Fertility Magazine • Volume 18
Transcripción
Fertility Magazine • Volume 18
V O LUME 1 8 LifeGlobal® global® Blastocyst Fast Freeze®: Optimal simplicity and effectiveness with successful outcomes by Toni Di Berardino, BSc, MSc Guidelines for the design of an IVF Laboratory by Gloria Calderón, PhD & Nuno Costa Borges, PhD Review: Reproductive Microbiome and its effect on IVF by Aami Mezezi, MS The Use of global® for Time-lapse Videographic Analysis of Human Embryo Development by Don Rieger, PhD The Patient's Corner ESHRE 2015, Lisbon IVFs First & Leading ‘One Solution Medium®’ Over 15 Years of Published Clinical Results Proven Consistency Proven Performance > 150 Independent Published Clinical Studies global® Family – A Unified Approach to Human Embryo Culture global® global® for Fertilization global® w/ HEPES global® total® w/ HSA LG PGD global® total® global® total® for Fert. w/ HSA w/ HEPES w/ HSA Biopsy Medium LiteOil® LifeGuard® Oil Best Decision Less Risk. Less Worries. 4-Well GPS® Less Time. μDrop GPS® Universal GPS® embryo GPS® GP 4-Well GPS Dish ® Key Features and Big Benefits embryo corral® S ® D is e r a hw •4 large wells with GPS bottoms for easy viewing and manipulation •GPS bottom for embryo settling in the center of the wells for quick location of the embryo •Wells are designed to reduce shadows and have concise focal positioning •4 large wells will hold up to 300 microliters of media and oil •4 smaller outer wells hold up to 100 microliter of media and oil •Wide well design allows quick viewing and oil overlay of each well as well as the entire dish •No pyrogenics, clean medical grade •CE and ISO Registered, FDA 510(k) Cleared •1-cell MEA and < 0.03 LAL Tested Applications •Holding oocytes prior to ICSI •Large Volume Culture and Group Culture •Assisted Hatching using laser •Denuding before ICSI •Cryopreservation and Thawing T: 1-800-720-6375 ¿ F: 1-519-826-6947 ¿ Intl.: 001-519-826-5800 ¿ [email protected] ¿ www.LifeGlobalGroup.com Fertility Magazine • Volume 18 • www.FertMag.com – Page 1 Contents Fertility Magazine The First Magazine In FertilityTM The Featured Article LifeGlobal global® Blastocyst Fast Freeze®: Optimal simplicity and effectiveness with successful outcomes. by Toni Di Berardino, BSc, MSc.........................................................................6 Featured On The Cover Review: Reproductive Microbiome and its effect on IVF by Aami Mezezi, MS.................................................................12 The Use of global® for Time-lapse Videographic Analysis of Human Embryo Development by Don Rieger, PhD..................................................................20 Guidelines for the design of an IVF Laboratory by Gloria Calderón, PhD & Nuno Costa Borges, PhD........52 Instructions to Contributors To submit an Article, Abstract or Ad email us at: [email protected] Note: Articles and Abstracts must be accompanied by a photo of the author(s). Page 2 – Fertility Magazine • Volume 18 • www.FertMag.com Toni Di Berardino, BSc, MSc Our New LifeGlobal Group facility and operation in Florida Features 28 Advanced maternal age and assisted reproductive technologies: developmental competence, clinical outcomes and future strategies by Lynne C. O’Shea, PhD 37 High-magnification selection of spermatozoa prior to oocyte injection: confirmed and potential indications by F Boitrelle, B Guthauser, L Alter, M Bailly, M Bergere, R Wainer, F Vialard, M Albert, and J Selva 45 The spatial arrangement of blastomeres at the 4-cell stage and IVF outcome by Goedele Paternot, Sophie Debrock, Diane De Neubourg, Thomas M D’Hooghe, Carl Spiessens 54 Cassandra’s prophecy: why we need to tell the women of the future about age-related fertility decline and ‘delayed’ childbearing by Jane Everywoman 62 New Products 64Conferences ASRM 2014 – Hawaii, October 2014 ESHRE 2014 – Munich, July 2014 Editor in Chief: Monica Mezezi, MBA Editor(s): Don Rieger, PhD Assistant Editor: Michael West Design: Debrah Frank Editorial Office: LifeGlobal Group, LLC 24 Norwich St. E. Guelph, ON, Canada N1H 2G6 T: 1-800-720-6375 F: 1-519-826-6947 Intl.: 001-519-826-5800 [email protected] www.LifeGlobalGroup.com www.IVFonline.com www.FertMag.com Fertility Magazine and all its associates ©2015, All Rights Reserved. Covers, contents, images, ads in print or web form are copryight protected and reprinting or reproduction of any kind is expressly prohibited without the written permission of Fertility Magazine. Fertility Magazine does not knowingly accept false or misleading advertising, articles, opinions or editorial, nor does the publisher assume any responsibility for the consequences that occur should any such material appear, and assumes no responsibility for content, text, opinions or artwork of advertisements appearing in Fertility Magazine in print or web form. Some of the views expressed by contributors may not be the representative views of the publisher. Fertility Magazine • Volume 18 • www.FertMag.com – Page 3 Free Subscription Free Subscription Subscribe today to Fertility Magazine …”The First Magazine In FertilityTM” By joining us today you will receive the latest in: • International News • Scientific Information• Patient Corner’s • New Products All the information needed to keep you up-to-date in Fertility. Join Now …Join Today Call US/Canada: 1-800-720-6375, International: 001-519-826-5800, email: [email protected], or Fax the form below to: 1-519-826-6947. Yes …Sign me up! Receive Fertility Magazine FREE …”The First Magazine In FertilityTM” Name: Address: City: State: Email address: Send it to a Friend at email: Page 4 – Fertility Magazine • Volume 18 • www.FertMag.com Zip Code: Country: The Consistency of global® Medium Don Rieger, PhD May 15, 2013 The following graphs show the results of the quality control measurements of the same 24 sequential lots of global® medium manufactured between May, 2012 and April, 2013 0HDQ &29 &/ 0HDQ &29 &/ 2VPRODULW\P2V0 S+ Third-party measurements of the pH of 24 sequential lots of global®. With 10 mg/ml protein, at 37°C and 5.5% CO2 ,QGLYLGXDO/RWV The osmolarity of 24 sequential lots of global® measured by freezing-point depression 0HDQ &29 &/ $OOORWV(8PO EHORZDVVD\VHQVLWLYLW\ (QGRWR[LQ(8PO ,QGLYLGXDO/RWV ,QGLYLGXDO/RWV Third-party measurements of the endotoxin content of 24 sequential lots of global® ([SDQGHG%ODVWVDWKRI&XOWXUH ,QGLYLGXDO/RWV The results of third-party 1-cell mouse embryo assays of 24 sequential lots of global® Fertility Magazine • Volume 18 • www.FertMag.com – Page 5 THE FEATURED ARTICLE LifeGlobal global® Blastocyst Fast Freeze®: Optimal simplicity and effectiveness with successful outcomes. by Toni Di Berardino, BSc, MSc Director of Clinical & Technical Support LifeGlobal® Group, Guelph, Ontario, Canada You can contact Toni Di Berardino at [email protected] T he cryopreservation of human oocytes, zygotes, cleavage stage embryos and blastocysts is an integral part of every human IVF program. The starting point can be found in the early seventies with the collaboration of three scientists: David Whittingham, Stanley Leibo and Peter Mazur. This collaboration led to the live birth of mice after thawing and transferring mouse embryos previously frozen at –196°C (Whittingham et al., 1972). Their work soon became the model for the cryopreservation of cow embryos (Wilmut et al., 1973), sheep embryos (Willadsen et al., 1976) and rabbit embryos (Whittingham et al., 1976). For human cryopreservation, the breakthrough came ten years later with the first report of human pregnancy following cryopreservation, thawing and transfer of an eight cell embryo (Trounson et al., 1983) and a human blastocyst (Cohen et al., 1985). Embryo and blastocyst cryopreservation has numerous applications in reproductive medicine. Cryopreservation of supernumerary embryos and blastocysts has been highlighted as a method to reduce multiple gestations but also to have subsequent transfers if the first attempt was to fail (Gerris et al., 2003, Kolibianakis et al., 2007). Cryopreservation has become one of the most important techniques in assisted reproductive technology (ART). Approximately 40,000 cryopreserved embryo transfer cycles were performed in 2013 in the US alone (SART 2013). Currently there are published data that show improved pregnancy and implantation rates by transferring blastocysts in non-stimulated cycles (Shapiro et al., 2011, 2014). In cases when the risk of ovarian hyperstimulation syndrome becomes apparent after oocyte retrieval, freezing all the embryos is certainly an option, as demonstrated by Wada et al., (1992). With the increased application of preimplantation genetic screening, immediate cryopreservation of biopsied blastocysts and transfer of euploid embryos is now common in many laboratories (Scott et al., 2013, Roy et al., 2014). Two different methods have been established successfully to cryopreserve oocytes, embryos and blastocysts: slow rate freezing and vitrification. Although Toni Di Berardino, BSc, MSc the basic objective of slow freezing and vitrification by rapid cooling is the same, that is to protect cells from cooling effects, intracellular ice formation, dehydration and the toxic effects at both high and low temperatures, the procedures are somewhat different. During slow rate freezing, embryos and blastocysts are exposed to relatively low concentrations of permeable and non-permeable cryoprotectants, loaded into a straw, sealed, and placed into a controlled rate freezer in order to avoid intracellular ice crystal formation. Although thousand of babies have been born from this technique, results have not always been consistent, especially when handling blastocysts. Vitrification, by rapid cooling, was first applied for cryopreservation of mammalian embryos in 1985 (Rall et al., 1985). Vitrification is a process by which cells can be frozen in such a way that a glass-like or vitrified state is obtained. The strategy of vitrification results in the total elimination of ice crystal formation both within the cells being vitrified, and in the surrounding solution. The success rate of recently developed vitrification methods for the cryopreservation of human oocytes, embryos and blastocysts are encouraging (Liebermann et al., 2006, Kuwayama et al., 2007) and vitrification is now widely applied in many IVF laboratories. Most of the vitrification methods currently available in the market are, in principle, the same. They involve the exposure of oocytes or embryos to high concentrations of cryoprotectants (CPAs) for a brief period of time, followed by loading into small carrier devices, that may or may not be sealed, and then immediate submersion and storage in liquid nitrogen. The majority of the studies support the implementation of a vitrification system by emphasizing its advantages such as its simple, inexpensive and rapid procedure leading to a higher survival pregnancy and implantation rates. However, two major drawbacks of vitrification are: the technical skill of Embryo and blastocyst cryopreservation has numerous applications in reproductive medicine. Page 6 – Fertility Magazine • Volume 18 • www.FertMag.com THE FEATURED ARTICLE the individual performing vitrification and the possible risk of bacterial or viral contamination of the biological sample during cooling when using an open device. Successful vitrification of oocytes and blastocysts critically depends on the proficiency of the staff involved in the vitrification. Therefore a vitrification methodology that is easy to learn and implement is crucial. The risks of (cross) contamination of the biological sample (Bielanski, 2012; Morris, 2005) or presence of toxic compounds during cooling as well as during long-term storage (Yan et al., 2011) remain contentious. Modified strategies have been developed in order to ensure protection during cooling and storage. The use of sterilized LN (Cobo et al., 2011; Parmegiani et al., 2010) and the storage ofthe straws in vapor (Cobo et al., 2010a) are seen asoptions if open carrier devices are used. Another alternative is the use of closed carrier devices (Vanderzwalmen et al., 2009, Whitney et al., 2012 Schiewe et al., 2013), which allows protection of the sample during the cooling procedure and subsequent storage. As stated by Vajta et al., (2006), vitrification is used to solidify a solution containing a sample and a cryoprotectant, without any ice crystal formation, and then to return it to the liquid state, again without ice formation. However, neither high cryoprotectant concentrations nor high cooling rates are absolutely required for vitrification. The key to survival is whether the intracellular contents vitrify. The survival of cryopreserved cells maybe more dependent on the rate at which the specimenis warmed, rather than the rate at which it is cooled (Leibo and Pool, 2011). Furthermore, the fundamentalprinciples of cryobiology suggest that the mechanisms leading to high survival of vitrified cells are similar, ifnot identical to the mechanisms resulting in survival of slow-frozen cells (Leibo 2012). Stachecki and his colleagues (Stachecki et al., 2008, Stachecki and Cohen 2008), described the S3 (‘safe, simple and successful’) system for vitrification of blastocysts. Most notably, the system uses ethylene glycol and glycerol as cryoprotectants, and the blastocysts were loaded into standard 0.25 mL freezing straws, rather than the small volume devices used for DMSO-based vitrification systems. Stachecki et al., (2008) reported that across five participating clinics, overall survival was 88.7%, and overall implantation rate was 42.8%. This demonstrated that blastocysts can be cryopreserved using a simple protocol while achieving excellent blastocyst survival, and good implantation rates. LifeGlobal developed the global® Blastocyst Fast Freeze® and Fast Freeze® Thawing kits, based on the S3 system, using global® w/ HEPES as the base medium. The blastocysts are held in a standard 0.25 mL straw and thus do not require specialized small holding devices. There is considerably more time to perform all the steps with this method, making it less stressful for the embryologist. Another benefit of this method and media is that blastocoel reduction or collapse is not necessary. The global® Blastocyst Fast Freeze® and Fast Freeze® Thawing kits are manufactured in GMP-certified facilities (ISO 9001: 2008 & ISO 13485: 2003) and are FDA 510k registered (K092963). Lopes et al., (2015) cultured surplus cleavage-stage embryos to the blastocyst stage and then compared global® Blastocyst Fast Freeze® with small-device vitrification. There were no differences between global® Blastocyst Fast Freeze® and small-device vitrification for immediate survival following thaw (88 vs. 90%), survival at 24 h post-thaw (76 vs. 72%), or re-expansion at 24 h post-thaw (67 vs. 55%). The proportion of live cells was not different among global® Blastocyst Fast Freeze® (91.6%), small-device vitrification (90.1%), and control (unfrozen) (95.8%) blastocysts. The study demonstrated that global® Blastocyst Fast Freeze® is effective for the cryopreservation of blastocysts, thatethylene glycol and glycerol are efficient cryoprotectants and that blastocyst vitrification can be easily achieved using a large volume device which is sealed, as demonstrated earlier by Stachecki et al., (2008). Reed et al., (2015) demonstrated that, in clinical practice, the transition from a slow freezing protocol to global® Blastocyst Fast Freeze® and Fast Freeze® Thawing Kits for the cryopreservation of blastocysts was easy, and less technically demanding than small-device vitrification. Blastocyst survival rate was significantly greater (P<0.05) with global® Blastocyst Fast Freeze® (96.9%) than for both small-device vitrification (84.4%) and slow-rate freezing (89.7%). The implantation rate for biopsied (euploid) blastocysts was significantly greater for global® Blastocyst Fast Freeze® (47.6%) than for small-device vitrification (24.0%). LifeGlobal has conducted many national and international workshops to introduce the principle and practical aspects of global® Blastocyst Fast Freeze® system. The response of the attendees has always been positive and it generally agreed that the blastocyst vitrification could be effectively achieved using large volume device, using longer loading and exposure times and without the collapse of the blastocoel. In conclusion, the global® Blastocyst Fast Freeze® system is a simple and effective approach to the cryopreservation of human blastocyst. Blastocyst survival and implantations rates have been shown to be as good as those for smalldevice vitrification. The use of standard freezing straws with global® Blastocyst Fast Freeze® is technically much simpler, very much less costly, and, because of the larger thermal mass, less susceptible to inadvertent warming. References Bielanski, A. A review of the risk of contamination of semen and embryos during cryopreservation and measures to limit cross-contamination during banking to prevent disease transmission in ET practices. Theriogenology. 2012; 77: 467– 482 Cobo, A., Romero, J.L., Pérez, S., de los Santos, M.J., Meseguer, M., and Remohi, J. Storage of human oocytes in the vapor phase of nitrogen. Fertil. Steril. 2010; 94: 1903–1907 Fertility Magazine • Volume 18 • www.FertMag.com – Page 7 THE FEATURED ARTICLE Cobo, A., Castello, D., Weiss, B., Vivier, C., DeLa Macorra, A., Kramp, F., 2011. Highest liquid nitrogen quality for vitrification process: Micro bacteriological filtration of LN2. In: 16th World Congress on In Vitro Fertilization, Tokyo, P-052, pp. 289 Cohen, J., Simons, R.F., Fehilly, C.B., Fishel, S.B., Edwards, R.G., Hewitt, J., Rowlant, G.F., Steptoe, P.C., Webster, J.M. Birth after replacement of hatching blastocyst cryopreserved at expanded blastocyst stage.Lancet. 1985 Mar 16; 1(8429):647 Gerris, J., De Neubourg, D., De Sutter, P., Van Royen, E., Mangelschots, K., Vercruyssen, M. Cryopreservation as a tool to reduce multiple birth. Reprod Biomed Online. 2003 Oct;7(3):286-94. Review Kolibianakis, E.M., Venetis, C.A., Tarlatzis, and B.C. Cryopreservation of human embryos by vitrification or slow freezing: which one is better? Curr Opin Obstet Gynecol. 2009; 21: 270–4. Kuwayama, M. Highly efficient vitrification for cryopreservation of human oocytes and embryos: the cryotop method. Theriogenology. 2007; 67: 73–80 Leibo, S.P. and Pool, T.B. The principal variables of cryopreservation: solutions, temperatures, and rate changes. Fertil. Steril. 2011; 96: 269–276 Leibo, S.P. The Alpha consensus meeting on cryopreservation key performance indicators and benchmarks: proceedings of an expert meeting. Reprod. Biomed. Online. 2012; 25: 146-167 Liebermann, J. and Tucker, M.J. Comparison of vitrification and conventional cryopreservation of day 5 and day 6 blastocysts during clinical application. Fertil. Steril. 2006; 86: 20–26 Lopes, A.S., Frederickx, V., Vankerkhoven, G., Campo, R., Puttermans P, Gordts, S. Survival, re-expansion and cell survival of human blastocysts following vitrification and warming using two vitrification systems. J. Assist. Reprod. Genet. 2015; 32: 83-90 Morris, G. The origin, ultrastructure, and microbiology of the sediment accumulating in liquid nitrogen storage vessels. Cryobiology. 2005; 50: 231–238 Parmegiani, L., Cognigni, G.E., Bernardi, S., Cuomo, S., Ciampaglia, W., Infante, F.E., Tabarelli de Fatis, C., Arnone, A., Maccarini, A.M., and Filicori, M. Efficiency of aseptic open vitrification and hermetical cryostorage of human oocytes. Reprod. Biomed. Online. 2011; 23: 505–512 Rall, W.F. and Fahy, G.M. Ice-free cryopreservation of mouse embryos at −196 °C by vitrification. Nature. 1985; 313: 573–575 Reed, M.L., Said, A., Thompson, D.J., Caperton, C.L. Largevolume vitrification of human biopsied and non-biopsied blastocysts: a simple, robust technique for cryopreservation. J. Assist. Reprod. Genet. 2015; 32: 207-214 Roy, T.K., Bradley, C.K., Bowman, M.C., McCarthur, S.J., Singleembryo transfer of vitrified-warmed blastocysts yields equivalent live-birth rates and improved neonatal outcomes compared with fresh transfers. Fertil. Steril. 2014; 101 No 5: 1294-1301 Schiewe MC, Nugent N, Zozula S, Whitney J and Anderson RE (2013) Prospective incorporation of vitrified embryo transfer (VFET) cycles into standard patient care: time to re-educate physicians & patients. Fertil Steril100, S282 (Abstract P-461) Page 8 – Fertility Magazine • Volume 18 • www.FertMag.com Scott, R.T., Upham, K.M., Forman, E.J., Hong, K.H., Scott, K.L., Taylor, D., Tao, X., Treff, N.R. Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial. Fertil. Steril. 2013; 100 No 3: 697-703 Shapiro, B.S., Daneshmand, S.T., Garner, F.C., Aguirre, M, Hudson, C., Thomas, S. Evidence of impaired a endometrial receptivity after ovarian stimulation for in vitro fertilization: a prospective randomized trial comparing fresh and frozenthawed embryo transfer in normal responders. Fertil. Steril. 2011; 96: 344-348 Shapiro, B.S., Daneshmand, S.T., Garner, F.C., Aguirre, M, Hudson, C. Freeze all can be a superior therapy to another fresh cycle in patients with prior fresh blastocyst implantation failure. Reprod Biomed Online. 2014; 29: 286-290 Stachecki, J.J., Garrisi, J., Sabino, S., Caetano, J.P.J., Wiemer, K.E., Cohen, J. A new safe, simple and successful vitrification method for bovine and human blastocysts. Reprod Biomed Online. 2008; 17: 360–7 Stachecki JJ, Cohen J. S3 vitrification system: a novel approach to blastocyst freezing. J Clin Embryol. 2008; 11: 5–14 Trounson, A. and Mohr, L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature. 1983; 305: 707–709 Vajta, G. and Nagy, Z.P. Are programmable freezers still needed in the embryo laboratory? Review on vitrification. Reprod. Biomed. Online. 2006; 12: 779–796 Vanderzwalmen, P., Ectors, F., Grobet, L., Prapas, Y., Panagiotidis, Y., Vanderzwalmen, S., Stecher, A., Frias, P., Liebermann, J., and Zech, N.H. Aseptic vitrification of blastocysts from infertile patients, egg donors and after IVM. Reprod. Biomed. Online. 2009; 19: 700–707 Wada I, Matson PL, Troup SA, Hughes S, Buck P, Lieberman BA. Outcome of treatment subsequent to the elective cryopreservation of all embryos from women at risk of the ovarian hyperstimulation syndrome. Hum Reprod. 1992 Aug; 7(7):962-6 Whitney J, Nugent N, Duggan K, S. Zozula, Anderson RE and Schiewe MC (2012) Controlled ovarian hyperstimulation (COH) cycles may benefit from vitrification of all Day 5 PGSbiopsied blastocysts.Fertil Steril98 Suppl., S185 (Abstract P-249) Whittingham, D.G., Leibo, S.P., Mazur, P. Survival of mouse embryos frozen at -196 degrees and -269 degrees C. Science. 1972; 178(4059):411-414 Whittingham, D.G., Adam, C.E. Low temperature preservation of rabbit embryos. J.Reprod.Fertil. 1976; 47(2): 269-274 Willadsen, S.M., Polge, C., Rowson, L.E., Moor, R.M. Deep freezing of sheep embryos. J.Reprod.Fertil. 1976; 46(1): 151-154 Wilmut I and Rowson LE. Experiments on the low temperature preservation of cow embryos. Veterinary Record. 1973; 92(26): 686-90 Yan, J., Suzuki, J., Yu, X.M., Qiao, J., Kan, F., and Chian, R.C. Effects of duration of cryo-storage of mouse oocytes on cryosurvival, fertilization and embryonic development following vitrification. J. Assist. Reprod. Genet. 2011; 28: 643–64 μDrop GPS® Dishware Embryo Culture Dish Safe, Effective and Easy to Use New Design Features • Precise 20 µl micro-wells with GPS® feature for rapid location and visualization • Enhanced optics • Better orientation and identification •EmbryoAddressTM helps to quickly identify and track embryos • 32 mm inner ‘oil ring’ for VOC protection • Uses up to 75% less oil than a conventional 60 mm dish • Improved safety (no droplet collapsing or mixing) New Breathable Packaging for all GPS® Dishware • Fill the outer wells with oil for better temperature stability • Reduces off-gassing time out of the incubator • Less VOCs introduced into the laboratory • Works well with both oil-overlay and oil-underlay methods • Tested and validated to maintain sterility for the • Designed to ensure safe embryo culturing entire 5-year shelf life of the dish • For use with both standard and mini incubators 4-Well GPS® Universal GPS® embryo GPS® embryo corral® The best dish for PGD cases. Standardize ART and other sensitive culture by using GPS® Dishware embryo GPS® Dish Traditional Petri Dish Úrunning droplets Ú droplets mixing Ú droplets flattening Ú poor temperature control Ú losing track of marked droplet Ú difficulty locating embryos Ú sealing of dish lid & increased pH ü ü ü ü ü ü ü ü ü ü ‘drop-less’ environment micro wells designed to enhance embryo culture no droplets collapsing and mixing save time and money by reducing set-up time, observation time, testing and handling better heat conservation enhanced safety easily locate and observe embryos new lid design for better gas exchange standardization of culture, uniform volumes 1-cell MEA and LAL testing by independent company Fertility Magazine • Volume 18 • www.FertMag.com – Page 9 global ® Based on Pure Science The leading scientifically based and clinically proven ‘Uninterrupted Single Solution Culture Medium®’. ‘Uninterrupted Time-Lapse Culture Medium®’ v The First leading scientifically based ‘Single Solution Medium®’. v Proven to work with any type of VOC controlled environment as a continuous culture. v Over 150 Independent Publications using global® medium. v Over 15 years of Consistent Superior Results worldwide. Let the Embryos Choose! ® Page 10 – Fertility Magazine • Volume 18 • www.FertMag.com “We are extremely happy with Global medium combined with the Embryoscope. Nice blastocyst formation and our pregnancy rates have been over 70% with Day 5 transfer. We use it as a continuous culture medium from Day 1 onwards with no media exchange.” “We transitioned from using it from D1 thru 6 with a half change on Day 3 to using it for “uninterrupted culture” from Day 1-Day 6. Equivalent results both ways.” Nina Desai, PhD, HCLD, Cleveland Clinic (global® user since 2001. No commercial ties with the company.) 1. Uninterrupted culture of human embryos in global® or global® total® • • • • • • • Campbell A, Fishel S, Bowman N, Duffy S, Sedler M and Hickman CFL (2013) Modelling a risk classification of aneuploidy in human embryos using non-invasive morphokinetics. Reprod Biomed Online 26, 477-85. Costa-Borges N, Bellés M, Herreros J, Teruel J, Ballesteros A, Pellicer A and Calderón G (2013) Single medium culture in a time-lapse incubator until the blastocyst stage with or without medium renewal on Day-3: a prospective randomised study with donor oocytes. Human Reprod. 28 Suppl. 1, i184 (Abstract P-167). Semeniuk L, Mazur P, Mikitenko D, Nagorny V and Zukin V (2013) Time-lapse and aCGH, is there any connection between ploidy and embryo cleavage timing on early stages of embryo development? Fertil Steril 99 Supplement, S6 (Abstract O-5). Cruz M, Garrido N, Herrero J, Perez-Cano I, Munoz M and Meseguer M (2012) Timing of cell division in human cleavage-stage embryos is linked with blastocyst formation and quality. Reprod Biomed Online 25, 371-381. Silva MM, Llanos BA, David Gumbao, Marcos J, Sanchez A, Nicolas M, Olmedilla LF and Gutierrez JL (2012) Optimization of clinical outcomes in an oocyte donation programme. Reprod Biomed Online 24 Suppl 1., S7 (Abstract PP-6). Bellver J, Mifsud A, Grau N, Privitera L and Meseguer M (2013) Similar morphokinetic patterns in embryos derived from obese and normoweight infertile women: a time-lapse study. Hum Reprod 28, 794-800. Munoz M, Cruz M, Humaidan P, Garrido N, Perez-Cano I and Meseguer M (2013) The type of GnRH analogue used during controlled ovarian stimulation influences early embryo developmental kinetics: a time-lapse study. Eur J Obstet Gynecol Reprod Biol 168, 167-72. 2. Time-lapse culture of human embryos in global® or global® total® • • • • • • • • • • • • • Basile N, Morbeck D, Garcia-Velasco J, Bronet F and Meseguer M (2013) Type of culture media does not affect embryo kinetics: a time-lapse analysis of sibling oocytes. Hum Reprod 28, 634-641. Bellver J, Mifsud A, Grau N, Privitera L and Meseguer M (2013) Similar morphokinetic patterns in embryos derived from obese and normoweight infertile women: a time-lapse study. Hum Reprod 28, 794-800. Campbell A, Fishel S, Bowman N, Duffy S, Sedler M and Hickman CFL (2013) Modelling a risk classification of aneuploidy in human embryos using non-invasive morphokinetics. Reprod Biomed Online 26, 477-85. Campbell A, Fishel S, Bowman N, Duffy S, Sedler M and Thornton S (2013) Retrospective analysis of outcomes after IVF using an aneuploidy risk model derived from time-lapse imaging without PGS. Reprod Biomed Online 27, 140-6. Costa-Borges N, Bellés M, Herreros J, Teruel J, Ballesteros A, Pellicer A and Calderón G (2013) Single medium culture in a time-lapse incubator until the blastocyst stage with or without medium renewal on Day-3: a prospective randomised study with donor oocytes. Human Reprod. 28 Suppl. 1, i185 (Abstract P-167). Cruz M, Garrido N, Herrero J, Perez-Cano I, Munoz M and Meseguer M (2012) Timing of cell division in human cleavage-stage embryos is linked with blastocyst formation and quality. Reprod Biomed Online 25, 371-381. Desai NN, Ploskonka S, Goldberg J, Austin C and Falcone T (2013) Morphokinetic analysis of embryos from patients having a day 5 transfer: preliminary results with the embryoscope. Fertil Steril 100, S120 (Abstract O-392). Martinez-Burgos M, Losada C, Pareja S, Agudo D and Bronet F (2013) Effects of low O2 concentration in extended embryo culture using benchtop incubators (Embryoscope and MINC). Fertil Steril 100, S251 (Abstract P-360). Munoz M, Cruz M, Humaidan P, Garrido N, Perez-Cano I and Meseguer M (2013) The type of GnRH analogue used during controlled ovarian stimulation influences early embryo developmental kinetics: a time-lapse study. Eur J Obstet Gynecol Reprod Biol 168, 167-72. Nakahara T, Iwase A, Goto M, Harata T, Suzuki M, Ienaga M, Kobayashi H, Takikawa S, Manabe S, Kikkawa F and Ando H (2010) Evaluation of the safety of time-lapse observations for human embryos. J Assist Reprod Genet 27, 93-6. Ramirez JM, Fernandez FG, Bueno AS, Brandt M, Fernandez JAG and Lopez EG (2012) Importance of multinucleation at 2-cell stage: study in a time-lapse incubator. Fertil Steril 98 Suppl., S-169 (Abstract P-196). Semeniuk L, Mazur P, Mikitenko D, Nagorny V and Zukin V (2013) Time-lapse and aCGH, is there any connection between ploidy and embryo cleavage timing on early stages of embryo development? Fertil Steril 99 Suppl, S6 (Abstract O-5). Silva MM, Llanos BA, David Gumbao, Marcos J, Sanchez A, Nicolas M, Olmedilla LF and Gutierrez JL (2012) Optimization of clinical outcomes in an oocyte donation programme. Reprod Biomed Online 24 Suppl 1., S7 (Abstract PP-6). Fertility Magazine • Volume 18 • www.FertMag.com – Page 11 ARTICLES Review: Reproductive Microbiome and its effect on IVF. by Aami Mezezi, MSc Post Graduate Student at Georgetown University You can contact Aami Mezezi at [email protected] M ore than 5 million babies have been born from IVF since the technique was first introduced in 1978 (www.eshre.eu). Many factors have been evaluated in an attempt to increase the IVF success rate; but little attention has been paid to the microbiome of the reproductive tract and its role in fertility. This relationship demands increased focus especially considering that it has been reported that up to 40% of patients undergoing IVF have abnormal flora somewhere along the reproductive tract (Leitech et al., 2007). Hyman et al. (2012) examined the correlation between the vaginal microbiome and IVF success rates and found that the microbiome on the day of embryo transfer was a factor in pregnancy outcome. In particular, this study focused on the transition of the microbiome through the course of an IVF treatment. Thirty one (31) women with no signs of vaginal infection were enrolled in the study. Vaginal swabs, followed by 16rRNA pyrosequencing of microbial cultures, were performed at various times during IVF treatment, including on the day of embryo transfer. Interestingly, a transition in the microbial flora occurred in most of the women during the course of IVF treatments. The study concluded that a vaginal microbiome, consisting predominantly of the lactobacilli species, L. crispatus, L. gasseri, and L. jensenii at the time of embryo transfer, had a statistically significant relationship to the live birth rate. Pyrosequencing of the V1 and V3 regions of the 16s rRNA on Lactobacilli species in vaginas with normal flora, has shown that L. crispatus, L. gasseri, and L. jensenii are the three predominant lactobacilli species (Jakobbson and Forsum, 2008). The study also concluded that all of the women who did conceive had a substantial decrease in their E2 levels in-between the time of hCG administration and embryo transfer. This particular observation has been previously observed and is now considered a necessary event for successful IVF treatments (Kim et al., 2010). How is this related to the microbiome? Due to the small sample size, the conclusions of this study are preliminary and must be further investigated through future research. However, the observations were consistent with previous findings and the same time raised a number of key questions. Of particular interest is the fact that a transition of the vaginal microbiome was observed along the course of an IVF treatment and that the transitions were seemingly related to hormonal changes, particularly with respect to variations in estradiol. Fluctuations in estradiol levels are seen in women Page 12 – Fertility Magazine • Volume 18 • www.FertMag.com Aami Mezezi, MSc undergoing IVF. In fact, women undergoing IVF may have estradiol (E2) levels eight times higher than normal (Kushnir et al., 2009). In a study performed on the vaginal microbiome in rats it was shown that following an ovariectomy the subsequent decrease in E2 led to a drastic decrease in the number of Lactobacilli species in the lower genital tract. However, a normal colony of Lactobacilli species was reestablished following exogenous replacement of E2 thus establishing the link between estrogen status and Lactobacilli colonization in the vaginal microbiome (Bezirtzoglou et al., 2004). Jakobsson and Forsum (2008) were the first to note changes in the human vaginal microbiome related to changes in E2 levels. Their study showed that women who had vagina flora dominated by the three normal Lactobacilli species underwent very little transition through the course of IVF treatment; while women whose flora was dominated by L. delbrueckii, L. rhamnosus or L. vaginalis transitioned to a normal lactobacilli flora as their E2 levels rose. These studies all point to E2 levels as having a positive effect on establishing and maintaining a normal and healthy vaginal microbiome dominated by L. crispatus, L. gasseri and/or L. jensenii. The most likely explanation for this observation is the increase of vaginal glycogen content in direct response to an increase in E2 (Mirmonsef et al., 2014). Glycogen is a preferred energy source by Lactobacilli. Therefore, there are known fluctuations in the numbers of lactobacilli and in turn the pH of the vagina as estrogen levels fluctuate naturally over the reproductive life cycle of a woman or more artificially during the course of an IVF treatment. Another possible consideration that must be addressed is the potential association between progesterone resistance and the vaginal microbiome. Progesterone is critical for endometrial function, blastocyst implantation and prevention of preterm birth and administration of progesterone is commonly provided to infertile patients to increase pregnancy rates during IVF (Usadi et al., 2008). It is also the only pharmacological prevention for preterm birth (Stout et al., 2014). However, some patients display progesterone resistance and it has been hypothesized that the vaginal microbiome could determine the efficacy of progesterone treatment. A recent study involving 10 patients ARTICLES at risk of pre-term birth and on progesterone therapy suggested that the vaginal microbiome may help determine the efficacy of progesterone treatment (Stout et al., 2014). A more elaborate study is needed to further establish the microbiome as a causative factor in progesterone resistance and to further determine the possible mechanisms at play. However, a possible mechanism linking the status of the vaginal microbiome to progesterone resistance may reside in the role of the vaginal microbiome in the inflammatory response. It has been demonstrated that cytokine levels change with respect to the vaginal microbiome (Hodges et al., 2005), and inflammation has been hypothesized as a cause for progesterone resistance (Sirota et al., 2014). Eckert et al. (2003) showed that IVF patients with a decreased population of normal Lactobacilli species in their vaginal flora had increased vaginal inflammation, decreased conception rates, and increased early pregnancy loss. A more comprehensive study is needed to better understand the mechanisms involved in a possible relationship between the vaginal microbiome, inflammation and progesterone resistance. Prophylactic antibiotics and glucocorticoids are commonly administered during IVF procedures in order to prevent infections and control inflammation. The role of these pharmacological interventions on the vaginal microbiome and IVF success rates is poorly understood. Although it has been shown that commonly used antibiotics such metronidazole reduce virulent bacteria, their effects on lactobacilli species in the normal vaginal flora can be detrimental (Egbase et al., 1996). Importantly, the Lactobacilli species L. iners, once believed to be part of the normal vaginal flora, is now thought be an indicator of a transition from a normal to an abnormal flora (Vasquez et al., 2002)., Jokobsson and Forsum (2008) found an increased presence of L. iners during times of artificially high estrogen levels during IVF treatments, and following administration of metronidazole (Jokobsson and Forsum, 2012). This is a critically important finding that must be further investigated as it could a) help explain why a decrease in estrogen levels between the time of hCG administration and embryo transfer has been shown to be a significant factor for conception following IVF and b) lead us to re-visit which antibiotics are being prescribed during IVF treatments due to their possibly negative effect on normal flora. It has also been demonstrated that the microbial flora of the uterine cervical canal could also be a determinant in IVF success rates. In a study of 204 ART patients, Salim et al., (2002) found that either a sterile cervical culture or Lactobacilli only positive culture had pregnancy rate of 30.7% compared to a pregnancy rate of 16.3% in women who had a cervical culture containing pathogenic microorganisms. Research findings have highlighted the importance of a normal vaginal and cervical flora on IVF success rates, particularly at the time of embryo transfer. These observations may suggest the clinical use of probiotics as a treatment to improve IVF success rates. At present, the majority of IVF clinics do not have procedures in place to screen for vagina flora prior to IVF. However, Gliboa (2005) concluded that the probiotic treatment they administered had no effect on either vaginal colonization or pregnancy rates. This particular study included 107 women into either a treatment group that received 2 probiotic supplements or a control group that received no supplementation. Intravaginal probiotic supplementation with 3 billion live L. acidophilus, Bifidobacterium bifidum and Bifidobacterium longum bacteria was administered immediately following oocyte retrieval. Day 3 embryo transfers were then performed, allowing only 3 days for probiotic supplementation to have an effect on colonization. Vaginal swabs and gram staining was performed to determine the microflora composition before and after probiotic treatment. Although Gliboa (2005) did not show any positive effect of intravaginal probiotic therapy, the sample size of the study was small and the number and types of probiotic species administered could be altered to produce a more beneficial effect. For example, it was found that colonizing the transfer tip catheter with L. crispatus during embryo transfer in healthy women of reproductive age led to increased implantation and live birth rates (Sirota et al., 2014). Therefore, the use of probiotics as a clinical therapy during IVF has promise and must be further evaluated. The microbiome of the female reproductive tract should become an important consideration in IVF treatments. Recent preliminary research findings have illustrated the importance of a normal vaginal and cervical flora composed primarily of lactic acid producing Lactobacilli species L. crispatus, L. gasseri, and L. jensenii, in successful IVF outcomes. The presence of a normal flora has been shown to have a significant and positive effect on implantation and , live birth rates, reduced inflammation and prevention of preterm birth. The composition of the reproductive tract microbiome is hormone dependent with increased E2 and P4 levels showing a positive effect on establishing and maintaining a normal Lactobacilli flora. However, artificially high E2 levels following hCG administration and before embryo transfer have a seemingly negative effect on the microbial flora and IVF success rate. Therefore, there appears to be time dependence to the hormonal effect on Research findings have highlighted the importance of a normal vaginal and cervical flora on IVF success rates, particularly at the time of embryo transfer. Fertility Magazine • Volume 18 • www.FertMag.com – Page 13 ARTICLES microbial status which could be due to other hormones and interacting factors in play at this particular physiological time-point. It also appears as if the microbiome itself could influence hormone status. Recent findings have given credence to the hypothesis that abnormal vaginal flora could be a causative factor in progesterone resistance. The possibility that the vaginal microbiome could help determine progesterone therapy efficacy could be of great interest to the field of IVF and is an avenue that should be further researched. Although significant correlations appear to exist between; the reproductive tract microbiome, hormone status and IVF success rate, the mechanisms underlying these relationships have yet to be determined., one possible consideration to further investigate is the role of the microbiome on the local and systemic inflammatory response. Cytokine levels have been proven to change with respect to transitions in the vaginal microflora and abnormal flora in the reproductive tract has been shown to lead to an increase in proinflammatory cytokines. The use of prophylactic antibiotics during IVF could also have a potentially detrimental effect on the female vaginal microbiome.. Antibiotics could also help select for and increase the amount of resistant pathogenic microbes. Screening of the vaginal microbiome of women undergoing IVF should become a common clinical practice, particularly on or near the day of embryo transfer. Research should focus on the possible solutions to mitigate the various factors effecting microbiome flora, implantation and delivery rates during an IVF cycle. References Bezirtzoglou E, Voidarou Ch, Papadaki A, Tsiotsias A, Kotsovolou O, et al. Hormone therapy alters the composition of the vaginal flora in ovariectomized rats. Microb Ecol. 2008; 55:751–9. Eckert LO, Moore DE, Patton DL, Agnew KJ, Eschenbach DA. Relationship of vaginal bacteria and inflammation with conception and early pregnancy loss following in-vitro fertilization. Infect Dis Obstet Gynecol. 2003; 11(1):11–17. [PubMed: 12839628]. Egbase PE, al-Sharhan M, al-Othman S, al-Mutawa M, Udo EE, Grudzinskas JG. Incidence of microbial growth from the tip of the embryo transfer catheter after embryo transfer in relation to clinical pregnancy rate following in-vitro fertilization and em- bryo transfer. Hum Reprod 1996;11(8):1687–1689. Hodges S, Barrientes F, Desmond R, Schwebke J: Local and Systemic Styokine Levels in Relation to Changes in Vaginal Flora. Journal of Infectious Disease 2006: 193; 556-562. Hyman RW, Herndon CN, Jiang H, et al. The dynamics of the vaginal microbiome during infertility therapy with in vitro fertilization- embryo transfer. J Assist Reprod Genet 2012;29(2):105–115. Jakobsson T, Forsum U. Changes in the predominant human Lactobacillus flora during in vitro fertilisation. Ann Clin Microbiol Antimicrob. 2008;7:14–21. Kim YJ, Ku SY, Jee BC, et al. Dynamics of early estradiol production may be associated with outcomes of in vitro fertilization. Fertil Steril 2010;94(7):2868–2870. Kushnir, M. M., Naessen, T., Kirilovas, D., Chaika, A., Nosenko, J., Mogilevkina, I., Rockwood, A. L., Carlstrom, K. and Bergquist, J. (2009) Steroid profiles in ovarian follicular fluid from regularlymenstruating women and women after ovarian stimulation. Clin Chem, 55, 519-526. Leitich H, Kiss H. Asymptomatic bacterial vaginosis and intermediate flora as risk factors for adverse pregnancy outcome. Best Pract Res Clin Obstet Gynaecol 2007;21(3):375–390. Mirmonsef P, Hotton AL, Gilbert D, Burgad D, Landay A, et al. (2014) Free Glycogen in Vaginal Fluids Is Associated with Lactobacillus Colonization and Low Vaginal pH. PLoS ONE 9(7). Stout M, LaRosa P, Shannon W, Weinstock G, Sodergen E, Macones G, Tuuli M: Role of the vaginal microbiome in the efficacy of progesterone for prevention of preterm birth. American Jounral of Obstetrics & Gynecology, 2014. Sirota I, Zarek S, Segars J: Potential Influence of the Microbiome on Infertility and Assisted Reproductive Technolgy. Semin Reprod Med 2014; 32; 35-42. Usadi RS, Groll JM, Lessey BA, et al. Endometrial development and function in experimentally induced luteal phase deficiency. J Clin Endocrinol Metab 2008;93(10):4058–4064. Vasquez, A., T. Jakobsson, S. Ahrne, U. Forsum, and G. Molin. 2002. Vaginal lactobacillus flora of healthy Swedish women. J. Clin. Microbiol. 40:2746–2749. LifeGuard® Oil LifeGuard® Oil is a high viscosity, quality-tested human embryo culture oil Prescreened for purity and batch controlled Thoroughly washed to remove the endotoxins and harmful chemicals LifeGuard® Oil protects against temperature fluctuation Page 14 – Fertility Magazine • Volume 18 • www.FertMag.com Protects from pH drift and impact from toxins Processed and sourced specifically for IVF and ART embryo culture Sterile filtered (SAL 10–3) 1-cell MEA and Endotoxin Tested Optimal viscosity for embryo culture 24 month minimum shelf life A unique LG PGD Biopsy Medium formulation based on global® medium LG PGD Biopsy Medium is ready-to-use and designed to maintain embryos during the biopsy procedure, allowing ease in removal of blastomeres. LG PGD Biopsy Medium was designed along with the staff of Reprogenetics, led by Santiago Munné, in conjunction with our ongoing development of global® based on Simplex Optimization and the entire line of LifeGlobal media. LG PGD Biopsy Medium Advantages • LG PGD Biopsy Medium is calcium- and magnesium-free to break the cadherin bonds between the blastomeres and thereby allow for the removal of one or two blastomeres for PGD. • LG PGD Biopsy Medium contains sucrose to cause a mild shrinkage of the blastomeres and thereby facilitate removal of the blastomere(s) for PGD. • LG PGD Biopsy Medium contains all the other components of global® embryo culture medium including energy substrates and amino acids. This reduces the stress on the embryo and promotes better development of the embryo after it is returned to culture. • LG PGD Biopsy Medium is HEPES-buffered for use outside of a CO2 incubator, and contains gentamicin and HSA so that it is ready to use. • LG PGD Biopsy Medium was designed in consultation with Santiago Munné and his scientific staff at Reprogenetics, one of the foremost PGD laboratories in the world. embryo GPS® Dish – The best dish for PGD cases LG PGD Biopsy Medium Stringent Quality Control: • Proven consistent results • Accommodating schedules with fresh overnight deliveries • Stringent third party QC testing includes: pH, Osmolality, LAL Endotoxin, Two cell MEA tested, Sterility Test Storage Requirements: Store at 2-8°C and protected from light. The formulation of LG PGD Biopsy Medium is consistent with the findings of Hill and Li (The Clinical Embryologist 7, 11-12, 2004) who conclude that: “We believe this step – that of using embryo biopsy media that contain essential amino acids and nutrients found in the primary culturing milieu – to be critical in maximizing clinical outcomes.” Shelf Life: No more than 10 weeks from the date of production when stored unopened at 2-8°C and protected from light. Composition: Sodium Chloride, Amino Acids, Potassium Chloride, EDTA, Potassium Phosphate, Phenol Red, Sodium Bicarbonate, Gentamicin Sulfate, Glucose, HEPES, Sodium Lactate, Sucrose, Sodium Pyruvate, Human Serum Albumin Recommended Product For Culture: LG PGD Biopsy Medium is based on global® medium. For optimal embryo development, we recommend that the embryos be cultured in global® medium both before (Day 1-3), and after (Day 3-5), the biopsy procedure. Ordering Information: LPGG-020........ LG PGD Biopsy Medium, 20 ml T: 1-800-720-6375 ¿ F: 1-519-826-6947 ¿ LPGG-050........... LG PGD Biopsy Medium, 50 ml Intl.: 001-519-826-5800 ¿ [email protected] ¿ www.LifeGlobalGroup.com Fertility Magazine • Volume 18 • www.FertMag.com – Page 15 Effect of Coda® Air Filtration on Chemical and Clinical Pregnancy Rates (Battaglia et al., Fertil Steril 75, Suppl. 1, 6S, 2001) ECOTM 1500 ECOTM 1200 T: 1-800-720-6375 Page 16 – Fertility Magazine • Volume 18 • www.FertMag.com CodaAir® 900 CodaAir® 800 Intl.: 001-519-826-5800 Aero® 700 F: 1-519-826- Effect of Follicular Phase Particulate Air Pollution on Pregnancy Loss First Trimester Loss (%) Q1-3 (Low Air Pollution) Q4 (High Air Pollution) 40 P = 0.001 P = 0.047 30 20 10 0 Natural Conception IVF (Perin et al., Fertil. Steril., 2009, in press) % of Embryos or Transfers Carbon-activated gas filtration during in vitro culture increased pregnancy rate following transfer of in vitro-produced bovine embryos 50 Control 40 Coda 30 P < 0.05 N.S. 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Page 18 – Fertility Magazine • Volume 18 • www.FertMag.com Polar Body Biopsy Reproductive BioMedicine Online An international journal devoted to biomedical research on human conception and the welfare of the human embryo VISIT OUR RESOURCE CENTERS on www.rbmonline.com IFFS-UIT Resource Center Serono Symposia International Foundation Resource Center Bob Edwards Resource Center Affiliated Societies Alpha (Scientists in Reproductive Medicine) American College of Embryology (ACE) The Global Chinese Association for Reproductive Medicine (GCARM) International Society for Fallopian Tubes and Reproductive Surgery (ISFT-RS) International Society for In Vitro Fertilization (ISIVF) Mediterranean Society for Reproductive Medicine (MSRM) Preimplantation Genetic Diagnosis International Society (PGDIS) Turkish Society of Reproductive Medicine (TSRM) www.rbmonline.com Fertility Magazine • Volume 18 • www.FertMag.com – Page 19 ARTICLES The Use of global® for Time-lapse Videographic Analysis of Human Embryo Development by Don Rieger, PhD Vice President, Research and Development, LifeGlobal, LLC You can contact Don Rieger at [email protected] 1. The Early History of Cinematography in Biomedical Research “As a matter of fact we can safely say that the motion picture originated in the biological laboratory.” (Rosenberger 1929) Most people would tend to think of motion pictures as primarily an entertainment medium that is occasionally used for educational purposes and, even less often, for scientific investigations. However, as noted by Rosenberger (1929), motion pictures were first used as a tool for the analysis of movement of humans and other animals, in particular the work of Eadweard J. Muybridge and ÉtienneJules Marey. (See also, Ruddock 2001) Muybridge was a photographer and a bona fide eccentric. His interest in biology was sparked (and financially supported) by A. Leland Stanford, a railroad tycoon and, later, governor of the state of California, who wanted to know whether the four feet of the running horse were ever off the ground at the same time. Muybridge invented a multi-camera apparatus to photograph successive images of Stanford’s prize race horse running, and showed that all four feet were, in fact, off the ground at the same time (Muybridge 1882). The history of this work has more the flavour of a way to settle a bar bet rather than a serious scientific study. Nonetheless, Muybridge went on to apply his technique to a wide variety of studies of the movement of humans and other animals at the University of Pennsylvania (Marks et al. 1888), and is recognized as the “Father of the Motion Picture.” It is perhaps interesting to note that Stanford later established Stanford University, the site of recent videographic studies of human embryo development (Wong et al. 2010; Chavez et al. 2012). Marey, by contrast, was a highly-accomplished physiologist and educator at the Collège de France, in Paris. The major focus of his career was in developing techniques and devices to make objective and graphic measurements of a wide variety of physiological processes (Marey 1876; 1886). Marey (1879) used his “graphic method” to demonstrate that all four feet of a horse were off the ground at the same time, which inspired Muybridge to develop his photographic method to confirm it. Marey, in turn, was inspired by Muybridge’s work to develop his own photographic equipment for the study of movement (Marey 1882; 1884). Following these early studies, cinematography was applied to numerous aspects of animal biology and medicine including x-ray cinematography of the heart, diaphragm, stomach and joints (Groedel 1909), nystagmus Page 20 – Fertility Magazine • Volume 18 • www.FertMag.com Don Rieger, PhD (Lucchetti 1912), mental and nervous diseases (Weisenburg 1912), and medical (Anonymous (BMJ) 1910;Taylor 1918) and public health (Moree 1916) education. Ruddock (2001) notes that photography was developed in 1839, and was quickly applied to medical science, especially for photomicrography (within the same year). It is therefore not at all surprising that medical scientists similarly attached cinematographic cameras to microscopes for the study of living specimens (micro-cinematography). Early among these was Comandon (1909), who combined a Zeiss “ultra-microscope” with a Pathé motion picture camera to film blood-borne trypanosomes and spirocytes, and polynuclear blood cells. Comandon was concerned with the effects of heat from the lights on the specimens and of ambient vibration on the images, which will strike a chord with modern embryologists. Micro-cinematography was subsequently applied to the study of ciliary motion (Buytendijk 1912; Gray 1930), bacterial penetration through capillary spaces (Mudd & Mudd 1924), capillary blood flow (Crawford & Rosenberger 1926a; b), and bacterial growth (Bayne-Jones & Tuttle 1927; Rogers & Greenbank 1930). Alexis Carrel, a pioneer of mammalian cell culture (Carrel & Burrows 1911; Carrel 1912) used micro-cinematography to study the locomotion of the macrophage (Carrel & Ebeling 1926). Comandon and Jolly (1917) filmed the division of newt red blood cells. They took exposures of one frame every 2 3/10 seconds or every 5 seconds, and then viewed the films at the normal rate of 16 frames/second, resulting in an acceleration of 36.8 or 80 times normal speed. They referred to this as “chronophotographie,” what we call time-lapse photography. They described the advantages of time-lapse cinematography as follows (my translation): “Marey also indicated the use of time-lapse photography in the study of phenomena which, because of their extreme slowness, are difficult to appreciate by direct observation: attention wearies, the eye tires, and the changes are imperceptible. …Sometimes the movement is too rapid, sometimes it is too slow. …With cinematographic projection, the movement can be accelerated and rendered perceptible to the eye.” ARTICLES 2. Time-lapse Studies of Pre-implantation Embryo Development In essence, time-lapse photography serves two functions, to capture and determine the timing of discrete events that would not likely be seen by intermittent observation, and to allow the visualization of processes that would otherwise seem to be unconnected events. Both of these functions are highly useful for the study of early embryonic development, as was shown by Lewis and Gregory (1929) in their seminal time-lapse study of development of the rabbit embryo. The rabbit was a good choice for this work because it is an induced ovulator, and therefore the time of fertilization is closely related to the time of mating. They collected zygotes at 21⅔-23½ hours, and late morulae at 67-71 hours, after mating. The embryos were cultured in blood plasma or modified plasma on microscope slides on the microscope stage in a warm box. The zygotes divided to 2 cells by 24½-24¾ hours, to 4 cells by 28-30 hours, and to 8 cells by 39-42 hours, after mating. The very narrow spans of time for the various cleavages are remarkably similar to those seen in modern-day time-lapse micro-videographic studies of human embryo development (see below). The embryos collected at the morula stage survived for as long as 10 days in culture and developed into blastocysts, expanded, and herniated through the zona pellucida. The blastocysts went through (a process of) repeated cycles of expansion and contraction prior to and during herniation through the zona pellucida, as was later shown for hatching of cattle (Massip & Mulnard 1980) , mouse (Bin & Mulnard 1980), hamster (Gonzales & Bavister 1995), and horse and human (Gonzales et al. 1996) blastocysts. The pioneering study of Lewis and Gregory (1929) was followed by numerous timelapse studies of embryo development in various species. An extensive review of the literature is beyond the scope of this paper, but a number of salient studies are listed in Table 1. Table 1. Time-lapse studies of in-vitro embryo development. Reference Species Observation(s) Cassini (1961) Mouse Development from 2-cell to blastocyst Cole (1967) Mouse Blastocyst hatching Brackett (1972) Rabbit Fertilization and development to morula Massip et al. (1982) Cattle Blastocyst expansion and hatching Massip et al. (1983) Cattle Atypical blastocyst hatching resulting in twin half-blastocysts Cohen et al. (1988) Human Morphological assessment of cleavage-stage embryos Selwood and Smith (1990) Marsupials Cleavage and development to blastocyst Gonzales et al. (1995) Hamster Cleavage, and timing of cleavage Gonzales and Bavister (1995) Hamster Blastocyst hatching Gonzales et al. (1996) Cattle, horse, human Blastocyst expansion, contraction and hatching, trophectoderm projections and locomotion of hatched blastocysts Payne et al. (1997) Human Polar body extrusion and pronuclear formation Holm et al. (1998) Cattle Developmental kinetics of early cleavage with respect to viability Van Blerkom et al. (2001) Human Fragmentation and subsequent development Peippo et al. (2001) Cattle Effect of sex and glucose on developmental kinetics Holm et al. (2002) Cattle Effect of serum on developmental kinetics Lequarre et al. (2003) Cattle Effect of oxygen on cell-cycle duration Mateusen et al. (2005) Pig Relationships among developmental kinetics, fragmentation and apoptosis Zaninovic et al. (2005) Human Development from zygote to hatched blastocyst Mio and Maeda (2008) Human Fertilization, development to blastocyst and hatching Gendelman et al. (2010) Cattle Effect of season on developmental kinetics Lopes et al. (2010) Cattle Relationship of cleavage to oxygen consumption Wale and Gardner (2010) Mouse Effect of oxygen on cell-cycle duration 3. Time-lapse Evaluation of Human Embryo Development Comparisons of culture of human embryos under timelapse videography with conventional culture have shown no differences in fertilization rate, embryo development, development to blastocyst, blastocyst quality or ongoing pregnancy rate (Cruz et al. 2011; Barrie et al. 2012; Kirkegaard et al. 2012b). These reports indicate that time-lapse videographic monitoring of human embryo development is a safe and effective technique. Most notably, Meseguer et al. (2012b) found that clinical pregnancy rates were better for embryos cultured in a time-lapse incubator than for those cultured in a conventional incubator. They attributed this to both a more stable culture environment and the use of Fertility Magazine • Volume 18 • www.FertMag.com – Page 21 ARTICLES morphokinetic parameters for embryo selection for transfer. Morphokinetic is defined as “a temporally demonstrable change in shape or form” (Daneo-Moore & Higgins 1972). Much of the interest in time-lapse videography of human embryos is as an approach to predicting development in vitro, and, ultimately, after transfer. A number of studies have shown that various morphokinetic measurements are related to subsequent in-vitro development. Development to the blastocyst stage and blastocyst quality have been shown to be related to the time of syngamy and timing of the early cleavage divisions (Wong et al. 2010; Cruz et al. 2012; Dal Canto et al. 2012; Hashimoto et al. 2012; McEvoy et al. 2012). Iwata et al. (2010) found that compaction before the 8-cell stage was associated with developmental arrest and multinucleation. Yumoto et al. (2012) observed that, for previously frozen-thawed embryos, blastocyst collapse was detrimental to hatching. Clinical outcome following transfer has also been related to morphokinetic measurements. Azzarello et al. (2012) observed that no live births resulted from embryos that experienced early pronuclear breakdown. Ramirez et al. (2012) found that early appearance of two pronuclei was associated with multinucleation and reduced implantation. Implantation has also been shown to be related to the timing of early cleavage events (Rubio et al. 2012; Chamayou et al. 2013). It is not at all surprising that successful clinical outcome can be related to the timing and other characteristics of early cleavage because they set the stage for subsequent development. However, in view of the fact that the major onset of activation of the human embryonic genome does not occur until the 4-8-cell stage (Telford et al. 1990), it would seem unwise to suggest that early cleavage events, alone, should be used for embryo selection for transfer. Evaluation of morphokinetics to the blastocyst stage would include evaluation of events controlled by expression of the embryonic genome, and thus be a much better indicator of viability. Time-lapse videography also offers much promise for fundamental studies of the early human embryo. For example, both the dose of FSH (Munoz et al. 2012) and the type of GnRH analogue used (Munoz et al. 2013) have been related to the morphokinetics of the early embryo, pointing out the significance of ovarian stimulation to early development. Mio et al. (2012) )have proposed a novel mechanism for the block to polyspermy, based on time-lapse observations of fertilization. Freour et al. (2013) have shown that maternal smoking is related to delays in early cleavage. Conversely, Bellver et al. (2013) found no effect of maternal obesity on morphokinetic patterns. Kirkegaard et al. (2012c) showed that cleavage-stage blastomere biopsy resulted in delayed compaction and a change in the mechanism of hatching. Chavez et al. (2012) found that the timing of early cleavage was disturbed in aneuploid embryos. Conversely, Semeniuk et al. (2013) found no relationship between ploidy and the timing of early cleavage events. Campbell et al. (2013) similarly found that ploidy was not related to early cleavage events, but aneuploidy was associated with delayed compaction and blastocyst formation. The demonstration of the effects of culture under Page 22 – Fertility Magazine • Volume 18 • www.FertMag.com atmospheric oxygen concentration (20%) on cell-cycle kinetics in cattle (Lequarre et al. 2003), mouse (Wale and Gardner 2010), and human (Kirkegaard et al. 2013) embryos is a particularly notable example of the potential value of time-lapse videography. Despite overwhelming evidence to show the deleterious effects of culture under 20% oxygen on embryo development and viability, culture under reduced oxygen is not yet universally practiced in human ART (Gardner 2005; Bontekoe et al. 2012). Perhaps the highly objective and precise observations from time-lapse studies will serve to finally put to rest the unphysiological practice of culture of human embryos under 20% oxygen. More extensive reviews of the literature are provided by Meseguer et al. (2012a), Kirkegaard et al. (2012a), Herrero and Meseguer (2013) and Wong et al. (2013). 4. The Use of global® for Time-lapse Evaluation of Human Embryo Development As noted by Herrero and Meseguer (2013), timelapse imaging of embryo development offers a significant advantage over standard culture because the embryos can be monitored without removing them from the stable gas and temperature conditions. This, in essence, is consistent global® medium with the philosophy of the use of ® and the global family of media in which stress on the embryo is minimized by maintaining it in the same chemical background throughout culture and other ART procedures (see Biggers and Summers 2008). Given the extensive history of the success of global® for human embryo culture from the zygote to the blastocyst stage, time-lapse imaging of embryos in global® is a natural fit. The results of a number of time-lapse imaging studies are described below in which the embryos were cultured in global®. Unless otherwise indicated, the medium was renewed or refreshed on Day 3. As previously discussed (Rieger 2012), our general recommendation is that embryos be moved to fresh medium under fresh oil on Day 3 (2step culture), in order minimize the possibility of exposure to volatile organic contaminants. It is certainly possible to culture human embryos from the zygote to the blastocyst stage without renewing the medium (1-step culture), providing that the environmental and other conditions in the laboratory are appropriate. (Reed et al. 2009; 2010; Keskintepe 2012; Singh et al. 2012) In this regard, time-lapse culture may be particularly suitable for 1-step culture because there is a significant reduction in the possibility of exposure of the embryos to environmental VOCs compared with culture in conventional incubators. Cruz et al. (2012) cultured embryos from the zygote stage in global® in a time-lapse incubator for 5 days, and then compared the morphokinetic data between embryos that developed to the blastocyst stage (66.2%) with those that did not. 33.8%). Development to 2, 3, 4 and 5-cells, and to morula was significantly longer for embryos that did not develop to blastocyst. Of the 247 blastocysts that were transferred, 136 (49.6%) implanted (Figure 1). Silva et al. (2012) incubated embryos from donor oocytes in global® in a time-lapse incubator up to the blastocyst stage, and then performed laser-assisted hatching ARTICLES before transfer. As shown in Figure 2, this resulted in an implantation rate of 52.4%. Bellver et al. (2013) compared the development of embryos from obese infertile, normal weight infertile, and normal weight fertile women during culture in global® in a time-lapse incubator over 5 days. The timing of cleavage of the embryos was not different between embryos from obese and normal weight infertile women, but was slower than cleavage of those from normal weight fertile women (Figure 3). Munoz et al. (2013) compared the development of embryos derived from donor cycles after ovarian stimulation using GnRH agonists with hCG triggering, or GnRH antagonists with GnRH agonist triggering. The embryos were cultured to Day 3 or Day 5 before transfer. As shown in Figure 4, early cleavage events were delayed in the GnRH agonists/hCG triggering group. The implantation rate was greater in the GnRH antagonists/GnRH agonist triggering group, and approached statistical significance (P = 0.084). There was no difference in miscarriage rate between the treatment groups (P = 0.524). Costa-Borges et al. (2013) evaluated embryo development in a time-lapse incubator during culture in global total® 120 Developed to blast (N=552) Mean time (h) 72 24 0 80 % of Embryos Did not develop to blast (N=282) 96 48 for 5 days. The medium was either renewed on Day 3 (2-step culture) or not (1-step culture). There was no difference in any of the morphokinetic parameters measured, blastocyst development or quality, or implantation rate between 2-step and 1-step culture (Figure 5). Campbell et al. (2013) cultured embryos from the zygote stage until the blastocyst stage in global® in a time-lapse incubator and then performed trophectoderm biopsy in order to determine ploidy by comparative genomic hybridization. Morphokinetic parameters were compared between embryos determined to be euploid, or to have single or multiple aneuploidy. There were no differences in the timing of the early cleavage events among the three groups. The first indication of compaction and/or blastocyst formation was delayed in the single or multiple aneuploidy groups compared with the euploid group (Figure 6). Semeniuk et al. (2013) cultured embryos from the zygote stage until the blastocyst stage in global® in a 1-step protocol in a time-lapse incubator, and then performed trophectoderm biopsy in order to determine ploidy by comparative genomic hybridization. There were no differences in the timing of the early cleavage events between euploid and aneuploid embryos (Figure 7). 16 60 40 20 80 Obese Infertile 60 40 20 0 Clin. Preg. Rate Impl. Rate Miscarriage Rate Figure 2. Clinical outcomes for embryos cultured in a time-lapse incubator (Silva et al. 2012). global® in Normal Wt Fertile Normal Wt Infertile 60 Mean time (h) % of Embryos or Transfers 0 t3 t4 t5 tM Blastocysts Impl. Rate Morphokinetic Event Figure 1. Comparison of the times of morphokinetic events between embryos that did or did not reach the blastocyst stage, and blastocyst development and implantation rates for embryos cultured in global® in a time-lapse incubator (Cruz et al. 2012). t2 40 20 0 t2 t3 t4 t5 s2 Cc2 Cc3 Figure 3. Comparison of the times of morphokinetic events between embryos derived from oocytes from obese infertile, normal-weight infertile, and normal-weight fertile women. The embryos were cultured in global® in a time-lapse incubator (Bellver et al. 2013). Fertility Magazine • Volume 18 • www.FertMag.com – Page 23 ARTICLES 96 Mean time (h) 16 GnRH Agonist GnRH Antagonist 72 48 24 0 t2 t3 t4 16 16 16 16 16 16 % of Patients or embryos 120 t5 t6 t7 t8 t9+ tM Morphokinetic Event 30 20 16 10 0 tB tEB 16 40 Implantation Rate Miscarriage Rate Figure 4. The effect of ovarian stimulation protocol on the times of morphokinetic events and clinical outcomes for embryos cultured in global® in a time-lapse incubator (Munoz et al. 2013). 2-step 96 1-step 16 72 48 16 16 16 16 16 16 16 16 80 16 16 60 16 40 20 24 0 16 % of Embryos Mean time (h) 120 t2 t3 t4 0 t5 t9+ tM tCM tB tEB tHB Morphokinetic Event Blastocysts G.Q. Blasts Impl Rate Figure 5. Comparison of the times of morphokinetic events, blastocyst development, and implantation rates between embryos cultured in global total® with either medium renewal on Day 3 (2-step culture) or not (1-step culture) (Costa-Borges et al. 2013). Mult. Aneuploid Euploid 96 72 16 48 24 0 16 t2 16 16 60 16 Euploid 48 Mean time (h) Median time (h) 120 16 16 36 Aneuploid 16 16 16 16 24 12 t3 t5 t8 tSC tM tSB tB Morpholokinetic Event tEB tHB Figure 6. Effect of ploidy on the times of morphokinetic events global® in a time-lapse incubator for embryos cultured in (Campbell et al. 2013). Page 24 – Fertility Magazine • Volume 18 • www.FertMag.com 0 t2 t3 t4 Morphokinetic Event t5 Figure 7. Effect of ploidy on the times of morphokinetic events for embryos cultured in global® in a time-lapse incubator (Semeniuk et al. 2013). ARTICLES 5. Discussion and Conclusions 1. Time-lapse videography of embryos throughout early development has been shown to have no deleterious effects on the in-vitro development of the embryos or on clinical outcomes after transfer. Conversely, time-lapse culture may, in itself, be advantageous because the embryos can be monitored without removing them from the incubator. 2. The measurement of various morphological parameters throughout culture offers the promise of an effective addition to the armament of techniques for selection of a single embryo for transfer with the maximum potential to produce a healthy baby. However, this will almost certainly require morphokinetic analysis up to and including the blastocyst stage. 3. Time-lapse videography of embryo development is also potentially an important tool for the study of more fundamental aspects of ART, including ovarian stimulation, fertilization, culture, and embryo manipulation. The deleterious effect of environmental oxygen concentrations (20%) on the morphokinetics of the early embryo is one notable example. 4. global® medium has been shown to be safe and effective for time-lapse culture of human embryos. Given appropriate air quality, 1-step culture in global® has been shown to be highly effective for human embryos. 5. global® is particularly suitable for time-lapse embryo culture. References Anonymous (BMJ) (1910) Special Correspondence: Berlin, Scientific cinematographs Brit. Med. J. 1910, 598. Azzarello A, Hoest T and Mikkelsen AL (2012) The impact of pronuclei morphology and dynamicity on live birth outcome after time-lapse culture. Hum Reprod 27, 2649-57. 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Wong CC, Loewke KE, Bossert NL, Behr B, De Jonge CJ, Baer TM and Reijo Pera RA (2010) Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage. Nat Biotechnol 28, 111521. Yumoto K, Iwata K, Sargent CH, Kai Y, Imajyo A, Iba Y and Mio Y (2012) Dynamic Analysis of the Relationship Between the Collapse of Blastulation and Hatching in Human Blastocysts Using Time-Lapse Cinematography. Fertil Steril 97 Suppl., S30 (Abstr P58). Zaninovic N, Bodine R, Gosden RG, Rosenwaks Z and Veeck L (2005) The secret lives of human preembryos: from fertilization to hatched blastocyst. Fertil Steril 84 Suppl.1, S476 (Abstract VP-3). Fertility Magazine • Volume 18 • www.FertMag.com – Page 27 ARTICLES Advanced maternal age and assisted reproductive technologies: developmental competence, clinical outcomes and future strategies. By Lynne C. O’Shea, PhD Rotunda IVF, Dublin, Ireland. University College Dublin, Belfield, Dublin, Ireland. You can contact Lynne O’Shea at [email protected] or [email protected] Lynne C. O’Shea, PhD Abstract Background: In recent decades the number of women over 40 seeking assisted reproductive technology (ART) interventions in order to become pregnant has increased dramatically. This is due to an increase in the average age at which women are choosing to have their first child while additionally, many couples are choosing to have a second family later in life. However, as with natural conception, ART success rates decrease with maternal age. The decrease in developmental competence of aged oocytes has pushed the field of assisted reproduction to develop additional management strategies and new techniques for optimising fertility outcomes in older women. Methods: In the present study, detailed analysis was performed on our clinical data of women between 40 and 45 years of age, who have undergone ART (16 year retrospective analysis) at our tertiary referral ART clinic. The percentage of patients in this age group was analysed over time, in addition to follicle recruitment, % oocyte yield (and oocyte numbers), embryonic quality, positive hCG (pregnancy rate), clinical pregnancy rate (presence of intrauterine sac), and rate of preclinical pregnancy loss. Published literature to date was also analysed, in conjunction with our clinic’s experience in treating patients of advanced maternal age, in order to identify both current and emerging treatment strategies. Results: Results from our clinic show that women greater than 43 years of age have a significantly reduced reproductive potential, compared even to women in the 40 to 42 years age group. Woman in the 43-45 age group showed reduced fertilization rates (53.73 vs. 58.82%), reduced positive hCG rates (11.51 vs.19.03%) and clinical pregnancy rates (5.04 vs.12.52%) and increased rates of preclinical pregnancy loss (56.23 vs.34.23%), compared to women in the 40-42 age group. Previously published reports showed that the chance of achieving a clinical pregnancy in women greater than 43 years of age is less than 2%. Conclusions: With the age at which couples are choosing to have children constantly increasing, novel patient management and treatment strategies need to be developed for women of increased reproductive age. Several emerging ART techniques, including oocyte/embryo donation, ‘social’ oocyte freezing, preimplantation genetic screening and ovarian tissue freezing, are being established to enhance clinical outcomes in patients of increased maternal age. Keywords: infertility, advanced age, ART, maternal age, over 40. Background There is a well-established link between advanced maternal age and reduced reproductive potential. This natural decrease in fertility in women is caused by several factors including reduced oocyte numbers, diminished oocyte and embryo quality, and an increase in miscarriage rate [1-3]. In addition, pregnancy at this later stage in life involves increased maternal and fetal risks including: miscarriage, hypertension, preeclampsia, gestational diabetes, placenta praevia, placental abruption, caesarean section, genomic disorders, premature birth, low foetal Page 28 – Fertility Magazine • Volume 18 • www.FertMag.com birth weight and neonatal morbidity [4]. In recent decades, the number of women over 40 seeking assisted reproductive technology (ART) interventions, in order to become pregnant, has dramatically increased. This is due to an increase in the average age at which women are choosing to have children; while additionally, many couples are choosing to have a second family later in life [5]. However, as with natural conception, ART success rates decrease with maternal age. In addition to a reduction in oocyte numbers retrieved during a standard ART cycle, maternal age has a detrimental effect on oocyte competence [6-8]. One major factor evident in aged oocytes ARTICLES is an increase in aneuploidy [9, 10]; with chromosomal abnormalities being a major determinant of subsequent embryonic development. In addition to morphological and structural anomalies evident in aged oocytes, they also possess altered gene expression profiles, and an increased potential to undergo apoptosis [11, 12]. Studies have shown alterations in the ability of aged oocytes to stockpile the maternal RNAs that are required to sustain both oocyte and embryo development prior to embryonic genome activation [13, 14]. The decrease in reproductive potential observed in couples of increased age has pushed the field of assisted reproduction to develop additional management strategies and new techniques for optimising fertility outcome. This has led to the establishment of oocyte donor programmes and ‘social’ oocyte-freezing in younger women, in addition to improved genetic testing (preimplantation genetic diagnosis) and advanced methods for determining ovarian reserved (AMH). However, in conjunction with the medical implications of increased reproductive age, societal and ethical implications need to also be considered when employing ART. Most ART clinics have self-imposed limits on the age of patients they are willing to provide with treatment; with vast variation between clinics and countries, and limited regulation in place. The aim of the present study was to perform a detailed analysis of our clinical data on women greater than 40 years of age, who have undergone ART at our tertiary referral ART clinic, between 1997 and 2013. We analysed our own data in the context of published data, in order to determine specific outcomes and review both current and emerging strategies for improving fertility in women greater than 40 years of age. Methods Study design A retrospective analysis of clinical and laboratory data was performed by collecting data from our tertiary referral ART academic program. All IVF/ICSI cycles carried out on women between 40 and 45 years of age, at the Human Assisted Reproduction Ireland (HARI) clinic, Rotunda Hospital, Ireland between January 1997 and January 2013 were identified. All patient treatment data is prospectively collected in a custom made, FilemakerPro-based electronic database. Our clinic has an age cut-off limit for ART of 45 years of age. Patients between 40 and 45 years of age at egg collection were selected for analysis. The percentage of patients in this age group were analysed over time. Due to previous studies demonstrating that the chance of achieving a clinical pregnancy in women greater than 43 years of age is significantly diminished, we also split our patients into two age groups for comparative analysis; 40-42 and 4345. The following parameters were then analysed: follicle recruitment, % oocyte yield (oocyte/follicle ratio), positive hCG, clinical pregnancy rate (ultrasound confirmation of a gestational sac at 7 weeks) and preclinical pregnancy loss (absence of intrauterine sac at 7 week ultrasound scan following previously positive urinary hCG test). Ovarian stimulation and IVF/ICSI treatment Ovarian stimulation was carried out as previously described [15, 16]. Briefly, female patients underwent one of three different types of ovarian stimulation protocol employed during the assessment period; namely using urinary (Menopur, Ferring, Ireland) or recombinant FSH (Puregon, MSD, Ireland) in combination with GnRH antagonist, GnRH agonist or a buserelin nasal spray (Suprecur, Sanofi-Aventis). Final oocyte maturation was induced by injection of 10,000 IU hCG (Pregnyl, MSD, Ireland), as soon as three follicles of 18 mm or more were observed on ultrasound scan. Oocyte retrieval was carried out under sedation using transvaginal ultrasound-guided puncture of ovarian follicles 36 h after hCG administration. IVF and ICSI treatments were carried out as previously described [15]. Statistical analysis Groups of interest were compared using MannWhitney U-test or Wilcoxon’s matched pairs rank sum test was used to compare mean values and Pearson chisquare or McNemar chi-square analysis for comparison of proportional values. All statistical analyses were carried out using SPSS statistics, version 20 (IBM Corporation, 2011, Armonk, New York, USA). Differences between groups were considered significant when p-values were less than 0.05. Systemic literature review: clinical data for ART in women over 40 years of age A systematic literature review was performed by searching PubMed, MEDLINE, CINAHL and The Cochrane Library for data relating to ART in women of advanced reproductive age. Phrases and key words searched for included: <fertility> and <assisted reproductive technology> in <advanced maternal age> or <older women>, including specific end points such as <fertilization>, <pregnancy>, <clinical pregnancy>, <live birth>, <in vitro fertilization>, <intracytoplasmic sperm injection>, <miscarriage>, <pregnancy loss>, <aneuploidy>, <pregnancy complications>, etc. Data was extracted from literature sources and collated, in conjunction with annual reports on ART success rates form the U.S. Centers for Disease Control and Prevention (http://www.cdc.gov/ ART). Fertility Magazine • Volume 18 • www.FertMag.com – Page 29 ARTICLES Results Our clinical data: ART in women between 40 and 45 years of age For the study period, we identified a total of 2068 egg collections from women between 40 and 45 years of age. The average age of the female patients on the date of egg collection was 41.14 ± 1.28 years and the average age of the male partner was 41.62 ± 5.63 years. Figure 1 demonstrates the percentage of patients, in the 40 to 45 year old age group, undergoing IVF/ICSI in our clinic from 1997 to 2013. The proportion of patients in this age group has grown significantly over this 17 year period; increasing from 7.2% of patients in 1997 to 19.8% in 2013. Table 1 presents the patient characteristics, including hormone profiles. Table 2 presents the parameters for oocyte retrieval and oocyte competence. The number of follicles aspirated and oocytes retrieved was significantly lower in patients in the 43-45 age group compared to the 40-42 age group (Table 2). The rate of mature (MII) oocytes was similar in both groups. The percentage of normal fertilization (2PN zygotes) was significantly lower in the 43-45 age group compared to the 40-42 age group and this corresponded to a lower fertilization rate for both ICSI and IVF fertilized oocytes. Table 3 presents the embryo transfer parameters and clinical outcomes. There was no difference in embryo transfer rates between the two age groups, or the mean number of embryos transferred per group. The pregnancy rate (positive hCG) per cycle and per embryo transfer was significantly lower in the 43-45 age group compared to the 40-42 age group. Clinical pregnancy rate was also significantly lower in the 43-45 age group, while the rate of preclincial pregnancy loss was markedly increased (Table 3). Systemic literature review: clinical data for ART in women greater than 40 years of age Table 4 presents the clinical data collated from the published literature for female patients over 40 years of age undergoing ART. These studies report a clinical pregnancy rate of 0–29.15% in women over 40 undergoing ART and a cut-off for ART efficiency after 43 years of age; with a less than 2% chance of achieving a clinical pregnancy in women beyond this age. Live birth rates observed in several of the studies appear to be compounded by high miscarriage rates (16.85 - 85.3%). The data also show that miscarriage rate increases with age, with a miscarriage rate of 50-85.3 % in women greater than 43 years of age. Discussion Women over 40 represent the fastest growing population of patients seeking ART in our clinic, with numbers increasing dramatically from 17 years ago. This corresponds with a proportionate increase in numbers worldwide [5]. With the age at which couples are choosing to have children constantly increasing, novel patient management and treatment strategies need to be developed for women of increased reproductive age. The ability to get pregnant following ART relies largely on the quality of oocytes retrieved and the subsequent development of high quality embryos for transfer. Although oocyte maturation rate in our study was similar between both groups, fertilization rate was greatly reduced in women in the 43-45 age group, compared to 40-42. This corresponds to data previous reported by Cabry Figure 1. The percentage of patients between 40 and 45 years of age undergoing egg collection at our clinic between 1997 and 2013 inclusive. Page 30 – Fertility Magazine • Volume 18 • www.FertMag.com ARTICLES Table 1. Baseline characteristics of patient parameters, in women between 40 and 45 years of age, undergoing IVF/ICSI. Parameter 40-45 years Number of cycles 2068 Number of cycles where no eggs were retrieved 225 Average maternal age (years ± SD) 41.14 ± 1.28 Average paternal age (years ± SD) 41.62 ± 5.63 Infertility duration (year ± SD) 4±3 Previous failed cycle 1.73 ± 1.94 Day 3 AMH (IU/L)* 9.03 ± 11.35 Day 3 E2 (IU/L) 196.23 ± 203.39 Day 3 FSH (IU/L) 9.15 ± 4.28 Day 3 LH (IU/L) 6.23 ± 10.44 *AMH data from March 2010-January 2013 Table 2. Oocyte retrieval and competence, in women over 40 years of age undergoing IVF/ICSI. Parameter Follicles aspirated (mean ± SD) 40-42 years a 9.44 ± 5.38 a Oocytes retrieved (mean ± SD) 7.93 ± 4.82 Oocyte yield (%) 84.07a (10141/12062) a 43-45 years Total (40-45 years) 7.57 ± 4.62 b 8.51 ± 5.0 6.28 ± 4.27 b 7.11 ± 4.55 82.84a (1381/1667) a 83.93 (11522/13729) Mature (metaphase II) oocytes (ICSI only) (%) 78.76 (2558/3248) 78.34 (416/531) 78.70 (2974/3779) Normal fertilization (all) (%) 58.82a (5965/10141) 53.73b (742/1381) 58.21 (6707/11522) a b Normal fertilization (IVF) (%) 60.18 (4148/6893) 55.53 (472/850) 59.67 (4620/7743) Normal fertilization (ICSI) (%) 55.92a (1817/3248) 50.85b (270/531) 55.23 (2087/3779) Number of fertilized oocytes/cycle a,b,c a 3.41 (5965/1750) b 2.33 (742/318) 3.24 (6707/2068) Superscripts of different alphabetic characters across rows indicate significant differences (P < 0.05) between groups, whereas presence of identical characters indicates lack of significant differences. Table 3. Clinical outcomes, in women over 40 years of age undergoing IVF/ICSI. Parameter 40-42 years 43-45 years Total (40-45 years) Embryo transfer performed (%) 89.49a (1566/1750) 87.42a (278/318) 89.17 (1844/2068) Transferred embryos (mean ± SD) 2.12 ± 0.93a 2.02 ± 0.92a 2.07 ± 0.93 a b Positive hCG/cycle (%) 17.03 (298/1750) 10.06 (32/318) 15.96 (330/2068) Positive hCG/embryo transfer (%) 19.03a (298/1566) 11.51b (32/278) 17.90 (330/1844) Clinical pregnancy rate/embryo transfer (%) 12.52a (196/1566) 5.04b (14/278) 11.39 (210/1844) Preclinical pregnancy loss (%) 34.23a (102/298) 56.23b (18/32) 36.36 (120/330) a,b,c Superscripts of different alphabetic characters across rows indicate significant differences (P < 0.05) between groups, whereas presence of identical characters indicates lack of significant differences. et al. [17] and demonstrates how the ability of an oocyte to undergo nuclear maturation is not always a reliable predictor of oocyte quality and subsequent developmental potential. Maternal age is known to be the biggest factor affecting clinical outcome following ART. Our data show that female patients greater than 42 years of age have a diminished prognosis following IVF/ICSI compared to patients between 40 and 42 years of age. This corresponds to previously published data which clearly shows a cut-off for success following ART in women to be 44 years of age (Table 4). The limitations of the studies performed to date reside in their retrospective nature, in addition to changing protocols over time. In addition, many variables associated with assisted reproductive technologies such as day of Fertility Magazine • Volume 18 • www.FertMag.com – Page 31 ARTICLES Table 4. Reported clinical outcomes from ART in women over 40. Reference Our clincal data Cabry et al., 2014 [17] Gleicher et al., 2014 [18] Niinimaki et al., 2012 [19] Nikolaou & Marinakis 2011 [20] Tsafrir et al., 2009 [21] Hourvitz et al., 2009 [22] Maternal Age (years) Clinical Pregnancy (%) Miscarriage Rate (%) Live Births (%) 40-42 12.52 43-45 5.04 41.5* 8.9 16.85 7.4 40-41 29.15 0 29.15 42-45 11.56 77.51 2.6 46-53 0 0 0 (SET) 19.5 43.59 11.0 (DET) 23.5 42.13 13.6 40-44 40-42 7.7 43-44 5.4 >44 1.9 40 13.9 34.53 9.1 45 2.8 75 0.7 42 7.7 43 5.4 85.3 3.1 44 1.9 Spandorfer et al., 2007 [23] 45.4* 21.2 Van Disseldorp et al., 2007 [24] 40-43 8 42 7.7 43 5.4 Ciray et al., 2006 [25] Klipstein et al., 2005 [26] Ron-El et al., 2000 [27] 44 1.9 ≥45 0 40 3 41-42 18.8 43 9.6 44 1.6 41 14 50 7 42 9 77.78 2 43 26 50 13 44 0 0 0 * mean maternal age, SET: single embryo transfer, DET: double embryo transfer. transfer, embryo quality and type of protocol are not taken into account when analysing results. However, studies such as these are not without their relevance and the correlation of similar studies performed worldwide provides a reliable indication of the effect advanced maternal aging is having on reproductive potential following ART. Current strategies Over the past decade many patient management and treatment strategies have been developed for women over 40 years of age. First, patients must be counselled on both the reduced clinical success rates for ART and increased pregnancy risks and miscarriage rates associated Page 32 – Fertility Magazine • Volume 18 • www.FertMag.com with increased maternal age. Here we show a significant reduction in pregnancy rate and clinical pregnancy rate, in addition to a substantial increase in the rate of preclincal pregancy loss (increase from 34.23 % in 40-42 year olds to 56.23% in 43-45 year olds at 7 week scan). It is known that a large proportion of miscarriages that occur in the first trimester of pregnancy are due to chromosomal abnormalities, a known factor associated with oocytes from women of increased reproductive age [28]. Due to the low success rates observed following ART, women of increased reproductive age often undergo the transfer of multiple embryos in order to obtain a pregnancy. One previous study reported that the optimum number of embryos for transfer in order to achieve an appropriate ARTICLES pregnancy and live birth rate in women over 40 is five [29], while many others have suggested 3 embryos to be sufficient [25, 26, 30]. Due to the increased risks associated with multiple births, and the increased pregnancy risk the patient is already subject to, this strategy should be considered very carefully before being initiated. Emerging strategies Preimplantation genetic screening (PGS) is one strategy which is currently being used by clinics to identify genetic and chromosomal abnormalities in embryos. This has been shown to allow for improved embryo selection techniques, leading to increased pregnancy rates and reduced implantation rates in women over 40 years of age [31-33]. Another strategy currently in focus for patients of increased age is the option of oocyte and embryo donation. Several studies have shown that many infertility issues associated with increased maternal age can be overcome by oocyte donation [34, 35]. Nevertheless, patient age also has an effect on uterine receptivity and implantation; as evident in the large meta-analysis performed by Vernaeve et al. [36] which showed that pregnancy rates were significantly reduced in patients over 45 years of age using donor oocytes. In such cases surrogacy may also be an option. The Spanish experience shows cumulative pregnancy rates after 4 cycles of embryo transfer from donor eggs reaching nearly 94%, with clinical pregnancy rates of 53.4% per cycle and live birth rates of 42.6% [35]. Therefore, oocyte donation is a very promising option for women over 40 years of age wishing to get pregnant. In addition, oocyte and embryo donation eliminates the risk of ovarian stimulation in the recipient. Before undergoing either oocyte or embryo donation patients need to be extensively counselled about the ethical and legal issues associated with such procedures. Patients need to be aware of the possibility of their child having full siblings following embryo donation, or half siblings in the case of oocyte donation. At present in many countries, including Ireland, there is no legislation in place to cover the legal issues arising from surrogacy, oocyte or embryo donation. This leads to ambiguity over who the legal parents of a child born from such procedures are. Due to the increased success rates associated with transferring embryos generated from young oocytes to women over 40, one option for women is to ‘self-preserve’ their own fertility by banking their eggs when they are in their 20’s and 30’s. With the development of vitrification, oocyte cryopreservation has vastly improved, however to date data are still limited [37]. It has been shown that the retrieval of 55 metaphase II oocytes is needed for women aged between 38 and 43 years of age to achieve a pregnancy following vitrification [38]. The large number of oocytes needed, in addition to the risks associated with ovarian stimulation and egg collection may deter women from availing of this ‘back-up plan’ for their future fertility. Furthermore, the cryopreservation of oocytes does not guarantee a pregnancy and the maternal risks associated with a pregnancy at advanced age are all still present. The ethics of offering such a service at present, giving the cost and the low rate of success, need to be questioned. Ovarian tissue freezing is a novel strategy under development in order to preserve fertility in women. This allows storage of a large pool of primordial follicles and negates the need for hormone treatment at the time of retrieval [39, 40]. For these reasons it is currently used as an option for cancer patients with time and/or hormone sensitive malignancies. Furthermore, this procedure not only has the potential to restore fertility but has also been shown to restore endocrine function, and could potentially radically postpone the onset of menopause [41]. These current and emerging advances in ART techniques may allow women to achieve pregnancy much later in life. However, when considering ART at an advanced age, it is necessary to take into account the severe risks associated with such a pregnancy [4]. One study observed that 63% women over 50 who achieved a pregnancy through embryo donation needed hospitalization, compared to 22% in women aged between 45 and 49 [34]. In such women, surrogacy may be the safer option. Conclusions Women of age greater than 42 have a significantly reduced reproductive potential, compared to women who are between 40 and 42 years of age. This corresponds to a reduced fertilization rate, pregnancy rate and clinical pregnancy rate, in addition to an increased miscarriage rate. With the life expectancy of children being born today expanding above 100 years of age, the demand for ART strategies to prolong reproductive potential and/or postpone the onset of menopause will continue to rise. Acknowledgements I wish to thank the embryology staff, nurses, patient support team and physicians at Human Assisted Reproduction Ireland (HARI), Rotunda Hospital, Dublin, Ireland who have contributed to the care of the patients. References 1. 2. 3. 4. 5. Ottolenghi, C., et al., Aging of oocyte, ovary, and human reproduction. Ann N Y Acad Sci, 2004. 1034: p. 117-31. Broekmans, F.J., et al., Female reproductive ageing: current knowledge and future trends. Trends Endocrinol Metab, 2007. 18(2): p. 58-65. te Velde, E.R. and P.L. Pearson, The variability of female reproductive ageing. Hum Reprod Update, 2002. 8(2): p. 141-54. Suchartwatnachai, C., et al., Cost-effectiveness of IVF in women 38 years and older. Int J Gynaecol Obstet, 2000. 69(2): p. 143-8. Craig, B.M., et al., A generation of childless women: lessons from the United States. Womens Health Issues, 2014. 24(1): p. e21-7. Fertility Magazine • Volume 18 • www.FertMag.com – Page 33 ARTICLES 6. Toner, J.P., Age = egg quality, FSH level = egg quantity. Fertil Steril, 2003. 79(3): p. 491. 7. Malhi, P.S., et al., Oocyte developmental competence in a bovine model of reproductive aging. Reproduction, 2007. 134(2): p. 233-9. 8. Tatone, C., Oocyte senescence: a firm link to age-related female subfertility. Gynecol Endocrinol, 2008. 24(2): p. 59-63. 9. Kuliev, A., J. Cieslak, and Y. Verlinsky, Frequency and distribution of chromosome abnormalities in human oocytes. Cytogenet Genome Res, 2005. 111(3-4): p. 193-8. 10. Pellestor, F., T. Anahory, and S. Hamamah, Effect of maternal age on the frequency of cytogenetic abnormalities in human oocytes. Cytogenet Genome Res, 2005. 111(3-4): p. 206-12. 11. Fujino, Y., et al., DNA fragmentation of oocytes in aged mice. Hum Reprod, 1996. 11(7): p. 1480-3. 12. Tatone, C., et al., Age-associated changes in mouse oocytes during postovulatory in vitro culture: possible role for meiotic kinases and survival factor BCL2. Biol Reprod, 2006. 74(2): p. 395-402. 13. Hamatani, T., et al., Age-associated alteration of gene expression patterns in mouse oocytes. Hum Mol Genet, 2004. 13(19): p. 2263-78. 14. Steuerwald, N.M., et al., Maternal age-related differential global expression profiles observed in human oocytes. Reprod Biomed Online, 2007. 14(6): p. 700-8. 15. Mocanu, E., et al., Odds of ovarian hyperstimulation syndrome (OHSS) - time for reassessment. Hum Fertil (Camb), 2007. 10(3): p. 175-81. 16. Mocanu, E.V., et al., First Irish birth following IVF therapy using antagonist protocol. Ir J Med Sci, 2010. 179(3): p. 455-7. 17. Cabry, R., et al., Management of infertility in women over 40. Maturitas, 2014. 78(1): p. 17-21. 18. Gleicher, N., et al., The “graying” of infertility services: an impending revolution nobody is ready for. Reprod Biol Endocrinol, 2014. 12(1): p. 63. 19. Niinimaki, M., et al., Elective single-embryo transfer in women aged 40-44 years. Hum Reprod, 2013. 28(2): p. 331-5. 20. Marinakis, G. and D. Nikolaou, What is the role of assisted reproduction technology in the management of age-related infertility? Hum Fertil (Camb), 2011. 14(1): p. 8-15. 21. Tsafrir, A., et al., What should be the first-line treatment for unexplained infertility in women over 40 years of age - ovulation induction and IUI, or IVF? Reprod Biomed Online, 2009. 19 Suppl 4: p. 4334. 22. Hourvitz, A., et al., Assisted reproduction in women over 40 years of age: how old is too old? Reprod Biomed Online, 2009. 19(4): p. 599-603. 23. Spandorfer, S.D., et al., Outcome of in vitro fertilization in women 45 years and older who use autologous oocytes. Fertil Steril, 2007. 87(1): p. 74-6. 24. van Disseldorp, J., et al., Cumulative live birth rates following IVF in 41- to 43-year-old women presenting with favourable ovarian reserve characteristics. Reprod Biomed Online, 2007. 14(4): p. 455-63. 25. Ciray, H.N., et al., Outcome of 1114 ICSI and embryo transfer cycles of women 40 years of age and over. Reprod Biomed Online, 2006. 13(4): p. 516-22. 26. Klipstein, S., et al., One last chance for pregnancy: a review of 2,705 in vitro fertilization cycles initiated in women age 40 years and above. Fertil Steril, 2005. 84(2): p. 435-45. 27. Ron-El, R., et al., Outcome of assisted reproductive technology in women over the age of 41. Fertil Steril, 2000. 74(3): p. 471-5. 28.Rubio, C., et al., Chromosomal abnormalities and embryo development in recurrent miscarriage couples. Hum Reprod, 2003. 18(1): p. 182-8. Page 34 – Fertility Magazine • Volume 18 • www.FertMag.com 29. Combelles, C.M., et al., Optimum number of embryos to transfer in women more than 40 years of age undergoing treatment with assisted reproductive technologies. Fertil Steril, 2005. 84(6): p. 1637-42. 30. Tsafrir, A., et al., Retrospective analysis of 1217 IVF cycles in women aged 40 years and older. Reprod Biomed Online, 2007. 14(3): p. 348-55. 31. Klipstein, S., Preimplantation genetic diagnosis: technological promise and ethical perils. Fertil Steril, 2005. 83(5): p. 1347-53. 32.Heng, B.C., Advanced maternal age as an indication for preimplantation genetic diagnosis (PGD)--the need for more judicious application in clinically assisted reproduction. Prenat Diagn, 2006. 26(11): p. 1051-3. 33. Taranissi, M., et al., Influence of maternal age on the outcome of PGD for aneuploidy screening in patients with recurrent implantation failure. Reprod Biomed Online, 2005. 10(5): p. 628-32. 34. Sauer, M.V., R.J. Paulson, and R.A. Lobo, Oocyte donation to women of advanced reproductive age: pregnancy results and obstetrical outcomes in patients 45 years and older. Hum Reprod, 1996. 11(11): p. 2540-3. 35. Remohi, J., et al., Pregnancy and birth rates after oocyte donation. Fertil Steril, 1997. 67(4): p. 717-23. 36. Vernaeve, V., et al., [Clinical factors associated with the outcome of oocyte donation]. Gynecol Obstet Fertil, 2007. 35(10): p. 1015-23. 37. Smith, G.D., et al., Prospective randomized comparison of human oocyte cryopreservation with slow-rate freezing or vitrification. Fertil Steril, 2010. 94(6): p. 2088-95. 38. Stoop, D., et al., Clinical validation of a closed vitrification system in an oocyte-donation programme. Reprod Biomed Online, 2012. 24(2): p. 180-5. 39. Bath, L.E., et al., Spontaneous conception in a young woman who had ovarian cortical tissue cryopreserved before chemotherapy and radiotherapy for a Ewing’s sarcoma of the pelvis: case report. Hum Reprod, 2004. 19(11): p. 2569-72. 40. Rosendahl, M., et al., Cryopreservation of ovarian tissue for fertility preservation: no evidence of malignant cell contamination in ovarian tissue from patients with breast cancer. Fertil Steril, 2011. 95(6): p. 2158-61. 41. Rosendahl, M., et al., Cryopreservation of ovarian tissue for a decade in Denmark: a view of the technique. Reprod Biomed Online, 2011. 22(2): p. 162-71. The Most Dependable IVF Aspiration Pump Single Vac Pioneer Pro-Pump® Dual Vac Pioneer Pro-Pump® “Uninterrupted TimeLapse Culture MediumTM” Fertility Magazine • Volume 18 • www.FertMag.com – Page 35 ARTICLES global® Blastocyst Fast Freeze® Easy-To-Use and a High Performance System Does not require special Vitrification device and uses regular sealable straws. global® Blastocyst Fast Freeze® Kit Easy three-step Fast Freeze® FREE Samples Available for your evaluation and comparison studies. (3–6 months FREE if your data is shared with our scientific team.) FREE consultation by our technical experts. [email protected] global® Blastocyst Fast Freeze® Thawing Kit Page 36 – Fertility Magazine • Volume 18 • www.FertMag.com www.LifeGlobalGroup.com ARTICLES High-magnification selection of spermatozoa prior to oocyte injection: confirmed and potential indications F Boitrelle*, B Guthauser, L Alter, M Bailly, M Bergere, R Wainer, F Vialard, M Albert, J Selva Department of Reproductive Biology, Cytogenetics and Gynaecology, Poissy General Hospital, F-78303 Poissy, rance; EA 2493, Versailles University of Medicine and Science, F-78000 Versailles, France *Corresponding author address: Department of Reproductive Biology, Cytogenetics and Gynaecology, Poissy General Hospital, F-78303 Poissy, France. E-mail address: [email protected] (F Boitrelle). Florence Boitrelle, MD Florence Boitrelle, MD is working as an embryologist at Poissy General Hospital’s assisted reproduction unit since 2009. She is currently working on her PhD thesis on the links between sperm morphology, nuclear quality, acrosome quality, genetics and epigenetics at the University of Versailles Saint-Quentin-en-Yvelines. Abstract Intracytoplasmic morphologically selected sperm injection (IMSI) involves the use of differential interference contrast microscopy at high magnification (at least ·6300) to improve the observation of live human spermatozoa (particularly by showing sperm head vacuoles that are not necessarily seen at lower magnifications) prior to intracytoplasmic sperm injection (ICSI) into the oocyte. However, a decade after IMSI’s introduction, the technique’s indications and ability to increase pregnancy and/or birth rates (relative to conventional ICSI) are subject to debate. In an attempt to clarify this debate, this work performed a systematic literature review according to the PRISMA guidelines. The PubMed database was searched from 2001 onwards with the terms ‘IMSI’, ‘MSOME’ and ‘high-magnification, sperm’. Out of 168 search results, 22 relevant studies reporting IMSI outcomes in terms of blastocyst, pregnancy, delivery and/ or birth rates were selected and reviewed. The studies’ methodologies and results are described and discussed herein. In view of the scarcity of head-to-head IMSI versus ICSI studies, the only confirmed indication for IMSI is recurrent implantation failure following ICSI. All other potential indications of IMSI require further investigation. KEYWORDS: human spermatozoa, IMSI, MSOME, outcome, pregnancy rate, vacuole This article was published in Reproductive BioMedicine Online, Vol 28, 2014, p6-13, High-magnification selection of spermatozoa prior to oocyte injection: confirmed and potential indications. Copyright Elsevier. It is reprinted here with permission. Introduction Since its first use in the early 1990s (Palermo et al., 1992), intracytoplasmic sperm injection (ICSI) has become a powerful tool for infertile couples – particular in cases of severe male infertility and low sperm counts. In ICSI, the ‘best-looking’ live spermatozoon is chosen for its motility, viability and gross morphology, using Hoffman contrast microscopy and a magnification of x200 or x400. Although the fertilization and clinical pregnancy rates associated with ICSI are high (Palermo et al., 2009), it has been shown that ejaculate characteristics (e.g. normal spermatozoa or mild or severe oligoasthenoteratozoospermia) (Loutradi et al., 2006) and the morphology of the individually selected spermatozoon may affect post-ICSI fertilization, implantation and pregnancy rates (De Vos et al., 2003). These results can be explained (at least in part) by the fact that, even though a spermatozoon’s morphology is slightly correlated with its chromatin condensation or DNA integrity, the selection of normal spermatozoa during ICSI does not enable spermatozoa with nuclear defects to be excluded (Abu Hassan Abu et al., 2012; Avendaño et al., 2009). Hence, over the last decade, some researchers have tried to improve sperm observation with higher-resolution microscopy techniques. Their objective has been to establish correlations between the morphology of a viable (and subsequently injectable) spermatozoon and its inherent quality (in terms of chromosomal content, degree of chromatin condensation and/or DNA integrity). The most studied of these novel techniques is motile sperm organelle Fertility Magazine • Volume 18 • www.FertMag.com – Page 37 ARTICLES morphology examination (MSOME), which uses differential interferential contrast microscopy and high magnification (>x6300), first described by Bartoov et al. (2001). This observation technique reportedly enables better assessment of a spermatozoon’s morphology and the visualization of sperm head vacuoles. The latter structures are not visible (particularly when they are small) at a conventional ICSI-like magnification (using Hoffman contrast and a magnification of x200–x400) (Bartoov et al., 2001). Nevertheless, since the introduction of IMSI, more attention has been given to the pre-ICSI detection of spermatozoa that contain vacuoles. Over the last decade, many researchers have evaluated IMSI (i.e. the MSOME-based selection of a spermatozoon and then its injection into the oocyte) and compared it with the gold-standard technique, ICSI. However, IMSI’s superiority over ICSI (in terms of pregnancy or delivery rates) is still subject to debate. The only meta-analysis of this topic was performed 3 years ago (Setti et al., 2010). It included three studies and, by pooling all the IMSI results, did not take account of the specific indication. In fact, the studies in this field differ significantly in terms of: (i) their design (e.g. randomized versus nonrandomized studies, or the comparison of IMSI results with previous ICSI results for the same couples versus other couples matched according to various criteria); (ii) the ICSI magnification used; (iii) the sprm morphology designated as ‘normal’ at an IMSIlike magnification; (iv) the sperm classification; and (v) the criteria used to assess the outcome (e.g. clinical pregnancy and delivery rates per couple, per transfer or per cycle). Hence, the objective of the present literature review was to assess the outcomes for IMSI vs. ICSI and determine the clinical situations in which the use of this assisted reproduction technology is likely to be of greatest value. Materials and methods This work performed a systematic review of the relevant literature, according to the PRISMA guidelines (Moher et al., 2009). The PubMed database was searched for work published between 2001 and March 2013 with the following search terms: ‘IMSI’, ‘MSOME’ and ‘high-magnification, sperm’. The publications’ titles, abstracts and reference lists were viewed and only relevant publications (i.e. those reporting on IMSI outcomes in terms of blastocyst, pregnancy, delivery and/or live birth rates) in English were selected and included. This review examined, compared and discussed study methodologies and results, including patient characteristics, the magnifications used for IMSI and ICSI (when stated) and the pregnancy and/or delivery rates associated with IMSI and ICSI. The results were subdivided into currently accepted indications of IMSI (i.e. clinically relevant indications confirmed by several studies, including at least two randomized clinical trials with a large sample size) and potential indications (i.e. those requiring additional research). Page 38 – Fertility Magazine • Volume 18 • www.FertMag.com Results and discussion Literature retrieved The PubMed search identified a total of 168 publications (58 using the term ‘IMSI’, 28 using the term ‘MSOME’ and 82 using the terms ‘high-magnification, sperm’) indexed between 2001 and March 2013. After viewing the publications’ titles, abstracts and reference lists, 24 studies which directly compared IMSI and ICSI were retrieved. Following the exclusion of two publications not written in English, a total of 22 studies were included in this review. Indication for IMSI In most studies, ICSI was indicated because of the presence of at least one male factor for infertility (oligoand/or asthenoand/or teratozoospermia) (see, for example, Table 1). The indication of ICSI was not specified in three studies and varied in one other study. The only confirmed indication of IMSI is recurrent implantation failure following ICSI. Outcomes after IMSI The outcomes of IMSI following ICSI failure are summarized in Table 1. In two studies, IMSI was directly compared with ICSI in couples matched for the number of previous ICSI failures (Bartoov et al., 2003; Oliveira et al., 2011). Bartoov et al. studied a total of 100 couples with an mean (range) of 4.1 (2–8) previous ICSI failures. When compared with ICSI (performed at a magnification of x200 or x400, n = 50 couples), IMSI (with selection of normal spermatozoa with no more than one small vacuole occupying <4% of the sperm head area, n = 50 couples) yielded a significantly higher clinical pregnancy rate per couple (30% versus 66%, respectively, P < 0.01), a lower miscarriage rate (33% versus 9%, P < 0.01) and a higher delivery rate per couple (20% versus 60%, P < 0.01). Furthermore, IMSI yielded even higher clinical pregnancy and delivery rates per couple (50%, in both cases) in a group of 12 additional, unmatched couples with more than eight ICSI failures (although the technique was not compared directly with ICSI; Bartoov et al., 2003). Oliveira et al. studied 200 couples having undergone IMSI (with selection of normal spermatozoa with no more than one small vacuole occupying <4% of the sperm head area) and found that clinical pregnancy and live birth rates per cycle tended to be higher and miscarriage rates tended to be lower than those of the 100 couples having undergone ICSI (performed at a magnification of x400), although these differences were not statistically significant (Oliveira et al., 2011). The comparison of these two studies suggest that sperm abnormalities that are not visible at x200 might indeed be detected at magnification x400. Hence, IMSI’s superiority over ICSI at ·400 might be less obvious than ARTICLES IMSI’s superiority over ICSI at x200. Hazout et al. (2006) compared outcomes for IMSI and ICSI (x200) in a total of 125 couples acting as their own controls. After at least two previous failed ICSI attempts, each couple underwent two further attempts: an additional round of ICSI (x200) and then IMSI (with selection of vacuole-free spermatozoa). When compared with the ICSI cycle, IMSI was associated with a significantly higher clinical pregnancy rate per transfer (2% versus 38%, respectively, P < 0.001) and a significantly lower miscarriage rate (data not reported). Furthermore, IMSI led to a significantly higher delivery rate per transfer (34% for IMSI and 0% for ICSI, P < 0.001) and a significantly higher live birth rate per embryo transferred (18% versus 0%, respectively, P < 0.001) than ICSI did (Hazout et al., 2006). Very recently, a prospective but non-randomized study compared IMSI and ICSI outcomes in patients with more than two ICSI failures (El Khattabi et al., 2013). This was the only study to report that IMSI (with selection of the ‘best available’ spermatozoon, according to Vanderzwalmen et al., 2008) yielded much the same results (in terms of clinical pregnancy and live birth rates per cycle) as ICSI performed at x200 (24% versus 26% and 21% versus 22%, respectively). However, the fact that patients were not matched for the number of previous ICSI failures may have been a source of bias. The mean number of attempts for the IMSI patients (4.8) was indeed significantly greater than that for the ICSI patients (4.1; P = 0.0001). Randomized studies (and particularly those with large sample sizes) provide the most robust evidence when comparing IMSI and ICSI outcomes. Knez et al. (2011) performed a randomized trial in patients with no blastocyst Fertility Magazine • Volume 18 • www.FertMag.com – Page 39 ARTICLES formation in previous ICSI failures. The researchers compared IMSI (with selection of the best spermatozoon, according to Cassuto et al. (2009), n = 37) and ICSI (performed at magnification of both x200 and x400, n = 20). There was a nonsignificant trend towards a higher number of cycles with at least one blastocyst in the IMSI group than in the ICSI group (50% versus 35%, respectively). Likewise, a nonsignificant trend towards a higher clinical pregnancy rate per cycle was achieved in the IMSI group, when compared with the ICSI group (25% versus 8%, respectively). Given that several factors (e.g. low oocyte quality) could be responsible for absence of blastocyst formation and that the study’s sample size was small, it remains to be determined whether IMSI is indeed better than ICSI in this precise indication of no blastocyst formation. Another randomized study (Antinori et al., 2008) of a larger number of couples (n = 446) compared 227 IMSI attempts (with selection of normal spermatozoa with no more than one small vacuole with a borderline diameter of 0.78 ± 0.18 lm) with 219 ICSI attempts (at an unspecified magnification). Overall, IMSI yielded a significantly higher clinical pregnancy rate per couple than ICSI (39% versus 27%, respectively; P = 0.004). For the 139 couples with at least two ICSI failures (Table 1), IMSI in 77 couples was associated with a 2-fold higher clinical pregnancy rate per couple than ICSI in 62 couples (30% versus 13%, respectively) and a 2-fold lower miscarriage rate (17% versus 38%). For couples with no previous ICSI failures (n = 123) or only one previous ICSI failure (n = 184), the clinical pregnancy rates per couple for IMSI and ICSI did not differ significantly (Antinori et al., 2008). Very recently, it was suggested that IMSI may be of value after just one previous ICSI failure (Klement et al., 2013). In fact, this group led by Berkovitz performed a randomized study of a very large number (449) of couples with one previous ICSI failure. The clinical pregnancy and delivery rates per cycle were significantly higher for the 127 couples randomized to IMSI (with selection of normal spermatozoa with no more than one small vacuole occupying <4% of the sperm head area) than for the 322 couples randomized to further ICSI (at a magnification of x200 or x400): 56% versus 38% (P = 0.002) and 28% versus 18% (P = 0.04), respectively. A multivariate analysis prompted these researchers to state that the ICSI-to-IMSI switch after the initial failure was associated with a 3-fold greater chance of clinical pregnancy and delivery. Furthermore, the study results also showed that IMSI was no better than ICSI when used in the first round of treatment. Other researchers have shown that IMSI is no more efficient than ICSI in unselected patients (i.e. regardless of the number of treatment attempts) (Balaban et al., 2011; De Vos et al., 2013). De Vos et al. (2013) recently analysed the outcomes of 350 attempts (including 125 IMSI cycles with the transfer of IMSI-only embryos and 139 ICSI cycles with the transfer of ICSI-only embryos) in a non-randomized study of 340 couples. The researchers reported that Page 40 – Fertility Magazine • Volume 18 • www.FertMag.com IMSI (with selection of vacuole-free spermatozoa, when available) yielded much the same results (in terms of clinical pregnancy rates per embryo transferred) as ICSI performed at a magnification of x400 (34% versus 37%, respectively). However, most of the patients included in this study were undergoing their first ICSI/IMSI attempt (188/350, 54%) or second attempt (72/350, 21%). Hence, this study provided additional evidence for the lack of superiority of IMSI in patients with no previous ICSI failures. Similarly, Balaban et al. (2011) compared the outcomes of IMSI (n = 87) and ICSI (n = 81) in a randomized study of 168 unselected couples (i.e. regardless of any previous ICSI or IVF failures). Clinical pregnancy rates per cycle (54% versus 44%) and live birth rates per cycle (44% versus 38%) did not significantly differ when comparing the IMSI group (with selection of normal spermatozoa with no more than one small vacuole with a borderline diameter of 0.78 ± 0.18 lm) and the ICSI group (for which the magnification was not stated in the report). In summary, the literature review results suggest that IMSI is only of value (in terms of higher clinical pregnancy and live birth rates) for patients with one or more previous ICSI failure and not for unselected patients or those undergoing their first treatment attempt. Given that (i) vacuoles were shown to be linked to chromatin condensation failure (Boitrelle et al., 2011; Boitrelle et al., 2013; Franco et al., 2012; Garolla et al., 2008; Perdrix et al., 2011), (ii) chromatin condensation failure is associated with recurrent abortions (Kazerooni et al., 2009; Talebi et al., 2012) and (iii) a growing body of evidence suggests that the degree of sperm chromatin condensation at the time of fertilization can influence early and late embryo development (Hammoud et al., 2011), the current work postulates that the higher pregnancy and delivery rates and lower miscarriage rates observed for IMSI after ICSI failure can be explained (at least in part) by the exclusion of spermatozoa containing sperm head vacuoles of nuclear origin. Potential indications of IMSI Teratozoospermia Teratozoospermia may be an indication for IMSI. In all but one of the studies reviewed below, ICSI and IMSI were indicated because of the presence of at least one male factor for infertility (oligo- and/or astheno- and/or teratozoospermia). The article by Berkovitz et al. (2006) did not specify an indication. It has already been shown that individual spermatozoa differ in their ability to produce an embryo capable of implanting. Indeed, the use of morphometrically normal spermatozoa with no vacuoles or less than two small vacuoles has been associated with significantly higher blastocyst rates than all other types of spermatozoa (i.e. those with more than two small vacuoles, those with one large vacuole and those with morphometric abnormalities) ARTICLES (Knez et al., 2012; Vanderzwalmen et al., 2008). In contrast, one study has reported lower blastocyst rates when vacuole-free spermatozoa were used for injection (relative to spermatozoa with vacuoles) (Tanaka et al., 2012); however, the sample size was small and the study population was not homogeneous because it included both patients with azoospermia and patients with normal sperm characteristics. Furthermore, concerning the ability of individual spermatozoa to lead to a pregnancy, the shape of the sperm head and the presence of vacuoles were reported as being significantly and positively correlated with the chance of achieving a pregnancy with IMSI (r = 0.38; P < 0.01; Bartoov et al., 2002). Berkovitz et al. (2006) reported on IMSI in 80 patients: when compared with spermatozoa with vacuoles or an abnormal morphology (so-called ‘second-choice’ spermatozoa), normal, vacuole-free spermatozoa yielded significantly higher clinical pregnancy and delivery rates per cycle (58% versus 26% and 53% versus 17%, respectively, both P < 0.01) and significantly lower miscarriage rates (10% versus 33%, respectively; P = 0.02) (Berkovitz et al., 2006). Hence, the morphology of individually selected spermatozoa seems to have an impact on pregnancy and delivery rates. This is why some researchers have tried to determine a threshold for the proportion of normal spermatozoa below which ICSI might be inefficient or, conversely, IMSI might be of value. Two studies have evaluated ICSI results as a function of the proportion of normal spermatozoa in the ejaculate, as assessed by MSOME (Bartoov et al., 2002; Falagario et al., 2012). It was shown that normalcy of the sperm nucleus (i.e. a normal shape and with less than one small vacuole occupying <4% of the sperm head area) was predictive of the clinical pregnancy rate after ICSI (Bartoov et al., 2002). Even though <20% of normal spermatozoa were found in the ejaculate with MSOME, no pregnancies were obtained after ICSI performed at a magnification of x200–x400 (Bartoov et al., 2002). Another study contributed additional data on this matter by reporting that the lower the proportion of normal spermatozoa in the ejaculate (as assessed by MSOME, with a threshold of 20%), the higher the risk of choosing a vacuolated spermatozoon with conventional ICSI and the lower the clinical pregnancy rate with conventional ICSI (Falagario et al., 2012). Hence, the proportion of normal spermatozoa (as assessed by MSOME) and the quality of the ICSI outcomes seem to decrease in parallel. Similarly, a group of researchers compared IMSI outcomes as function of the proportion of normal spermatozoa (as assessed by MSOME) in the ejaculate (Berkovitz et al., 2005). They reported that IMSI in which normal, vacuole-free spermatozoa were available for injection (n = 126 IMSI cycles) yielded a significantly higher clinical pregnancy rate per transfer and a significantly lower miscarriage rate relative to IMSI (n = 38 cycles) in which no normal spermatozoa were available (respectively 53% versus 18%, P < 0.01; 10% versus 57%; P = 0.02) (Berkovitz et al., 2005). This finding suggested that even in the absence of normal spermatozoa (according to MSOME), pregnancy rates were low but not null with IMSI. Hence, IMSI might be preferable to ICSI when few normal spermatozoa (as assessed by MSOME) are present in the ejaculate. Very recently, a prospective but non-randomized study (El Khattabi et al., 2013) compared IMSI and ICSI results in patients with teratozoospermia (defined as <10% of normal spermatozoa in a spermocytogram, according to David’s criteria (Auger et al., 2001)). In this study, IMSI (with selection of the ‘best available’ spermatozoon, according to Vanderzwalmen’s criteria (Vanderzwalmen et al., 2008)) yielded higher clinical pregnancy and live birth rates per cycle than ICSI performed at a magnification of ·200 (46% versus 26%, P = 0.001 and 38% versus 20%, P = 0.002, respectively). However, randomized ICSI versus IMSI studies constitute the only way of robustly testing the value of IMSI in cases of teratozoospermia. The only study of this type to date was performed recently in patients with isolated teratozoospermia (defined as <14% of normal spermatozoa in a spermocytogram, according to Kruger’s strict criteria; Knez et al., 2012). In this randomized study, 52 couples underwent IMSI (with selection of the ‘best available’ spermatozoon, according to Vanderzwalmen’s criteria) and 70 underwent ICSI (performed at a magnification of x200–x400). The clinical pregnancy rates per couple were significantly higher for IMSI than for ICSI (48% and 24%, respectively; P < 0.05). However, Knez et al. did not state threshold values for the number or proportion of normal spermatozoa (as assessed by MSOME) below which IMSI (rather than ICSI) could be indicated because the couples were not matched by the degree of teratozoospermia (Knez et al., 2012). In contrast, one can legitimately question whether IMSI is indicated in some types of teratozoospermia. It appears that IMSI was ineffective (or no more efficient than ICSI, at least) in patients with a high proportion of spermatozoa with enlarged heads (Chelli et al., 2010), since normal spermatozoa selected at both ICSI-like and IMSIlike magnifications were potentially aneuploid. In cases of globozoospermia, however, IMSI might enable the selection of spermatozoa with a small acrosomal bud. Indeed, IMSI enabled a successful pregnancy for a couple in which the male displayed almost total globozoospermia (99% of the spermatozoa were round-headed), in the absence of assisted oocyte activation (Sermondade et al., 2011). In summary, IMSI might be indicated in some cases of teratozoospermia. However, given that only one randomized study observed higher clinical pregnancy rates for IMSI than for ICSI in patients with teratozoospermia and the threshold for the number of morphometrically normal spermatozoa (as assessed by MSOME) below which IMSI might produce higher clinical pregnancy and delivery rates than ICSI remains to be determined, further studies of the potential value of IMSI in patients with teratozoospermia are required. Fertility Magazine • Volume 18 • www.FertMag.com – Page 41 ARTICLES IMSI and spermatozoa with nuclear abnormalities IMSI for older women The use of IMSI might help to avoid the selection of spermatozoa with nuclear abnormalities such as chromatin condensation failure, DNA fragmentation and an abnormal chromosomal content. First, given that the nuclear origin of sperm head vacuoles has been linked to chromatin condensation failure, a high proportion of non-condensed chromatin could be considered as an indication for IMSI. No data are available but this question deserves to be evaluated in large-scale, randomized trials. A second potential indication of interest is sperm DNA fragmentation. Indeed, it has been shown that spermatozoa judged to be normal at ICSI-like magnifications can present DNA fragmentation and that normal vacuole-free spermatozoa selected at an IMSI-like magnification are less DNA fragmented than normal spermatozoa selected at an ICSI-like magnification (Hammoud et al., 2013). However, only one study has compared IMSI and ICSI outcomes in patients with high levels of sperm DNA fragmentation (Hazout et al., 2006), in which 72 couples with two or more previous ICSI failures were subdivided according to the proportion of spermatozoa with DNA fragmentation in the male’s whole semen: normal (i.e. <30% of spermatozoa with DNA fragmentation, n = 51), moderately elevated (30–40%, n = 11) and greatly elevated (>40%, n = 10). Overall, IMSI yielded significantly higher clinical pregnancy, delivery and birth rates per transfer than ICSI (performed at a magnification of x200) did, with values of 38% versus 2%, 34% versus 0% and 18% versus 0%, respectively (P < 0.001 for all comparisons). For patients with normal proportions of DNA-fragmented spermatozoa, the birth rate was 19% for IMSI and 0% for ICSI (P < 0.001). The superiority of IMSI over ICSI (in terms of birth rates) was particularly obvious in the group with the highest percentage of DNAfragmented spermatozoa (29% versus 0%, respectively; P < 0.01), although the small sample size reduced the statistical significance (Hazout et al., 2006). Given that the sample size was small and the study was not randomized, it remains to be seen whether IMSI is indicated in cases of sperm DNA fragmentation. This potential indication deserves to be evaluated in large-scale randomized trials. Thirdly, sperm aneuploidy may be considered. Although two studies have reported that spermatozoa with large vacuoles are more likely to be aneuploid than normal, vacuole-free spermatozoa (Garolla et al., 2008) or spermatozoa from whole semen (Perdrix et al., 2011), IMSI was found to be no more efficient than ICSI for selecting euploid spermatozoa in patients with a high proportion of aneuploid spermatozoa (e.g. patients with a high proportion of spermatozoa with enlarged heads (Chelli et al., 2010) or translocations (Cassuto et al., 2011; Chelli et al., 2013)). Hence, it has not been proved that IMSI can be used to efficiently select euploid spermatozoa. Indeed, one can even consider that the opposite is true (i.e. a demonstrated lack of efficiency). Interestingly, only one research group has evaluated IMSI in older women. In a randomized study of patients with a mean age of 37, preimplantation genetic diagnosis showed that ICSI (n = 60) was associated with significantly higher sex chromosome aneuploidy in the embryo and a significantly greater proportion of chaotic embryos (i.e. with two or more chromosomal number abnormalities) relative to IMSI (n = 60). The researchers postulated that, in older women, oocytes were less able to repair the injected spermatozoon’s DNA and hence that use of IMSI to select spermatozoa with fewer nuclear abnormalities could be of value for aged oocytes and older women (Figueira et al., 2011). This indication also deserves to be evaluated in largescale, randomized trials. Page 42 – Fertility Magazine • Volume 18 • www.FertMag.com IMSI for everyone Some authors go as far as to suggest that IMSI can be used for all couples in assisted reproduction programmes. They argue that, relative to ICSI, IMSI increases the likelihood of obtaining a healthy, normal child (Berkovitz et al., 2007). In the latter study, children (n = 176) born after ICSI had a significantly greater risk of major congenital malformations than those born after IMSI (n = 181; 8% versus 3%, respectively; P = 0.02). However, the value of 8% is the highest post-ICSI malformation rate ever reported and casts doubt on the reliability of the study data (for a metaanalysis, see Wen et al., 2012). Hence, one cannot conclude as to the potential value of IMSI for reducing congenital malformations; only randomized studies in a large number of patients are capable of providing robust information. Conclusion A decade after the introduction of IMSI, this technique continues to divide assisted reproduction professionals. There are few confirmed indications of IMSI, partly because few randomized, head-to-head studies have been performed. 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Pregnancy rates are higher with intracytoplasmic morphologically selected sperm injection than with conventional intracytoplasmic injection. Fertil. Steril. 80, 1413–1419. Berkovitz, A., Eltes, F., Paul, M., Adrian, B., Bartoov, B., 2007. The chance of having a healthy normal child following intracytoplasmic morphologically-selected sperm injection (IMSI) treatment is higher compared to conventional IVFICSI treatment. Fertil. Steril. 88 (Suppl. 1), S20. Boitrelle, F., Ferfouri, F., Petit, J.M., Segretain, D., Tourain, C., Bergere, M., Bailly, M., Vialard, F., Albert, M., Selva, J., 2011. Large human sperm vacuoles observed in motile spermatozoa under high magnification: nuclear thumbprints linked to failure of chromatin condensation. Hum. Reprod. 26, 1650–1658. Boitrelle, F., Albert, M., Petit, J.M., Ferfouri, F., Wainer, R., Bergere, M., Bailly, M., Vialard, F., Selva, J., 2013. Small human sperm vacuoles observed under high-magnification are pocket-like nuclear concavities linked to chromatin condensation failure. Reprod. Biomed. Online 27, 201–211. Cassuto, N.G., Bouret, D., Plouchart, J.M., Jellad, S., Vanderzwalmen, P., Balet, R., Larue, L., Barak, Y., 2009. A new real-time morphology classification for human spermatozoa: a link for fertilization and improved embryo quality. Fertil. Steril. 92, 1616–1625. Cassuto, N.G., Le Foll, N., Chantot-Bastaraud, S., Balet, R., Bouret, D., Rouen, A., Bhouri, R., Hyon, C., Siffroi, J.P., 2011. Sperm fluorescence in situ hybridization study in nine men carrying a Robertsonian or a reciprocal translocation: relationship between segregation modes and high-magnification sperm morphology examination. Fertil. Steril. 96, 826–832. Chelli, M.H., Albert, M., Ray, P.F., Guthauser, B., Izard, V., Hammoud, I., Selva, J., Vialard, F., 2010. Can intracytoplasmic morphologically selected sperm injection be used to select normal-sized sperm heads in infertile patients with macrocephalic sperm head syndrome? Fertil. Steril. 93, 1347. Chelli, M.H., Ferfouri, F., Boitrelle, F., Albert, M., Molina-Gomes, D., Selva, J., Vialard, F., 2013. High-magnification sperm selection does not decrease the aneuploidy rate in patients who are heterozygous for reciprocal translocations. J. Assist. Reprod. Genet. 30, 525–530. De Vos, A., Van De Velde, H., Joris, H., Verheyen, G., Devroey, P., Van Steirteghem, A., 2003. Influence of individual sperm morphology on fertilization, embryo morphology, and pregnancy outcome of intracytoplasmic sperm injection. Fertil. Steril. 79, 42–48. De Vos, A., Van de Velde, H., Bocken, G., Eylenbosch, G., Franceus, N., Meersdom, G., Tistaert, S., Vankelecom, A., Tournaye, H., Verheyen, G., 2013. Does intracytoplasmic morphologically selected sperm injection improve embryo development? A randomized sibling-oocyte study. Hum. Reprod. 28, 617–626. El Khattabi, L., Dupont, C., Sermondade, N., Hugues, J.N., Poncelet, C., Porcher, R., Cedrin-Durnerin, I., Le´vy, R., Sifer, C., 2013. Is intracytoplasmic morphologically selected sperm injection effective in patients with infertility related to teratozoospermia or repeated implantation failure? Fertil. Steril. 100, 62–68. Falagario, D., Brucculeri, A.M., Depalo, R., Trerotoli, P., Cittadini, E., Ruvolo, G., 2012. Sperm head vacuolization affects clinical outcome in ICSI cycle. A proposal of a cut-off value. J. Assist. Reprod. Genet. 29, 1281–1287. Figueira Rde, C., Braga, D.P., Setti, A.S., Iaconelli Jr., A., Borges Jr., E., 2011. Morphological nuclear integrity of sperm cells is associated with preimplantation genetic aneuploidy screening cycle outcomes. Fertil. Steril. 95, 990–993. Franco Jr., J.G., Mauri, A.L., Petersen, C.G., Massaro, F.C., Silva, L.F., Felipe, V., Cavagna, M., Pontes, A., Baruffi, R.L., Oliveira, J.B., Vagnini, L.D., 2012. Large nuclear vacuoles are indicative of abnormal chromatin packaging in human spermatozoa. Int. J. Androl. 35, 46–51. Garolla, A., Fortini, D., Menegazzo, M., De Toni, L., Nicoletti, V., Moretti, A., Selice, R., Engl, B., Foresta, C., 2008. Highpower microscopy for selecting spermatozoa for ICSI by physiological status. Reprod. Biomed. Online 17, 610–616. Hammoud, S.S., Nix, D.A., Hammoud, A.O., Gibson, M., Cairns, B.R., Carrell, D.T., 2011. Genome-wide analysis identifies changes in histone retention and epigenetic modifications at developmental and imprinted gene loci in the sperm of infertile men. Hum. Reprod. 26, 2558–2569. Hammoud, I., Boitrelle, F., Ferfouri, F., Vialard, F., Bergere, M., Wainer, B., Bailly, M., Albert, M., Selva, J., 2013. Selection of normal spermatozoa with a vacuole-free head (·6300) improves selection of spermatozoa with intact DNA in patients with high sperm DNA fragmentation rates. Andrologia 45, 163– 170. Hazout, A., Dumont-Hassan, M., Junca, A.M., Cohen Bacrie, P., Tesarik, J., 2006. High-magnification ICSI overcomes paternal effect resistant to conventional ICSI. Reprod. Biomed. Online 12, 19–25. Kazerooni, T., Asadi, N., Jadid, L., Kazerooni, M., Ghanadi, A., Ghaffarpasand, F., Kazerooni, Y., Zolghadr, J., 2009. Evaluation of sperm’s chromatin quality with acridine orange test, chromomycin A3 and aniline blue staining in couples with unexplained recurrent abortion. J. Assist. Reprod. Genet. 26, 591–596. Klement, A.H., Koren-Morag, N., Itsykson, P., Berkovitz, A., 2013. Intracytoplasmic morphologically selected sperm injection versus intracytoplasmic sperm injection: a step toward a clinical algorithm. Fertil. Steril. 99, 1290–1293. Knez, K., Zorn, B., Tomazevic, T., Vrtacnik-Bokal, E., VirantKlun, I., 2011. The IMSI procedure improves poor embryo development in the same infertile couples with poor semen quality: a comparative prospective randomized study. Reprod. Biol. Endocrinol. 29, 123. Fertility Magazine • Volume 18 • www.FertMag.com – Page 43 ARTICLES Knez, K., Tomazevic, T., Zorn, B., Vrtacnik-Bokal, E., VirantKlun, I., 2012. Intracytoplasmic morphologically selected sperm injection improves development and quality of preimplantation embryos in teratozoospermia patients. Reprod. Biomed. Online 25, 168–179. Loutradi, K.E., Tarlatzis, B.C., Goulis, D.G., Zepiridis, L., Pagou, T., Chatziioannou, E., Grimbizis, G.F., Papadimas, I., Bontis, I., 2006. The effects of sperm quality on embryo development after intracytoplasmic sperm injection. J. Assist. Reprod. Genet. 23, 69–74. Moher, D., Liberati, A., Tetzlaff, J., Altman, D.G.PRISMA Group, 2009. Reprint – preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. Phys. Ther. 89, 873–880. Oliveira, J.B., Cavagna, M., Petersen, C.G., Mauri, A.L., Massaro, F.C., Silva, L.F., Baruffi, R.L., Franco Jr., J.G., 2011. Pregnancy outcomes in women with repeated implantation failures after intracytoplasmic morphologically selected sperm injection (IMSI). Reprod. Biol. Endocrinol. 9, 99. Palermo, G., Joris, H., Devroey, P., Van Steirteghem, A.C., 1992. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340, 17–18. Palermo, G.D., Neri, Q.V., Takeuchi, T., Rosenwaks, Z., 2009. ICSI: where we have been and where we are going. Semin. Reprod. Med. 27, 191–201. Perdrix, A., Travers, A., Chelli, M.H., Escalier, D., Do Rego, J.L., Milazzo, J.P., Mousset-Sime´on, N., Mace´, B., Rives, N., 2011. Assessment of acrosome and nuclear abnormalities in human spermatozoa with large vacuoles. Hum. Reprod. 26, 47–58. Sermondade, N., Hafhouf, E., Dupont, C., Bechoua, S., Palacios, C., Eustache, F., Poncelet, C., Benzacken, B., Le´vy, R., Sifer, C., 2011. Successful childbirth after intracytoplasmic morphologically selected sperm injection without assisted oocyte activation in a patient with globozoospermia. Hum. Reprod. 26, 2944–2949. Setti, A.S., Ferreira, R.C., Paes de Almeida Ferreira Braga, D., de Ca´ssia Sa´vio Figueira, R., Iaconelli Jr., A., Borges Jr., E., 2010. Intracytoplasmic sperm injection outcome versus intracytoplasmic morphologically selected sperm injection outcome: a meta-analysis. Reprod. Biomed. Online 21, 450– 455. Talebi, A.R., Vahidi, S., Aflatoonian, A., Ghasemi, N., Ghasemzadeh, J., Firoozabadi, R.D., Moein, M.R., 2012. Cytochemical evaluation of sperm chromatin and DNA integrity in couples with unexplained recurrent spontaneous abortions. Andrologia 44 (Suppl. 1), 462–470. Tanaka, A., Nagayoshi, M., Tanaka, I., Kusunoki, H., 2012. Human sperm head vacuoles are physiological structures formed during the sperm development and maturation process. Fertil. Steril. 98, 315–320. Vanderzwalmen, P., Hiemer, A., Rubner, P., Bach, M., Neyer, A., Stecher, A., Uher, P., Zintz, M., Lejeune, B., Vanderzwalmen, S., Cassuto, G., Zech, N.H., 2008. Blastocyst development after sperm selection at high magnification is associated with size and number of nuclear vacuoles. Reprod. Biomed. Online 17, 617–627. Wen, J., Jiang, J., Ding, C., Dai, J., Liu, Y., Xia, Y., Liu, J., Hu, Z., 2012. Birth defects in children conceived by in vitro fertilization and intracytoplasmic sperm injection: a meta-analysis. Fertil. Steril. 97, 1331–1377. • 510(k) Cleared • Ready-To-Use Page 44 – Fertility Magazine • Volume 18 • www.FertMag.com ARTICLES The spatial arrangement of blastomeres at the 4-cell stage and IVF outcome Goedele Paternot*, Sophie Debrock, Diane De Neubourg, Thomas M D’Hooghe, Carl Spiessens Leuven University Fertility Center, UZ Leuven, Gasthuisberg, Campus Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium *Corresponding author. E-mail addresses: [email protected] (G Paternot), [email protected] (S Debrock), [email protected] (D De Neubourg), [email protected] (TM D’Hooghe), [email protected] (C Spiessens). Goedele Paternot, PhD Goedele Paternot obtained her Masters in biomedical sciences in 2007 at the Catholic University of Leuven, Belgium. In 2011, she finished her PhD in medical sciences entitled ‘‘Selecting the embryo with the highest implantation potential regarding the morphology and developmental potential in a clinical setting’’ at the Leuven University Fertility Centre (ISO 9001:2008 certified). The main research topics of the centre are embryo scoring and the optimization of in-vitro culture conditions, quality of patient care and endometriosis. Abstract This study compared the developmental and implantation potential of tetrahedrally arranged versus nontetrahedrally arranged 4-cell-stage embryos. If the cleavage planes of a 4-cell-stage embryo were perpendicularly orientated, blastomeres were defined as tetrahedrally arranged, while embryos with parallel-orientated cleavage axes were considered as non-tetrahedral embryos. The 4-cell-stage embryos (n = 862) examined in this study were obtained from 299 patients aged <36 years. A total of 299 embryos were transferred as a single-embryo transfer on day 3. This study showed that tetrahedral embryos developed into a 8-cell-stage embryo on day 3 more frequently (307, 45% versus 42, 24%; P < 0.0001) and also developed more frequently into good-quality embryos (461, 67% versus 67, 38%; P < 0.0001) and into excellent-quality embryos (290, 42% versus 34, 19%; P < 0.0001). Tetrahedral embryos had a significantly higher implantation potential (98, 38% versus 9, 21%; P = 0.038), ongoing pregnancy rate (84, 33% versus 7, 16%; P = 0.032) and live birth rate (84, 33% versus 7, 16%; P = 0.032). In conclusion, tetrahedral 4-cell-stage embryos on day 2 developed into embryos of better quality on day 3 with a higher implantation potential and live birth rate compared with non-tetrahedral 4-cell-stage embryos. KEYWORDS: 4-cell-stage embryos, embryo quality, embryo selection, IVF, live birth rate, spatial arrangement This article was published in Reproductive BioMedicine Online, Vol 28, 2014, p198-203, The spatial arrangement of blastomeres at the 4-cell stage and IVF outcome. Copyright Elsevier. It is reprinted here with permission. Introduction The mechanisms behind the development of mammalian embryos from a single cell, the fertilized oocyte, into a complex structure of a blastocyst consisting of different cell lineages has been debated for the last few years (Zernicka-Goetz, 2006). The mouse model has been used to understand the mechanisms underlying the earliest stages of development by different research groups. As mentioned by Cooke et al. (2003), clear definitions are needed for terms like polarity and poles when assessing development poles and planes. While polarity defines an ongoing axis of symmetry throughout pre- and post-implantation embryo development, the term ‘‘pole’’ (animal or vegetal) defines two distinguishing parts of the oocyte from which the A–V plane is formed by a meridional cleavage (Cooke et al., 2003). It has been stated that cell fate decisions in the embryo are crucial for the further development (Edwards and Beard, 1997; Zernicka-Goetz et al., 2009). The first cell fate decisions in the embryo can be explained by different points of views. A recent review focused on the different opinions (Johnson, 2009) and concluded that a combination of the early asymmetric hypothesis (Johnson and Ziomek, 1981), the inside–outside hypothesis (Tarkowski and Wroblewska, 2006) and the polarization hypothesis (Plusa et al., 2005) can explain the differentiation processes and the formation of the axis of symmetry in early development. In addition, studies on transcription factors confirmed the complexity of the process indicating the presence of different mechanisms (Plachta et al., 2011; Vermilyea et al., 2011; Zernicka-Goetz, 2011). Fertility Magazine • Volume 18 • www.FertMag.com – Page 45 ARTICLES Studies on basic embryology of embryos revealed that the oocyte consists of an animal and vegetal pole (Antczak and Van Blerkom, 1997, 1999; Edwards and Beard, 1997; Edwards, 2002). Although not morphologically visible, this polarization was found based on the differential expression of molecular factors (Antczak and Van Blerkom, 1997, 1999; Edwards and Beard, 1997; Van Blerkom, 2007). Maternal proteins like leptin and STAT3 are distributed differentially and are mainly expressed in the cortex of the animal pole (Antczak and Van Blerkom, 1997, 1999). Differences in distribution in human embryos was confirmed by the detection of high concentrations of animal-distributed leptin/STAT3 in one blastomere of a 4-cell embryo, in two blastomeres of the 8-cell embryo and finally to trophectoderm only (Antczak and Van Blerkom, 1999). Controlled cleavage planes leading to correct distribution of proteins might have an influence on important developmental aspects of the embryo (Hansis and Edwards, 2002). Indeed, the pattern of inheritance of polarized domains in daughter cells seems to be determined by how successive equatorial or meridional planes of cell division are oriented with respect to the domains (Antczak and Van Blerkom, 1999). The first meridionally orientated cleavage results in two daughter cells with the same polarity, meaning that both cells contain animal and vegetal cytoplasm. One cell divides meridionally orientated while the other cell divides equatorially (or after the second cleavage plane rotated through 90 degrees) which result in four cells with different polarity (Edwards and Hansis, 2005; Gulyas, 1975). The two daughter cells resulting from the meridionally orientated cleavage have again the full polarity (animal and vegetal cytoplasm). The daughter cells resulting from equatorially cleavage differ in polarity since one cell will contain mostly animal cytoplasm while the other contains mostly vegetal cytoplasm. These specific cleavage planes result in a tetrahedral arrangement of the blastomeres (Edwards, 2002). On the other hand, other researchers have claimed that the pattern of the second cleavage is random (Louvet-Valle´e et al., 2005) and, as a consequence, other topologies were found in 4-cell-stage embryos. However, as mentioned by Gardner (2006) the validity of the latter study is questionable due to the method used. The only clear experimental evidence for the second cleavage was published by Gardner, who described tetrahedral and non-regular tetrahedral 4-cellstage embryos in mice (Gardner, 2002). The last group was subdivided into two groups based on the positioning of the polar body (either in contact with all the blastomeres or lay between only two) (Gardner, 2002) which might result in blastulation failure (Gardner, 2002; Ebner et al., 2012). The current manuscript focuses on the arrangement of blastomeres at the four cell stage (tetrahedral versus non-tetrahedral arrangement). In turn, this characteristic can be useful in embryo selection in a non-invasive way. Tetrahedral 4-cell-stage embryos have been described with a meridional axis and an equatorial axis during the second cleavage (Cauffman et al., 2010). Where the two Page 46 – Fertility Magazine • Volume 18 • www.FertMag.com cleavage planes are oriented otherwise, a non-tetrahedral arrangement of blastomeres may occur (Cauffman et al., 2010; Ebner et al., 2012). This can result in a different distribution of cytoplasm in the daughter cells (for example different distribution of membrane associated or cortical factors (Edwards, 2005) or mitochondria (Van Blerkom, 2007). The aim of this study was to compare in a large set of embryos the developmental and implantation potential of tetrahedral versus non-tetrahedral 4-cell-stage embryos. Materials and methods Patients The 4-cell-stage embryos examined in this study resulted from 299 patients aged <36 years, who received a single-embryo transfer on day 3 at the Leuven University Fertility Centre. Patients entered their first (n = 254) or second (n = 45) IVF/intracytoplasmic sperm injection (ICSI) cycle and were only included once. The stimulation protocol used in this study has been published (Debrock et al., 2010). Patients were excluded when the cycle included biopsy for preimplantation genetic diagnosis or if donor spermatozoa/donor oocytes were used. In total, the dataset contained 862 4-cell-stage embryos of which 299 embryos were transferred on day 3. The study was approved by Institutional Review Board of the University Hospitals Leuven (ML4564, 16 November 2007). Assisted reproduction treatment After oocyte retrieval, the oocytes were washed through four wells and placed in a 4-well dish containing 500 μl fertilization medium (Cook medium; Sydney IVF, Queensland, Australia; or GM501 medium; Gynemed Lensahn Germany) at 37°C, pH 7.25–7.35 per well, under mineral oil. Spermatozoa used for the IVF procedure were prepared using standard density gradient procedures (Isolate; Irvine Scientific, USA). Sperm samples used for ICSI were diluted and were centrifuged twice for 10 min at 300 g. Standard IVF/ICSI procedures were performed 2–6 h after oocyte retrieval. During the IVF procedure, oocytes were inseminated with 300,000 progressively motile spermatozoa per well (5 oocytes in 0.5 ml). In the case of ICSI cycles, injected oocytes were incubated together in a 20 ll culture medium droplet under oil. On day 1 (16–20 h after insemination/injection), fertilization was evaluated. Only normal fertilized oocytes (2 pronuclei) were cultured individually in a 20 ll droplet of culture medium covered with mineral oil. Embryo evaluation Image sequences were recorded of each embryo on day 1 (16–20 h after insemination/injection), day 2 (41–44 h after insemination/injection) and day 3 (66–71 h after ARTICLES insemination/injection) using a computer-assisted scoring system (CellCura FertiMORPH; CellCura Software Solutions, Copenhagen, Denmark). The semi-automatic embryo quality assessment using this system has been recently described by Paternot et al. (2011). Based on the 26 sequential images of the same embryo, the system calculates the total cytoplasmic reduction (which can be interpreted as the degree of fragmentation) and the size of blastomeres. The criteria for distinguishing between a blastomere and a fragment were based on the findings by Hnida et al. (2005) and Johansson et al. (2003). For this study, embryos evaluated by this system with 4 cells on day 2 (44% of the day 2 embryos in the study period) were included and additionally evaluated based on the blastomere arrangement within the zona pellucida. If the cleavage planes were perpendicularly orientated, blastomeres were defined as tetrahedrally arranged (tetrahedral embryos), while embryos with rather parallelly orientated cleavage axes were considered as non-tetrahedral embryos. In cases where the cleavage axes were not exactly perpendicular, the embryos were considered as non-tetrahedral embryos (Figure 1). In addition, the embryos were evaluated using the standard scoring system of the Leuven University Fertility Centre. This embryo evaluation is based on the assessment by an embryologist who evaluated the number and size of blastomeres and the degree of fragmentation. On day 3, embryos with 7, 8 or 9 equally or approximately equally sized blastomeres (<50% difference) and a degree of fragmentation <25% were defined as good-quality embryos. Embryos with 8 equally or slightly unequally sized blastomeres and <10% fragmentation were defined as excellent-quality embryos. On day 3 of development, the best embryo was chosen for transfer based on the standard scoring system. Embryos with at least 6 equally or slightly unequally sized blastomeres and <25% of fragmentation on day 3 were cryopreserved. Utilization rate was defined as the number of embryos available for cryopreservation and embryo transfer over the total number of embryos. A positive b-human chorionic gonadotrophin result was defined as <25 IU/l 14 days after embryo transfer. Implantation rate was defined as the number of gestational sacs observed divided by the number of embryos transferred. Live birth rate was defined as the number of deliveries divided by the number of embryos transferred (Zegers-Hochschild et al., 2009). Statistical analysis The developmental and implantation potential of tetrahedral versus non-tetrahedral embryos was compared using a Fisher Exact chi-squared test with a significance level of 0.05. Results The characteristics of the patients and cycles are listed in Table 1. Fertility Magazine • Volume 18 • www.FertMag.com – Page 47 ARTICLES Developmental capacity nontetrahedral embryos of tetrahedral versus A total of 862 4-cell-stage embryos were evaluated based on the blastomere arrangement including 684 (79%) tetrahedral embryos 178 (21%) non-tetrahedral embryos (Table 2). The percentage of tetrahedral and non-tetrahedral embryos was similar with respect to the type of insemination and the type of culture medium. A total of 459 (80%) and 225 (78%) tetrahedral and 114 (20%) and 64 (22%) nontetrahedral embryos were found in the IVF and ICSI groups, respectively. In the Cook medium, a total number of 436 (79%) embryos were tetrahedral and 113 embryos (21%) were non-tetrahedral embryos which was comparable to the GM501 medium, in which 248 (79%) embryos were tetrahedral and 65 (21%) were non-tetrahedral embryos. When compared with non-tetrahedral embryos, tetrahedral embryos developed more frequently into the 8-cell-stage embryo on day 3 (307, 45% versus 42, 24%, P < 0.0001) and also developed more frequently into goodquality embryos (461, 67% versus 67, 38%; P < 0.0001) and excellent-quality embryos (290, 42% versus 34, 19%; P < 0.0001; Table 2). On day 3, 299 embryos were transferred (256 tetrahedral versus 43 non tetrahedral embryos), 370 embryos were cryopreserved (309 tetrahedral and 61 non-tetrahedral embryos) and 193 embryos were discarded (119 tetrahedral and 74 non-tetrahedral embryos). The utilization rate was significantly higher for the tetrahedral embryos (565, 83%) compared with the non-tetrahedral embryos (104, 58%; P < 0.0001; Table 2). Implantation potential of tetrahedral versus nontetrahedral embryos Since no significant differences were found in the live birth rate of embryos of patients entering their first (77/254, 30%) or second (14/45, 31%) IVF/ICSI cycle, the transferred embryos were analysed as one group (Table 3). A total number of 256 (86%) patients had a single-embryo transfer with tetrahedral blastomere arrangement on day 2 while 43 (14%) patients received an embryo that had a non-tetrahedral blastomere arrangement on day 2. When compared with non-tetrahedral embryos, tetrahedral Page 48 – Fertility Magazine • Volume 18 • www.FertMag.com embryos had a significantly higher implantation rate (98, 38% versus 9, 21%; P = 0.038), pregnancy rate (84, 33% versus 7 16%; P = 0.032) and live birth rate (84, 33% versus 7 16%; P = 0.032; Table 3). The number of biochemical pregnancies (18, 5% versus 2, 7%) and miscarriages (13, 5% versus 2, 5%) were comparable between both groups (Table 3). Discussion The aim of this study was to compare in a large set of embryos the developmental and implantation potential of tetrahedral versus non-tetrahedral 4-cell-stage embryos. An advantage of this study is the use of single-embryo transfers on day 3, which makes it possible to link the orientation of the cleavage planes to the implantation potential of the transferred embryo. In addition, this study contained a large set of 4-cell-stage embryos, which make the results of the developmental potential analysis reliable. The study of Ebner et al. (2012) found fewer embryos with a suboptimal blastomere configuration in IVF cycles compared with ICSI cycles or ICSI cycles including testicular spermatozoa and assumed that the sperm centrosome composition or function might be affected and as a consequence lead to an abnormal blastomere configuration. This could not be confirmed in the current study since no differences were found between IVF and ICSI cycles nor between cycles with or without male infertility. A full comparison between both studies is, however, difficult since Ebner et al. (2012) focused on a specific type of nontetrahedral embryos (planar embryos) while this study did not define subgroups. A significantly higher embryo quality (good and excellent quality) was found for the tetrahedral embryos. In contrast, Ebner et al. (2012) reported only a positive impact on blastocyst development and not on embryo quality on days 2 and 3. Cauffman et al. (2010) also found a higher proportion of good- and top-quality blastocysts when embryos had a tetrahedral shape on day 2 compared with the non-tetrahedral group of embryos. In line with the results reported by Ebner et al. (2012), this study found a higher implantation and live birth rate when a tetrahedral embryo was transferred. This is in contrast with the results described by Cauffman et al. (2010), with the same study design as this study, who found no differences in implantation potential in the group of single- ARTICLES embryo transfers on day 3 (n = 60). The same was found for the single-blastocyst transfers on day 5 (n = 53). This difference could be due to a low number of nontetrahedral transferred embryos (19 on day 3 and 9 on day 5) in the study of Cauffman et al. (2010). This latter finding could be due to the fact that these embryos had a lower quality on day 3 and were therefore not chosen for transfer. The question remains, however, as to why the nontetrahedral group had a lower developmental potential. One hypothesis, as mentioned by Louvet-Valle´e et al. (2005), is that the zona pellucida exerts a constraining effect on the blastomeres. This would indicate that blastomeres of non-tetrahedral embryos are more compressed within the zona pellucida than in tetrahedral embryos, which can in turn affect cell fate or vitality. Based on the finding that the distribution of maternal proteins and regulatory domains are important for embryo development (Antczak and Van Blerkom, 1999; Hansis and Edwards, 2002), another hypothesis is that the distribution of maternal proteins is disturbed in this type of embryos. A nontetrahedral embryo can result from two cleavage patterns. When the 2-cell-stage embryo cleaves with both cleavage axes meridionally orientated, blastomeres retain the polarity of the mother cells and the embryo may recover from the disturbed distribution. However, when a 2-cellstage embryo cleaves with equatorially orientated axes, this can be more detrimental to embryo development since fundamental studies on cell differentiation and cell replacement have found that cells with predominant animal or vegetal cytoplasm have a lower developmental competence compared with fully potent cells (Edwards and Hansis, 2005). This is, however, hypothetical and has to be confirmed in molecular studies. In addition, it is based on the findings of Antczak and Van Blerkom (1999), which are so far not confirmed by others. Ebner et al. (2012) postulated that the mitotic spindle might have been affected which in turn might have led to the observed cleavage anomaly. Another hypothesis is that the contact between blastomeres in non-tetrahedral embryo is lower compared with tetrahedral embryos (Ebner et al. 2008), which can influence the developmental potential. Indeed, it has been stated that an increase in the number of contact points will facilitate compaction because of a larger number of tight junctions available (Ding et al., 1999). The aim of the current study was not to give an answer on the general molecular mechanisms of the blastomere arrangement in human embryos since it focused on the use of the spatial arrangement of a 4-cellstage embryo as a non-invasive morphological characteristic to improve the embryo selection. In conclusion, this study shows that tetrahedral 4-cell-stage embryos have a higher developmental and implantation potential. This characteristic should be included in the embryo evaluation. In the case of selecting a single embryo for transfer, one should choose an embryo with a tetrahedral arrangement over a non-tetrahedrally arranged embryo. References Antczak, M., Van Blerkom, J., 1997. Oocyte influence on early development: the regulatory proteins leptin and STAT 3 are polarized in mouse and human oocytes and differentially distributed within the cells of the preimplantation stage embryo. Mol. Hum. Reprod. 3, 1067–1086. Antczak, M., Van Blerkom, J., 1999. Temporal and spatial aspects of fragmentation in early human embryos: possible effects on developmental competence and association with the differential elimination of regulatory proteins from polarized domains. Hum. Reprod. 14, 429–447. Cauffman, G., Verheyen, G., Haentjens, P., Devroey, P., Liebaers, I., Van de Velde, H., 2010. Developmental Capacity and Pregnancy Rate of Tetrahedral Versus Non-tetrahedral 4-Cell Stage Human Embryos. Oral Presentation, Annual Meeting ESHRE Rome, (O-153). Cooke, S., Tyler, J.P.P., Driscoll, G.L., 2003. Meiotic spindle location and identification and its effect Fertility Magazine • Volume 18 • www.FertMag.com – Page 49 ARTICLES on embryonic cleavage plane and early development. Hum. Reprod. 18, 2397–2405. Ding, T., Rana, N., Dmowski, W.P., 1999. Intracytoplasmic sperm injection into zona-free human oocytes results in normal fertilization and blastocyst development. Hum. Reprod. 14, 476–478. Debrock, S., Melotte, C., Spiessens, C., Peeraer, K., Vanneste, E., Meeuwis, L., Meuleman, C., Frijns, J.P., Vermeersch, J.R., D’Hooghe, T.M., 2010. Pre-implantation genetic screening for aneuploidy of embryos after in vitro fertilization in women aged at least 35 years: a prospective randomized trial. Fertil. Steril. 93, 364–373. Ebner, T., Shebl, O., Moser, M., Sommergruber, M., Tews, G., 2008. Developmental fate of ovoid oocytes. Hum. Reprod. 23, 62– 66. Ebner, T., Maurer, M., Shebl, O., Moser, M., Mayer, R.B., Duba, H.C., Tews, G., 2012. Planar embryos have poor prognosis in terms of blastocyst formation and implantation. Reprod. Biomed. Online 25, 267–272. Edwards, R.G., 2002. Aspects of the molecular regulation of early mammalian development. Reprod. Biomed. Online 6, 97–113. Edwards, R.G., 2005. Genetics in polarity in mammalian embryos. Reprod. Biomed. Online 11, 104–114. Edwards, R.G., Beard, H.K., 1997. Oocyte polarity and cell determination in early mammalian embryos. Mol. Hum. Reprod. 3, 863–905. Edwards, R.G., Hansis, C., 2005. Initial differentiation of blastomeres in 4-cell human embryos and its significance for early embryogenesis and implantation. Reprod. Biomed. Online 11, 206–218. Gardner, R.L., 2002. Experimental analysis of second cleavage in the mouse. Reprod. Biomed. Online 17, 3178–3189. Gardner, R.L., 2006. Weaknesses in the case against prepatterning in the mouse. Reprod. Biomed. Online 12, 144–149. Gulyas, B.J., 1975. A reexamination of cleavage patterns in eutherian mammalian eggs: rotation of blastomere pairs during second cleavage of the rabbit. J. Exp. Zool. 193, 235–248. Hansis, C., Edwards, R.G., 2002. Cell differentiation in the preimplantation human embryo. Reprod. Biomed. Online 6, 215–220. Hnida, C., Agerholm, I., Ziebe, S., 2005. Traditional detection versus computer-controlled multilevel analysis of nuclear structures from donated human embryos. Hum. Reprod. 20, 665–671. Johansson, M., Hardarson, T., Lundin, K., 2003. There is a cutoff limit in diameter between a blastomere and a small anucleate fragment. J. Assist. Reprod. Genet. 20, 309–313. Page 50 – Fertility Magazine • Volume 18 • www.FertMag.com Johnson, M.H., 2009. From mouse egg to mouse embryo: Polarities, axes and tissues. Annu. Rev. Cell Dev. 25, 483–512. Johnson, M.H., Ziomek, C.A., 1981. The foundation of two distinct cell lineages within the mouse morula. Cell 24, 71–80. Louvet-Valle´e, S., Vinot, S., Maro, B., 2005. Mitotic spindles and cleavage planes are oriented randomly in the two-cell mouse embryo. Curr. Biol. 15, 464–469. Paternot, G., Debrock, S., D’Hooghe, T.M., Spiessens, C., 2011. Computer-assisted embryo selection: a benefit in the evaluation of embryo quality? Reprod. Biomed. Online 23, 347–354. Plachta, N., Bollenbach, T., Pease, S., Fraser, S.E., Pantazis, P., 2011. Oct4 kinetics predict cell lineage patterning in the early mammalian embryo. Nat. Cell Biol. 13, 117–123. Plusa, B., Frankenberg, S., Chalmers, A., Hadjantonakis, A.K., Moore, C.A., Papalopulu, N., Papaioannou, V.E., Glover, D.M., Zernicka-Goetz, M., 2005. Downregulation of Par3 and aPKC function directs cells towards the ICM in the preimplantation mouse embryos. J. Cell Sci. 118, 505–515. Tarkowski, A.K., Wroblewska, J., 2006. Development of blastomeres of mouse eggs isolated at the 4-and 8-cell stage. J. Embryol. Exp. Morphol. 18, 155–180. Van Blerkom, J., 2007. Translocation of the subplasmalemmal cytoplasm in human blastomeres: possible effects on the distribution and inheritance of regulatory domains. Reprod. Biomed. Online 14, 191–200. VerMilyea, M.D., Maneck, M., Yoshida, N., Blochberger, I., Suzuki, E., Suzuki, T., Spang, R., Klein, C.A., Perry, A.C.F., 2011. Transcriptome asymmetry within mouse zygotes but not between early embryonic sister blastomeres. EMBO J. 30, 1841–1851. Zegers-Hochschild, F., Adamson, G.D., de Mouzon, J., Ishihara, O., Mansour, R., Nygren, K., Sullivan, E., van der Poel, S., 2009. The international committee for monitoring assisted reproductive technology (ICMART) and the World Health Organization (WHO) revised glossary on ART technology. Hum. Reprod. 24, 2683–2687. Zernicka-Goetz, M., 2006. The first cell-fate decisions in the mouse embryo: destiny is a matter of both chance and choice. Curr. Opin. Genet. Dev. 16, 406–412. Zernicka-Goetz, M., 2011. Proclaiming fate in the early mouse embryo. Nat. Cell Biol. 13, 112–114. Zernicka-Goetz, M., Morris, S.A., Bruce, A.W., 2009. Making a firm decision: multifaceted regulation of cell fate in the early mouse embryo. Nat. Rev. Genet. 10, 467–477. Don’t leave it to chance ® be prepared with Coda . The Coda ® Inline ® Filter patented technology is proven to effectively remove VOCs and CACs. They are CE certified and manufactured in a clean room. Each Coda ® Inline ® Filter contains a HEPA filter and can last between 3-6 months. All Coda ® Inline ® Filters are FDA 510(k) cleared, ISO 13485:2003 medical certified and made in the USA. Coda® Xtra Inline® Filter – Green with Aldasorb® Coda® Xtra Inline® Filter – Blue Coda® Regular Inline® Filter – Purple Pure activated carbon and Aldasorb ® Pure activated carbon Pure activated carbon Removes 98% of VOCs and aldehydes from gas 20 times absorptive capacity 10 times absorptive capacity Smaller footprint 25 times absorptive capacity T: 1-800-720-6375 ¿ F: 1-519-826-6947 Reduced weight ¿ Intl.: 001-519-826-5800 ¿ [email protected] ¿ www.LifeGlobalGroup.com Fertility Magazine • Volume 18 • www.FertMag.com – Page 51 PATIENT’S CORNER Guidelines for the design of an IVF Laboratory by Gloria Calderón, PhD & Nuno Costa Borges, PhD Embryotools SL, Barcelona, Spain You can contact Gloria Calderón at [email protected] and Nuno Borges at [email protected]. D esigning a new human IVF laboratory is not an easy task. Several important factors must be taken into account in order to construct a high standard IVF laboratory, which we know to be the key to success of any ART center. One of the most important factors to be considered is the location of the site. Specialists working in human IVF, are aware of the importance of air quality when working with human gametes and embryos. Air quality can vary enormously depending on the geographic location of the lab. Commonly, a clinic located in a small village with little traffic, and surrounded by parks or a forest, will give the site one of the best external air qualities. On the contrary, if the site is located in a crowded city, the air quality might not be optimal. Therefore, when the lab is designed, an appropriate ventilation system must be considered, in order to protect and isolate human gametes and embryos from external pollution. In this sense, the first recommended step would be to measure the concentrations of volatile organic compounds (VOC) in the external air. After performing a deep examination of the results, we should be able to design, with the help of engineers, an appropriate ventilation system that covers the needs of the chosen site. It is also important to emphasize that IVF labs and operating rooms should not share ventilation systems or air flow. The embryology and cryopreservation laboratories, should have the highest positive pressure with airflow towards the operating rooms and sperm laboratory. Another important aspect to take into account is the size and shape of the building, because this will define the final layout of the center, including the size and location of the IVF Laboratories. The number of IVF cycles that the center is expected to carry out within its first 10 years is also valuable information during the design phase. This allows us to better determine how to dimension the IVF laboratories, avoiding the need for disruption of renovation and extension of the clean areas due to the lack of space in the following years. Another very important design aspect to consider is the materials used for construction. We must always avoid the use of plastics, glue, wood, paint, and in general, all components that could potentially generate long-duration VOC emissions. Taking care of these details will help us to preserve the quality of the air. In recent years, the use of prefabricated modules for the construction of IVF laboratories has become very common Gloria Calderón, PhD Nuno Borges PhD and convenient. These modules offer several advantages over classic construction, including antibacterial finishes, layout and size high flexibility, and maximum asepsis. They also provide active support for the air treatment system, as their structure has been designed for compartmentalization, allowing for perfect integration of any kind of equipment. Moreover, it is highly advantageous to use prefabricated SMS® (solid mineral surfaces), panels that are bacteriostatic, uniform, non-porous and very easy to clean or disinfect. As mentioned before, in order to maintain stable air quality conditions inside the lab, operating rooms and laboratories must be sealed and positively pressurized, in order to protect these spaces from the other areas of the building. Therefore, it is recommended the installation of an independent ventilation system, together with VOC and HEPA filters, as well as the conventional filters types with biggest porous size at the beginning of the ventilation system. Additionally, a UV light system should be included at the end of the ventilation system for a more efficient removal of VOCs and better protection against possible bacterial, viruses or fungus contamination. The ideal layout plan of an IVF lab should include at least 3 different rooms: one for cryopreservation, a second one for sperm preparation, and a third one for embryology work. The embryology lab should be connected through a door with the cryopreservation lab. The cryopreservation lab should be wall to wall with the liquid nitrogen tank room. This will not only save liquid nitrogen, but will also provide a direct connection of the liquid nitrogen to the tank storage of gametes and embryos. The sperm lab can One of the most important factors to be considered is the location of the site. Page 52 – Fertility Magazine • Volume 18 • www.FertMag.com PATIENT’S CORNER be located inside the clean area, but not necessarily next door to the embryology or cryopreservation lab. It is also advisable to design a storage room close to the IVF area, with enough space to keep all material and disposables needed in all labs. In all laboratories it is convenient to avoid the use of excessive furniture inside the IVF area, and all surfaces should be made of stainless steel and glass. The location of the equipment inside the laboratory is also another key point that must be considered during the design of a new lab. We recommend organizing the lab in individual working units/workstations. Each of these should include an anti-vibration table with the inverted microscope and adapted micromanipulation system; a vertical laminar flow hood with heated surface and a working incubator. Regarding incubators, we should tend to use bench top incubators for gamete and embryo culture, because they are more stable than regular cell culture incubators. They can be located on one of the laboratory walls on shelves, stacked at different heights to save space. Our last recommendation would be to install an alarm system to continuously monitor the equipment, in particular, all incubators, storage tanks and refrigerators with all media. Last, but certainly not least, the budget available for the project needs to be sufficient to provide for fully equipped laboratories with high quality and state-of-the art equipment. We believe that by following these simple but crucial rules, the performance of your IVF lab can improve. global® DMSO Blastocyst Vitrification Easy-To-Use and a High Performance System global® DMSO Blastocyst Vitrification Kit global® DMSO Blastocyst Warming Kit Fertility Magazine • Volume 18 • www.FertMag.com – Page 53 PATIENT’S CORNER Cassandra’s prophecy: why we need to tell the women of the future about age-related fertility decline and ‘delayed’ childbearing Jane Everywoman1 C/O Duck End Farm, Park Lane, Dry Drayton, Cambridge CB23 8DB, UK E-mail address: [email protected] Abstract This anonymized paper describes the author’s experience of age-related infertility and unintended childlessness. It outlines her journey from diagnosis to treatment success and clinical pregnancy through assisted reproduction using oocyte donation, followed by subsequent early miscarriage. It makes subjective observations about treatment she received and presents her impressions of how discourses of knowledge dissemination, communication and care were constructed in the organizations she encountered. It sets her own reflections alongside broader observations on the challenges facing women today when planning a family and draws attention to what she perceives to be the misleading myths and misunderstandings concerning reproduction that these women are now subject to. In the light of this, it offers some suggestions for modified public health messages and new approaches to sex education and health screening that may consequently help to empower tomorrow’s women (and men) to take full control over their reproductive lives in the 21st century. The paper takes as its mascot the figure of Cassandra, daughter of King Priam and Queen Hecuba. She was loved by Apollo, but resisted him. In consequence, he rendered useless the gift of prophecy that he had bestowed on her by causing her predictions never to be believed. KEYWORDS: abortion, assisted reproduction, infertility, IVF, oocyte donation, premenopause 1Pseudonym This article was published in Reproductive BioMedicine Online, Vol 27, 2013, p4-10, Cassandra’s prophecy: why we need to tell the women of the future about age-related fertility decline and ‘delayed’ childbearing. Copyright Elsevier. It is reprinted here with permission. A personal story At the age of 32, I suddenly began to suffer considerable, cyclical gynaecological discomfort. By age 37, my weight was inexorably increasing and my menstrual flow became lighter and would begin unpredictably between day 21 and 35. I had the occasional night sweat and hot flush. Trips to the doctor where these symptoms were discussed alongside other things elicited no further comments. During this time, I had started trying to conceive because, even though married 5 years before, at age 27, I had earlier lacked the emotional and financial confidence to have a baby. As a school teacher and academic researcher, I had made many sacrifices to gain several higher degrees and professional certifications. By 32, I had a full-time job, a husband and a comfortable home – things that were hard and time consuming to attain – and I felt the conditions were exactly right to begin the family that was always firmly on my agenda, if not for a long time at the top of it. After the first time of unprotected sex on cycle day 14, I looked forward to my forthcoming offspring. To my surprise, no baby was conceived. I had assumed, like many of my contemporaries (Edwards, 2011), that sex without contraception always resulted in pregnancy. This was because since childhood I had been educated to believe that ‘all’ acts of unprotected intercourse would inevitably Page 54 – Fertility Magazine • Volume 18 • www.FertMag.com result in a live birth – which, if it was unplanned, was naturally morally suspect and to be avoided until ‘the right time’, which was always left undefined. Months passed and no baby materialized. Time and time again, pregnancy announcements came from family, friends and colleagues and irritated my profound and unrelenting pain. I forgot what it was like not to be constantly and secretly sad, and carrying the burden of loss of control blanched the colour from every aspect of my life all the time. At around age 34/35, I consulted a doctor, who told me that the hormonal imbalances that I informed her I suspected I had (but could not make sense of), would be easily ‘corrected’ by a pregnancy. Another GP told me to ‘wait 2 years’, because women are only assessed as infertile if they have been trying unsuccessfully for that long. Respectful around doctors, I went away, misconstrued his advice and waited for 24 attempts at trying to conceive, which actually turned into three chronological years, possibly because of my unacknowledged reactive depression and busy work schedule. During this time, my husband and I also attended a family planning and women’s health clinic because, we assumed, our issues were surely relevant to their mission. Yet we were turned away. At smear tests, nurses in whom I confided told me to ‘have patience’. Another GP consultation at age 37 where I again talked of my PATIENT’S CORNER lack of pregnancy alongside other issues simply elicited the response ‘go home and relax’. Still no baby resulted. I purchased ovulation sticks which apparently showed I was ovulating. What I was unaware of at the time is that these devices detect merely hormonal surges. They do not prove an egg has been released nor measure its potential to develop into a live birth and therefore they are, in some contexts, misleading. I felt ashamed, vaguely and constantly unwell and very confused, especially since the clinicians in whom I placed my trust seemed unconcerned. I disclosed nothing to close family or friends because of the very private nature of my struggles. I assumed it was something I was doing wrong, and given that women are so urgently told to use contraception at the very earliest opportunity to prevent pregnancy, I believed that it must have been my inadequacy alone. The reason I felt I had plenty of time for childbirth was also everywhere around me. No one in my wide and international social and professional circle had given birth before about 33. In media reports, numerous celebrities were also getting pregnant for the first time at 40+, often with twins (Freedman, 2007; Mamamia, 2007; Morrell, 2008; Sager, 2010). Some were admitting to the use of IVF treatment, but rarely did they (understandably) disclose how many pregnancies were lost in the process, whether donor eggs from a much younger woman were used, the toll taken on their own or their baby’s bodies, what the total cost was, or the number of attempts needed for a live birth (Mail Foreign Service, 2010). Therefore I ‘knew’ that IVF would easily ‘cure’ me – if only I had the courage to go to a clinic – and, since no GP had formally referred me, I still assumed all I had to do was wait, and I was also terrified at the prospect of the cost. No one had explained to me how the process would work, how long it might take, how it would be paid for, what tests might be done and what criteria would be used to make me eligible for treatment. As I still menstruated – to me an obvious sign of fertility – I firmly believed my pregnancy was just around the corner. Having never been given a diagnosis, I wonder, with hindsight, if my symptoms were those of perimenopause, the transitional period of hormonal shifts that can, in some women, occur years before final menopause (Corio, 2000). Tragically, this had been suggested to me by a herbalist I consulted only in desperation. However, because for me she had less authority than a formally-qualified clinician, I doubted her. Aged 42 now, I expect my menopause to happen around age 43–45 – just within the ‘normal’ spectrum. I know this now because late in 2008 at the age of 39 I finally attended a private IVF clinic through selfrecommendation. Two quick but expensive blood tests told me I had a raised FSH and low anti-Mu¨llerian hormone. They revealed my ovaries were almost ‘closed down’ and my egg reserve was virtually completely empty. I had nothing to make a baby with, no matter how regularly I had sex. The clinic doctor informed me my menopause was imminent and only the use of donor eggs would lead to pregnancy. He said, ‘Treatment here costs £8000 per cycle including drugs and you’ll probably need three cycles for it to work. Our waiting list is about 2+ years and it costs £1500, refundable against future treatment, to put your name on the list now.’ Since then, my personal IVF experience means that my husband and I have haemorrhaged money as well as blood. Our life savings are around £18,000 lighter, for the NHS would not treat me because at age 39/40 I was by then ineligible for funding (although no one ever pointed this prospect out to me when I had previously sought advice). I have had two gynaecological operations, endless vaginal scans and blood tests whose results were essentially meaningless as there is currently no formal consensus on what they reveal (Rai et al., 2005). I managed unexpectedly quickly to get a first attempt at donor egg IVF through egg sharing at a second clinic (where it also cost a nonrefundable £450 simply to place my name on the waiting list), but it failed. A second attempt a year later gave a positive pregnancy test. Two weeks afterwards, a missed miscarriage was diagnosed. After this, I was told by a nodoubt well-meaning clinician that all I needed to do was simply ‘try harder’ to get pregnant. This puzzled me, since I had exhausted my life savings to get that far and taken the most powerful drugs I had ever used in order to conceive. I wondered what more I could possibly have done. Making sense of my experience was made all the more difficult because staff, perhaps desperate to convince me (and reassure themselves) that the process does work sometimes, bombarded me with anecdotes of unexpected IVF ‘miracle’ babies, who had been born to patients with very poor prognoses. This merely intensified my sense of personal failure. As I passed out during the operation to remove the products of conception, the anaesthetist told me that it was better that the embryo had miscarried because it would not have been viable. I was crying as I drifted into unconsciousness and struggled to tell him that it was a donor egg from some anonymous woman, and I had no more money to pay for another. Becoming conscious one hour later, the nurse by my side told me that miracles do happen as her relative at age 44 gave birth to her first child that year. I was crying even before I had opened my eyes. Once recovered from the miscarriage, I was bound to try a third cycle with embryos cryopreserved from the previous attempt (if only to rule out a pregnancy if I subsequently wanted to adopt). Mere ‘shadows of possibility’, treatment with these resulted in an initial negative result, which was subsequently reinterpreted as a ‘weak positive’. I was then instructed to continue medications until another blood test could determine whether there was an ectopic pregnancy. To pay for all this, my husband and I took on two jobs each, and for 2 years juggled these with appointments for time-consuming treatments and tests in several different places. Although infertility consumed every element of our lives for what has ultimately amounted to 10 years, I did Fertility Magazine • Volume 18 • www.FertMag.com – Page 55 PATIENT’S CORNER not confess to a soul outside of my clinics that I was unable to do what I perceived everyone could do effortlessly – get pregnant – and my sense of shame cut into my heart. This was even more so since I personally felt that clinical staff in IVF centres tended to hint the lack of success was my failure rather than that of the science and technology they used. This is probably because from their perspective the treatments work (as they sometimes do), and it is only a deficiency in women’s stamina that makes them withdraw before their goal is achieved (Cross, 2010). Ultimately I had to resign from work in order to fully recover from my experiences and this merely exacerbated my sense of loss of control. This writing describes only my personal truth – a narrative that could be told in many ways. Some may dismiss me as naïve for, as one journalist has said, ‘it would be impossible . . . to exist in society and not know that having children became problematic in one’s mid30s’ (Williams, 2005). Whilst initially I would have agreed with this, I have reflected at length on my experiences and assumptions and have wondered about just how flawed or unrepresentative they might be and what can be learned from them. Although admitting to ignorance about reproduction in our information-saturated, highly sexualized culture is embarrassing, I am also beginning to suspect that I am definitely not alone and that assuming ‘everybody knows’ may be far too simplistic an explanation (Birrittieri, 2005, 2006; Bretherick et al., 2010; Bunting and Boivin, 2008; Cooke et al., 2010; Daly, 2011; Peterson et al., 2012). Indeed, my private tragedy transcends the personal and embraces the political domain, as it speaks for the asyet unidentified community of women in the developed world who inevitably and sadly will, in future years, trudge the arduous path I have traversed. I would argue that what women know and understand and what they relate to and use in their own lives may be subtly different things (Elkind, 1981). I did know about fertility ‘decline’ with age; but I did not really understand it in an adequate way, perceiving it merely as ‘risk’ easily managed through excellent prenatal care. I had no idea that late-age pregnancy is ultimately difficult because of a decline in egg quantity and quality – the key building blocks of life. Nor did I really believe the risk especially in the light of media reporting of first babies at age 40+ and the fact that no clinician outside my fertility clinic communicated to me that age was a relevant issue (Cohen, 2010). For me, highly educated and ‘well informed’, it was extremely difficult to relate to my own life all the competing, confused and fragmented threads of information that I had gleaned since childhood about pregnancy and fertility from a wide variety of sources and then to step back in order to see that in fact I really was ageing and moving fast towards chronological middle age and reproductive old age. For I still felt young, looked young and perceived myself to be in relatively good health. Thus, I feel it is reasonable to claim that I am as much Page 56 – Fertility Magazine • Volume 18 • www.FertMag.com a victim of a deficiency of information as I was solely responsible for my miserable plight. I lacked key information to make appropriate choices about reproduction as a result of the sex education I had received and, despite great effort, I failed to access it from public health agencies. I was also falsely given hope by the oblique reporting of ‘miracle’ babies born through IVF (Jenny, 2010). I therefore write because, as a teacher, I have a duty of care to ensure that the next generation of adults, who could be struggling with many of the issues I had to make sense of on my own, are well informed. For, statistics show that there is a global trend for delaying childbearing beyond 30 years of age and by 2007, 20% of British women had waited until after 35 to begin their family (Botting, 1992; BUPA, 2009; Campbell, 2010; Cooke et al., 2010). Moreover, infertility issues affect around one in six of the population (Templeton, 1992). Gustafsson (2001) estimates that as many as half of women in their thirties will have some sort of fertility problem and she also comments on the ‘spectacular’ increase in the percentage of women who have not yet given birth by 34 in Europe, of whom many, as a result, will be ‘ultimately childless’. Myths and misunderstandings A number of misunderstandings and myths may need to be addressed. First, it may be that teens and young adults are genuinely confused about what are, in the developed world, stark dissonances between chronological, reproductive and cultural age. A well-groomed celebrity can be 47, look 32 and be perceived as ‘young’ especially since life expectancy is around 70–80 years. As her egg reserve was fixed at birth and from that point began a slow decline, reproductively she is almost certainly unable to conceive her own genetic child, or indeed any child unless donor eggs from a much younger woman are used. When the journalist Kasey Edwards was told by her gynaecologist that she was approaching infertility, she responded ‘But I’m only 32 . . . Surely that’s not old’ (Edwards, 2011, p. 17). For the girls in schools now, 40 really is the new 20, a phrase increasingly in common parlance (Cougar Town, 2010). Second, such is the entrenched potency of the ‘family planning’ rhetoric and so extensive is contraceptive use in developed societies that anything to do with baby making carries with it a strong sense of personal control, as if implicit in every act of contraception is a guaranteed conception and live birth that we simply choose to defer (Marks, 2001). This may drive men and women to ‘delay’ families precisely because they partly assume that starting them is their decision alone. Posters in GP surgeries talk of ‘planning’ (not ‘hoping for’) a baby; ovulation monitors indicate when sex should occur to conceive; the habit of The Pill allows you to control when you will menstruate. As Kasey Edwards has written, ‘in sex-education classes in school, it’s implied that there is a one-to-one relationship between unprotected [intercourse] and getting knocked up’ PATIENT’S CORNER (Edwards, 2011, p. 105) and similar assumptions are not uncommon (Kardashian, 2011). Yet, in clinical reality, if 100 ova are exposed to spermatozoa at ovulation, only around 31% will become a live birth (Leridon, 1977, p. 79; Macklon et al., 2002). As one author has pointed out, these discourses mean that for some women reproduction is viewed as so inevitable it is relegated to the lowest point on their ‘to do’ list. She admitted ‘I didn’t consider motherhood as any sort of accomplishment, because . . . anybody could do it . . . that’s hardly an achievement’ (Edwards, 2011, p. 10). Third, it seems that there exist key misunderstandings of what IVF treatment can actually achieve (Adashi et al., 2000; Hashiloni-Dolev et al., 2011). It may be that many perceive it (before they try it) as a failsafe ‘cure’ for infertility (Parkin, 2007). As one journalist admitted, ‘I honestly thought that you start to worry at 38 and then you go to doctors at 39 and then you would have treatment to help you get pregnant’ (Bewley et al., 2009, p. 348). Thus, many do not view it as a treatment that must work in harmony with one’s own body and that relies on viable eggs or spermatozoa being available. Furthermore, it is in reality a treatment with a rate of ‘success’, which, overall, currently hovers around the 20–30% mark worldwide (Bewley, 2005; Throsby, 2001). For the 39,879 women who had IVF in the UK in 2008, there were only 12,211 successful live births resulting from 50,687 cycles of treatment (HFEA, 2011). Therefore, it is also reasonable to describe it as an unreliable and unpredictable process which does not work for the majority trying it and when it does, its success is often the result of multiple, timeconsuming, expensive attempts. It is a stark ‘truth’ that, if around 4 million IVF babies were born since treatment first began, many more millions of couples who were treated were left empty handed (Brown, 1979; Groskop, 2010; Nobel Prize, 2010). Indeed, as one clinician has admitted, ‘We would probably have said 20 years ago that by 2008 we will be able to pick the best embryo to transfer but it is still beyond our grasp’ (Bewley et al., 2009, p. 330). Moreover, IVF is regularly perceived by the lay person as a treatment specifically for older women. As one woman has reported ‘. . . with all these medical breakthroughs women can have children later and later . . . it means I can put off having children until my forties. It’s such a relief’ (Hewlett, 2002a, p. 184). As the Princeton-educated actress Brooke Shields exclaimed when told she would need IVF, ‘Isn’t that for older women? I’m only thirty-six’ (Shields, 2005, pp. 8–9). Hewlett (2001) and Hewlett (2002b) reported that 90% of women aged 28–40 who participated in her 2001 study stated that they believed that reproductive technologies would allow them to get pregnant into their forties. IVF does tend to be used more by older women because it is precisely that group that struggles most to conceive. Yet after around 36/37, ‘success rates’ plummet and by the age of 40 they are almost negligible for patients using their own eggs (HFEA, 2011). Thus, it is possible to argue that IVF is in reality a treatment that usually works best on younger people and it cannot be the guaranteed, failsafe back-up plan for older women as two recent movies imply (The Back-up Plan, 2010; The Switch, 2011). Similarly, it is quite possible that miscarriage rates are also greatly misunderstood. Unfortunately, these can occur in one in four or five pregnancies overall and in one in two women over 40 years old (Holman et al., 2000; KlugerBell, 2000; Leridon, 1977, pp. 54, 63–65, 75–79; Miscarriage Association, 2010). Although seldom discussed, upon disclosure a woman can often find that they are far more common than she originally anticipated. Such an experience can delay pregnancy plans and, whilst waiting for recovery from them, fertility still continues its inexorable decline. Moreover, many women may be uncertain of the wide individual variation in levels of fertility across the population and the fact that it is not something fixed and unchanging, but a characteristic that briefly blossoms and then declines in females and males (although comparatively ‘slowly’ in the latter) (Faddy et al., 1992). For example, analysis of school curriculum materials reveals that it is often unhelpfully characterized as something ‘without an end’ and universally guaranteed, rather than something that is, for all women, finite (Kisby Littleton, 2012). Neither are women aware that some members of the medical community have suggested that subfertility and infertility can occur at least a decade or more before menopause, which can vary widely between about 40 and 60 years (Lobo, 2003; te Velde, 1997). Whilst some women do give birth easily in their late thirties and beyond, what many of them (or those ‘planning’ to emulate them) will probably not really understand is that it is ultimately biological serendipity that permitted this and that personal volition only plays a secondary role. New directions for education In Greek mythology, the beauty of Cassandra (daughter of Queen Hecuba and King Priam) caused Apollo to grant her the gift of prophecy. When his love was not returned, Apollo placed a curse on her so that no one would ever believe her predictions until they had come to pass (Harvey, 1986, p. 92). She thus possessed a combination of deep understanding and powerlessness and I know how she must have felt. Although positive reproduction experiences are thankfully common, similar stories to mine have been, and will continue to be, replayed around the developed world as women are educated to expect fulfilling careers, may have to work for economic reasons, anticipate meeting the perfect partner and continue to struggle with work–life balance and the decision to have a baby whilst they are still enmeshed in career structures suffused with patriarchal values (Barker, 2009; Harris, 2005; Hewlett, 1988). Although in the short term we cannot change many of the very complex issues that lead to this situation, we can modify the subsequent public health message that the women of the future experience at the school and adult level – if we have the will to do it. Fertility Magazine • Volume 18 • www.FertMag.com – Page 57 PATIENT’S CORNER Twenty-first century culture tells women that they can ‘outsmart Mother Nature’ (Tampax, 2011a, 2011b). At present, a ‘two-dimensional’ version of reproduction is also disseminated in schools – man + woman + unprotected sex = guaranteed baby – and, until some discover otherwise, most carry this notion into adulthood (Skoog Svanberg et al., 2006). Most adults know the options for dealing with unplanned parenthood; most know how to nourish a developing baby in utero and we mostly have complete faith in the medical profession to care for our baby and young child. Most dangerously, many feel a strong moral sense that, if a family should be ‘delayed’, it is simply personal choice, a lack of a suitable partner and is no-one else’s business. Most harbour a vague notion that, if they are so unlucky as to experience a problem conceiving, an IVF clinic can provide a ‘cure’ (Dickson, 1992). These beliefs have validity, but together they may not make up the whole story that couples need to know now. Thus although my account does not have the epistemological authority of a formal study, it is still possible to make some suggestions on the basis of it (DasGupta and Charon, 2004). While it should not be necessary to cause anxiety around reproduction, it may be that now more balanced and honest information needs to be disseminated, first in schools and then later also supported by official public health messages disseminated at the grassroots level and directed at young adults – not through media headlines, ill-informed TV documentaries or websites of dubious quality (Marriott et al., 2008). This information should be designed to empower the women who will grow up in cultures very different from those in which ‘developed’ society’s original messages about sex education were first formulated (Hall, 2009). It may be that these women should be encouraged to take adequate notice of their natural menstrual patterns if they have used chemical contraception for a long time (Grigoriadis, 2010), and also be sensitively warned of the relative frequency and potential disruption of early miscarriage (Tough et al., 2007). They need to be briefed about the wide spectrum of ages of menopause and the fact that a woman’s egg supply is fixed at birth and the implications of the decline in quantity and quality of eggs should be made crystal clear to them in a timely fashion. The telling changes that may occur in the transitional phase before a woman’s final period should be clearly outlined, lest women walk into perimenopause with their eyes closed before their family is complete (Feinmann, 2008). It may be that, at the same time, honest, more balanced and easily accessible accounts (beyond statistics or abridged stories of straightforward patient success) of the current limitations of the fertility industry (including egg-freezing technology) would be helpful so that these women do not assume they can fully rely on it to conquer Nature’s plan if the need should arise (Extend Fertility, 2011; Parkin, 2007). Most crucially, it may be that for these women the debate about ‘delayed childbearing’ needs to be moved Page 58 – Fertility Magazine • Volume 18 • www.FertMag.com from the moral sphere where it usually sits (Frostrup, 2006) and be repositioned within the biological one too. Whether it is desirable or not for older women to bear children, because fertility potential varies and ultimately ceases in women, and to a lesser extent men, not all couples can have the choice. Indeed, it may be that women cannot avoid ‘delayed’ childbearing until they are made more fully aware of the true concept of it and are (re)introduced to the basic notion of ‘optimum time for birth’ (van NoordZaadstra et al., 1991; Maheshwari et al., 2008). These basic notions could also be set forth alongside the traditional warnings about teenage pregnancy currently disseminated at the school level (Allen, 2007; Boseley, 2008; Dickson, 1992; Kisby Littleton, 2012; Peterson et al., 2012). They could also be reinforced carefully in nurse-led fertility support sessions offered to women who have not conceived by age 29/30, where current fertility myths are also challenged (Asthana and Hill, 2009). These information sessions would be a modified form of the ‘fertility MOTs’ already suggested, for they would avoid the potential complacency, expense and possible inaccuracies that could result from the formal assessment of ovarian reserve through blood testing (Hussell, 2010; Macrae, 2009; Visser et al., 2006). How can people be expected to properly ‘know’ what appears to be currently formally untold? Clearly, what are considered to be ‘our’ reproductive choices are to some extent cultural illusions divorced from the reality that human reproduction is ultimately dependent on biology’s plans. In the light of all this, it is important this paper is not interpreted as yet another piece telling women at what age to have a baby, nor a judgement on those that delay childbirth, or even a criticism of the fertility industry. It is none of these. Rather, it is a call for appropriate education so that people become fully empowered to make reproductive decisions for themselves. It is also a hint that perhaps infertility clinics could further integrate medical, social and psychological models of care and learn from palliative care. Relentless positivity and descriptions of others’ success does not always assuage emotional pain; but sensitive validation of that pain is usually reassuring to its sufferer. Perhaps, at the beginning of the third millennium, women who consider themselves ‘emancipated’ should be helped to re-engage in a more respectful dialogue with Mother Nature alongside the one they have with Science, and all of us should be made to listen more carefully to Cassandra as well. For, reproductive health and rights are not only about the choice not to have a baby, but they are also about the choice to have one too. Acknowledgements I am grateful to Professor Susan Bewley, Professor Bill Ledger, Antonia Rodriguez, Irenee Daly and Kate Bentley for their sensitivity during preparation of this article. 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A fertile gesture. The Guardian, 1 October 2005. <http://webcache.googleusercontent.com/ search?q=cache:zAC9bb7wzTQJ:www.guardian.co.uk/ lifeandstyle/2005/oct/01/familyandrelationships.amily3±sus an±bewley±contactandcd=2andhl=enandct=clnkandgl=uk> (accessed 12 Nov 2010). GPS® Dishware: Specially Designed & Tested for IVF • Breathable Packaging for all GPS® Dishware • Simplifies embryology set-up • 1-cell MEA and LAL tested by independent company • Easily locate and observe embryos • No droplets collapsing nor mixing • Designed to enhance embryo culture FDA 510(k) Cleared μDrop GPS Universal GPS® 4-Well GPS ® embryo GPS® The most secure dish for PGD cases. ® embryo corral® MY AIR QUALITY IS GOOD… CODA® • • • • VOCs are too small to be removed by HEPA filtration alone. VOCs can have significant detrimental effects on human embryos. CodaAir® effectively removes VOCs. Coda® is an important line of defense in case of mechanical failure of any central air system in the IVF laboratory. 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Single Vac Pioneer Pro-Pump® Dual Vac Pioneer Pro-Pump® Fertility Magazine • Volume 18 • www.FertMag.com – Page 63 CONFERENCES ASRM American Society for Reproductive Medicine Steering Reproductive Medicine to the Forefront of Global Public Health October 17-21, 2015 Baltimore, Maryland www.asrm.org FIGO 2015 Vancouver Convention Centre 1055 Canada Place Vancouver, BC, V6C 0C3 Canada www.figo2015.org Page 64 – Fertility Magazine • Volume 18 • www.FertMag.com CONFERENCES Welcome to PCRS 2016 Annual Meeting March 9 - 13, 2016 Rancho Las Palmas 41-000 Bob Hope Drive Rancho Mirage, CA 92270 www.pcrsonline.org ESHRE 2016 European Society of Human Reproduction and Embryology 32nd Annual Meeting July 3-6, 2016 HELSINKI, Finland www.eshre.eu Fertility Magazine • Volume 18 • www.FertMag.com – Page 65 CONFERENCES Conferences and Workshops India – February 2014 Thailand – May 2014 ESHRE 2014 – Munich, July 2014 Page 66 – Fertility Magazine • Volume 18 • www.FertMag.com CONFERENCES Attended by LifeGlobal® Group ALPHA 2014 – May 2014 ASRM 2014 – Hawaii, October 2014 Red Lara Meeting – March 2015 Fertility Magazine • Volume 18 • www.FertMag.com – Page 67 Less Stress is a beautiful thing! • Less disturbance of the embryo • Less work load • Less dishes & oil The system that’s good for both embryo and embryologist. global® w/ HS total® 86 A 45 HGGT-100 , 100 ml HGGT-150 320C May 29, 2015 REF LOT Expiration Date EC REP Rev F blast 96h 1-cell MEA > 80% EU/ml 0.5 Endotoxin (LAL) < os from zygote to For culture of embrytransfer. blastocyst, embryo in Sulfate (10 μg/ml) ) /ml Contains: Gentamic um Albumin (10 mg Human Ser ction. g. after openin inje Not to be used for dium within 7 days Discard unused me Only Rx A STERILE 8°C LifeGlobal Eur Aseptic Filtered T: 32-2 227 ope, Rue de la Pre 1129 F: Belgium LifeGlo 32-2 218 sse 4, 1000 Brussels 2°C bal 3141 T: 1-800- Group, LLC, 393 720 37 US sales@ -6375 F: 1-5 Soundview Rd, Guilford, CT 064 0 LifeGlo bal.com 19-826-6947 580 Intl.: 001-519-826www.L ifeGloba lGroup.com global® for Fert. w/ HSA total® 7988 HGTF-100 , 100 ml HGTF-141 215C May 26, 2015 REF LOT Expiration global® total® w/ HSA Date EC REP Rev F blast 96h 1-cell MEA > 80% EU/ml 0.5 Endotoxin (LAL) < 0 IVF. 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Canisters, contains a higher volume of activated carbon, • Installation requires only hours – not days! potassium permanganate, and high quality HEPA filtration than • Simple filter replacement by any any other system found on the staff member – no expensive market. visits from a specialist is required. ® •Coda Positive Pressure Unit • Filter replacement occurs offers continuous air circulation outside of the lab, reducing the and purification while introduction of contaminants and maintaining a positive pressure preventing lost time that occurs environment. during a shutdown. ® •Coda 4-Stage Filter System offers multiple lines of defense against particulates, biologicals and volatile organic compounds (VOCs). T: 1-800-720-6375 ¿ F: 1-519-826-6947 ¿ PROVEN FOR SAFE USE ECONOMICAL • Safe and Clinically Proven for Best performance and results in IVF laboratories for over 15 years. • The total cost of installation is approximately 1/4 of competitors products price. • Highly effective 4-Stage filtration system with high performance removing particulates, VOCs and biological compounds. • Inexpensive filter replacement. •Coda® systems do not contain UV technology that has been proven to create ozone which is detrimental to embryo development. •Coda® Technology has helped to improve air quality in almost 1000 ART centers worldwide. •Coda® purification technology achieves particulate counts (0.5 μm to 5.0 μm) that are compliant with ISO Class 7 (10,000) standards, or better. Intl.: 001-519-826-5800 ¿ [email protected] • Slim design saves space outside of the IVF laboratory. • Reduced cost – no significant laboratory remodeling required. • Items recommended for a Regular Size IVF lab: –1-2 CodaAir® Positive Pressure Units placed inside the adjacent room or hallway bringing purified air inside the IVF lab with positive pressure –1 CodaAir® 800 and/or 1 CodaAir® 900 –1-2 AC Eco Wall Units easily control temperature and humidity ¿ www.LifeGlobalGroup.com • global ® total ® w/ HSA – 510(k) Cleared • Ready-To-Use; same global ® formulation + HSA • global ® – over 150 Independent Publications; over 15 years of Proven Records of lot-to-lot Consistency and Superior Results; over 1 million live births • Excellent customer service and established cold chain delivery worldwide. • Free Clinical & Technical Support to all global ® users worldwide • Why take a chance with anything other than global ® ? Your patients deserve the best culture system. Contact us for FREE Samples [email protected] T: 1-800-720-6375 • Intl.: 001-519-826-5800 F: 1-519-826-6947 • www.LifeGlobalGroup.com