Preimplantation genetic diagnosis of spinal muscular atrophy

After Duchenne muscular dystrophy, spinal muscular atrophy (SMA) is the most common severe neuromuscular disease in childhood. Since 1995, homozygous deletions in exon 7 of the survival motor neuron (SMN) gene have been described in >90-95% of SMA patients. However, the presence of a highly homologous SMN copy gene complicates the detection of exon 7 deletions. This paper describes the adjustment and evaluation of an established SMN exon 7 polymerase chain reaction (PCR) protocol at the single cell level, and the first preimplantation genetic diagnosis (PGD) of SMA with this PCR protocol. To determine PCR efficiency and allelic loss, 200 leukocytes of normal individuals, SMA carriers and patients, and 25 blastomeres were tested. The PCR efficiency of the SMN exon 7 and the adjacent copy gene sequence, tested in the leukocytes, were 90% and 91% respectively. No allelic loss was detected. One out of 25 blastomeres tested revealed a negative PCR signal for the SMN exon 7 sequence. All 25 showed the copy gene sequence. PGD of SMA was offered to a couple with an affected child homozygous for the SMN exon 7 deletion. After intracytoplasmic sperm injection, four and five embryos could be genotyped for the SMN exon 7 in two cycles respectively. After embryo transfer in the second PGD cycle an ongoing gemelli pregnancy was achieved. This study demonstrates that PGD for SMA is feasible when a previous child is homozygous for the SMN exon 7 deletion

Toxicity testing of human assisted reproduction devices using the mouse embryo assay.

Contains fulltext : 81090.pdf (publisher's version ) (Closed access)Systems to assess the toxicity of materials used in human assisted reproduction currently lack efficiency and/or sufficient discriminatory power. The development of 1-cell CBA/B6 F1 hybrid mouse embryos to blastocysts, expressed as blastocyst rate (BR), is used to measure toxicity. The embryos were divided into control and test groups, and were exposed to either control medium or to a potentially toxic test medium. Inferences on toxicity were based on differences in BR between the two groups. The mouse embryo assay followed a stratified (mouse), randomized (embryo), and balanced (equal number of embryos per group and per mouse) design. The number of embryos needed was calculated using power analysis. The basal BR of the hybrid strain was determined in a historical population. Sixty-nine mouse embryos per group were required to detect toxic materials with sufficient sensitivity and to account for the considerable inter-mouse variation in blastocyst development. Fifty-two samples, divided over batches of seven different products were tested before use in the study IVF centre and five of these were found to be toxic. This test system, presented as the Nijmegen mouse embryo assay (NMEA), can be used to detect embryo-toxic materials in daily IVF practice, and this report may provide a starting point for standardization

Deformation of a single mouse oocyte in a constricted microfluidic channel

Single oocyte manipulation in microfluidic channels via precisely controlled flow is critical in microfluidic-based in vitro fertilization. Such systems can potentially minimize the number of transfer steps among containers for rinsing as often performed during conventional in vitro fertilization and can standardize protocols by minimizing manual handling steps. To study shape deformation of oocytes under shear flow and its subsequent impact on their spindle structure is essential for designing microfluidics for in vitro fertilization. Here, we developed a simple yet powerful approach to (i) trap a single oocyte and induce its deformation through a constricted microfluidic channel, (ii) quantify oocyte deformation in real-time using a conventional microscope, and (iii) retrieve the oocyte from the microfluidic device to evaluate changes in their spindle structures. We found that oocytes can be significantly deformed under high flow rates, e.g., 10 μl/min in a constricted channel with a width and height of 50 and 150 μm, respectively. Oocyte spindles can be severely damaged, as shown here by immunocytochemistry staining of the microtubules and chromosomes. The present approach can be useful to investigate underlying mechanisms of oocyte deformation exposed to well-controlled shear stresses in microfluidic channels, which enables a broad range of applications for reproductive medicine