Embryo quality evaluated by the embryo morphology is a critical parameter in human in-vitro fertilization (IVF) and embryo transfer. It determines which and how many embryos will be replaced, as pregnancy rates are directly related to number and quality of transferred embryos. This retrospective analysis included 1301 IVF and embryo transfer cycles to identify which factors influenced embryo quality. Embryo quality did not correlate with maternal age, causes of infertility, ovarian stimulation parameters or embryo cohort size. However, the mean score of transferred embryos was significantly higher for patients with more than five embryos compared to fewer than five embryos (P < 0.001), irrespective of maternal age. Patients tended to produce a similar embryo quality from cycle to cycle, r = 0.33 (P < 0.001) for the embryo cohort and r= 0.47 (P < 0.001) for the transferred embryos. Poor embryo morphology probably reflects oocytes with compromised development competence and could be an independent factor of infertility. Furthermore, a large embryo cohort was the main factor increasing the chances of at least one good embryo in the cohort.

The aim of this study was to determine whether the early stages of embryo development, as assessed by the zygote score (Z-score), could be influenced by the injection of spermatozoa that had been preselected on the basis of their binding to hyaluronic acid (HA). A total of 407 sibling metaphase II oocytes, belonging to 44 different patients, were injected in a prospective randomized way, with either hyaluronic acid bound (HA(+) ) or non-bound (HA(-)) spermatozoa. The fertilization rate (75-70%), the percentage of the different Z-scores (Z 1: 22-24%, Z 2: 22-22.5%, Z 3: 44- 45%, Z 4: 12-8.5%), the mean score of the transferred embryos (3.76 +/- 1.29, 3.78 +/- 1.1) and the number of embryos at the 4-cell stage 45 h after injection (77-76%) were not different between the two groups. The ongoing pregnancy rate in this study (>20 weeks of gestation) was 36.4% per replacement, the implantation rate 28% and the twin pregnancy rate 44% (7/16). Although binding to HA did not apparently influence the Z-score, this agent continues to be used for the immobilization of spermatozoa prior to injection, on the basis that it is a natural product that can easily be metabolized by the oocyte via normal biological mechanisms.

The objective of this study was to optimize the use of testicular biopsies in 14 patients with obstructive azoospermia. Testicular specimens were retrieved from six patients (group I) and cultured at 32 and 37 degrees C for up to 20 days; changes in percentage motile spermatozoa were compared. In four men of group I, one portion of the specimen was frozen at retrieval, and changes in post-thaw motility after 24 h of culture at 37 degrees C were recorded. In the other eight patients (group II), testicular specimens were frozen at retrieval and after 72 h culture at 37 degrees C. Pre and post-freezing motility and post-thaw recovery rate were compared. No significant differences were observed until day 8 in the improvement of motility between 32 and 37 degrees C in-vitro culture. Maximum motility was reached, under both conditions, between 48 h and 72 h. Post-thaw 24 h culture at 37 degrees C of specimens frozen at retrieval did not improve motility; however, 72 h pre-freezing culture significantly improved initial motility (P: < 0.01), post-thaw motility (P: < 0.01) and post-thaw recovery rate (P: < 0. 001). The higher recovery rate of samples frozen 3 days after retrieval allows more economical use of the tissue that is available.

Chromosomal mosaicism has been reported in in vitro-cultured embryos at early cleavage stages, as well as in morulae and blastocysts. We have assessed the incidence and pattern of mosaicism during in vitro development of human embryos from early-cleavage stages to morula and blastocyst. Fifty spare embryos were fixed for fluorescence in situ hybridization (FISH) analysis for chromosomes X, Y, 13, 18, and 21 on days 2 or 3 (4- to 10-cell stage) (n = 16), on day 4 (morula stage) (n = 14), on day 5 (pre-expanded blastocyst) (n = 5), and the expanded blastocyst stages (n = 15). Blocked embryos (no cleavage observed within the last 24 hr) were not included. A total of 2367 cells were analyzed. Four early-cleavage stage embryos were found uniformly diploid; all of the others were mosaic for the chromosomes analyzed (mean diploid nuclei 48.3% +/- 28.7). All of the embryos at more advanced developmental stages, except one fully normal morula, had mosaic chromosome constitutions, with an increase in the percentage of diploid cells in morulae, pre-expanded, and expanded blastocysts, respectively (mean diploid nuclei 78.6% +/- 11.7, 66.0% +/- 20.8, 79.6% +/- 12.8), in comparison with earlier stages. Hypotheses about the origin of mosaicism and embryo regulation mechanisms will be discussed.

The aim of the present study was to verify whether culturing testicular tissue, to obtain a higher percentage of motile spermatozoa and a better post-thaw recovery rate, affected the ratio between single/double-stranded sperm DNA and, consequently, DNA sensitivity to damage. Testicular biopsy samples from men with obstructive and secretory azoospermia, candidates for assisted reproductive treatment, were cultured for 72 h. The percentage of motile spermatozoa and the single/double stranded DNA ratio were assessed on the day of retrieval (day 0) and again on day 3. The single/double stranded DNA ratio was measured by the acridine orange (AO) staining method. Spermatozoa were classified as green (double-stranded chromatin) or red fluorescing (single-stranded chromatin). In obstructive azoospermia, median motility was 22% (range 10-44%) on day 0 and 50% (range 38-63%) on day 3 (P < 0.01). The median percentage of red stained spermatozoa was 53.5% (range 0.1-88%) on day 0 and 20% (range 2.7-99.9%) on day 3 (P < 0.05). No changes were observed in secretory azoospermia. The culture procedure from obstructive azoospermia not only increased the post-thaw recovery rate, as previously observed, but also reduced the portion of spermatozoa containing single-stranded DNA, thereby increasing the availability of double-stranded DNA spermatozoa for ICSI use.