Large nuclear vacuoles are indicative of abnormal chromatin packaging in human spermatozoa.

The aim of this investigation was to determine the presence of abnormal sperm chromatin packaging in spermatozoa with large nuclear vacuoles (LNV) selected via high magnification by analysing the pattern of chromomycin A3 (CMA3) staining. A prospective observational study was designed to analyse semen samples obtained from 66 men undergoing infertility diagnosis and treatment. The numbers of cells with normal (dull yellow staining of the sperm head/CMA3-negative) and abnormal (bright yellow fluorescence of the sperm head/CMA3-positive) chromatin packaging were determined on slides with normal and LNV spermatozoa. The presence of bright yellow fluorescence (CMA3-positive) was significantly higher (p < 0.0001) in spermatozoa with LNV than in normal spermatozoa (719/1351; 53.2% vs. 337/835; 40.3%, respectively), reflecting a higher percentage of abnormal chromatin packaging in spermatozoa with large LNV. Our data support the hypothesis that the presence of LNV reflects the presence of abnormal chromatin packaging, which may facilitate sperm DNA damage. As sperm nuclear vacuoles are evaluated more precisely at high magnifications using motile sperm organelle morphology examination (MSOME), the present results support the use of high-magnification sperm selection for intracytoplasmic sperm injection (ICSI).

Relationship between DNA damage and sperm head birefringence.

Birefringence or double refraction is the decomposition of a ray of light into two rays when it passes through an anisotropic material such as quartz. Sperm cells have been demonstrated to be optically anisotropic. The objective of this study was to evaluate the relationship between the pattern of human sperm head birefringence (SHBF) and DNA damage. A total of 26 patients with normal semen were included. DNA damage (fragmentation and denaturation) was evaluated in the sperm head in the context of birefringence, both total (SHBF-T) and partial (SHBF-P), by terminal deoxyribonucleotidyl transferase (TdT)-mediated dUDP nick-end labelling assay and acridine orange fluorescence, respectively. Positive DNA fragmentation in spermatozoa with SHBF-T (205/1053; 19.5%) was significantly higher (P<0.0001) than in spermatozoa that presented SHBF-P (60/820; 7.3%). However, the percentage of denatured DNA in spermatozoa with SHBF-T (824/1256; 65.6%) was not significantly different from the ones with SHBF-P (666/1009; 66.0%). In conclusion, the data support a positive relationship between spermatozoa with total SHBF in their head and increased DNA fragmentation.

Significance of extruded nuclear chromatin (regional nuclear shape malformation) in human spermatozoa: implications for ICSI.

The aim of this study was to determine the extent of DNA fragmentation and the presence of denatured single-strand or normal double-strand DNA in spermatozoa with extruded nuclear chromatin (ENC) selected by high magnification. Fresh semen samples from 55 patients were prepared by discontinuous isolate concentration gradient. Spermatozoa with normal nucleus (NN) and ENC were selected at 8400× magnification and placed on different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double-stranded DNA was identified by the acridine orange fluorescence method. DNA fragmentation was not significantly different (p = 0.86) between spermatozoa with ENC (19.6%) and those with NN (20%). However, the percentage of spermatozoa with detectable denatured-stranded DNA in the ENC spermatozoon group (59.1%) was significantly higher (p < 0.0001) than in the NN group (44.9%). The high level of denatured DNA in spermatozoa with ENC suggests premature decondensation and disaggregation of sperm chromatin fibres. The results show an association between ENC and DNA damage in spermatozoa, and support the routine morphological selection and injection of motile spermatozoa at high-magnification intracytoplasmic sperm injection.

Motile sperm organelle morphology examination is stricter than Tygerberg criteria.

The present study aimed to evaluate the correlation between the motile sperm organelle morphology examination (MSOME) and a well-known sperm morphology classification (Tygerberg criteria). For MSOME, spermatozoa were analysed at x8400 magnification by inverted microscope equipped with Nomarski differential interference contrast optics, Uplan Apo x100 oil/1.35 objective lens and variable zoom lens. By Tygerberg criteria, the semen underwent morphological evaluation as described in the literature. Regression analysis demonstrated significant positive correlation between percentage of normal sperm forms by Tygerberg criteria and by MSOME (r = 0.83, P < 0.0001). However, the incidence of normal spermatozoa by Tygerberg criteria (9.4%) was significantly higher (P < 0.0001) than under MSOME (3.3%). Despite the highly positive correlation, MSOME is a much stricter criterion of sperm morphology classification, since it identifies vacuoles and chromatin abnormalities that are not evaluated with the same precision by the analysis of Tygerberg criteria. MSOME should be included among the routine criteria for semen analysis. In addition, MSOME should be used for selection of spermatozoa for intracytoplasmic sperm injection based on the already published literature, as this is a good selection tool.

Relationship between visualization of meiotic spindle in human oocytes and ICSI outcomes: a meta-analysis.

The objective of this meta-analysis was to investigate the influence of meiotic spindle visualization in human oocytes on intracytoplasmic sperm injection (ICSI) outcomes. Search strategies included on-line surveys of databases (MEDLINE, EMBASE, Science Citation Index, Cochrane Controlled Trials Register and Ovid). The fixed effect was used for odds ratio. Ten trials fulfilled the inclusion criteria comparing in-vitro and clinical ICSI outcomes with or without visualization of meiotic spindle in fresh and in-vivo matured oocytes. According to the meta-analysis, the results showed statistically significant higher fertilization rate (P < 0.0001) when the meiotic spindle was viewed than when it was not. Moreover, the percentage of pro-nuclear-stage embryos with good morphology (P = 0.003), cleavage rate (P < 0.0001), percentage of day-3 top-quality embryos (P = 0.003) and percentage of embryos that reached the blastocyst stage (P < 0.0001) were statistically significantly better among embryos derived from oocytes in which meiotic spindle was viewed compared with those in which meiotic spindle was not observed. However, these differences were not observed in the clinical pregnancy or implantation rates. This observation has clinical relevance mainly in countries where there is a legal limit on the number of oocytes to be fertilized. However, additional controlled trials are needed to further confirm these results.

Development and population growth of Hydra viridissima Pallas, 1766 (Cnidaria, Hydrozoa) in the laboratory.

Hydras, the most representative freshwater Cnidaria, are of common occurrence in bodies of water in every continent except Antarctica. This study was planned with the aim of maintaining a population of Hydra viridissima in laboratory culture to enable the determination of the individual and population growth-rates of this species, as well as its population doubling time and generation time, with a view to employing these common animals as test-organisms in ecotoxicological assays. The organisms were maintained in reconstituted water at 20 +/- 2 degrees C, illuminated at 800 lux with a photoperiod of 12 hours light: 12 hours dark, and were fed on neonates of the cladoceran Ceriodaphnia silvestrii (3 or 4 neonates per hydra, 3 times a week). The individual growth-rate (k) of the species was 0.43, the maximum length of the column 2.53 mm and the generation time 6.6 +/- 1.5 days on average. The hydra population showed an intrinsic growth-rate (r) of 0.0468, according to the fitted curve, and a doubling time of 14.8 +/- 2.63 days. Hydra viridissima is easy to grow in the laboratory and performs well in the conditions used in this study. It is thus a promising candidate test-organism for ecotoxicological studies.

Development and population growth of Hydra viridissima Pallas, 1766 (Cnidaria, Hydrozoa) in the laboratory / Desenvolvimento e crescimento populacional de Hydra viridissima Pallas, 1766 (Cnidaria, Hydrozoa) em laboratório

Hydras, the most representative freshwater Cnidaria, are of common occurrence in bodies of water in every continent except Antarctica. This study was planned with the aim of maintaining a population of Hydra viridissima in laboratory culture to enable the determination of the individual and population growth-rates of this species, as well as its population doubling time and generation time, with a view to employing these common animals as test-organisms in ecotoxicological assays. The organisms were maintained in reconstituted water at 20 ± 2 °C, illuminated at 800 lux with a photoperiod of 12 hours light: 12 hours dark, and were fed on neonates of the cladoceran Ceriodaphnia silvestrii (3 or 4 neonates per hydra, 3 times a week). The individual growth-rate (k) of the species was 0.43, the maximum length of the column 2.53 mm and the generation time 6.6 ± 1.5 days on average. The hydra population showed an intrinsic growth-rate (r) of 0.0468, according to the fitted curve, and a doubling time of 14.8 ± 2.63 days. Hydra viridissima is easy to grow in the laboratory and performs well in the conditions used in this study. It is thus a promising candidate test-organism for ecotoxicological studies.

As hidras, os mais representativos Cnidaria de água doce, são organismos comuns em corpos de água doce de todos os continentes, exceto na Antártida. O objetivo do presente estudo foi manter o cultivo em laboratório da espécie Hydra viridissima, determinando-se o crescimento individual e populacional desta espécie, assim como seu tempo de duplicação da população e de geração, visando sua utilização como organismo-teste em ensaios ecotoxicológicos. Os organismos foram mantidos em água reconstituída, a uma temperatura de 20 ± 2 °C, com luminosidade de 800 lux e fotoperíodo de 12 horas luz/ 12 horas escuro, e foram alimentados com neonatas do Cladocera Ceriodaphnia silvestrii (3 a 4 neonatas por indivíduo). A taxa de crescimento individual (k) obtida foi de 0,43, o comprimento máximo da coluna das hidras foi de 2,53 mm e o tempo de geração foi, em média, de 6,6 ± 1,5 dias. A espécie apresentou uma taxa intrínseca de crescimento populacional (r) igual a 0,0468 para a curva ajustada e um tempo de duplicação da população de 14,8 ± 2,63 dias. A espécie Hydra viridissima é de fácil cultivo em laboratório e apresentou um bom desempenho nas condições testadas, sendo, portanto, um potencial organismo-teste para estudos ecotoxicológicos.

The effects of male age on sperm DNA damage in an infertile population.

The objective was to investigate the influence of age on sperm DNA damage. Semen samples were collected from 508 men in an unselected group of couples attending infertility investigation and treatment. DNA fragmentation in spermatozoa was measured by TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick-end labelling (TUNEL) assay; at least 200 spermatozoa in randomly selected areas of microscope slides were evaluated using a fluorescent microscope and the percentage of TUNEL positive spermatozoa was determined. The number of cells with red fluorescence (TUNEL positive) was expressed as a percentage of the total sample [DNA fragmentation index (DFI)]. Age was treated as a continuous variable for regression and correlation analysis. The following male age groups were used: Group I: < or =35 years, Group II: 36-39 years, and Group III: > or =40 years. DFI was significantly lower in Group I than in Group II (P = 0.034) or III (P = 0.022). There was no difference in DFI between Groups II and III. In addition, regression analysis demonstrated a significant increase in sperm DFI with age (P = 0.02). TUNEL assay clearly demonstrates an increase in sperm DNA damage with age.

Implantation failures: success of assisted hatching with quarter-laser zona thinning.

Implantation failure after IVF is one of the factors associated with a reduced chance of pregnancy for some patients. Assisted hatching methodologies are designed to facilitate the embryo's escape from the zona pellucida, and this strategy has been suggested as a means of improving pregnancy rates in patients with previous implantation failure. The aim of this prospective and randomized study was to evaluate the efficacy of quarter-laser zona thinning assisted hatching (qLZT-AH) in improving the implantation of embryos in patients with previous implantation failure. A total of 150 patients with a history of previous implantation failure were treated with intracytoplasmic sperm injection, and allocated into two groups: group 1, only one previous implantation failure, and group 2, repeated implantation failures. The patients in each group were randomized at the time of embryo transfer into a control group (no qLZT-AH) or experimental group where qLZT-AH was performed. For patients with repeated implantation failures, the implantation rate in those who received laser-thinned embryos was significantly higher (P = 0.02) than in those whose embryos were not laser-thinned (10.9 and 2.6% respectively). However, this difference was not observed in patients who presented with only one previous implantation failure. The data demonstrate that qLZT-AH is an effective strategy for improving the implantation of embryos in patients with repeated implantation failures.