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INSTRUCTIONSONTHEANNOTATIONOFPDFFILES To view, print and annotate your article you will need Adobe Reader version 7 (or higher). This program is freely available for a whole series of platforms that include PC, Mac, and UNIX and can be downloaded from http://get.adobe.com/reader/. The exact system requirements are given at the Adobe site: http://www.adobe.com/products/reader/techͲspecs.html. PDFANNOTATIONS AdobeReaderversion7,8or9
AdobeReaderversionX
TomakeannotationsinthePDFfile,gotothemainAdobe TomakeannotationsinthePDFfile,openthePDFfileusing toolbarandchangethecursorfromahandsymboltothe AdobeReaderX,clickon‘Comment’. in IfthisoptionisnotavailableinyourAdobeReadermenus thenitispossiblethatyourAdobeAcrobatversionislower thanXorthePDFhasnotbeenpreparedproperly. When you open the PDF file using Adobe Reader, the Commentingtoolbarshouldbedisplayedautomatically;if not, click on ‘Tools’, select ‘Comment & Markup’ (or ‘Commenting’ in version 7), then click on ‘Show Comment This opens a task pane and, below that, a list of all &Markuptoolbar’(or‘Commentingtoolbar’inversion7, Comments in the text. These comments initially show all or‘ShowCommentingbar’ontheMac).Iftheseoptionsare thechangesmadebyourcopyeditortoyourfile. not available in your Adobe Reader menus then it is possiblethatyourAdobeAcrobatversionislowerthan7or thePDFhasnotbeenpreparedproperly. normalcursorbyclickingonthe‘Select’button themenubaratthetop(versions7and8).
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(Changingthedefaultsetting,Adobeversion9)
HOWTO... Action Inserttext
AdobeReaderversion7,8or9
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onthe Clickthe‘TextEdits’button Commenting tool bar. Click to set the cursor location in the text and simply start typing. The text will appear in a commenting box. You may also cutͲandͲpaste text from another file into the commentingbox.Closetheboxbyclickingon‘x’in thetoprightͲhandcorner.
Clickthe‘InsertText’icon ontheComment toolbar.Clicktosetthecursorlocationinthetext and simply start typing. The text will appear in a commentingbox.YoumayalsocutͲandͲpastetext fromanotherfileintothecommentingbox.Close
Clickthe‘TextEdits’button onthe Commenting tool bar. To highlight the text to be replaced, click and drag the cursor over the text. Then simply type in the replacement text. The replacementtextwillappearinacommentingbox. YoumayalsocutͲandͲpastetextfromanotherfile into this box. To replace formatted text (an equation for example) please Attach a file (see below).
on the Click the ‘Replace (Ins)’ icon Comment tool bar. To highlight the text to be replaced, click and drag the cursor over the text. Then simply type in the replacement text. The replacementtextwillappearinacommentingbox. YoumayalsocutͲandͲpastetextfromanotherfile into this box. To replace formatted text (an equation for example) please Attach a file (see below).
Clickthe‘TextEdits’button onthe Commentingtoolbar.Clickanddragoverthetext to be deleted. Then press the delete button on yourkeyboard.Thetexttobedeletedwillthenbe struckthrough.
on the Click the ‘Strikethrough (Del)’ icon Commenttoolbar.Clickanddragoverthetextto bedeleted.Thenpressthedeletebuttononyour keyboard. The text to be deleted will then be struckthrough.
on the Click on the ‘Highlight’ button Commentingtoolbar.Clickanddragoverthetext. To make a comment, double click on the highlightedtextandsimplystarttyping.
on the Click on the ‘Highlight Text’ icon Commenttoolbar.Clickanddragoverthetext.To make a comment, doubleclick on the highlighted textandsimplystarttyping.
onthe Clickonthe‘AttachaFile’button Commentingtoolbar.Clickonthefigure,tableor formatted text to be replaced. A window will automaticallyopenallowingyoutoattachthefile. To make a comment, go to ‘General’ in the ‘Properties’ window, and then ‘Description’. A graphic will appear in the PDF file indicating the insertionofafile.
on the Click on the ‘Attach File’ icon Comment tool bar. Click on the figure, table or formatted text to be replaced. A window will automaticallyopenallowingyoutoattachthefile. A graphic will appear indicating the insertion of a file.
on Clickonthe‘NoteTool’button theCommentingtoolbar.Clicktosetthelocation of the note on the document and simply start typing.Donotusethisfeaturetomaketextedits.
on the Click on the ‘Add Sticky Note’ icon Commenttoolbar.Clicktosetthelocationofthe noteonthedocumentandsimplystarttyping.Do notusethisfeaturetomaketextedits.
theboxbyclickingon‘_’ corner.
inthetoprightͲhand
Replacetext
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Highlighttext/ makea comment
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HOWTO... Action Review
AdobeReaderversion7,8or9 AdobeReaderversionX To review your changes, click on the ‘Show’ Your changes will appear automatically in a list below the Comment tool bar. Navigate by on the Commenting tool clickingonacorrectioninthelist.Alternatively, button bar.Choose‘ShowCommentsList’.Navigateby double click on any markͲup to open the clickingonacorrectioninthelist.Alternatively, commentingbox. double click on any markͲup to open the commentingbox.
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To undo any changes made, use the right click buttononyourmouse(forPCs,CtrlͲClickforthe Mac). Alternatively click on ‘Edit’ in the main Adobe menu and then ‘Undo’. You can also delete edits using the right click (CtrlͲclick on theMac)andselecting‘Delete’.
To undo any changes made, use the right click buttononyourmouse(forPCs,CtrlͲClickforthe Mac). Alternatively click on ‘Edit’ in the main Adobe menu and then ‘Undo’. You can also delete edits using the right click (CtrlͲclick on theMac)andselecting‘Delete’.
PLEASE DO NOT ATTEMPT TO EDIT THE ARTICLE TEXT ITSELF
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Assessment of 1,2-propanediol (PrOH) genotoxicity on mouse oocytes by comet assay Anais Berthelot-Ricou, M.D.,a Jeanne Perrin, M.D., Ph.D.,a,b Carole di Giorgio, Ph.D.,a Michel de Meo, Ph.D.,a Alain Botta, M.D., Ph.D.,a and Blandine Courbiere, M.D., Ph.D.a,c a Laboratoire de Biogenotoxicologie et Mutagenese Environnementale, Federation de Recherche CNRS 3098 ECCOREV, Facultes de Medecine et Pharmacie, Universite de la Mediterranee, b CECOS–Laboratoire de Biologie de la Reproduction, Pr. Grillo, AP-HM La Conception, and c P^ole de Gynecologie-Obstetrique et de Medecine de la Reproduction, Pr. Gamerre, AP-HM La Conception, Marseille, France
Objective: To assess the genotoxicity of 1,2-propanediol (PrOH) on mouse oocytes by comet assay. Design: In vitro assay using murine model. Setting: Biogenotoxicology research laboratory. Animal(s): CD1 female mice. Intervention(s): Three 40-oocyte groups were exposed to different PrOH concentrations (5%, 7.5%, and 15%). Each concentration was tested during both long and short exposures (1–2 hours and 1–5 minutes) in comparison with control groups. DNA damage was evaluated by a single-cell gel electrophoresis assay, also called ‘‘comet assay,’’ and analyzed with Komet software. Main Outcome Measure(s): DNA damage was quantified as Olive tail moment (OTM). Interpretation was done on OTM with the use of c2. Result(s): High PrOH concentrations (7.5% and 15%) induced significant DNA damage on mouse oocytes. The OTM c2 values were 4.16 0.40 and 6.80 0.4 with 7.5% PrOH at 1 and 2 hours, respectively, 24.35 1.60 with 15% at 1 hour, and too drastic to calculate with 15% at s hours. After 1 and 5 minutes, the OTM c2 values were, respectively, 5.19 0.26 and 6.06 0.42 with 7.5%, and 7.53 0.33 and 16.81 0.67 with 15%. Conclusion(s): High concentrations of PrOH (7.5% and 15%) induced significant DNA damage on mouse oocytes, whatever the exposure duration. These results should be interpreted with caution, because additional data are needed to evaluate PrOH genotoxicity and DNA oocyte reparation after exposure to high PrOH concentrations. (Fertil Steril 2011;-:-–-. 2011 by American Society for Reproductive Medicine.) Key Words: 1,2-Propanediol, PrOH, cryoprotectant, mouse, oocytes, genotoxicity, DNA damage, comet assay
The cryopreservation of spermatozoa or embryo is widely performed in reproductive medicine. However, oocytes are more difficult to cryopreserve, because they are the largest cells in the body and are very sensitive to ice crystals formed during freezing (1). There are two main techniques for cryopreserving cells or tissues: standard ‘‘slow’’ freezing, or vitrification by rapid cooling (2, 3). For slow cooling, an optimal cooling rate specific to a given cell line is used, but this process induces intracellular ice crystals that can be harmful. Vitrification traps all the aqueous solutions in an amorphous, so-called vitreous solid phase, preventing any ice crystal formation (4). Oocyte cryopreservation by vitrification has improved survival cell rates after thawing (5, 6). This improvement is due to rapid cooling, in combination with a high concentration (7.5% and 15%) of cryoprotectants to give an efficient glassforming mixture with water (7). At high concentration, cryoprotecReceived November 16, 2010; revised June 30, 2011; accepted July 19, 2011. A.B.-R. has nothing to disclose. J.P. has nothing to disclose. C.d.G. has nothing to disclose. M.d.M. has nothing to disclose. A.B. has nothing to disclose. B.C. has nothing to disclose. decine, France, and Supported by a grant from the Agence de la Biome veloppement a study grant to A.B.-R. from the Association pour le De dicales au Centre Hospitalier des Recherches Biologiques et Me gional de Marseille and Schering Plough. Re ^ le de Gyne cologie– Reprint requests: Blandine Courbiere, M.D., Ph.D., Po trique et de Me decine de la Reproduction, AP-HM La Conception, Obste 147 Bd. Baille, 13005 Marseille, France (E-mail: blandine.courbiere@ ap-hm.fr).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2011.07.1106
tants, such as dimethylsulfoxide (DMSO) (8), ethyleneglycol (EG) (9), and 1,2-propanediol (PrOH) (10), rapidly penetrate into cells, create hydrogen bonds with H2O molecules, and transform all the aqueous solutions into a vitreous solid phase, without any ice crystal formation (7). Since 1999, vitrification-cryopreservation has led to several hundred live births with reassuring obstetrical and perinatal outcomes (2, 11–14). However, little is known about the possible long-term consequences on human live births after oocyte vitrification, and Edgar and Gook highlighted the lack of well controlled clinical trials (15). In our previous work (16), we studied the genotoxicity of DMSO, EG, and PrOH on mammalian somatic proliferating cell lines (Chinese hamster ovary [CHO]), a validated and commonly used model in genetic toxicology (17). Our results showed that DMSO and EG long exposures were not genotoxic for such mammalian cells. On the other hand, long exposure to PrOH induced in vitro DNA damage on CHO cells (16). These results suggested that PrOH may cause long-term adverse effects in eukaryotic cells. Moreover, some studies assessed the effects of PrOH on mouse oocytes after slow freezing and observed that PrOH caused significantly more zona pellucida hardening and cellular degeneration, and led to proteome alterations, which could result in a higher risk of aneuploidy than vitrification techniques using DMSO and EG (18, 19). Indeed, although genotoxicity in somatic cells can lead to degenerative disorders and cancers at the individual level, genotoxic events occurring in germ cells may lead to chromosome abnormalities in future generations (20, 21). Additional studies of PrOH
Fertility and Sterility Vol. -, No. -, - 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
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genotoxicity therefore seem to be necessary on germinal cells with short exposure to PrOH in vitrification protocols. We previously described a comet assay on nondepellucidated mouse oocytes (22). The comet assay, or single-cell gel electrophoresis assay, is a simple and rapid genotoxicity test to observe and quantify DNA damage and to assess the genotoxic activity of potential carcinogenic agents in a wide range of cells (23). The principle underlying the comet assay is that denatured DNA fragments can be measured migrating out of the cell nucleus during electrophoresis. The image obtained is a ‘‘comet’’ with a distinct head consisting of intact DNA and a tail containing relaxed DNA loops or broken pieces of DNA (24). Although the comet assay has been successfully applied to human sperm in the context of male infertility and assisted reproduction technologies (ART) (25), female germ cells have been poorly investigated, and few techniques exist for genotoxic risk assessment in oocytes. The aim of the present study was to test the genotoxicity of PrOH with comet assay on mouse oocytes. Our first objective was to assess the genotoxicity of PrOH after long exposures, as in classic genotoxicity assays (16). Our second objective was to test PrOH genotoxicity after short exposures in the same experimental conditions as in human oocyte vitrification protocols.
MATERIALS AND METHODS PrOH was from Fisher Scientific, and all other chemicals were from Sigma, unless stated otherwise.
Animals Prepubescent (
