Obtaining metaphase spreads from single blastomeres for PGD of chromosomal rearrangements

Abstract

It has previously been shown that it is possible to obtain metaphase chromosomes from single blastomeres converted into metaphase in the cytoplasm of a mouse zygote. This method is highly labour intensive and cannot be performed outside the preimplantation genetic diagnosis (PGD) laboratory, so to overcome these limitations, a method was developed for obtaining metaphase spreads from single biopsied blastomeres using different chemicals. The substances tested were calyculin A, caffeine, paclitaxel and colcemid in a total of 496 disaggregated and 234 biopsied blastomeres from day 3 embryos. It was demonstrated that the optimal method involved a combined use of 'selective biopsy' (selection of the biopsied blastomere according to morphological criteria) and exposure to caffeine. This resulted in shortening the mean incubation time of biopsied blastomeres, with a metaphase formation rate of 80%. The method is simple for obtaining metaphases from single blastomeres, and may be implemented in clinical practice of PGD for structural rearrangements.

Conversion and non-conversion approach to preimplantation diagnosis for chromosomal rearrangements in 475 cycles

“…Of the 227 cycles involving blastomere nucleus conversion, 133 were performed by the fusion technique and 94 by the chemical approach ( Figure 1). The latter was performed according to the procedure reported earlier (Shkumatov et al, 2007), involving morphological selection of the largest blastomere with 1-2 large nucleoli within the cell nucleus. Upon embryo biopsy, each blastomere was contained in microdrops of Global culture medium (LifeGlobal, USA) supplemented with Plasmanate (Bayer Biological, USA) 10% v/v, containing caffeine (Sigma, USA) (1 mmol/l) and a low dose of colcemid (Sigma) (<0.1 g/ml) under mineral oil.…”

“…To avoid human blastomere fusion with mouse oocytes as well as to simplify the process of conversion from interphase nuclei to metaphase, the chemical conversion method has been recently developed, which is robust and highly reproducible for practical purposes (Shkumatov et al, 2007). The present paper describes the first report of the clinical application and outcome of this chemical conversion method for PGD of translocations as it pertains to the authors overall experience of 475 PGD cycles for chromosomal rearrangements.…”

Role of Preimplantation Genetic Diagnosis (PGD) in Current Infertility Practice

“…More recently, a chemical conversion method has been developed for blastomeres, utilizing caffeine and low dose colcemid or electrical stimulation. [22][23][24][25][26] Although these approaches were originally not always consistently successful, recent experience has been more promising. Kuliev et al 25 report that 75.7% of 1451 blastomeres analyzed by conversion methods yielded diagnostic results, albeit with the addition of FISH methods in 12.7% of the 1451.…”

Origin of Aneuploidy and Strategies Underlying Clinical Application of Preimplantation Genetic Testing for Chromosomal Disorders (PGT-A and PGT-SR)

“…However, to avoid human embryonic cells fusing with mouse oocytes and to simplify the process of conversion from interphase nuclei to metaphase, a chemical conversion method was introduced that appeared to be more robust and highly reproducible, as described in detail in Chap. 3 and reported elsewhere [94].…”