected (84) samples appeared the negative impact of bacterial infection on sperm parameters and P1/P2 ratios. The percentages of P1/P2 ratio abnormality were significantly higher in infected patients. Sperm concentration, motility, progression and chromatin condensation were significantly lower in infected patients (p < .010). Depending on these results, we concluded that the bacterial infections have significant negative effects on sperm chromatin condensation and protamine P1/P2 ratio. Moreover, the negative relationship between the bacterial infections and sperm parameters, such as concentration, motility and progressive motility, has been shown.

Cigarette smoking induces only marginal changes in sperm DNA methylation levels of patients undergoing intracytoplasmic sperm injection treatment.

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.

Alterations in DNA methylation patterns and gene expression in spermatozoa of subfertile males.

This study was designed to evaluate the DNA methylation patterns and gene expression in spermatozoa from subfertile males. Thirty samples were subjected to 450K arrays as a screening study to evaluate the variation in sperm DNA methylation levels between cases and controls groups, and then three CpG sites (cg07227024, cg05813498 and cg23081194) have the highest difference in methylation levels and located within ALS2CR12, GAA and UBE2G2 genes respectively; these were selected for further analysis using deep bisulphite sequencing and qPCR in 136 samples (64 proven fertile males "controls" and 72 subfertile males "cases"). A significant difference in the methylation level was found between cases and controls at two CpGs, six CpGs and three CpGs in ALS2CR12, GAA and UBE2G2 gene-related amplicon respectively. Besides, the qPCR results showed a significant change in the expression levels of GAA, UBE2G2 and ALS2CR12 gene in cases compared to the controls (p ≤ .00001). In conclusion, the methylation levels at CpGs in GAA, UBE2G2 and ALS2CR12 gene amplicons were significantly different in subfertile compared to proven fertile males. In addition, a significantly different was showed in the expression levels of GAA, UBE2G2 and ALS2CR12 genes in subfertile males compared to proven fertile males.

Aberrations in sperm DNA methylation patterns of males suffering from reduced fecundity.

The purpose of this study was to evaluate the aberrations in sperm DNA methylation patterns of males suffering from reduced fecundity. A total of 108 males (65 males suffering from reduced fecundity as cases and 43 proven fertile males as a control) were included in the study. Thirty samples were subjected to 450K arrays as a screening phase, and then, three CpG sites located in the following genes: TYRO3, CGß and FAM189A1 were selected to validate on 78 samples using deep bisulphite sequencing. A significant difference in the methylation level was found between cases and controls at all CpGs in TYRO3 gene-related amplicon (CpG1, p ≤ .003, CpG2, p ≤ .0001, CpG3, p ≤ .003 and CpG4, p ≤ .030) and CpG1 in CGß gene-related amplicon (p ≤ .0001). Besides, a significant difference was found at two CpGs (CpG1, p ≤ .004 and CpG2, p ≤ .002) tested in the FAM189A1 gene-related amplicon. A significant correlation was found between the methylation level at CpG1 in the FAM189A1 gene and the different types of sperm motility. In conclusion, an alteration in the methylation levels of sperm DNA from males with reduced fecundity was showed. In addition, a relationship between variations in the methylation level of these CpGs and sperm motility has been observed.

Aberrant DNA methylation patterns of human spermatozoa in current smoker males.

The purpose of this study was to investigate the impact of current cigarette smoking on sperm DNA methylation patterns. A total of 108 males (51 current smokers and 57 never smoked males) were included in the study. Using 450 BeadChip Arrays, the differentially methylated CpGs between current smokers (n=15) and never smoked males (n=15) were identified. Out of significantly 11 CpGs identified, 2 CpGs namely cg07869343 and cg19169023, which are located in the MAPK8IP3 and TKR genes were selected for further analysis. Using deep bisulfite sequencing in an independent cohort of current smokers (n=36) and never smoked males (n=42), 6 and 1 CpGs showed a significant difference in the MAPK8IP (CpG3, CpG5, CpG6, CpG7, CpG8, and CpG21) and in the TKR (CpG4) were identified, respectively (P≤0.05). Our results indicate that cigarette smoking causes biochemical changes in the sperm DNA methylation in many regions and could adversely affect semen parameters.

Impact of cigarette smoking on histone (H2B) to protamine ratio in human spermatozoa and its relation to sperm parameters.

Smoking is strongly associated with abnormalities in histone-to-protamine transition and with alteration of protamine expression in human spermatozoa. A proper protamine to histone ratio is, however, essential for sperm chromatin maturity and DNA integrity. Alterations in these sperm nuclear proteins were observed in infertile men. The present prospective study is aimed at evaluating the possible relationship among smoking, semen quality and the histone-to-protamine transition ratio in mature spermatozoa. Histone H2B and protamine 1 (P1) and 2 (P2) were quantified using acid-urea polyacrylamide gel electrophoresis in the spermatozoa of 35 smokers and 19 non-smokers. Levels of lipid peroxidation marker malondialdehyde (MDA) were measured in seminal plasma by thiobarbituric acid assay. Cotinine concentrations were determined in seminal plasma using an enzyme-linked immunosorbent assay. Histone H2B levels in smokers (292.27 ± 58.24 ng/10(6)) were significantly higher (p = 0.001) than that of non-smokers (109.1 ± 43.70 ng/10(6)), besides, a significant difference (p > 0.0001) was found for the P1 and P2 ratio between smokers (1.71 ± 0.071) and non-smokers (1.05 ± 0.033). The H2B/(H2B+P1 + P2) ratio (0.29 ± 0.71) of smokers were significantly higher (p = <0.0001) than that of non-smokers (0.12 ± 0.01). The concentrations of MDA (µm) (7.13 ± 1.15) and cotinine (ng/mL) (60.44 ± 31.32) in seminal plasma of smokers were significantly higher (p = 0.001) than those in the samples of the non-smoker group (4.42 ± 1.16 and 2.01 ± 2.84 respectively). In addition, smokers showed significantly (p ≤ 0.002) lower sperm count, motility (p = 0.018), vitality (p = 0.009) and membrane integrity (p = 0.0001) than non-smokers. These results reveal that patients who smoke possess a higher proportion of spermatozoa with an alteration of the histone to protamine ratio than patients who do not smoke, and suggest that cigarette smoking may inversely affect male fertility.

Protamine contents and P1/P2 ratio in human spermatozoa from smokers and non-smokers.

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.

Influence of heat stress on the cortisol and oxidant-antioxidants balance during oestrous phase in buffalo-cows (Bubalus bubalis): thermo-protective role of antioxidant treatment.

In the present study, the effect of heat stress, which is commonly observed in the animals of Upper Egypt area in summer, as well as the effect of antioxidant treatment as a thermo-protective was examined. In this study, the animals (n = 120) were divided into winter group (n = 40, bred during winter) and summer group (n = 80, bred during summer) as well as, animals in the summer group were divided into first subgroup animals (n = 40) and injected with Viteselen intramuscularly (15 ml) twice weekly for 10 weeks and second subgroup animals (n = 40) were not treated (as control). Serum levels of progesterone (P4), oestradiol (E2), cortisol, superoxide dismutase (SOD), lipid peroxidase (LPO) and nitric oxide (NO) were measured. The pregnancy rate of all animals was detected rectally. The levels of oestradiol and the activity of the antioxidant SOD were decreased in serum of animals in behavioural oestrus during summer as compared with those in winter. During the same time period the levels of oxidants such as LPO and NO were increased in the serum of animals again in the phase of oestrus. In another group of animals treated by intramuscular injection with 15 ml viteselen (antioxidant) twice weekly for 6 weeks during hot months, the activities of serum SOD showed an increase and the levels of oxidants and cortisol decreased. Moreover, the levels of oestradiol were increased during the oestrous behaviour. The pregnancy rate was decreased in animals under heat stress and the pregnancy rate was enhanced dramatically when these animals received antioxidants during the heat stress. This means that the heat-stress in Upper Egypt may affect the fertility of animals and pregnancy rate and this effect may be through an increased production of free radicals and decreased production of antioxidants as well as increased levels of cortisol. Treatment of animals or supplementation with antioxidants before the beginning of months of heat-stress and also during the stress period may correct the infertility due to heat-stress through the decrease in cortisol secretion and a decrease in the oxidative stress. These results resulted in an increase in pregnancy rate in treated animals.

Reactive oxygen species, total antioxidant concentration of seminal plasma and their effect on sperm parameters and outcome of IVF/ICSI patients.

OBJECTIVE: The purpose of this study was to determine and compare the concentration of reactive oxygen species (ROS) and total antioxident (TAS) in seminal plasma of IVF (in vitro fertilization) and ICSI patients, to establish their effect on sperm quality (count, vitality, HOS, morphology, maturity, DNA strand breaks) and assess the fertilization potential of spermatozoa and IVF/ICSI outcome. METHOD: IVF/ICSI patients (n = 48) 26 IVF and 22 ICSI were included in this study. A spermiogram was generated from each patient one-hour post ejaculation and smears were made from each semen sample to evaluate the morphology, sperm maturity (Chromomycin CMA3) and DNA strand breaks (Terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling, TUNEL-assay). RESULTS: In both groups a negative correlation was found between ROS concentration in seminal plasma and sperm vitality (r= -0.111; P = 0.453); membrane integrity and morphology (-0.141; P = 0.340) and fertilization rate (r = -0.0290; P=0.045). However, TAS in seminal plasma correlated positive with fertilization rate (r = 0.081; P = 0.584). In addition, an inverse correlation was found between sperm DNA strand breaks (TUNEL-test) and spermatozoa global and progressive motility, vitality, and membrane integrity. Furthermore, the mean percentage of normal condensed spermatozoa (CMA3) was significantly higher (P = 0.0001) in patients undergoing IVF compared to ICSI. Spermatozoa of male ICSI patients were more susceptible to acid denaturation (acridine orange staining) compared to spermatozoa of male IVF patients (P = 0.041). However, ROS concentration was higher in IVF patients compared to ICSI patients (94.73 +/- 102.84 vs. 54.78 +/- 39.83 micromol/l, whilst TAS levels (1.43 +/- 0.28 vs. 1.53 +/- 0.22) and fertilization rate (67. 26 vs. 67.26) were similar in both groups. CONCLUSION: ROS concentration and other sperm parameters were higher in IVF compared to ICSI patients. TAS concentration was comparable between the two groups. However, the fertilization rate was smilar in IVF and ICSI patients. Therefore, ROS concentration in seminal plasma affects the quality of spermatozoa but does not affect the fertilization rate in IVF/ICSI cycles.

Comparison of reactive oxygen species concentration in seminal plasma and semen parameters in partners of pregnant and non-pregnant patients after IVF/ICSI.

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.

[Body mass index, protein metabolism profiles and impact on IVF/ICSI procedure and outcome]. / Body Mass Index, Proteinstoffwechselprofile und Ergebnisse bei Patientinnen unter IVF-/ICSI-Behandlung.

OBJECTIVE: Higher risks of infertility have been found in overweight women. The purpose of the present study was to explore whether protein metabolism profiles related to body mass index (BMI) and to find out whether these parameters should affect IVF/ICSI outcome. PATIENTS AND METHODS: 52 patients were enrolled in this study. All patients underwent an ovarian stimulation either with recombinant follicle stimulating hormone (Gonal-F) or human menopausal gonadotropin (Menogon) after pituitary down-regulation with Goserelin (Zoladex) or Triptorelin (Decapeptyl Gyn). Five blood samples were taken: before treatment, at the beginning of ovarian stimulation, on the day of HCG injection for the ovulation induction, on the day of follicle aspiration and 14 days after embryo transfer. The blood samples were analysed with regard to the serum concentrations of total protein, albumin, total bilirubin and urea. According to the BMI values the patients were divided into two groups: BMI < 25 kg/m (2) (GI, n = 28) and BMI > 25 kg/m (2) (GII, n = 24). The results of IVF/ICSI outcome were compared in both groups. RESULTS: In both groups, the serum concentrations of total protein, albumin, total bilirubin and urea decreased during ovarian stimulation. In GII, albumin concentration decreased significantly on the day of follicle aspiration (46.0 +/- 2.3 g/l versus 43.5 +/- 2.5 g/l, p < 0.001) and 14 days after embryo transfer (46.8 +/- 2.5 g/l versus 44.7 +/- 2.3 g/l, p < 0.002), whereas the concentration of total bilirubin was not significantly decreased on the day of HCG injection (0.57 +/- 0.29 mg/dl versus 0.49 +/- 0.26 mg/dl, p = 0.11). Furthermore, pregnancy rate in women with BMI < 25 kg/m (2) was 46.4 % and in women with BMI > 25 kg/m (2) 33.1 % (p = 0.34). CONCLUSIONS: Serum concentrations of albumin and total bilirubin are influenced by BMI. Excess weight defined as BMI > 25 kg/m (2) has a negative impact on IVF outcome leading to decreased chances of pregnancy.

[Effects of ovarian stimulation on serum concentrations of lipids and ketone bodies in patients undergoing IVF/ICSI treatment]. / Effekte der ovariellen Stimulation auf die Serumkonzentrationen von Lipiden und Ketonkörper bei Patientinnen unter IVF-/ICSI-Behandlung.

OBJECTIVE: The ovarian stimulation increase the estradiol concentration in comparison to the one observed in the menstrual cycle. The aim of the study was to determine the influence of ovarian stimulation on serum concentrations of lipids and ketone bodies in patients undergoing IVF/ICSI treatment. PATIENTS AND METHODS: 50 patients were included in this study. All patients underwent an ovarian stimulation either with recombinant follicle stimulating Hormone (Gonal-F, n = 28) or human menopausal gonadotropin (Menogon, n = 22) after pituitary down-regulation with Goserelin or Triptorelin. Five blood samples were taken: before treatment, at the beginning of ovarian stimulation, on the day of HCG injection for the ovulation induction, on the day of follicle aspiration and 14 days after embryo transfer. Afterwards, the samples were analysed with regard to the concentrations of triglyceride, total-cholesterol, HDL-/LDL-cholesterol, acetoacetate, beta-hydroxybutyrate and alpha-ketoglutarate. RESULTS: During the ovarian stimulation, total-cholesterol andA'LDL-cholesterol decreased significantly (p(Chol) < or = 0.001; p(LDL) < or = 0.001), whereas the concentration of HDL-cholesterol increased significantly (p(HDL) < or = 0.001). Furthermore, a significant increase of beta-hydroxybutyrate and alpha-ketoglutarate has been detected (p(HBS) = 0.039; p (KG) = 0.023). CONCLUSIONS: The observed changes in serum concentrations of lipids and ketone bodies began with the application of gonadotrophins for stimulation and persisted until the end of the treatment. Therefore, the increase of estradiol, stimulated by gonadotrophins, seems to be responsible for the changes in the serum concentrations of lipids and ketone bodies. All women undergoing ovarian stimulation should be monitored for long-term thrombogenic and atherogenic risks.

[Association between ovarian stimulation regime and protein metabolism of patients undergoing IVF/ICSI therapy]. / Zusammenhang zwischen ovarieller Stimulations-Therapie und Proteinstoffwechsel bei Patientinnen unter IVF/ICSI-Behandlung.

The aim of this study was to determine the relationship between the ovarian stimulation protocol (with HMG or FSH) after down-regulation with GnRH anologa and protein (total protein and albumin) as well as bilirubin and urea in serum. Furthermore, it was intended to find out the effect of these parameters on IVF/ICSI outcome. 50 patients were included in this study. All patients underwent controlled ovarian hyperstimulation for assisted reproduction therapy either with FSH (Gonal-F) or HMG (Menogon). Ovulation induction was induced by human chorionic gonadotrophine (HCG, Predalon) 10 000 IU i. m. The protein concentration (total protein, albumin) as well as bilirubin and urea concentrations were determined before down-regulation with GnRHa, at the beginning of ovarian stimulation with FSH or HMG, on the day of ovulation induction with HCG, during oocyte retrieval and fourteen days after embryo transfer. The age, body mass index and etiology of infertility showed no significant difference between patients stimulated with HMG or FSH. Total protein and albumin concentration decreased significantly (p=0.001) from 77.45 +/- 5.90 g/L and 47.02 +/- 3.41 g/L to 74.60 +/- 4.6 g/L and 45.04 +/- 2.39 g/L, respectively at the time of oocyte retrieval. Whereas, no significant change with bilirubin and urea concentration was observed. However, the mean concentration of total protein, albumin, bilirubin of patients who become pregnant was higher of those who did not. In conclusion, this study shows that total protein and albumin concentration in plasma decreased during the follicular phase significantly with the application of exogenous gonadotrophins and steroid hormones in comparison to the value before down-regulation. The mean value of total protein, albumin, bilirubin of patients who become pregnant was higher (but not significant) of those who did not. However, the high individual variation in the present results shows that these parameters are not useful as a predictor of IVF/ICSI outcome.

Fibroblast growth factor (FGF), intracellular adhesion molecule (sICAM-1) level in serum and follicular fluid of infertile women with polycystic ovarian syndrome, endometriosis and tubal damage, and their effect on ICSI outcome.

PROBLEM: The objective of this study was to determine the concentration of fibroblast growth factor (FGF) and soluble intracellular adhesions molecule (sICAM-1) in serum and follicular fluid (FF) of polycystic ovary (PCO), endometriosis and tubal factor infertility and male factor infertility patients, and to investigate the relationship between these parameters and the outcome of intracytoplasmic sperm injection (ICSI). METHOD OF STUDY: The concentration of FGF and sICAM-1 in serum and FF were determined in patients undergoing controlled ovarian hyperstimulation (COH) for ICSI therapy for various etiology of infertility and the results of cytokines concentration and ICSI outcome were compared between the groups. Twenty patients with PCO (G.I), 17 with endometriosis (G.II), 19 with tubal damage (G.III) and 19 with male factor infertility (G.IV) were enrolled in this study. Quantitative determination of levels of FGF and sICAM-1 was performed using enzyme-linked immunosorbent assays (ELISAs). RESULTS: The FGF level in serum of PCO patients (G.I) were 4.8 +/- 2.3 and in FF were 104.0 +/- 39.0 pg/mL. The corresponding values in the endometriosis patients group (G.II) were 5.9 +/- 3.1 and 125.4 +/- 74.9 pg/mL. The concentration of FGF in tubal factor infertility group (G.III) in serum was significantly higher (P = 0.009) than those observed in the PCO group (G.I) 7.4 +/- 4.5 pg/mL, whereas the concentration in FF was at the same level like the other groups investigated, 128.7 +/- 75.9 pg/mL. Besides, the sICAM-1 (pg/ml) concentration in FF showed a significant difference between the groups investigated (G.I, 175.3 +/- 52.8; G.II 194.4 +/- 32.2; G.III 233.1 +/- 54.3; and G.IV 215.1 +/- 54.4 ng/mL; P = 0.003). The sICAM-1 levels in serum were not significantly different between the groups (217.0 +/- 42.9; 216.3 +/- 73.6; 254.8 +/- 79.6; 237.56 +/- 78.4 ng/ml; P = 0.267). The fertilization rate was significantly higher in G.III (66.0 +/- 23.89%) in comparison to G.II (38.8 +/- 33.9%; P = 0.014) or G.IV (38.7 +/- 22.7%; P = 0.012). The pregnancy rates were similar in all groups (30, 35.3 and 35.0, 38.6%, respectively). CONCLUSION: Both, FGF and sICAM-1 are present in serum and FF of patients undergoing controlled ovarian hyperstimulation for ICSI therapy. The FGF concentration in serum differs significantly between the groups investigated, whereas, no significant difference could be observed in the FF concentration of FGF. On the other hand, the sICAM in serum showed no significant difference between the groups, whereas, sICAM in FF demonstrated a significant difference between the patient groups investigated. On the whole, the ICSI outcome was not related to serum or FF concentrations of FGF or sICAM-1. Therefore, the mean concentration of FGF and sICAM-1 in serum and in FF could not be used to predict the fertilization rate in an ICSI program.

Comparison between cytokine concentration in follicular fluid of poor and high responder patients and their influence of ICSI-outcome.

OBJECTIVE: The aim of this study was to compare the cytokine concentration in follicular fluid (FF) of low and high responder intracytoplasmic sperm injection (ICSI) patients and to find out the impact of these cytokines in FF on ICSI outcome. DESIGN: The levels of insulin-like growth factor (IGF)-I, IL-6, IL-8, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), granulocyte-macrophage-colony stimulating factor (GM-CSF) were measured from low and high responder ICSI patients, the results were compared between the two groups and their influence on ICSI outcome was analysed. MATERIAL AND METHODS: A total of 49 low (G.I) and 34 high (G.II) responder patients were enrolled in this study. FF was collected at the time of oocyte retrieval and measured either by enzyme-linked immunosorbent assay (IL-6, IL8, EGF, PDGF, GM-CSF) or radio immuno assay (IGF-I). RESULTS: The concentration of IL-6 (pg/mL), IL-8 (pg/mL), IGF-I (ng/mL), PDGF (pg/mL), EGF (pg/mL), GM-CSF (pg/mL) in G.I was 6.0 +/- 4.3, 288.1 +/- 139.2, 0.416 +/- 0.089, 249.8 +/- 150.1, 9.12 +/- 5.5 and 1.45 +/- 2.10 and the corresponding value in G.II was 7.4 +/- 4.8, 208.6 +/- 64.0, 0.431 +/- 0.094, 387.6 +/- 36.0, 8.9 +/- 5.4 and 1.8 +/- 3.3, respectively. Only the PDGF concentration showed a significant (P = 0.007) difference between the two groups. Besides, negative correlations were found between PDGF and fertilization rate (r = -0.287; P = 0.046) of G.I. The mean number of retrieved (6.4 +/- 2.3 versus. 15.7 +/- 5.4) and fertilized (3.6 +/- 1.6 versus 7.0 +/- 4.5) oocytes differ significantly (P = 0.001) between the two groups. The fertilization rate was significantly higher in G.I than in G.II (60.9 +/- 25.1 versus 43.4 +/- 20.7%). CONCLUSION: There was no significant difference between IGF-I, IL-6, IL-8, EGF and GM-CSF concentrations of low and high responder patients. Besides, PDGF was significantly (P = 0.007) higher in high responder compared with low responder patients. Moreover, in poor responder patients, a negative correlation was found between PDGF and fertilization rate. However, the cytokine levels in FF of the patients undergoing controlled ovarian hyperstimulation for ICSI could not be used as a marker of oocyte fertilization and implantation potential.