- Email: [email protected]
New culture devices in ART
Placenta 32 (2011) S248eS251
Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
New culture devices in ART L. Rienzi a, *, G. Vajta b, F. Ubaldi a a b
G.EN.E.R.A. Centre for Reproductive Medicine, Clinica Valle Giulia, Via G. De Notaris 2b, 00197 Rome, Italy BGI Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen 518083, China
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 26 June 2011
During the past decades, improvements in culture of preimplantation embryos have contributed substantially in the success of human assisted reproductive techniques. However, most efforts were focused on optimization of media and gas components, while the established physical conditions and applied devices have remained essentially unchanged. Very recently, however, intensive research has been started to provide a more appropriate environment for the embryos and to replace the rather primitive and inappropriate devices with more sophisticated and practical instruments. Success has been reported with simple or sophisticated tools (microwells or microchannels) that allow accumulation of autocrine factors and establishment of a proper microenvironment for embryos cultured individually or in groups. The microchannel system may also offer certain level of automation and increased standardization of culture parameters. Continuous monitoring of individual embryos by optical or biochemical methods may help to determine the optimal day of transfer, and selection of the embryo with highest developmental competence for transfer. This advancement may eventually lead to adjustment of the culture environment to each individual embryo according to its actual needs. Connection of these techniques to additional radical approaches as automated ICSI or an ultimate assisted hatching with full removal of the zona pellucida after or even before fertilization may result in devices with high reliability and consistency, to increase the overall efficiency and decrease the workintensity, and to eliminate the existing technological gap between laboratory embryology work and most other fields of biomedical sciences. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: In vitro Embryo Human IVF Microchannel Monitoring
1. Introduction For long, in vitro culture of human preimplantation stage embryos was regarded by embryologist as an inconvenient must. It was required for selection of the developmentally most competent embryo for transfer. However, a short in vitro culture did not allow proper evaluation, requiring multiple embryo transfers with high risk for multiple pregnancies, while the suboptimal in vitro environment decreased the quality of all embryos and compromised chances of full term development. Efforts to improve culture conditions have resulted in a substantial breakthrough around the millennium, with widespread application of new approaches including the use of simplified media, radical elimination of toxic components, introduction of sequential culture systems and decreasing the atmospheric oxygen level during the whole culture period. As the result, culture to the blastocyst stage has become a suggested and achievable goal, allowing single blastocyst transfer with acceptably high pregnancy
* Corresponding author. Tel.: þ39 06 3269791; fax: þ39 06 32697979. E-mail address: [email protected] (L. Rienzi). 0143-4004/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2011.06.018
and birth rates. The principles and achievements of this approach have been outlined in several reviews [1e4], and its possibilities are still far from fully exploited.
2. Current devices: principles and limitations In sharp contrast with the widespread research focusing on the optimum culture media, very little attention has been paid for the devices used for embryo culture. The routine procedure is to place media in disposable polystyrene multiwell or Petri dishes, in 10e80 ml drops covered with oil and equilibrate these dishes overnight in the proper gas mixture at core temperature to stabilize pH, temperature and proper saturation with gasses. The next day, embryos are placed individually or in small groups in the dishes and incubated again for days, while pronuclear development, cleavage, compaction and blastulation are monitored with routine daily or bi-daily control under an external microscope [1,2,4]. In most systems, medium change on Day 3 after fertilization is also suggested to provide optimum composition for all developmental stages [5], although the theoretical background and practical value of this approach have recently been questioned [6].
L. Rienzi et al. / Placenta 32 (2011) S248eS251
The benefits of the above mentioned system are obvious, simple preparation, easy handling, identification and monitoring and affordable price. However, these factors are positive features principally for the embryologists, and much less for the embryos. Although the in vivo environment should not be always regarded as optimal, it is worth to consider the fundamental differences that exist between the oviduct and the drop in the culture dish. First of all, embryos in vivo develop in the virtual space of oviduct, a narrow tube lined by the dynamic layer of microvilli; are smoothly moved forward, mildly soaked by the largely unknown constituents of pregnant human’s oviductal fluid, and subjected to changing gravity position time to time. In contrast, the in vitro environment exposes embryos to a swimming pool of media, placed on artificial surface and covered by oil; autocrine factors are permanently diluted and some of them diffused in the oil layer; change in composition of media occurs only once during the culture period, and not necessarily according to the proper need; no dynamic movements are ensured, and the only common factor, the darkness is also drastically interrupted time to time causing potential, although mostly undetected damage. Most dishes were not developed for embryological purposes, and the standard embryo culture system is based on monolayer culture methods developed more than 50 years ago for primary cultures of somatic cells and cell lines. Controversially, some special dishes designed recently for embryological purposes did not result in breakthroughs and their application remains modest. Special surface treatments of dishes do not seem to have obvious benefits, while drastic differences between batches of products from the same company may occur due to inconsistencies during manufacturing and exposition of the product to toxic effects during transportation and storage. The change is inevitable, and should have happened long ago. However, the limited number of cycles performed in an average IVF laboratory restricts the need for more sophisticated equipments and devices. The established background systems and the everyday routine also hamper the application of more creative solutions. Accordingly, new initiatives were and are always regarded with reservation. This mentality discouraged large industrial producers from investment into an uncertain market, while sporadic efforts of small ventures were foredoomed to failure due to imperfections and the lack of appropriate server instruments. Additionally, all devices which make contact with live human biological material should be considered as medical devices (the term device also includes media in several countries), and should therefore meet the requirements of specific regulations. There are well-know agencies including the US FDA, Health Canada, the Australian TGA, and several European Institutions (NB-MED, MEDDEV etc) responsible for the regulations, and supervising all related issues. Procedures and devices in human assisted reproduction may also need to pass through specific test including mouse embryo assays or sperm survival tests. Due to these complex tasks only systems and devices with convincing benefits and affordable price have the chance to compete with the existing systems. Fortunately, this principle seems to become accepted by several groups with high intellectual and financial potential, and serious efforts are being performed to implement a radically new embryo culture system. This review is an effort to outline the possible solutions and extensions, to stimulate further research and openness toward acceptation of the new approaches. 3. Individual culture in minimum amount of solution Data regarding the minimum required amount of medium for a preimplantation human embryo are most controversial. According
S249
to Gardner and Lane [5], 50 ml drops for 4 human embryos is the minimum requirement, provided the medium is renewed every 48 h. Slight variations of this volume/embryo ratio are applied worldwide, although most laboratories prefer individual culture of embryos for easy identification and follow up. Unfortunately this density is insufficient to provide the supportive effect derived from autocrine and paracrine factors, and to establish a stable and appropriate microenvironment around the embryos. Group or communal culture with high embryo/volume ratio increases developmental competence in many mammalian species including humans [reviewed in Ref. [7,8]]. Concerns regarding the supposed negative effect of dead or dying embryos were not justified [9,10]. Theoretically, this group effect can be reproduced by a radical decrease of the volume embryos are cultured in, although this arrangement contradicts the calculated minimum required amount of media. Sporadic empirical evidences, however, indicate the viability of this approach. Ultra-microdrop culture, i.e. 1.5e2 ml volume for culture of 3e9 (!) embryos together for 2e3 days was reported to improve both in vitro and in vivo developmental rates (Ali, pers. comm). According to Roh et al. [11] 1 ml culture medium was enough to support 2 mouse embryos for the full period of in vitro development.
3.1. The WOW and the GO system Similar observations have lead to the establishment of new culture systems that combine or mimick the communal effect while allow individual identification of embryos. The Well of the Well or WOW system consist of small microwells produced on the bottom of a culture dish. The volume is approx. 0.05 ml, and the inverted sugar loaf shape offers appropriate space for safe culture of a single human or domestic animal embryo [4,7,12]. Although the content of the well is openly connected with the medium above, embryo development was higher than in the traditional individual cultures, and identical with that in large groups (30% vs. 50% blastocyst rates in cattle, respectively; approximate values). According to an initial human trial, similar improvement in blastocyst rates can be achieved in WOWs compared to traditional cultures (56 vs. 37%, respectively), and promising pregnancy and birth rates after culture in WOWs were also reported [13]. A modified version of the WOW is now applied in the two timelapse systems developed for monitoring embryo development, the Primo Vision and the Embryoscope [14e16]. Based on the success of the WOW, another, more innovative approach has been developed by Thouas et al. [17], the Glass Oviduct or GO system. An open ended 2 ml sterile capillary with 200 mm inner diameter is loaded manually by immersing one end into an oil-covered drop containing embryos. Due to the capillary effect, a small oil column enters the capillary followed by the medium with the embryos. Subsequently, upon retraction, another small oil layer closes the solution column. The capillary is cultured vertically for the entire culture period undisturbed in a carbon dioxide incubator. Compared to the traditional culture methods the GO system has improved the qualitative parameters of mouse embryos (total cell number, hatching rates), and was also found efficient for culturing zona intact and zona free cattle embryos ([18], Vajta, unpublished). The GO system can be also described as an extremely simplified and static version of the microchannel system. More sophisticated and purpose-designed versions of microchannels have been regarded as the greatest promise to establish a multipurpose automated system for in vitro production of preimplantation embryos.
S250
L. Rienzi et al. / Placenta 32 (2011) S248eS251
3.2. Microchannels Controversial opinions exist regarding the use of microchannel technology in laboratory embryology. The topics always in the spotlight of IVF conferences, but the extremely slow advancement creates the impression that this approach is ‘the future that never arrives’. The technique has been developed from the inkjet printers of the 80’s of the last century to a multidisciplinary approach used in various fields of physics, chemistry, microtechnology and biotechnology, including most elements of the lab-on-a-chip systems. Solutions in small channels (from 100 nm to several hundred micrometers) show a special behavior, flow remains laminar, no turbulence occurs and components mingle by diffusion only. This feature allows delicate and controllable manipulations, especially if the system is equipped with computer-regulated pneumatic valves and pumps. Eventually, an integrated and sophisticated system can be developed similar to those in computer microchips. The usual microchannel device consist of the following parts: a glass microscopic slide base; a plastic (for example polydimethylsiloxane) layer with the channels and valves; and connections to mechanical or pneumatic pumps. Fortunately the range of microfluidic channels is covering the size of human spermatozoa, oocytes and even expanded blastocysts. Sporadic and isolated attempts performed in the past decade have demonstrated that microchannels are suitable to perform almost all steps of human IVF including in vitro maturation of oocytes, selection of motile spermatozoa, in vitro fertilization by insemination and embryo culture in small volumes with or without medium change. Moreover, the system is also capable to perform more complicated manipulations including cumulus removal, zona thinning or zona removal, and for special purposes, fluorescent associated cell sorting [see reviewed in Ref. [19e22]]. Initial steps to assemble the isolated steps into a production line have also been successfully performed, accordingly there is a chance to make complex procedures completely automated including the whole human IVF [20,21]. The only unproven step that should be adopted to the microchannel system is the intracytoplasmic sperm injection: however, it may be replaced with alternative solutions, or performed in a semi-automated way [23]. Theoretically, there are several possibilities to simplify this delicate and complicated procedure including complete removal of the zona before injection and zona free growth of embryos for the whole culture period as suggested earlier [4], or complete omission of the careful
orientation of oocytes before injection e the latter approach has been found feasible without compromising the overall efficiency [24,25]. 3.3. Vision of a complex automated embryo production system Once established, the system can also be expanded with other equipment as a video camera with low magnification and high resolution to monitor all step of the procedure, and allowing detailed analysis of embryo development on timelapse recordings. Such purpose-designed instruments are already available and proved their value in embryo selection [14,15]. Further extensions may include various sensors measuring parameters in the tiny amount of culture medium surrounding the embryos including but not restricted to amino acid and sugar uptake, respiration, metabolomic features and gene expression characteristics (Fig. 1). The enormous amount of information derived from the morphological (including phase-contrast) pictures and timelapse videos together with the biochemical parameters may provide an invaluable help to determine the optimal time of embryo transfer; to select the best embryo(s), and to compare various versions of culture methods and parameters. Eventually, the microchannel system may also be applied to adjust the media and other parameters of single embryos according to their individual needs, to compensate deviations in metabolism or handicap in certain structural parameters. However, caution is suggested while using this approach. It should be considered that embryos are autonomous living beings with proven ability to establish their proper microenvironment even under compromised conditions. On the other hand, their adaptation ability to the everchanging environment may be limited, and continuous or frequently repeated flushings even with the most sophisticated solutions may cause more problem than benefit. A careful consideration of this principle, and proper use (instead of abuse) of the enormous possibilities offered by the microchannel system may help to find the right compromise and to bridge the existing gap between the technology level of laboratory embryology and that of other prominent branches of science. An ideal system should also reduce risk of mistakes providing secure identification of the biological material during each stage of a patient’s cycle. This is already possible by using Radio Frequency Identification (RFID) technology to monitor all critical steps carried out in the laboratory (RI WitnessÔ). In a futurist vision MicroLabeling codification could be considered [26].
Fig. 1. Vision of the automated IVF machine based on microchannels. See detailed explanation in the text (adapted and revised from an idea of Thorir Hardarson).
L. Rienzi et al. / Placenta 32 (2011) S248eS251
4. Conclusion After thirty years of standstill, there are signs of forthcoming advancement in the application of new devices for culturing and manipulating mammalian embryos and oocytes. The automation of the whole IVF procedure based on microchannel systems is a realistic perspective, although requires considerable multidisciplinary efforts, creativity and investment. The foreseeable benefits include standardization, consistency, and improvement in overall efficiency based also on better evaluation and selection of embryos, and individual adjustments of culture conditions according to the specific needs of a single embryo. Conflict of interest statement Dr. Laura Rienzi and Dr. Filippo Maria Ubaldi declare no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within three (3) years of beginning the work submitted that could inappropriately influence (bias) their work. References [1] Summers MC, Biggers JD. Chemically defined media and culture of mammalian preimplantation embryos: historical perspective and current issues. Hum Reprod Update 2003;9:557e82. [2] Quinn P. The development and impact of culture media for assisted reproductive technologies. Fertil Steril 2004;81:27e9. [3] Leese HJ, Baumann CG, Brison DR, McEvoy TG, Sturmey RG. Metabolism of the viable mammalian embryo: quietness revisited. Mol Hum Reprod 2008;14: 667e72. [4] Vajta G, Cobo A, Rienzi L, Yovich J. Culture of mammalian embryos. Can we perform better than nature? Reprod Biomed Online 2010;20:453e69. [5] Gardner DK, Lane M. Culture of the mammalian preimplantation embryo. In: Gardner DK, Lane M, Watson AJ, editors. A laboratory guide to the mammalian embryo. Oxford, New York: Oxford University Press; 2004. p. 41e61. [6] Biggers JD, Summers MC. Choosing a culture medium: making informed choices. Fertil Steril 2008;90:473e83. [7] Vajta G, Peura TT, Holm P, Páldi A, Greve T, Trounson AO, et al. New method for culture of zona-included and zone-free embryos: the Well of the Well (WOW) system. Mol Reprod Dev 2000;55:256e64. [8] O’Neill C. The potential roles for embryotrophic ligands in preimplantation embryo development. Hum Reprod Update 2008;14:275e88.
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[9] Hagemann LJ, Weilert LL, Beaumont SE, Tervit HR. Development of bovine embryos in single in vitro production (sIVP) system. Mol Reprod Dev 1998;51: 123e47. [10] Holm P, Booth PJ, Schmidt MH, Greve T, Callesen H. High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myoinositol with or without serum. Theriogenology 1999;52:683e700. [11] Roh S, Choi Y-J, Min B-M. A novel microtube culture system that enhances the in vitro development of parthenogenetic murine embryos. Theriogenology 2008;69:262e7. [12] Booth PJ, Tan SJ, Reipurth R, Holm P, Callesen H. Simplification of bovine somatic cell nuclear transfer by application of a zona-free manipulation technique. Cloning Stem Cells 2001;3:139e50. }rösi T, Du Y, Nakata K, Ieda S, Kuwayama M, et al. The Well-of-the[13] Vajta G, Ko Well system: an efficient approach to improve embryo development. Reprod Biomed Online 2008;17:73e81. [14] Pribenszky C, Molnár M, Conceicao J, Vajta G. Prediction of the in vitro developmental competence of early cleavage stage mouse embryos with a purpose-designed timelapse equipment. Reprod Biomed Online 2010;20: 371e9. [15] Pribenszki C, Mátyás S, Kovács P, Losonczi E, Zádori J, Vajta G. Pregnancy achieved by transfer of a single blastocyst selected by time-lapse monitoring. Reprod Biomed Online 2010;21:533e6. [16] Cruz M, Gadea B, Garrido N, Pedersen KS, Martínez M, Pérez-Cano I, et al. Embryo quality, blastocyst and ongoing pregnancy rates in oocyte donation patients whose embryos were monitored by time-lapse imaging. J Assist Reprod Genet; 2011 Mar 11 [Epub ahead of print]. [17] Thouas GA, Jones GM, Trounson AO. The ‘GO’ system e a novel method of microculture for in vitro development of mouse zygotes to the blastocyst stage. Reproduction 2003;126:161e9. [18] Vajta G, Lewis IM, Hyttel P, Thouas GA, Trounson AO. Somatic cell cloning without micromanipulators. Cloning 2001;3:89e95. [19] Beebe DJ, Wheeler M, Zeringue H, Walters E, Raty S. Microfluidic technology for assisted reproduction. Theriogenology 2002;57:125e35. [20] Vajta G, Kragh PM, Mtango NR, Callesen H. Handmade cloning approach: potentials and limitations. Reprod Fertil Dev 2005;17:97e112. [21] Smith GD, Takayama S. Gamete and embryo isolation and culture with microfluidics. Theriogenology 2007;68S:S190e5. [22] Thompson JG. Culture without the petri dish. Theriogenology 2007;67: 16e20. [23] Wang W, Liu X, Gelinas D, Ciruna B, Sun Y. A fully automated robotic system for microinjection of zebrafish embryos. PLoS ONE 2007;2:e862. [24] Stoddart NR, Fleming SD. Orientation of the first polar body of the oocyte at 6 or 12 o’clock during ICSI does not affect clinical outcome. Hum Reprod 2000; 15:1580e5. [25] Woodward BJ, Montgomery SJ, Hartshorne GM, Campbell KH, Kennedy R. Spindle position assessment prior to ICSI does not benefit fertilization or early embryo quality. Reprod Biomed Online 2008;16:232e8. [26] Novo S, Barrios L, Santaló J, Gómez-Martínez R, Duch M, Esteve J, et al. A novel embryo identification system with direct tagging of mouse embryos using silicon-based barcodes. Hum Reprod 2011;26:96e105.
Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
New culture devices in ART L. Rienzi a, *, G. Vajta b, F. Ubaldi a a b
G.EN.E.R.A. Centre for Reproductive Medicine, Clinica Valle Giulia, Via G. De Notaris 2b, 00197 Rome, Italy BGI Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen 518083, China
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 26 June 2011
During the past decades, improvements in culture of preimplantation embryos have contributed substantially in the success of human assisted reproductive techniques. However, most efforts were focused on optimization of media and gas components, while the established physical conditions and applied devices have remained essentially unchanged. Very recently, however, intensive research has been started to provide a more appropriate environment for the embryos and to replace the rather primitive and inappropriate devices with more sophisticated and practical instruments. Success has been reported with simple or sophisticated tools (microwells or microchannels) that allow accumulation of autocrine factors and establishment of a proper microenvironment for embryos cultured individually or in groups. The microchannel system may also offer certain level of automation and increased standardization of culture parameters. Continuous monitoring of individual embryos by optical or biochemical methods may help to determine the optimal day of transfer, and selection of the embryo with highest developmental competence for transfer. This advancement may eventually lead to adjustment of the culture environment to each individual embryo according to its actual needs. Connection of these techniques to additional radical approaches as automated ICSI or an ultimate assisted hatching with full removal of the zona pellucida after or even before fertilization may result in devices with high reliability and consistency, to increase the overall efficiency and decrease the workintensity, and to eliminate the existing technological gap between laboratory embryology work and most other fields of biomedical sciences. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: In vitro Embryo Human IVF Microchannel Monitoring
1. Introduction For long, in vitro culture of human preimplantation stage embryos was regarded by embryologist as an inconvenient must. It was required for selection of the developmentally most competent embryo for transfer. However, a short in vitro culture did not allow proper evaluation, requiring multiple embryo transfers with high risk for multiple pregnancies, while the suboptimal in vitro environment decreased the quality of all embryos and compromised chances of full term development. Efforts to improve culture conditions have resulted in a substantial breakthrough around the millennium, with widespread application of new approaches including the use of simplified media, radical elimination of toxic components, introduction of sequential culture systems and decreasing the atmospheric oxygen level during the whole culture period. As the result, culture to the blastocyst stage has become a suggested and achievable goal, allowing single blastocyst transfer with acceptably high pregnancy
* Corresponding author. Tel.: þ39 06 3269791; fax: þ39 06 32697979. E-mail address: [email protected] (L. Rienzi). 0143-4004/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2011.06.018
and birth rates. The principles and achievements of this approach have been outlined in several reviews [1e4], and its possibilities are still far from fully exploited.
2. Current devices: principles and limitations In sharp contrast with the widespread research focusing on the optimum culture media, very little attention has been paid for the devices used for embryo culture. The routine procedure is to place media in disposable polystyrene multiwell or Petri dishes, in 10e80 ml drops covered with oil and equilibrate these dishes overnight in the proper gas mixture at core temperature to stabilize pH, temperature and proper saturation with gasses. The next day, embryos are placed individually or in small groups in the dishes and incubated again for days, while pronuclear development, cleavage, compaction and blastulation are monitored with routine daily or bi-daily control under an external microscope [1,2,4]. In most systems, medium change on Day 3 after fertilization is also suggested to provide optimum composition for all developmental stages [5], although the theoretical background and practical value of this approach have recently been questioned [6].
L. Rienzi et al. / Placenta 32 (2011) S248eS251
The benefits of the above mentioned system are obvious, simple preparation, easy handling, identification and monitoring and affordable price. However, these factors are positive features principally for the embryologists, and much less for the embryos. Although the in vivo environment should not be always regarded as optimal, it is worth to consider the fundamental differences that exist between the oviduct and the drop in the culture dish. First of all, embryos in vivo develop in the virtual space of oviduct, a narrow tube lined by the dynamic layer of microvilli; are smoothly moved forward, mildly soaked by the largely unknown constituents of pregnant human’s oviductal fluid, and subjected to changing gravity position time to time. In contrast, the in vitro environment exposes embryos to a swimming pool of media, placed on artificial surface and covered by oil; autocrine factors are permanently diluted and some of them diffused in the oil layer; change in composition of media occurs only once during the culture period, and not necessarily according to the proper need; no dynamic movements are ensured, and the only common factor, the darkness is also drastically interrupted time to time causing potential, although mostly undetected damage. Most dishes were not developed for embryological purposes, and the standard embryo culture system is based on monolayer culture methods developed more than 50 years ago for primary cultures of somatic cells and cell lines. Controversially, some special dishes designed recently for embryological purposes did not result in breakthroughs and their application remains modest. Special surface treatments of dishes do not seem to have obvious benefits, while drastic differences between batches of products from the same company may occur due to inconsistencies during manufacturing and exposition of the product to toxic effects during transportation and storage. The change is inevitable, and should have happened long ago. However, the limited number of cycles performed in an average IVF laboratory restricts the need for more sophisticated equipments and devices. The established background systems and the everyday routine also hamper the application of more creative solutions. Accordingly, new initiatives were and are always regarded with reservation. This mentality discouraged large industrial producers from investment into an uncertain market, while sporadic efforts of small ventures were foredoomed to failure due to imperfections and the lack of appropriate server instruments. Additionally, all devices which make contact with live human biological material should be considered as medical devices (the term device also includes media in several countries), and should therefore meet the requirements of specific regulations. There are well-know agencies including the US FDA, Health Canada, the Australian TGA, and several European Institutions (NB-MED, MEDDEV etc) responsible for the regulations, and supervising all related issues. Procedures and devices in human assisted reproduction may also need to pass through specific test including mouse embryo assays or sperm survival tests. Due to these complex tasks only systems and devices with convincing benefits and affordable price have the chance to compete with the existing systems. Fortunately, this principle seems to become accepted by several groups with high intellectual and financial potential, and serious efforts are being performed to implement a radically new embryo culture system. This review is an effort to outline the possible solutions and extensions, to stimulate further research and openness toward acceptation of the new approaches. 3. Individual culture in minimum amount of solution Data regarding the minimum required amount of medium for a preimplantation human embryo are most controversial. According
S249
to Gardner and Lane [5], 50 ml drops for 4 human embryos is the minimum requirement, provided the medium is renewed every 48 h. Slight variations of this volume/embryo ratio are applied worldwide, although most laboratories prefer individual culture of embryos for easy identification and follow up. Unfortunately this density is insufficient to provide the supportive effect derived from autocrine and paracrine factors, and to establish a stable and appropriate microenvironment around the embryos. Group or communal culture with high embryo/volume ratio increases developmental competence in many mammalian species including humans [reviewed in Ref. [7,8]]. Concerns regarding the supposed negative effect of dead or dying embryos were not justified [9,10]. Theoretically, this group effect can be reproduced by a radical decrease of the volume embryos are cultured in, although this arrangement contradicts the calculated minimum required amount of media. Sporadic empirical evidences, however, indicate the viability of this approach. Ultra-microdrop culture, i.e. 1.5e2 ml volume for culture of 3e9 (!) embryos together for 2e3 days was reported to improve both in vitro and in vivo developmental rates (Ali, pers. comm). According to Roh et al. [11] 1 ml culture medium was enough to support 2 mouse embryos for the full period of in vitro development.
3.1. The WOW and the GO system Similar observations have lead to the establishment of new culture systems that combine or mimick the communal effect while allow individual identification of embryos. The Well of the Well or WOW system consist of small microwells produced on the bottom of a culture dish. The volume is approx. 0.05 ml, and the inverted sugar loaf shape offers appropriate space for safe culture of a single human or domestic animal embryo [4,7,12]. Although the content of the well is openly connected with the medium above, embryo development was higher than in the traditional individual cultures, and identical with that in large groups (30% vs. 50% blastocyst rates in cattle, respectively; approximate values). According to an initial human trial, similar improvement in blastocyst rates can be achieved in WOWs compared to traditional cultures (56 vs. 37%, respectively), and promising pregnancy and birth rates after culture in WOWs were also reported [13]. A modified version of the WOW is now applied in the two timelapse systems developed for monitoring embryo development, the Primo Vision and the Embryoscope [14e16]. Based on the success of the WOW, another, more innovative approach has been developed by Thouas et al. [17], the Glass Oviduct or GO system. An open ended 2 ml sterile capillary with 200 mm inner diameter is loaded manually by immersing one end into an oil-covered drop containing embryos. Due to the capillary effect, a small oil column enters the capillary followed by the medium with the embryos. Subsequently, upon retraction, another small oil layer closes the solution column. The capillary is cultured vertically for the entire culture period undisturbed in a carbon dioxide incubator. Compared to the traditional culture methods the GO system has improved the qualitative parameters of mouse embryos (total cell number, hatching rates), and was also found efficient for culturing zona intact and zona free cattle embryos ([18], Vajta, unpublished). The GO system can be also described as an extremely simplified and static version of the microchannel system. More sophisticated and purpose-designed versions of microchannels have been regarded as the greatest promise to establish a multipurpose automated system for in vitro production of preimplantation embryos.
S250
L. Rienzi et al. / Placenta 32 (2011) S248eS251
3.2. Microchannels Controversial opinions exist regarding the use of microchannel technology in laboratory embryology. The topics always in the spotlight of IVF conferences, but the extremely slow advancement creates the impression that this approach is ‘the future that never arrives’. The technique has been developed from the inkjet printers of the 80’s of the last century to a multidisciplinary approach used in various fields of physics, chemistry, microtechnology and biotechnology, including most elements of the lab-on-a-chip systems. Solutions in small channels (from 100 nm to several hundred micrometers) show a special behavior, flow remains laminar, no turbulence occurs and components mingle by diffusion only. This feature allows delicate and controllable manipulations, especially if the system is equipped with computer-regulated pneumatic valves and pumps. Eventually, an integrated and sophisticated system can be developed similar to those in computer microchips. The usual microchannel device consist of the following parts: a glass microscopic slide base; a plastic (for example polydimethylsiloxane) layer with the channels and valves; and connections to mechanical or pneumatic pumps. Fortunately the range of microfluidic channels is covering the size of human spermatozoa, oocytes and even expanded blastocysts. Sporadic and isolated attempts performed in the past decade have demonstrated that microchannels are suitable to perform almost all steps of human IVF including in vitro maturation of oocytes, selection of motile spermatozoa, in vitro fertilization by insemination and embryo culture in small volumes with or without medium change. Moreover, the system is also capable to perform more complicated manipulations including cumulus removal, zona thinning or zona removal, and for special purposes, fluorescent associated cell sorting [see reviewed in Ref. [19e22]]. Initial steps to assemble the isolated steps into a production line have also been successfully performed, accordingly there is a chance to make complex procedures completely automated including the whole human IVF [20,21]. The only unproven step that should be adopted to the microchannel system is the intracytoplasmic sperm injection: however, it may be replaced with alternative solutions, or performed in a semi-automated way [23]. Theoretically, there are several possibilities to simplify this delicate and complicated procedure including complete removal of the zona before injection and zona free growth of embryos for the whole culture period as suggested earlier [4], or complete omission of the careful
orientation of oocytes before injection e the latter approach has been found feasible without compromising the overall efficiency [24,25]. 3.3. Vision of a complex automated embryo production system Once established, the system can also be expanded with other equipment as a video camera with low magnification and high resolution to monitor all step of the procedure, and allowing detailed analysis of embryo development on timelapse recordings. Such purpose-designed instruments are already available and proved their value in embryo selection [14,15]. Further extensions may include various sensors measuring parameters in the tiny amount of culture medium surrounding the embryos including but not restricted to amino acid and sugar uptake, respiration, metabolomic features and gene expression characteristics (Fig. 1). The enormous amount of information derived from the morphological (including phase-contrast) pictures and timelapse videos together with the biochemical parameters may provide an invaluable help to determine the optimal time of embryo transfer; to select the best embryo(s), and to compare various versions of culture methods and parameters. Eventually, the microchannel system may also be applied to adjust the media and other parameters of single embryos according to their individual needs, to compensate deviations in metabolism or handicap in certain structural parameters. However, caution is suggested while using this approach. It should be considered that embryos are autonomous living beings with proven ability to establish their proper microenvironment even under compromised conditions. On the other hand, their adaptation ability to the everchanging environment may be limited, and continuous or frequently repeated flushings even with the most sophisticated solutions may cause more problem than benefit. A careful consideration of this principle, and proper use (instead of abuse) of the enormous possibilities offered by the microchannel system may help to find the right compromise and to bridge the existing gap between the technology level of laboratory embryology and that of other prominent branches of science. An ideal system should also reduce risk of mistakes providing secure identification of the biological material during each stage of a patient’s cycle. This is already possible by using Radio Frequency Identification (RFID) technology to monitor all critical steps carried out in the laboratory (RI WitnessÔ). In a futurist vision MicroLabeling codification could be considered [26].
Fig. 1. Vision of the automated IVF machine based on microchannels. See detailed explanation in the text (adapted and revised from an idea of Thorir Hardarson).
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4. Conclusion After thirty years of standstill, there are signs of forthcoming advancement in the application of new devices for culturing and manipulating mammalian embryos and oocytes. The automation of the whole IVF procedure based on microchannel systems is a realistic perspective, although requires considerable multidisciplinary efforts, creativity and investment. The foreseeable benefits include standardization, consistency, and improvement in overall efficiency based also on better evaluation and selection of embryos, and individual adjustments of culture conditions according to the specific needs of a single embryo. Conflict of interest statement Dr. Laura Rienzi and Dr. Filippo Maria Ubaldi declare no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within three (3) years of beginning the work submitted that could inappropriately influence (bias) their work. References [1] Summers MC, Biggers JD. Chemically defined media and culture of mammalian preimplantation embryos: historical perspective and current issues. Hum Reprod Update 2003;9:557e82. [2] Quinn P. The development and impact of culture media for assisted reproductive technologies. Fertil Steril 2004;81:27e9. [3] Leese HJ, Baumann CG, Brison DR, McEvoy TG, Sturmey RG. Metabolism of the viable mammalian embryo: quietness revisited. Mol Hum Reprod 2008;14: 667e72. [4] Vajta G, Cobo A, Rienzi L, Yovich J. Culture of mammalian embryos. Can we perform better than nature? Reprod Biomed Online 2010;20:453e69. [5] Gardner DK, Lane M. Culture of the mammalian preimplantation embryo. In: Gardner DK, Lane M, Watson AJ, editors. A laboratory guide to the mammalian embryo. Oxford, New York: Oxford University Press; 2004. p. 41e61. [6] Biggers JD, Summers MC. Choosing a culture medium: making informed choices. Fertil Steril 2008;90:473e83. [7] Vajta G, Peura TT, Holm P, Páldi A, Greve T, Trounson AO, et al. New method for culture of zona-included and zone-free embryos: the Well of the Well (WOW) system. Mol Reprod Dev 2000;55:256e64. [8] O’Neill C. The potential roles for embryotrophic ligands in preimplantation embryo development. Hum Reprod Update 2008;14:275e88.
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