Pronuclear zygote score following intracytoplasmic injection of hyaluronan-bound spermatozoa: a prospective randomized study.

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.

Ten years of Swiss National IVF Register FIVNAT-CH. Are we making progress?

In 2001, analysis of Swiss data collected since 1993 included 1001 treatment cycles with IVF, 2217 treatment cycles with intracytoplasmic sperm injection and 2160 treatment cycles with frozen-thawed embryos or zygotes. IVF cycle number has remained constant over the past 10 years, now representing only 18% of the total. ICSI treatment cycles have plateaued since 2001. Altogether, patients receive 1.56 treatment cycles per year, nearly constant since 1995. Mean maternal age has increased from 33.9 to 35.7 years, while mean number of recovered oocytes has increased by 1.3. Considerable improvement was seen in clinical pregnancy rate after 'fresh' treatment cycles since 2000. Mean number of replaced embryos in 'fresh' treatment cycles has fallen to below 2.5 since 1996, long before the legal imposition of the three-embryo transfer limit in 2001, and is still decreasing without affecting the consistent twin pregnancy rate of 19%. The frequency of ovarian hyperstimulation syndrome has increased three-fold. External audits have reduced the mean number of errors per data file by half, and increased the number of correct files by 20%. Data collected over this 10-year period show that despite the introduction of a restrictive law and increasing mean maternal age, the overall clinical pregnancy rate has continued to improve.

The use of an hydrogen peroxide multipurpose isolator for inhouse preparation of human embryo culture media: a unique successful Swiss randomized prospective study.

PURPOSE: To develop inhouse made (IHM) embryo culture medium with a Multipurpose Isolator and compare the embryo development in a prospective randomized study with commercial media. METHODS: Fertilization by intracytoplasmic single sperm injection (ICSI) of Metaphase II oocytes obtained after 96 controlled ovarian hyperstimulation cycles in patients not older than 37 years. Transfer of zygotes to IHM or commercial Cook Sydney IVF Cleavage medium (SIC) immediately after pronucleus observation. Evaluation of embryo cleavage and score, pregnancy, and implantation rate. RESULTS: From 100 zygotes cultured in SIC, 61% were at the 4 cell stage 45 h after ICSI compared to 77% (78/101) in the IHM, P < 0.05. The mean embryo score with IHM was 3.9 +/- 0.9 compared to 3.5 +/- 1.2 with SIC, P < 0.05. The clinical pregnancy rate per transfer was 38.9% (37/95), the implantation rate was 23% (46/200), and no differences were observed between the groups.

Incidence of chromosomal mosaicism in human embryos at different developmental stages analyzed by fluorescence in situ hybridization.

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.

Higher degree of chromosome mosaicism in preimplantation embryos from carriers of robertsonian translocation t(13;14) in comparison with embryos from karyotypically normal IVF patients.

PURPOSE: To compare the frequency and the degree of mosaicism in human embryos from Robertsonian translocation (RT) t(13;14) carriers, with embryos from karyotypically normal IVF patients METHODS: FISH analysis of embryos from PGD cycles for RT t(13;14), with probes for chromosomes 13, 14, and 18 (Group I) and of embryos from karyotypically normal IVF patients with probes for chromosomes 13, 18, 21, X, and Y (Group II). RESULTS: The incidence of abnormal mosaic embryos was significantly higher in group I (38/51) as compared with group II (6/45) (chi2: P < 0.01). Furthermore, in group I the percentage of diploid cells per embryo was lower for chromosome 13 and 14 in comparison with 18, while in group II no differences were observed between the five chromosomes analyzed. CONCLUSIONS: RT induces a high frequency of mosaicism specifically for the chromosomes implicated in the translocation; the analysis by FISH of two blastomeres is strongly recommended for these patients

Correlation between fluorescence in-situ hybridization analyses and in-vitro development to blastocyst stage of embryos from Robertsonian translocation (13;14) carriers.

BACKGROUND: Little is known about the extent and timing of selection against the embryos that are carriers of unbalanced translocations. METHODS: Fluorescence in-situ hybridization (FISH) with probes for chromosomes 13, 14 and 18 was performed, mostly on day 3, on 69 human embryos which were then allowed to develop further in culture to day 5, from five carriers of Robertsonian translocation (RT) t(13;14). RESULTS: Twelve normal/balanced blastocysts were replaced in seven consecutive cycles (day 5). Three cycles resulted in clinical pregnancies. The proportion of blastocysts displaying a normal/balanced karyotype was 56%, while only the 20% of blocked embryos were normal/balanced (chi(2): P < 0.05). All the embryos analysed on day 5, except one, displayed mosaicism. The percentages of diploid cells for chromosomes 13 and 14 were significantly lower than for chromosome 18 (chromosome 13: 49.0 +/- 28.0; chromosome 14: 53.0 +/- 31.8; chromosome 18: 75.7 +/- 20.4; Mann-Whitney test: P < 0.01). The embryos displaying vertical line 62% of diploid cells for at least two of the three chromosomes analysed, more frequently reached the blastocyst stage (blocked embryos: blastocysts chromosome 13: 43.1 +/- 30.3, 64.9 +/- 29.0; chromosome 18: 64.9 +/- 29.0, 83.0 +/- 12.9; Mann-Whitney test: P < 0.01). CONCLUSIONS: Normal/balanced embryos developed better but the proportion of abnormal blastocysts was still high. Preimplantation genetic diagnosis is recommended to select normal/balanced embryos from RT t(13;14) carriers.