Fluorescent in-situ hybridization (FISH) of decondensed sperm nuclei has been used directly to evaluate the enrichment efficiency of human sperm separation using Sephadex gel filtration and human serum albumin (HSA) gradients. Control and processed spermatozoa were fixed and their nuclei decondensed. In-situ hybridization was carried out with a Y-specific DNA probe (DYZ1). Sephadex filtration yielded 52.5% Y-chromosome-bearing spermatozoa, HSA separation resulted in 49.4% Y-chromosome-bearing spermatozoa and in the untreated control sample the percentage of Y spermatozoa was 49.3%. Statistical analysis revealed no significant differences between the selection methods employed and the controls, and no real enrichment for X- or Y-bearing spermatozoa was detected for any of the selection methods assayed. The usefulness of the protocols reported for selection of spermatozoa by sex chromosome in couples at risk for X-linked diseases is discussed.
Co-culture of human embryos (n = 384 cycles) to the blastocyst stage using Vero cell monolayers was carried out between August 1995 and December 1997. A total of 2868 zygotes were co-cultured and 1027 embryos reached the blastocyst stage (blastocyst formation rate 35.8%). The blastocysts were frozen in 43.7% of patients. A mean of 1.8 blastocysts was transferred per patient and 95 pregnancies were obtained (pregnancy rate/cycle 24.7%). The blastocyst implantation rate was 23.6%. Miscarriage occurred in 15 patients (15.7%) and ectopic pregnancy in three (3.1%) patients. The multiple pregnancy rate was 32.6%. No differences were observed in the blastocyst rate between poor, normal or high response patients. Blastocyst formation was significantly lower when frozen donor spermatozoa were used. Significantly higher pregnancy rates per transfer and blastocyst implantation rates were attained when embryos were transferred on days 5 or 6 compared with day 7. No advantage was observed when co-culture was used in first cycle IVF patients, in comparison with conventional day 2 replacements. The use of blastocysts for preimplantation genetic diagnosis (PGD) increases the diagnostic reliability and widens diagnostic possibilities. A total of 215 cycles with frozen-thawed co-cultured blastocysts were carried out, with a pregnancy rate of 22.7% per replacement.
Implantation failure rates after in-vitro fertilization (IVF) can be reduced in the IVF laboratory using two different techniques: assisted hatching and embryo co-culture on monolayer feeder cells. A review of the different methodologies and the results gained using these techniques is reported. Preliminary results obtained in the Reproductive Medicine Service of the Institut Universitari Dexeus, Barcelona, Spain using these techniques are also presented.
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