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Imaging meiotic spindles by polarization light microscopy: principles and applications to IVF

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Abstract

Meiotic spindles tether the chromosomes of oocytes and have been found to be structurally abnormal in older women. Conventional methods to image the meiotic spindle, such as immunostaining or transmission electron microscopy, require prior fixation, so they cannot be used clinically, and their utility in developmental studies is limited. Spindles can also be imaged non-invasively based on their birefringence, an inherent optical property of highly ordered molecules, such as microtubules, as they are illuminated with polarized light. Polarized light microscopy has been gainfully applied to embryology for decades, but recently a digital, orientation-independent polarized light microscope, the polscope, has demonstrated the exquisite sensitivity needed to image the low levels of birefringence exhibited by mammalian spindles. Its use of nearly circularly polarized light also produces orientation-independent measures of spindle birefringence, thus providing a method to quantify spindle architecture in living oocytes. The safety and utility of polscope imaging has been demonstrated in mammalian oocytes, including those from women undergoing ICSI. Spindle imaging with the polscope provides structural information closely related to the more invasive immunostaining method, and also enables study of the dynamic architecture of spindles. Profound effects of cooling on meiotic spindles have also been shown, and polscope imaging has been used to optimize thermodynamic stability of oocytes during ICSI. It has been shown that embryos derived from oocytes with normal, intact meiotic spindles exhibit superior development after fertilization and in-vitro culture. The mechanisms underlying age-related disruption of meiotic spindles in women remain unclear, but may relate to factors residing within the chromosomes themselves, since mice engineered to shorten their telomeres exhibit structurally abnormal spindles in their oocytes, and their embryos undergo cell cycle arrest and apoptosis, a phenotype remarkably similar to that observed in oocytes and embryos from older women. A time-lapse video of a mouse oocyte imaged by polscope may be purchased for viewing on the internet at www.rbmonline.com/Article/824 (free to web subscribers).

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Dr David Keefe serves as Medical Director of the combined IVF programmes at New England Medical Center in Boston, Massachusetts, and Women and Infants Hospital in Providence, Rhode Island. Dr Keefe also serves on the faculty as an Associate Professor at Tufts and Brown University Medical Schools. He graduated from Georgetown University School of Medicine and completed his residency in Obstetrics and Gynecology and fellowship in Reproductive Endocrinology/Infertility at Yale University School of

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Dr David Keefe serves as Medical Director of the combined IVF programmes at New England Medical Center in Boston, Massachusetts, and Women and Infants Hospital in Providence, Rhode Island. Dr Keefe also serves on the faculty as an Associate Professor at Tufts and Brown University Medical Schools. He graduated from Georgetown University School of Medicine and completed his residency in Obstetrics and Gynecology and fellowship in Reproductive Endocrinology/Infertility at Yale University School of Medicine. He received research training through an NIH-funded grant in the Reproductive Biology Training Programme at Northwestern University and as the Kennedy–Dannreuther Research Fellow at Yale. Previously, he also trained in psychiatry at Harvard and University of Chicago. His research at Brown focuses on reproductive ageing in women and on the molecular and cellular basis of oocyte dysfunction. Before joining the Faculty at Brown, Dr Keefe was at Yale, where he directed the Oocyte Donation Programme and directed a laboratory funded by a Clinical Investigator Award from the NIH.

Paper based on a contribution presented at the Serono Symposium ‘Towards Optimizing ART: a Tribute to Howard and Georgeanna Jones’ in Williamsburg, Virginia, USA, April 2002.

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