Effect of Ca2+/Mg2+-free medium on the biopsy procedure for preimplantation genetic diagnosis and further development of human embryos

In this study, the use of Ca²⁺/Mg²⁺-free medium for biopsy ofhuman embryos at the 4- to 10-cell stage on the third day ofdevelopment was evaluated. When compared with control mediumcontaining normal concentrations of Ca²⁺ and Mg²⁺ ions, theuse of Ca²⁺/Mg²⁺ -free medium allows an easier removal of blastomeresas illustrated by a lower rate of cell lysis as well as by ashorter time needed to perform the procedure. Subsequent embryodevelopment to the blastocyst stage is not affected by the choiceof biopsy medium, not even when embryos are exposed to the mediumfor 45 min. The use of Ca²⁺/Mg²⁺-free medium thus allows foran easier biopsy procedure during preimplantation genetic diagnosis,while it does not result in a loss of developmental potentialof the embryo to the blastocyst stage.     

Does Ca/Mg-free medium have an effect on the survival of the preimplantation mouse embryo after biopsy?

The way in which the different technical aspects of clinicalpreimplantation genetic diagnosis affect the survival of thebiopsied embryos is not well understood. One of these aspectsis the influence of Ca²⁺/Mg²⁺-free medium on the developmentalcapacity of the biopsied embryo and the biopsied cell itself.Therefore, we used an experimental design involving 4-cell mouseembryos to evaluate the effect of performing the biopsy procedureunder such conditions. Our results indicate that the use ofCa²⁺/Mg²⁺-free medium has no detrimental effect (at least inmouse embryos) on the viability of the biopsied embryos andtheir biopsied cells when used during relatively short timesof exposure.     

Comparison of blastocyst transfer with or without preimplantation genetic diagnosis for aneuploidy screening in couples with advanced maternal age: a prospective randomized controlled trial

BACKGROUND: It is generally accepted that the age-related increased aneuploidy rate is correlated with reduced implantation and a higher abortion rate. Therefore, advanced maternal age (AMA) couples are a good target group to assess the possible benefit of preimplantation genetic diagnosis for aneuploidy screening (PGD-AS) on the outcome after assisted reproductive technology (ART). METHODS: A prospective randomized controlled clinical trial (RCT) was carried out comparing the outcome after blastocyst transfer combined with PGD-AS using fluorescence in situ hybridization (FISH) for the chromosomes X, Y, 13, 16, 18, 21 and 22 in AMA couples (aged > or =37 years) with a control group without PGD-AS. From the 400 (200 for PGD-AS and 200 controls) couples that were allocated to the trial, an oocyte pick-up was performed effectively in 289 cycles (148 PGD-AS cycles and 141 control cycles). RESULTS: Positive serum HCG rates per transfer and per cycle were the same for PGD-AS and controls: 35.8% (19.6%) [%/per embryo transfer (per cycle)] and 32.2% (27.7%), respectively (NS). Significantly fewer embryos were transferred in the PGD-AS group than in the control group (P<0.001). The implantation rate (with fetal heart beat) was 17.1% in the PGD-AS group versus 11.5% in the control group (not significant; P=0.09). We observed a normal diploid status in 36.8% of the embryos. CONCLUSIONS: This RCT provides no arguments in favour of PGD-AS for improving clinical outcome per initiated cycle in patients with AMA when there are no restrictions in the number of embryos to be transferred.     

Pregnancies following blastocyst stage transfer in PGD cycles at risk for beta-thalassaemic haemoglobinopathies.

BACKGROUND: Preimplantation genetic diagnosis (PGD) usually involves blastomere biopsy 3 days post-insemination (p.i.), followed by genetic analysis and transfer of unaffected embryos later on day 3 or 4. We evaluate a strategy involving embryo biopsy on day 3 p.i., genetic analysis on day 4 and, following culture in blastocyst sequential media, transfer of unaffected embryos on day 5 p.i. METHODS: PGD cycles were initiated in 15 couples at risk of transmitting beta-thalassaemia major. Oocyte retrieval and ICSI were performed according to standard protocols. Embryo culture used blastocyst sequential media. Embryos were biopsied on day 3 p.i. using acid Tyrode's for zona drilling, and the single blastomeres were genotyped by a protocol involving nested polymerase chain reaction and denaturing gradient gel electrophoresis analysis. RESULTS: Forty of 109 (37%) embryos biopsied on day 3 p.i. developed to blastocysts by day 5 p.i., with at least one blastocyst available for transfer in 12 cycles (80%). Genotype analysis characterized 51/109 (47%) embryos unaffected for beta-thalassaemia major, of which 28 were blastocysts. Transfer of 37 day 5 p.i. embryos (blastocysts and non blastocysts) initiated eight clinical pregnancies. Implantation rate per embryo transferred was 12/37 (32%). CONCLUSIONS: Embryo biopsy on day 3, followed by delayed transfer until day 5 p.i. offers a novel and effective strategy to overcome the time limit encountered when performing PGD, without compromising embryo implantation.     

Mechanisms and targets involved in maternal and paternal age effects on numerical aneuploidy

Trisomy in the human appears to be predominantly associated with maternal age. The maternal-age effect, however, shows considerable variability across affected chromosomes. Chromosome-specific variation has been reported in the shapes of the maternal-age-effect curves, including very small effects for the large chromosomes (groups A and B), linear increases (chromosome 16), and exponential increases (chromosome 21). There is also variation among chromosomes in whether the segregation errors occur predominantly at maternal meiosis I, meiosis II, and/or postfertilization mitotic divisions. There is also limited epidemiological evidence for a paternal-age effect, which was recently supported by the findings of age-related increases in sperm aneuploidy using fluorescence in situ hybridization methods. The paternal-age effect is considerably smaller than the maternal and is more likely to involve meiotic II errors of the sex chromosomes, whereas the maternal-age effect is more likely to arise from meiotic I errors producing autosomal trisomies. These and other differences suggest that constitutional aneuploidy arises by multiple mechanisms that may affect (1) the nature and timing of an initiating lesion affecting the oocyte or sperm; (2) the cellular physiology at the time of the nondisjunction event at meiosis I, II, or postfertilization; and (3) the selection against specific chromosomal aneuploidies during embryonic development. Multidisciplinary research is needed to understand the maternal- and paternal-age effects on aneuploidy, to (1) identify and characterize the genes that control meiosis, recombination, and segregation; (2) identify the microenvironmental factors around the oocyte and male germ cells that are involved in the age effects; (3) develop a laboratory animal model for the age effects; (4) characterize the role of genetics, physiology, and environmental toxicology for the parental-age effects; and (5) identify cohorts of men and women of differing ages who have been exposed to high doses of candidate aneugens and conduct epidemiological investigations of aneuploidies transmitted to their offspring. © 1996 Wiley-Liss, Inc.