The enormous volume of the fertilized egg is attributable to the suppression of cleavage during oocyte growth and the unequal cleavages during the first and second meiotic divisions. The two products of these divisions are the diminutive polar bodies (PB), which contain a redundant set of chromosomes/chromatids plus cytoplasmic organelles. The PB have strictly limited but differential life spans; while viable they possess the genetic potential to support normal embryonic development after transfer to a cytoplast. In addition to the theoretical possibility of using this non-cloning technique to generate more embryos, polar bodies can be used for genetic testing. By cytogenetic analysis of both PB using fluorescent in-situ hybridization (FISH) or chromosome painting, partial or full chromosomal status in the oocyte can be predicted; this approach finds particular application for women of advanced reproductive age as well as with maternally inherited translocations and single gene defects. By studying both of the PB, potential problems of interpretation arising from allele dropout can be reduced; a heterozygous first polar body provides the least ambiguous result. Mitochondria segregate randomly during meiotic cleavages providing an opportunity also to use the PB to screen for mitochondrial mutations and deletions. Thus, the PB can serve useful diagnostic purposes, especially where pre-fertilization screening or avoidance of embryo biopsy is desirable.

organic compounds.In order to explore the source of CAC, samples were taken from outside air, laboratory spaces and procedure rooms, incubators, compressed CO2 tanks and sterile air flow hoods.Certain types of CAC were determined to have an internal source.The aim of this study was to assess whether the emissions of certain volatile organic compounds detected in incubators such as styrene and ethyl-benzene, could be traced to plastic ware used for culture medium preparation or culture processes.Design: Off-gassed samples were captured for a 24 hour period at 37°C from batches of virgin petri dishes and culture flasks in nitrogen purged vessels.Plastic ware was obtained from two manufacturers (A and B) commonly used for human gamete and embryo culture.Materials and Methods: Volatile organic compounds were analyzed by gas chromatography and mass spectroscopy using cryogenic concentration.Results: All of the tested batches released styrene at levels ranging from 350-3700 ng per sample.Ethyl-benzene and benzaldehyde were also present at levels between 24 to 100 ng/sample.The total CAC for the petri dishes was 2283 and 457 ng/sample for manufacturers A and B respectively.A total of 33 volatile organic compounds were detected in plastic ware; of these, 26 were emitted from dishes made by manufacturer A and 8 by manufacturer B (p<0.01).Flasks produced by manufacturer B however, had significantly higher rates of CAC (15413 ng/sample) than any petri dishes tested.High levels of C8-C10 branched hydrocarbons were detected.Conclusions: Sterile plastic ware may off-gas after packaging and the amount is dependent on brand and type of vessel.Levels of styrene, ethyl-benzene and benzaldehyde are proportional to the total loading of petri dishes per incubator, indicating that their source is derived from essential disposables used during gamete and embryo culture.In reviewing these findings and determining counter measures, certain processes used in the manufacturing or design of plastic ware should probably be considered.In addition, embryologists may assay batches using the method described here or air and rinse plastic ware prior to use.However, the removal of potentially offending material, may not be complete.Additionally, filter systems can be placed in incubators aimed at active removal of volatile organic compounds.

OBJECTIVES:Our purpose was to identify and evaluate practical methods within a preimplantation genetic diagnosis program that will increase the percentage of embryos for which a genetic diagnosis can be obtained, including clinical responses after failure of deoxyribonucleic acid amplification has occurred.STUDY DESIGN:Known human lymphoblast cell lines and human embryo blastomeres were evaluated in a single-cell, nested primer polymerase chain reaction system with primer sequences for the specific locus surrounding the four base pair insertion mutation on exon 11 of beta-hexosaminidase A-Tay-Sachs disease, the delta F508 mutation of cystic fibrosis, and the sex-determining region on the Y chromosome. Reamplification polymerase chain reaction with standard polymerase chain reaction and primer extension preamplification was performed in deoxyribonucleic acid preparations after previous polymerase chain reaction amplification attempts had resulted in failure of amplification.RESULTS:The amplification efficiency of Tay-Sachs disease, 51% (97/187), was significantly lower than that for cystic fibrosis, 85% (87/107), and for the sex-determining region on the Y chromosome, 85% (77/90). Tay-Sachs disease polymerase chain reaction amplification occurred in 51% of one-cell lymphoblasts, 89% of two-cell lymphoblasts, and 94% of samples when more than two cells were processed together. When previous amplification failure had occurred, standard Tay-Sachs disease polymerase chain reaction resulted in an amplification efficiency of 16% (three of 19), whereas primer extension preamplification polymerase chain reaction for Tay-Sachs disease resulted in amplification of 52% (31/59) lymphoblasts and 54% (13/24) of polyspermic human blastomeres. Four of six human blastomeres in which amplification failure occurred in a Tay-Sachs disease preimplantation genetic diagnosis cycle amplified by primer extension preamplification polymerase chain reaction, which increased the diagnostic information obtained from four to six of the seven embryos on which biopsy was performed.CONCLUSIONS:We suggest that practical approaches for consideration within a clinical preimplantation genetic diagnosis program to limit the net effect of amplification failure (i.e., reduced embryo transfer number) include increasing the deoxyribonucleic acid content in the polymerase chain reaction tube by using more than one blastomere and by using primer extension preamplification when the initial attempt at amplification fails.

Objective: To determine the ability to apply preimplantation genetic diagnostic techniques to screen for and prevent Tay-Sachs disease (TSD).Design: A couple, both carriers for the 4 base pair (bp) insertion in exon II of the P-hexosaminidase A gene, which results in TSD, underwent IVF, pre-embryo biopsy, polymerase chain reaction (PCR) DNA amplification of the biopsied blastomeres, and pre-embryo transfer. One to two blastomeres were aspirated using a biopsy pipette that was inserted through an opening in the zona formed with acidified phosphate buffer. Polymerase chain reaction was performed on the individual blastomeres for 20 cycles followed by an additional 30 cycles using nested primers. This yielded amplified DNA products of 272 and 276 bp for the normal and mutant gene, respectively. Heteroduplex formation was used for identification of normal, homozygous affected, and heterozygous preembryos.Results: Seven of 13 oocytes fertilized normally and were biopsied at the four- to eight-cell stages. Deoxyribonucleic acid amplification occurred in four of seven pre-embryos (one homozygous affected and three homozygous normal pre-embryos). The three normal pre-embryos that continued to cleave after biopsy were transferred on the evening of day 3 after retrieval. Subsequently, a single gestational sac was observed and the genetic diagnosis was confirmed at amniocentesis.Conclusion: A successful pregnancy and birth were accomplished after preimplantation genetic diagnostic screening for the prevention of TSD.