Phospholipase C-ζ-induced Ca2+ oscillations cause coincident cytoplasmic movements in human oocytes that failed to fertilize after intracytoplasmic sperm injection
ABSTRACT To evaluate the imaging of cytoplasmic movements in human oocytes as a potential method to monitor the pattern of Ca(2+) oscillations during activation.
Test of a laboratory technique.
University medical school research laboratory.
Donated unfertilized human oocytes from intracytoplasmic sperm injection (ICSI) cycles.
Microinjection of oocytes with phospholipase C (PLC) zeta (ζ) cRNA and a Ca(2+)-sensitive fluorescent dye.
Simultaneous detection of oocyte cytoplasmic movements using particle image velocimetry (PIV) and of Ca(2+) oscillations using a Ca(2+)-sensitive fluorescent dye.
Microinjection of PLCζ cRNA into human oocytes that had failed to fertilize after ICSI resulted in the appearance of prolonged Ca(2+) oscillations. Each transient Ca(2+) concentration change was accompanied by a small coordinated movement of the cytoplasm that could be detected using PIV analysis.
The occurrence and frequency of cytoplasmic Ca(2+) oscillations, a critical parameter in activating human zygotes, can be monitored by PIV analysis of cytoplasmic movements. This simple method provides a novel, noninvasive approach to determine in real time the occurrence and frequency of Ca(2+) oscillations in human zygotes.
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ABSTRACT: Since the establishment of in vitro fertilization, it became evident that almost half of the couples failed to achieve fertilization and this phenomenon was attributed to a male gamete dysfunction. The adoption of assisted fertilization techniques particularly ICSI has been able to alleviate male factor infertility by granting the consistent ability of a viable spermatozoon to activate an oocyte. Single sperm injection, by pinpointing the beginning of fertilization, has been an invaluable tool in clarifying the different aspects of early fertilization and syngamy. However, even with ICSI some couples fail to fertilize due to ooplasmic dysmaturity in relation to the achieved nuclear maturation marked by the extrusion of the first polar body. More uncommon are cases where the spermatozoa partially or completely lack the specific oocyte activating factor. In this work, we review the most relevant aspects of fertilization and its failure through assisted reproductive technologies. Attempts at diagnosing and treating clinical fertilization failure are described.Cell calcium 11/2013; 55(1). DOI:10.1016/j.ceca.2013.10.006 · 4.21 Impact Factor
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ABSTRACT: Intracytoplasmic sperm injection (ICSI) is a very efficient technique used in couples diagnosed with severe sperm abnormalities. Fortunately, in only a minority of the couples ICSI does not lead to normal fertilization, which means that, in those couples, no (or very few) embryos will be available for transfer. Failure of fertilization following conventional ICSI can be related to the oocyte or the spermatozoon. Only a few diagnostic tests are currently available to assess the reason for ICSI fertilization failure. The most often advised treatment is assisted oocyte activation (AOA), a highly specialized fertilization technique that can be added to conventional ICSI to overcome fertilization failure in those couples. Since the late 1990s, many centres have been performing AOA in this rare group of patients, with restoration of fertilization and pregnancy rates in many couples. However, until adequate scientific evidence is provided regarding its safety and efficacy, AOA cannot yet be considered an established treatment. This review tackles the mechanism of human oocyte activation and the relatively rare phenomenon of fertilization failure after ICSI. Next, we describe the current diagnostic approaches and focus on the application, efficiency and safety of AOA in human assisted reproduction.Reproductive biomedicine online 05/2014; 28(5). DOI:10.1016/j.rbmo.2014.01.008 · 2.98 Impact Factor
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ABSTRACT: studyquestion: Can the approach to, and terminology for, time-lapse monitoring of preimplantation embryo development be uniformly defined in order to improve the utilization and impact of this novel technology? summaryanswer: The adoption of the proposed guidelines for defining annotation practice and universal nomenclature would help unify time-lapse monitoring practice, allow validation of published embryo selection algorithms and facilitate progress in this field. what is known already: An increasing quantity of publications and communications relating to time-lapse imaging of in vitro embryo development have demonstrated the added clinical value of morphokinetic data for embryo selection. Several articles have identified similar embryo selection or de-selection variables but have termed them differently. An evidence-based consensus document exists for static embryo grading and selection but, to date, no such reference document is available for time-lapse methodology or dynamic embryo grading and selection. study design, size and duration: A series of meetings were held between September 2011 and May 2014 involving time-lapse users from seven different European centres. The group reached consensus on commonly identified and novel time-lapse variables. participants/materials, setting, methods: Definitions, calculated variables and additional annotations for the dynamic monitoring of human preimplantation development were all documented. main results and the role of chance: Guidelines are proposed for a standard methodology and terminology for the of use time-lapse monitoring of preimplantation embryo development. limitations, reasons for caution: The time-lapse variables considered by this group may not be exhaustive. This is a relatively new clinical technology and it is likely that new variables will be introduced in time, requiring revised guidelines. A different group of users from those participating in this process may have yielded subtly different terms or definitions for some of the morphokinetic variables discussed. Due to the technical processes involved in time-lapse monitoring, and acquisition of images at varied intervals through limited focal planes, this technology does not currently allow continuous monitoring such that the entire process of preimplantation embryo development may be visualized. wider implications: This is the first time that a group of experienced time-lapse users has systematically evaluated current evidence and theoretical aspects of morphokinetic monitoring to propose guidelines for a standard methodology and terminology of its use and study, and its clinical application in IVF. The adoption of a more uniform approach to the terminology and definitions of morphokinetic variables within this developing field of clinical embryology would allow practitioners to benefit from improved interpretation of data and the sharing of best practice and experience, which could impact positively and more swiftly on patient treatment outcome. † The authors consider that the first two authors should be regarded as joint first authors. & The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: firstname.lastname@example.org Human Reproduction, Vol.0, No.0 pp. 1–11, 2014 doi:10.1093/humrep/deu278 Hum. Reprod. Advance Access published October 24, 2014 Downloaded from http://humrep.oxfordjournals.org/ by Kamal Ahuja on October 27, 2014 study funding/competing interest(s): There was no specific funding for the preparation of these proposed guidelines. Meetings were held opportunistically during scientific conferences and using online communication tools. H.N.C. is a scientific consultant for ESCO,supplier of Miri TL. I.E.A. is a minor shareholder in Unisense Fertilitech, supplier of the EmbryoScope. Full disclosures of all participants are presented herein. The remaining authors have no conflict of interest. Key words: embryo assessment / embryo development / morphology / developmental kinetics / time-lapse monitoringHuman Reproduction 10/2014; DOI:10.1093/humrep/deu278 · 4.59 Impact Factor