The use of fluorescence in situ hybridization analysis on sperm: indications to perform and assisted reproduction technology outcomes.

PURPOSE: To determine the consequences of an altered sperm fluorescence in situ hybridization (FISH) result for ART outcomes and the indications for a sperm FISH analysis. METHODS: Data from 439 infertile men were collected. Bivariate analyses were performed to determine the association of men's age, seminal alterations, and sperm FISH indication, with the incidence of X, Y, 13, 18, and 21 sperm chromosomal abnormalities. A multivariate logistic regression analysis was performed to establish the most predictive variables for altered sperm FISH. Results from the IVF/ICSI cycles were collected for 248 out of 439 patients. Two distinct groups were established: 151 couples that used their own oocytes and 97 couples involved in egg donation programs. In both groups, ART outcomes were compared between normal and altered sperm FISH. RESULTS: Teratozoospermia and oligozoospermia were associated with sperm chromosome anomalies (p < 0.05). Indications for sperm FISH analysis with the highest predictability were teratozoospermia, male age, oligozoospermia, and implantation failure (AUC = 0.702). Embryo quality (p = 0.096), pregnancy rate (p = 0.054), and implantation rate (p = 0.089) were higher in own-oocytes couples with normal sperm FISH than in altered sperm FISH couples, although differences were not statistically significant. In donor-oocytes couples, in which high-quality embryos were transferred later than in own-oocytes couples (3.8 vs. 3.0 days), we did not identify differences in the ART outcome between normal and altered sperm FISH couples. In both groups, the possible interference of woman age was negligible. CONCLUSIONS: Sperm FISH is indicated in middle-aged oligoteratozoospermic patients with implantation failures in previous IVF/ICSI cycles. Sperm chromosome anomalies have a moderate detrimental impact on embryo quality, implantation, and pregnancy rates.

Oxidative stress in follicular fluid of young women with low response compared with fertile oocyte donors.

The aim of this study was to determine the concentrations of oxidative stress markers, antioxidant enzymes and cytokines in the follicular fluid of young women with low response in ovarian stimulation cycles compared with high responders and fertile oocyte donors of the same age, to assess the impact of oxidative stress on ovarian reserve. The activity of follicular fluid antioxidant enzymes glutathione transferase, glutathione reductase and glutathione peroxidase was significantly lower in young women with reduced ovarian reserve compared with that in high responders and oocyte donors. Follicular fluid concentrations of oxidative stress marker malondialdehyde combined with 4-hydroxyalkenals and nitric oxide were higher in low responders than in high responders and oocyte donors. Significant differences between low responders and donors in concentrations of IL-2, IL-6, IL-8 and vascular endothelial growth factor were observed, with higher concentrations in low responders. However, IL-10 concentration was lower in low responders than in high responders and donors. No significant differences were found in follicular fluid concentrations of tumour necrosis factor alpha between the three groups. These results demonstrate that different concentrations of oxidative stress markers, oxidant enzymes and cytokines in low responders compared with high responders and oocyte donors may negatively impact ovarian response.

Endometrial expression of selected genes in patients achieving pregnancy spontaneously or after ICSI and patients failing at least two ICSI cycles.

The objective of this study was to identify the endometrial gene expression profile in receptive phase, which could represent a useful prognostic tool for selecting IVF patients. Endometrial expression of 47 selected genes biopsied during the window of implantation in natural cycles was compared between patients who achieved a successful pregnancy spontaneously or after subsequent intracytoplasmic sperm injection (ICSI) cycles and patients who did not achieve a pregnancy after at least two failed ICSI cycles. The comparative analysis showed significantly different levels of expression in 19 genes, five implicated in apoptosis (CASP8, FADD, CASP10, APAF1, ANXA4), three in immunity (LIF, SPP1, C4BPA), five in transcriptional activity (MSX1, HOXA10, MSX2, HOXA11, GATA2), two in lipid metabolism (LEPR, APOD) and four in oxidative metabolism (AOX1, ALDH1A3, GPX3, NNMT). The evidence for these genes being differently expressed could represent the starting point of identifying the ideal receptive endometrial gene expression profile, which could be used in the future as a prognostic tool for IVF patients. Gene expression analysis technology has opened new important perspectives on the study of the physiological processes of different tissues and organs. Specifically for the endometrium, it would be really interesting to find out an endometrial gene expression profile of receptive phase, which could be used in future as a useful prognostic tool for selecting IVF patients. To achieve this aim, the objective of the present paper was the comparison of endometrial expression in natural cycles of 47 selected genes between the biopsies of patients who achieved a successful pregnancy, either spontaneously or after subsequent ICSI cycles, and those of patients who did not achieve a pregnancy after at least two failed ICSI cycles. The comparative analysis showed a significant different expression in 19 genes: five implicated in programmed cell death, known as apoptosis (CASP8, FADD, CASP10, APAF1, ANXA4), three in immunity (LIF, SPP1, C4BPA), five in transcriptional activity (MSX1, HOXA10, MSX2, HOXA11, GATA2), two in lipid metabolism (LEPR, APOD) and four in oxidative metabolism (AOX1, ALDH1A3, GPX3, NNMT). The evidence of these genes being differently expressed could represent the starting point of identifying the ideal receptive endometrial gene expression profile which could be used in the future as a prognostic tool for IVF patients.

Relationships between lipids levels in blood plasma, follicular fluid and seminal plasma with ovarian response and sperm concentration regardless of age and body mass index.

PURPOSE: There is a considerable literature supporting the role of lipids in fertility. However, little is known about their impact on male and female gametes. Our study aimed to investigate the relationships between lipids levels in serum, follicular fluid and seminal plasma with ovarian response and sperm concentration regardless of age and body mass index (BMI). METHODS: 51 follicular fluid and serum samples of IVF-ICSI cycles and 52 seminal plasma and serum samples of males in the infertility study were analyzed for cholesterol, triglycerides, and non-esterified fatty acids. The parameters used to assess gonadal response were number of mature oocytes in metaphase II and total motile sperm. Differences between groups were studied by means Principal Component Analysis, Kolmogorov-Smirnov test, Pearson correlation, Student's T, and multivariate linear regression. RESULTS: Using a multivariate linear regression model to exclude the effect of the age and BMI, we found that the lipid profile in follicular fluid and plasma influence inversely and significantly on ovarian response and the number of matured oocytes recovered. Moreover, we found that seminal lipid levels are predictors of seminal quality independent of plasma lipid values. CONCLUSION: Our current analysis demonstrates the association of low ovarian response and low number of motile sperms with abnormal lipids levels.

Seminal Plasma and Serum Afamin Levels Are Associated with Infertility in Men with Oligoasthenoteratozoospermia.

The plasma glycoprotein afamin has been previously identified as an alternative carrier protein for vitamin E in extravascular fluids such as plasma and cerebrospinal, ovarian follicular, and seminal fluids. However, to date, no study has established a relationship between afamin levels and infertility in women or men. The purposes of our study were (i) to assess the level of afamin in serum and seminal fluids in infertile men compared to healthy controls and (ii) to study the association between polymorphisms in afamin genes and male infertility. This observational, prospective study evaluated the afamin levels in serum and seminal fluids from infertile men (n = 39) and compared them to those in healthy controls (n = 30). We studied the association between single-nucleotide polymorphisms (SNPs) in the 5`-untranslated region (5`-UTR) of the afamin gene and infertility and analyzed a total of 1000 base pairs from the untranslated region of the afamin gene. Subjects with low sperm motility and low sperm concentration had higher median seminal afamin (18.9 ± 2.9 ng/mg of proteins) and serum afamin concentrations (24.1 ± 4.0 ng/mg of proteins) than subjects with normal sperm parameters (10.6 ± 1.4 ng/mg of proteins) (p < 0.02) (15.6 ± 1.4 ng/mg of proteins) (p < 0.002). A total of five different polymorphisms were found, including one deletion and four single-nucleotide polymorphisms (SNPs). A new transversion (A/T) (position 4:73481093) was identified in an oligoasthenoteratozoospermic patient and was associated with high levels of afamin in plasma and seminal fluids. The prevalence of this variant in our study in the case homozygous for TT is 0.985 (98.5%), and in the case heterozygous for TA is 0.015 (1.5%). Our results suggest that genetic variations in afamin might be associated with male infertility. These findings could significantly enhance our understanding of the molecular genetic causes of infertility.

Diagnostic accuracy of sperm DNA degradation index (DDSi) as a potential noninvasive biomarker to identify men with varicocele-associated infertility.

PURPOSE: Varicocele is a frequent cause of impaired testicular function that has been associated with increased levels of sperm DNA fragmentation (SDF). Sperm with degraded DNA (DDS), as observed using the sperm chromatin dispersion (SCD) test, represent a subpopulation of spermatozoa with extensive DNA and nuclear protein damage. The aim of this work was to determine the usefulness of sperm DNA degradation index (DDSi) as a novel noninvasive biomarker to identify infertile men with varicocele. METHODS: A total of 593 semen samples obtained from men attending infertility clinics were analyzed for SDF and DDS with the SCD test. These samples were classified as: (1) fertile donors; (2) infertile patients with least two failed assisted reproduction cycles; (3) leukocytospermia; (4) Chlamydia trachomatis infection; (5) testicular cancer, and (6) infertile men with varicocele. The DDSi was obtained by determining the proportion of DDS in the whole sperm population presenting with fragmented DNA. The diagnostic accuracy of DDSi was evaluated by correlation coefficient and receiver operating characteristics analyses. RESULTS: A positive correlation (r ≥ 0.52) was observed between the SDF and the frequency of degraded sperm in all patient groups. The sperm DNA degradation index (DDSi) was at least twice as higher in infertile men with varicocele (mean: 0.54) compared with other clinical conditions and fertile donors (means ranging from 0.02 to 0.21; P < 0.0001). A DDSi ≥ 0.33 identified patients with varicocele with 94 % accuracy. CONCLUSION: Although DDS is not pathognomonic of varicocele, the DDSi is a useful noninvasive biomaker to identify infertile individuals with varicocele when examining sperm DNA damage during a routine semen analysis. This finding may alert practitioners and laboratories performing semen analysis that in the presence of an abnormal DDSi it is likely that a given patient has varicocele. It is therefore strongly recommended that such patients be referred to urologists in order to undergo a full andrological examination and be properly counseled.

Can DNA fragmentation of neat or swim-up spermatozoa be used to predict pregnancy following ICSI of fertile oocyte donors?

This study compared the potential of assessing sperm DNA fragmentation (SDF) from neat semen and the subsequent swim-up (SU) procedure to predict pregnancy when conducting ICSI of fertile donor oocytes. Infertile females (n=81) were transferred embryos resulting from intracytoplasmic sperm injection (ICSI) of their partner's spermatozoa and proven donor oocytes. This model normalized the impact of female factor in putative sperm DNA repair. Semen was blindly assessed for SDF using Halosperm immediately following ejaculation (NS) and after swim-up at the time of ICSI fertilisation. There was a decrease in SDF values of the ejaculated semen sample following the swim-up protocol (P=0.000). Interestingly, pregnancy could be equally predicted from SDF values derived from either neat or swim-up semen samples. Receiver operator curves and the derived Youden's indices determined SDF cutoff values for NS and SU of 24.8% and 17.5%, respectively. Prediction of pregnancy from NS SDF had a sensitivity of 75% and a specificity of 69%, whereas for SU SDF was 78% and 73%, respectively. While increased levels of SDF negatively impact reproductive outcome, we have shown that a reduction in SDF following sperm selection using ICSI with proven donor oocytes is not mandatory for achieving pregnancy. This suggests that a certain level of DNA damage that is not detectable using current technologies could be impacting on the relative success of assisted reproductive technology (ART) procedures. Consequently, we propose a modification of the so called 'iceberg model' as a possible rationale for understanding the role of SDF in reproductive outcome.

An improved experimental model for understanding the impact of sperm DNA fragmentation on human pregnancy following ICSI.

Using donor oocytes of proven fertility, the effect of sperm DNA fragmentation (SDF) and motility on reproductive success was examined in 70 couples undergoing ICSI. Both SDF and sperm motility were assessed at the time of sperm injection and using the same sperm sample that was processed for ICSI. While there was no difference in the fertilization rate, cleavage rate, embryo quality, or sperm motility between pregnant and nonpregnant couples, the SDF of nonpregnant couples (SDF = 23.9%) was higher than that of pregnant couples (SDF = 17.0%; U Mann-Whitney 347; P = .002). Using a combination of the sensitivity and specificity measures from the production of ROC (receiver-operating characteristic) curves and the Youden index, we determined a threshold SDF value for our data set of 17% for predicting pregnancy (77.8% sensitivity and 71.1% specificity). Our results suggest that proven donor oocytes in combination with SDF assessment at the time of sperm injection represent a useful experimental model for reducing the confounding influences of sperm DNA repair by the oocyte and iatrogenic sperm damage.