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Simultaneous analysis of cytoskeletal patterns and chromosome positioning in human fertilization failures
João Ramalho-Santos1, Alexandra Amaral, Raquel Brito
1Center for Neuroscience and Cell Biology, Department of Zoology, University of Coimbra, Coimbra, Portugal. jramalho@ci.uc.pt
Objective:
To sequentially and reliably apply both tubulin immunocytochemistry (ICC) and fluorescence in situ hybridization (FISH) to human fertilization failures, thus providing a tool for a multiple analysis of arrest.
Design:
Analysis of human fertilization failures at several stages of arrest.
Setting:
Academic and clinical institutions.
Patient(S):
Consenting patients undergoing assisted reproduction technologies.
Intervention(S):
Failed fertilizations displaying signs of activation without pronuclear development, or with the absence of polar body emission or cleavage 48 hours after insemination or microinjection were analyzed. Fertilization failures were fixed and processed for ICC. After data was collected the same samples were then subjected to FISH analysis using probes for chromosomes X, Y, and 18.
Main Outcome Measure(S):
Simultaneous ICC and FISH data on the same sample.
Result(S):
Sequential application of straightforward standard ICC and FISH techniques was not possible, as the morphologic features had been altered, microtubular patterns were not preserved, and many samples were rendered opaque. Only chromatin at the cell surface or outside the oocyte/zygote, such as metaphase II spindles or polar body nuclei, could be routinely probed for FISH after ICC. However, an increase in detergent-induced sample permeabilization as well as the removal of several steps usually performed for FISH made it possible to directly compare microtubular patterns and chromosome position, regardless of chromatin status.
Conclusion(S):
Analysis of specific proteins by immunocytochemistry and of chromosome status/positioning by FISH can be carried out sequentially in human fertilization failures, irrespective of the stage of arrest.
