Time-Lapse Systems: A Comprehensive Analysis on Effectiveness.

Time-lapse systems have quickly become a common feature of in vitro fertilization laboratories all over the world. Since being introduced over a decade ago, the alleged benefits of time-lapse technology have continued to grow, from undisturbed culture conditions and round the clock, noninvasive observations to more recent computer-assisted selection of embryos through the development of algorithms. Despite the global uptake of time-lapse technology, its real impact on clinical outcomes is still controversial. This review aims to explore the different features offered by time-lapse technology, discussing incubation, algorithms, artificial intelligence and the regulation of nonessential treatment interventions, while assessing evidence on whether any benefit is offered over conventional technology.

DNA fragmentation in microorganisms assessed in situ.

Chromosomal DNA fragmentation may be a direct or indirect outcome of cell death. Unlike DNA fragmentation in higher eukaryotic cells, DNA fragmentation in microorganisms is rarely studied. We report an adaptation of a diffusion-based assay, developed as a kit, which allows for simple and rapid discrimination of bacteria with fragmented DNA. Intact cells were embedded in an agarose microgel on a slide, incubated in a lysis buffer to partially remove the cell walls, membranes, and proteins, and then stained with a DNA fluorochrome, SYBR Gold. Identifying cells with fragmented DNA uses peripheral diffusion of DNA fragments. Cells without DNA fragmentation show only limited spreading of DNA fiber loops. These results have been seen in several gram-negative and gram-positive bacteria, as well as in yeasts. Detection of DNA fragmentation was confirmed by fluoroquinolone treatment and by DNA breakage detection-fluorescence in situ hybridization. Proteus mirabilis with spontaneously fragmented DNA during exponential and stationary growth or Escherichia coli with DNA damaged after exposure to hydrogen peroxide or antibiotics, such as ciprofloxacin or ampicillin, was clearly detected. Similarly, fragmented DNA was detected in Saccharomyces cerevisiae after amphotericin B treatment. Our assay may be useful for the simple and rapid evaluation of DNA damage and repair as well as cell death, either spontaneous or induced by exogenous stimuli, including antimicrobial agents or environmental conditions.

Successful pregnancy and childbirth after intracytoplasmic sperm injection with calcium ionophore oocyte activation in a globozoospermic patient.

OBJECTIVE: To check the effectiveness of intracytoplasmic sperm injection (ICSI) combined with assisted oocyte activation (AOA) in a globozoospermic patient. DESIGN: Case report. SETTING: Instituto Valenciano de Infertilidad, Valencia, Spain. PATIENT(S): A patient with globozoospermia. INTERVENTION(S): ICSI was administered in 14 oocytes. ICSI combined with AOA, in which a small amount of calcium was injected followed by calcium ionophore exposure, was done in 9 oocytes. MAIN OUTCOME MEASURE(S): Fertilization rate and embryo quality was assessed in both groups. RESULT(S): Chemical activation increased fertilization rate (55.6% vs. 35.7%) and the number of embryos with less multinucleation on day 2 (0 vs. 60%). Two embryos generated from AOA were transferred into the uterus (on day 3), resulting in a pregnancy and a healthy newborn. CONCLUSION(S): The AOA with calcium ionophore treatment improved fertilization rate and quality of the embryos, and was found to be an effective method for AOA in this patient with a low fertilization rate after previous ICSI treatment.

Increased aneuploidy rate in sperm with fragmented DNA as determined by the sperm chromatin dispersion (SCD) test and FISH analysis.

Previous studies suggest that sperm DNA fragmentation may be associated with aneuploidy. However, currently available tests have not made it possible to simultaneously perform DNA fragmentation and chromosomal analyses on the same sperm cell. The recently introduced sperm chromatin dispersion (SCD) test allows users to determine this relationship. Semen samples from 16 males, including 4 fertile donors, 7 normozoospermic, 3 teratozoospermic, 1 asthenozoospermic, and 1 oligoasthenoteratozoospermic, were processed for DNA fragmentation analysis by the SCD test using the Halosperm kit. Three-color fluorescence in situ hybridization (FISH) was performed on SCD-processed slides to determine aneuploidy for chromosomes X, Y, and 18. Spermatozoa with DNA fragmentation showed a 4.4 +/- 1.9-fold increase in diploidy rate and a 5.9 +/- 3.5-fold increase in disomy rate compared to spermatozoa without DNA fragmentation. The overall aneuploidy rate was 4.6 +/- 2.0-fold higher in sperm with fragmented DNA (Wilcoxon rank test: P < .001 in the 3 comparisons). A higher frequency of DNA fragmentation was found in sperm cells containing sex chromosome aneuploidies originated in both first and second meiotic divisions. The observed increase in aneuploidy rate in sperm with fragmented DNA may suggest that the occurrence of aneuploidy during sperm maturation may lead to sperm DNA fragmentation as part of a genomic screening mechanism developed to genetically inactivate sperm with a defective genomic makeup.

Evidence of modified nuclear protein matrix in human spermatozoa with fragmented deoxyribonucleic acid.

Human spermatozoa were processed for determination of DNA fragmentation with use of an in situ diffusion assay, so that those cells containing DNA fragmentation produce extensive peripheral dissemination of DNA fragments after lysis in an agarose microgel. Quantification of specific protein staining confirmed that sperm cells without DNA fragmentation had almost complete removal of nuclear matrix proteins, whereas spermatozoa with DNA fragmentation tended to retain residual nucleoskeletal protein in a collapsed and condensed state. This result suggests that a modified nuclear protein matrix associates with fragmented sperm DNA.

The human sperm glutathione system: a key role in male fertility and successful cryopreservation.

The equilibrium of the creation and scavenging of free radicals is mandatory in the spermatozoa to fertilize and initiate a full-term pregnancy. The glutathione (GSH) enzymatic system studies have discovered its relationship with oxidative stress in the ejaculate and new strategies to regulate its activity in the semen could be developed. Intracellular sperm GSH system components are altered in infertile men, and these alterations seem to be linked to sperm morphology. We have been able to correlate embryo morphology on 8 cell embryos with the sperm expression of GPx family members; this relationship appears quite promising for discovery of molecular causes of male infertility. Oxidative stress imbalance potentially leads to damage of the structure of plasma membrane. The freezing and subsequent thawing of sperm is a physically stressful process carried out during routine procedures in assisted reproduction, which results in a highly variable and unpredictable reduction of motile sperm. Subsequently, oxidative status can positively or negatively affect the motility, viability, and fertilizing capacity of thawed sperm. A reserve of glutathione, together with GPx expression, is necessary to eliminate free radicals using GSH or GPx-4 like structural protein and seems to be essential for a good post thaw recovery.

Value of the sperm deoxyribonucleic acid fragmentation level, as measured by the sperm chromatin dispersion test, in the outcome of in vitro fertilization and intracytoplasmic sperm injection.

OBJECTIVE: To determine the prognostic value of sperm DNA fragmentation levels, as measured by the sperm chromatin dispersion (SCD) test, in predicting IVF and ICSI outcome. DESIGN: Double-blind prospective study. SETTING: University-affiliated private IVF setting. PATIENT(S): A total of 85 couples undergoing infertility treatment with IVF/ICSI. INTERVENTION(S): Analysis of DNA fragmentation by the SCD test in 170 aliquots obtained from the ejaculate and from the processed semen used for assisted reproductive technologies (ART). MAIN OUTCOME MEASURE(S): Percentage of spermatozoa with fragmented DNA was statistically correlated with embryo quality and reproductive success. RESULT(S): Fertilization rate was inversely correlated with DNA fragmentation (r = -0.245 P = .045). Higher DNA fragmentation rate gave an increased proportion of zygotes showing asynchrony between the nucleolar precursor bodies of zygote pronuclei (73.8% vs. 28.8% P < .001). In addition, the slower embryo development and worst morphology on day 6 was correlated with higher sperm DNA fragmentation (47.7% vs. 29.4% P = .044). We also observed a negative correlation between DNA fragmentation and the implantation rate (r = -0.250 P = .042). However, SCD test values were not statistically different in cycles that resulted in a pregnancy compared with those that did not (33.2 vs. 28.2 and 32.4 vs. 34.7). CONCLUSION(S): This is the first report that describes a correlation between sperm DNA integrity, as measured by the SCD test, and fertilization rate, embryo quality, and implantation rate in IVF/ICSI. The degree of DNA fragmentation was inversely correlated with fertilization rate, synchrony of the nucleolar precursor bodies' pattern in pronuclei, embryo ability to achieve blastocyst stage, and embryo morphological quality. Because SCD test values were correlated with embryo quality and blastocyst rate, the lack of correlation between sperm DNA fragmentation and pregnancy outcome in IVF might be due to embryo selection before transfer. The ability of the SCD test to predict the blastocyst rate after IVF/ICSI warrants further study.

Infertile men with varicocele show a high relative proportion of sperm cells with intense nuclear damage level, evidenced by the sperm chromatin dispersion test.

The frequency of sperm cells with fragmented DNA was studied in a group of 18 infertile patients with varicocele and compared with those obtained in a group of 51 normozoospermic patients, 103 patients with abnormal standard semen parameters, and 22 fertile men. The spermatozoa were processed to discriminate different levels of DNA fragmentation using the Halosperm kit, an improved Sperm Chromatin Dispersion (SCD) test. In this technique, after an acid incubation and subsequent lysis, those sperm cells without DNA fragmentation show big or medium-sized halos of dispersion of DNA loops from the central nuclear core. Otherwise, those spermatozoa containing fragmented DNA either show a small halo, exhibit no halo with solid staining of the core, or show no halo and irregular or faint stain of the remaining core. The latter, that is, degraded type, corresponds to a much higher level of DNA-nuclear damage. The varicocele patients showed 32.4% +/- 22.3% of spermatozoa with fragmented DNA, significantly different from the group of fertile subjects (12.6% +/- 5.0%). Nevertheless, this was not different from that of normozoospermic patients (31.3% +/- 16.6%) (P = .83) and with abnormal semen parameters (36.6% +/- 15.5%) (P = .31). No significant differences were found between the normozoospermic patients and the patients with abnormal semen parameters. Strikingly, the proportion of the degraded cells in the total of sperm cells with fragmented DNA was 1 out of 4.2 (23.9% +/- 12.9%) in the case of varicocele patients, whereas it was 1 out of 8.2 to 9.7 in the normozoospermic patients (11.1% +/- 9.9%) in the patients with abnormal sperm parameters (12.2% +/- 8.3%) and in the fertile group (10.3% +/- 7.2%). Thus, whereas no differences in the percentage of sperm cells with fragmented DNA were evident with respect to other infertile patients, individuals with varicocele exhibit a higher yield of sperm cells with the greatest nuclear DNA damage level in the population with fragmented DNA. This finding illustrates the value of assessing different patterns of DNA-nuclear damage within each sperm cell and the particular ability of the Halosperm kit to reveal them.

Value of the sperm chromatin dispersion test in predicting pregnancy outcome in intrauterine insemination: a blind prospective study.

BACKGROUND: Sperm DNA integrity has been used as a new marker of sperm quality in the prediction of pregnancy. Nevertheless, no previous study has been performed by analysing the same samples that were employed in assisted reproduction. The main objective of this work was to correlate sperm chromatin dispersion (SCD), measured by the SCD test, with semen parameters and pregnancy outcome in intrauterine insemination (IUI). METHODS: A total of 100 semen samples obtained from males of couples undergoing IUI were analysed by the SCD test before and after swim-up, and the results were correlated with semen parameters and pregnancy outcome. RESULTS: SCD was negatively correlated with sperm motility in both ejaculated and processed semen. Sperm recovered by swim-up did not show a significant improvement in DNA integrity. No correlation was found between SCD and pregnancy outcome in IUI. CONCLUSIONS: DNA dispersion, as measured by the SCD test, is not correlated with pregnancy outcome in IUI.

Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test.

OBJECTIVE: To improve the sperm chromatin dispersion (SCD) test and develop it as a simple kit (Halosperm kit) for the accurate determination of sperm DNA fragmentation using conventional bright-field microscopy. DESIGN: Method development, comparison, and validation. SETTING: Medical genetics laboratory, academic biology center, and reproductive medicine centers. PATIENT(S): Male infertility patients attending the Reproductive Medicine Center. A varicocele patient and a group of nine fertile subjects. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): [1] The quality of chromatin staining in relaxed sperm nuclear halos and tail preservation; [2] SCD scoring reproducibility; [3] comparison with the sperm chromatin structure assay in 45 samples; [4] frequency of sperm with DNA fragmentation after incubation with increasing doses of the nitric oxide donor sodium nitroprusside and in sperm samples for 9 fertile men, 46 normozoospermic patients, 23 oligoasthenoteratozoospermic patients, and a subject with varicocele. RESULT(S): The sperm nuclei with DNA fragmentation, either spontaneous or induced, do not produce or show very small halos of DNA loop dispersion after sequential incubation in acid and lysis solution. The improved SCD protocol (Halosperm kit) results in better chromatin preservation, therefore highly contrasted halo images can be accurately assessed using conventional bright-field microscopy after Wright staining. Moreover, unlike in the original SCD procedure, the sperm tails are now preserved, making it possible to unequivocally discriminate sperm from other cell types. The chi2 test did not detect significant differences in the mean number of sperm cells with fragmented DNA as scored by four different observers. The intraobserver coefficient of variation for the estimated percentage of spermatozoa with fragmented DNA ranged from 6% to 12%. There was good correlation between the SCD and the sperm chromatin structure assay DNA fragmentation index (intraclass correlation coefficient R: 0.85; percent DNA fragmentation index mean difference: 2.16 significantly higher for SCD). Using the Halosperm kit, a dose-dependent increase in sperm DNA damage after sodium nitroprusside incubation was detected. The percentage of sperm cells with fragmented DNA in the fertile group was 16.3 +/- 6.0, in the normozoospermic group, 27.3 +/- 11.7, and in the oligoasthenoteratozoospermic group, 47.3 +/- 17.3. In the varicocele sample, an extremely high degree of nuclear disruption was detected in the population of sperm cells with fragmented DNA. CONCLUSION(S): The improved SCD test, developed as the Halosperm kit, is a simple, cost effective, rapid, reliable, and accurate procedure, for routinely assessing human sperm DNA fragmentation in the clinical andrology laboratory.

Halosperm is an easy, available, and cost-effective alternative for determining sperm DNA fragmentation.

The characteristics of Halosperm make this kit a reasonable alternative to allow basic and clinical research on sperm DNA fragmentation in any basic laboratory around the world.

Structure of human sperm DNA and background damage, analysed by in situ enzymatic treatment and digital image analysis.

DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) is a procedure to detect and quantify DNA breaks in situ, on a cell-by-cell basis. A comparison between sperm nuclei versus peripheral blood leukocytes using this method demonstrated that the nucleoids from mature human sperm are 12.7 times more sensitive to alkaline denaturation than those from human peripheral blood leukocytes. To investigate the origin of this alkali sensitivity, different approaches were employed. First, free 3'-OH ends of background DNA breaks were labelled by Klenow polymerase, or by DNA polymerase I following the in situ nick translation assay. Second, the presence of abasic sites, the other recognized DNA lesions that lends to constitutive alkali sensitivity, and DNA breaks with blocked 3' ends, were determined by in situ exonuclease III digestion prior to the polymerase labelling. The results demonstrated that the sperm nucleoid contains approximately 2.5-fold higher density of background DNA breaks with 3'-OH ends, and also approximately 2.8-fold higher density of basal abasic sites and DNA breaks with blocked 3' termini, than leukocytes. These differences only partially explain the significant alkali sensitivity of sperm DNA. However, in situ digestion with mung bean nuclease before DNA break labelling showed that sperm DNA is 9-fold more enriched in segments of ssDNA than DNA from leukocytes. The high frequency of partially denatured regions may result from a greater torsional stress of DNA loops in sperm chromatin due to its higher degree of compaction. Moreover, these short unpaired ssDNA stretches should be included in the category of alkali-labile sites detected by all techniques that measure DNA breaks through an alkaline unwinding step. These results provide new insights into the nature of DNA packaging in sperm nuclei.

The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation.

Sperm DNA fragmentation is being increasingly recognized as an important cause of infertility. We herein describe the Sperm Chromatin Dispersion (SCD) test, a novel assay for sperm DNA fragmentation in semen. The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops that is observed in sperm with non-fragmented DNA, following acid denaturation and removal of nuclear proteins. This was confirmed by the analysis of DNA fragmentation using the specific DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) assay, which allows the detection of DNA breaks in lysed sperm nuclei. Sperm suspensions either prepared from semen or isolated from semen by gradient centrifugation were embedded in an agarose microgel on slides and treated with 0.08 N HCl and lysing solutions containing 0.8 M dithiothreitol (DTT), 1% sodium dodecyl sulfate (SDS), and 2 M NaCl. Then, the slides were sequentially stained with DAPI (4',6-diamidino-2-phenylindole) and/or the Diff-Quik reagent, and the percentages of sperm with nondispersed and dispersed chromatin loops were monitored by fluorescence and brightfield microscopy, respectively. The results indicate that all sperm with nondispersed chromatin displayed DNA fragmentation, as measured by DBD-FISH. Conversely, all sperm with dispersed chromatin had very low to undetectable DBD-FISH labeling. SCD test values were significantly higher in patients being screened for infertility than in normozoospermic sperm donors who had participated in a donor insemination program. The coefficient of variation obtained using 2 different observers, either by digital image analysis (DIA) or by brightfield microscopy scoring, was less than 3%. In conclusion, the SCD test is a simple, accurate, highly reproducible, and inexpensive method for the analysis of sperm DNA fragmentation in semen and processed sperm. Therefore, the SCD test could potentially be used as a routine test for the screening of sperm DNA fragmentation in the andrology laboratory.