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The future ivf lab

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A talk I gave in india, 2013, for ACE. …

A talk I gave in india, 2013, for ACE.

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  • In 1978 when Robert Edwards and Patrick Steptoe announced the birth of the first human fertilized in vitro from a small cottage hospital in Oldham a worldwide industry was born. Once recommended for the treatment of fallopian tube pathologies, the field of in vitro fertilization (IVF) has enveloped a spectrum of reproductive and genetic disorders creating social, moral, legal and religious issues in its wake. The nascent field of embryology has evolved, Assisted Reproductive Technologies (ART) have progressed, experience gained and techniques refined.We live in a society where never before has scientific data been so readily accessible and so quickly disseminated. Future developments will be due not only to the number of dedicated scientists involved in research but to the merging of multiple scientific fields. The advances of biological sciences, biotechnology, bio-engineering and informatics converge to create new innovations: many of which are of immediate relevance to the field of ART, and some of which
  • Regulations and the search for excellenceThe profession of clinical embryology has evolved considerably. Once research scientists were chosen to head IVF laboratories as they were experienced in culture methodologies and possessed knowledge which was passed to others through the traditional scientific apprenticeship. Today’s field requires a highly structured training programme with strict documentation.A call for licensing by National Authorities, or accreditation by organizations such as Eshre2 have put embryologists on a course of professional development targeting excellence. Rigorous Quality Management Systems have become a cornerstone of our work and are now mandatory or at least highly encouraged. Clinical embryology has become a sophisticated profession demanding rigorous standards and laboratory guidelines3,4, all of which have fine tuned the IVF laboratory into the highly standardized workplace it has become today.It is remarkable however that few IVF laboratories are actually built and desi
  • In Europe, Assisted Reproductive Technology is covered under the European Union Tissue and Cells Directive (EUTCD; 2004/23/EC) 8 which stipulates specific quality and safety requirements with a critical point in the doctrine being clean air. The surprise inclusion of IVF facilities into this directive has led to most units, at the very least, making at least some changes to the quality of air control in their laboratories. If embryonic development and implantation depends so highly upon its culture environment, the IVF industry would do well to follow the same stringent regulatory standards as are present in the electronic, biotechnology and pharmaceutical industries which use Cleanrooms for critical processes. The principal function of a cleanroom is to protect the “manufactured product” and to this end provides a controlled environmental with a particular emphasis upon monitoring air particles, microbial counts and contaminants. Such critical processes take place within a sealed room which is supplied th
  • In Europe, Assisted Reproductive Technology is covered under the European Union Tissue and Cells Directive (EUTCD; 2004/23/EC) 8which stipulates specific quality and safety requirements with a critical point in the doctrine being clean air. The surprise inclusion of IVF facilities into this directive has led to most units, at the very least, making at least some changes to the quality of air control in their laboratories. If embryonic development and implantation depends so highly upon its culture environment, the IVF industry would do well to follow the same stringent regulatory standards as are present in the electronic, biotechnology and pharmaceutical industries which use Cleanrooms for critical processes. The principal function of a cleanroom is to protect the “manufactured product” and to this end provides a controlled environmental with a particular emphasis upon monitoring air particles, microbial counts and contaminants. Such critical processes take place within a sealed room which is supplied through high efficiency particle air filters (HEPA). All furnishings, cleaning methods and garments must be in keeping with the cleanroom classification which is defined by the maximum permitted airborne particle concentration in accordance with international quality standards9.Such advancements in laboratory standards are essential to elevate the IVF lab to the biotechnology arena. Continuous monitoring, which is a prerequisite of the directive’s technical annex (EU 2006/86/EC annex 1 Equipment and Material) 10,will strengthen the quality management systems existing within IVF laboratories today. Technological advances in real time data monitoring offers the ability to monitor any equipment offering an analog or digital signal from a remote location giving the ability to detect malfunctions and ensure corrective action is taken.
  • Over the years more specialized equipment has been developed exclusively for use with human in vitro fertilization. Every conference exhibition showroom has become a showcase for new equipment dedicated for use by the clinical embryologist. State-of-the-art incubators, integrated laminar flow workstations and specialized micro-manipulator have certainly improved the way we work. Maintaining embryos safely in the IVF laboratory require optimum culture conditions. It is critical to embryo viability and pregnancy outcome to minimize stress and to optimize the in-vitro environment11-13. But after three decades of IVF is it still acceptable for dishes containing gametes to be transported manually within the laboratory?The innovation of closed workstations ensures constant environmental conditions providing an incubator-like environment during critical procedures. Whether small enclosed units designated for specific work, or larger units are preferred to enclose all procedures, these models offer the embryologist
  • Over the years more specialized equipment has been developed exclusively for use with human in vitro fertilization. Every conference exhibition showroom has become a showcase for new equipment dedicated for use by the clinical embryologist. State-of-the-art incubators, integrated laminar flow workstations and specialized micro-manipulator have certainly improved the way we work. Maintaining embryos safely in the IVF laboratory require optimum culture conditions. It is critical to embryo viability and pregnancy outcome to minimize stress and to optimize the in-vitro environment11-13. But after three decades of IVF is it still acceptable for dishes containing gametes to be transported manually within the laboratory?The innovation of closed workstations ensures constant environmental conditions providing an incubator-like environment during critical procedures.Whether small enclosed units designated for specific work, or larger units are preferred to enclose all procedures, these models offer the embryologist more time to complete tasks within a controlled environment avoiding deterioration in embryo quality.Find which lab
  • The advent of time-lapse imaging systems allows the “tireless embryologist” to monitoring critical stages of early embryonic development. These imaging devices, which are small enough to fit inside existing incubators or stand-alone composite models, are sophisticated, easily installed devices which will offer a growing range of applications (Fig.3 & 4).
  • In Europe, Assisted Reproductive Technology is covered under the European Union Tissue and Cells Directive (EUTCD; 2004/23/EC) 8 which stipulates specific quality and safety requirements with a critical point in the doctrine being clean air. The surprise inclusion of IVF facilities into this directive has led to most units, at the very least, making at least some changes to the quality of air control in their laboratories. If embryonic development and implantation depends so highly upon its culture environment, the IVF industry would do well to follow the same stringent regulatory standards as are present in the electronic, biotechnology and pharmaceutical industries which use Cleanrooms for critical processes. The principal function of a cleanroom is to protect the “manufactured product” and to this end provides a controlled environmental with a particular emphasis upon monitoring air particles, microbial counts and contaminants. Such critical processes take place within a sealed room which is supplied th
  • The introduction of intracytoplasmic sperm injection (ICSI) in 1992 revolutionized the treatment of male infertility50. The proportion of ICSI versus IVF procedures continues to increase, encompassing conditions other than male factor patients51, making it arguably one of the most important advances in ART. Gamete micromanipulation requires an experienced and highly skilled embryologist and the process is labor intensive. The laboratory outcomes of this manual operation are susceptible to human factors such as volume of work, stress and fatigue, as well as variations in performance between operators. Is it possible for this most sophisticated of all ART techniques could ultimately be automated? Significant advances have been made in both microelectromechanical systems (MEMS) and micro-robotics. The auto-injection of biological cells is being increasingly used within the field of transgenics52 and cell biology53; using principals which entail tracking, positioning, grasping and injecting material into a s
  • The introduction of intracytoplasmic sperm injection (ICSI) in 1992 revolutionized the treatment of male infertility50. The proportion of ICSI versus IVF procedures continues to increase, encompassing conditions other than male factor patients51, making it arguably one of the most important advances in ART. Gamete micromanipulation requires an experienced and highly skilled embryologist and the process is labor intensive. The laboratory outcomes of this manual operation are susceptible to human factors such as volume of work, stress and fatigue, as well as variations in performance between operators. Is it possible for this most sophisticated of all ART techniques could ultimately be automated? Significant advances have been made in both microelectromechanical systems (MEMS) and micro-robotics. The auto-injection of biological cells is being increasingly used within the field of transgenics52 and cell biology53; using principals which entail tracking, positioning, grasping and injecting material into a single cell. Improved technical aspects include MEMS-based cell holding devices and visual servo-control schemes making robotic micromanipulation an enticing prospect that may herald the introduction of automation of ICSI into the IVF laboratories of the future. Indeed, in light of recent animal model studies using micro-robotics might silence even the strongest skeptics. Nano-Newton force sensing and sub-pixel visual control have enhanced the dexterity and delicacy of embryo manipulation and oocyte microinjection in mice54,55, while oocyte and polar body orientation has been achieved through the use of motorized rotational microscopy stages, computer tracking and pattern recognition algorithms. One such system56 can precisely orientate oocytes up to 7200 per second with an accuracy of 0.240. The robotic immobilization and micromanipulation of spermatozoa is currently possible within 6-7 seconds by the use of sperm trajectories and velocity algorithms originally used for computer assisted sperm assessment (CASA) 57. In the first report of robotic ICSI (RICSI), using a hamster oocyte/ human sperm model, the robotic system demonstrated a high (90%) survival rate58. Already human trials are under way for RICSI- robotic intracytoplasmic sperm injection (Yu Sun personnel communication), and it may only be a matter of time before automation, in some form, gains confidence amongst the IVF community (Fig.6).
  • The introduction of intracytoplasmic sperm injection (ICSI) in 1992 revolutionized the treatment of male infertility50. The proportion of ICSI versus IVF procedures continues to increase, encompassing conditions other than male factor patients51, making it arguably one of the most important advances in ART. Gamete micromanipulation requires an experienced and highly skilled embryologist and the process is labor intensive. The laboratory outcomes of this manual operation are susceptible to human factors such as volume of work, stress and fatigue, as well as variations in performance between operators. Is it possible for this most sophisticated of all ART techniques could ultimately be automated? Significant advances have been made in both microelectromechanical systems (MEMS) and micro-robotics. The auto-injection of biological cells is being increasingly used within the field of transgenics52 and cell biology53; using principals which entail tracking, positioning, grasping and injecting material into a single cell. Improved technical aspects include MEMS-based cell holding devices and visual servo-control schemes making robotic micromanipulation an enticing prospect that may herald the introduction of automation of ICSI into the IVF laboratories of the future. Indeed, in light of recent animal model studies using micro-robotics might silence even the strongest skeptics. Nano-Newton force sensing and sub-pixel visual control have enhanced the dexterity and delicacy of embryo manipulation and oocyte microinjection in mice54,55, while oocyte and polar body orientation has been achieved through the use of motorized rotational microscopy stages, computer tracking and pattern recognition algorithms. One such system56 can precisely orientate oocytes up to 7200 per second with an accuracy of 0.240. The robotic immobilization and micromanipulation of spermatozoa is currently possible within 6-7 seconds by the use of sperm trajectories and velocity algorithms originally used for computer assisted sperm assessment (CASA) 57. In the first report of robotic ICSI (RICSI), using a hamster oocyte/ human sperm model, the robotic system demonstrated a high (90%) survival rate58. Already human trials are under way for RICSI- robotic intracytoplasmic sperm injection (Yu Sun personnel communication), and it may only be a matter of time before automation, in some form, gains confidence amongst the IVF community (Fig.6).
  • Culture media and Growth factorsHuman embryo culture media has improved significantly since the early days; originally adapted from simple balanced salt solutions or media based on chemical compositions in the oviduct as known at the time18. These formulations were effective but attempts at culture to blastocyst stage were poor; although a number of embryos reached this stage the viability was compromised with reported low implantation rates19. Most IVF labs today use specific sequential media that is based on the changing energy requirements and basic physiological principles identified in the works of Leese20, Quinn21 and Gardner22. Studies in both animals and humans indicate development in vitro is sub-optimal23. In light of epidemiological studies that demonstrate growth impairment24 and meta analysis of poor perinatal outcomes25 it is imperative that we strive to continue to improve media formulations.A new generation of media under development is now being supplemented with bioactive compounds to res
  • The static culture media we employ today bears little comparison to the micro-environment of the fertilized oocyte. Since its inception, in vitro fertilization has been conducted in polystyrene tubes and dishes bathed in a sea of culture media. Common culture practices may allow the accumulation of toxic substances such as ammonia34 and free radicals35 that may well be harmful to the embryos36 . Transferring embryos into ‘fresh’ culture media may alleviate such effects but removes secreted autocrine and paracrine factors which are beneficial to the embryo37.Novel attempts to imitate the dynamics of the reproductive tract have produced some encouraging results. Exposure of embryos to mechanical stimuli has improved the development of mouse and human embryos38 and enhanced development to the blastocyst stage after in vitro maturation (IVM) of porcine oocytes39 by employing a tilting embryo culture system to mimic the shear stress, compression and frictional force which embryos experience in vivo. micro
  • Another promising approach is the use of ‘Microfluidics’, which has ever increasing applications in chemical engineering and genetics40, may now hold significant promise for ART. This nascent technology utilizes the characteristics of fluid movements within micro and nano- environments by producing multiple streams of media through the same microchannels. This ‘lab-on-a-chip’ incorporating tiny channels, gates and pumps directing fluids are a prime example of how biotechnology is entering into the realms of ART. Microfluidics has a potentially broad spectrum of applications within the field of ART. Co-culture using embryos separated from endometrial cells by a thin polyester membrane in both mice and human embryonic cultures41,42 create a continuous perfusion system. While, microchannels afford the opportunity to bring oocytes and spermatozoa into close proximity, providing more favorable conditions for fertilization at lower concentrations of sperm43, which could potentially improve the efficiency with
  • Another promising approach is the use of ‘Microfluidics’, which has ever increasing applications in chemical engineering and genetics40, may now hold significant promise for ART. This nascent technology utilizes the characteristics of fluid movements within micro and nano- environments by producing multiple streams of media through the same microchannels. This ‘lab-on-a-chip’ incorporating tiny channels, gates and pumps directing fluids are a prime example of how biotechnology is entering into the realms of ART. Microfluidics has a potentially broad spectrum of applications within the field of ART. Co-culture using embryos separated from endometrial cells by a thin polyester membrane in both mice and human embryonic cultures41,42 create a continuous perfusion system. While, microchannels afford the opportunity to bring oocytes and spermatozoa into close proximity, providing more favorable conditions for fertilization at lower concentrations of sperm43, which could potentially improve the efficiency with which we fertilize oocytes. In creating a culture system that mimics the embryo’s more natural, dynamic environment, it is proposed that there may be a reduction of localized oxygen tension and a natural removal of harmful toxins. Such gentile agitation may help in the clearing of receptors or stimulate signal pathways which promote improved growth in culture44.  Microfluidic devices have been constructed from a variety of materials, but more recently polydimethylsiloxane [PDMS] has been selected due to its superior mechanical and optical properties, high permeability to gases and biocompatibility45 . Interest in a ‘Braille pin’ type pumping system is gaining popularity, reducing the complexity and inconvenience of older designs (Fig. 5). Controlled by computer, tiny piezoelectric pins are used to deform microchannels on a malleable microfluidic device, eliminating the need for pumps and connecting tubes46. Heo and colleagues recently used this technology to refresh media surrounding mouse embryos in a physiological pulsating manner and reported enhanced embryo development with blastocyst cell numbers, implantation and ongoing pregnancy rates that closely matched in vivo data47.
  • The major limitation for clinical IVF treatment today is the inability to predict which embryos are potentially the most viable. Despite a global tendency (voluntary, mandatory, or morally obligated) for single embryo transfer 59,60, the phenomenon of multiple pregnancies still prevails61, highlighting our inability to detect, select and transfer the most competent embryo. Over the years, embryologists have striven to select the best embryo from a cohort of potential candidates. Systems of grading by cell morphology62, developmental timing63,64, polar body orientation65, and pronuclear morphology66 have all contributed to pregnancy outcome but their value remains limited.Hopefully, a call for uniformity of terminology for grading oocytes and embryos formulated in an International consensus on embryo assessment will help to assess new technologies and to enhance future prognostic indicators67.There is great momentum in the pursuit to find the appropriate criteria which will give us the opportunity to ma
  • The emergence of the so-called “omic” sciences, including genomics, transcriptomics, proteomics and metabolomics, are improving our collective understanding of cellular processes in addition to providing diagnostic tools for the evaluation of pre-implantation human embryos. Proteomic assessment meanwhile remains in its infancy. Unlike DNA and RNA, proteins are not subject to amplification, and conventional analysis of this vast repertoire of macromolecules is technically challenging and labor intensive98 . However, recent developments in mass spectrometry have for the first time allowed us to identify protein expression related to morphology, and degenerating embryos exhibiting significant up-regulation of potential markers can be identified99.While pre-implantation genetic diagnosis (PGD) has been of benefit to countless couples at risk of transmitting a genetic disease since 199079, its cousin, pre-implantation genetic screening (PGS), a method for selecting euploid embryos for transfer in an atte
  • The emergence of the so-called “omic” sciences, including genomics, transcriptomics, proteomics and metabolomics, are improving our collective understanding of cellular processes in addition to providing diagnostic tools for the evaluation of pre-implantation human embryos. Proteomic assessment meanwhile remains in its infancy. Unlike DNA and RNA, proteins are not subject to amplification, and conventional analysis of this vast repertoire of macromolecules is technically challenging and labor intensive98 . However, recent developments in mass spectrometry have for the first time allowed us to identify protein expression related to morphology, and degenerating embryos exhibiting significant up-regulation of potential markers can be identified99.While pre-implantation genetic diagnosis (PGD) has been of benefit to countless couples at risk of transmitting a genetic disease since 199079, its cousin, pre-implantation genetic screening (PGS), a method for selecting euploid embryos for transfer in an attempt to increase the success rate of IVF, has not fulfilled its expectations and is now under scrutiny. To select the most viable embryos, many centers have screened for numerical chromosomal abnormalities using fluorescence in situ hybridization (FISH) 80, 81, although in the wake of several randomized controlled trials are not encouraging82. The inherent problems within the embryo, such as mosacism, coupled to the technical problems associated with FISH have been blamed for any positive influence on pregnancy outcomes83.The progress in the genetic analysis of embryos has relied upon joint developments within IVF and genetic laboratories. The use of lasers for embryo biopsy84, the success of blastocyst culture85, and the beneficial outcomes of cryopreservation by vitrification86 have extended the viable window for diagnosis. Simultaneously, advances in genetics have also fuelled the genetic testing of preimplantation embryos: two colored FISH evolved to multi-probe FISH87 and amplification of DNA by conventional PCR has since been replaced by fluorescent and multiplex PCR, becoming more reliable and more informative88.  The future of aneuploidy screening perhaps now rests upon the success of a new genetic screening tool that offers a more “global view” of the developing embryos. Whole genome amplifications have become very efficient, incorporating comparative genomic hybridization (arrayCGH) or single nucleotide polymorphism (SNP) arrays to yield high resolution molecular and cytogenetic analysis at the single cell level 89, 90 .     Applied to PGS, array technology offers the opportunity for a genome wide analysis, eliminating the necessity to generate patient specific DNA probes. Together with the renewed interest in blastocyst stage biopsy these techniques may provide a more favorable outcome, overcoming diagnostic problems such as moscaism91,92. Array technology also affords the ART community a first glimpse of the possibilities offered by genetic profiling. Complementary DNA (cDNA) microarray technology combined with cDNA amplification means that it is now possible to analyze the entire transcriptome within a single cell as a parallel to aneuploidy screening. Improved embryo selection could be accomplished by selecting favorable gene expression patterns in biopsied cells. DNA fingerprinting, namely matching gene expression profiles of blastocysts transferred to the resulting offspring, has provided us with the ultimate system for biometric identification, and could be used to predict positive diagnostic biomarkers for developmentally competent embryos93.
  • The emergence of the so-called “omic” sciences, including genomics, transcriptomics, proteomics and metabolomics, are improving our collective understanding of cellular processes in addition to providing diagnostic tools for the evaluation of pre-implantation human embryos. Proteomic assessment meanwhile remains in its infancy. Unlike DNA and RNA, proteins are not subject to amplification, and conventional analysis of this vast repertoire of macromolecules is technically challenging and labor intensive98 . However, recent developments in mass spectrometry have for the first time allowed us to identify protein expression related to morphology, and degenerating embryos exhibiting significant up-regulation of potential markers can be identified99.While pre-implantation genetic diagnosis (PGD) has been of benefit to countless couples at risk of transmitting a genetic disease since 199079, its cousin, pre-implantation genetic screening (PGS), a method for selecting euploid embryos for transfer in an attempt to increase the success rate of IVF, has not fulfilled its expectations and is now under scrutiny. To select the most viable embryos, many centers have screened for numerical chromosomal abnormalities using fluorescence in situ hybridization (FISH) 80, 81, although in the wake of several randomized controlled trials are not encouraging82. The inherent problems within the embryo, such as mosacism, coupled to the technical problems associated with FISH have been blamed for any positive influence on pregnancy outcomes83.The progress in the genetic analysis of embryos has relied upon joint developments within IVF and genetic laboratories. The use of lasers for embryo biopsy84, the success of blastocyst culture85, and the beneficial outcomes of cryopreservation by vitrification86 have extended the viable window for diagnosis. Simultaneously, advances in genetics have also fuelled the genetic testing of preimplantation embryos: two colored FISH evolved to multi-probe FISH87 and amplification of DNA by conventional PCR has since been replaced by fluorescent and multiplex PCR, becoming more reliable and more informative88.  The future of aneuploidy screening perhaps now rests upon the success of a new genetic screening tool that offers a more “global view” of the developing embryos. Whole genome amplifications have become very efficient, incorporating comparative genomic hybridization (arrayCGH) or single nucleotide polymorphism (SNP) arrays to yield high resolution molecular and cytogenetic analysis at the single cell level 89, 90 .     Applied to PGS, array technology offers the opportunity for a genome wide analysis, eliminating the necessity to generate patient specific DNA probes. Together with the renewed interest in blastocyst stage biopsy these techniques may provide a more favorable outcome, overcoming diagnostic problems such as moscaism91,92. Array technology also affords the ART community a first glimpse of the possibilities offered by genetic profiling. Complementary DNA (cDNA) microarray technology combined with cDNA amplification means that it is now possible to analyze the entire transcriptome within a single cell as a parallel to aneuploidy screening. Improved embryo selection could be accomplished by selecting favorable gene expression patterns in biopsied cells. DNA fingerprinting, namely matching gene expression profiles of blastocysts transferred to the resulting offspring, has provided us with the ultimate system for biometric identification, and could be used to predict positive diagnostic biomarkers for developmentally competent embryos93.
  • The emergence of the so-called “omic” sciences, including genomics, transcriptomics, proteomics and metabolomics, are improving our collective understanding of cellular processes in addition to providing diagnostic tools for the evaluation of pre-implantation human embryos. Proteomic assessment meanwhile remains in its infancy. Unlike DNA and RNA, proteins are not subject to amplification, and conventional analysis of this vast repertoire of macromolecules is technically challenging and labor intensive98 . However, recent developments in mass spectrometry have for the first time allowed us to identify protein expression related to morphology, and degenerating embryos exhibiting significant up-regulation of potential markers can be identified99.While pre-implantation genetic diagnosis (PGD) has been of benefit to countless couples at risk of transmitting a genetic disease since 199079, its cousin, pre-implantation genetic screening (PGS), a method for selecting euploid embryos for transfer in an attempt to increase the success rate of IVF, has not fulfilled its expectations and is now under scrutiny. To select the most viable embryos, many centers have screened for numerical chromosomal abnormalities using fluorescence in situ hybridization (FISH) 80, 81, although in the wake of several randomized controlled trials are not encouraging82. The inherent problems within the embryo, such as mosacism, coupled to the technical problems associated with FISH have been blamed for any positive influence on pregnancy outcomes83.The progress in the genetic analysis of embryos has relied upon joint developments within IVF and genetic laboratories. The use of lasers for embryo biopsy84, the success of blastocyst culture85, and the beneficial outcomes of cryopreservation by vitrification86 have extended the viable window for diagnosis. Simultaneously, advances in genetics have also fuelled the genetic testing of preimplantation embryos: two colored FISH evolved to multi-probe FISH87 and amplification of DNA by conventional PCR has since been replaced by fluorescent and multiplex PCR, becoming more reliable and more informative88.  The future of aneuploidy screening perhaps now rests upon the success of a new genetic screening tool that offers a more “global view” of the developing embryos. Whole genome amplifications have become very efficient, incorporating comparative genomic hybridization (arrayCGH) or single nucleotide polymorphism (SNP) arrays to yield high resolution molecular and cytogenetic analysis at the single cell level 89, 90 .     Applied to PGS, array technology offers the opportunity for a genome wide analysis, eliminating the necessity to generate patient specific DNA probes. Together with the renewed interest in blastocyst stage biopsy these techniques may provide a more favorable outcome, overcoming diagnostic problems such as moscaism91,92. Array technology also affords the ART community a first glimpse of the possibilities offered by genetic profiling. Complementary DNA (cDNA) microarray technology combined with cDNA amplification means that it is now possible to analyze the entire transcriptome within a single cell as a parallel to aneuploidy screening. Improved embryo selection could be accomplished by selecting favorable gene expression patterns in biopsied cells. DNA fingerprinting, namely matching gene expression profiles of blastocysts transferred to the resulting offspring, has provided us with the ultimate system for biometric identification, and could be used to predict positive diagnostic biomarkers for developmentally competent embryos93.
  • The emergence of the so-called “omic” sciences, including genomics, transcriptomics, proteomics and metabolomics, are improving our collective understanding of cellular processes in addition to providing diagnostic tools for the evaluation of pre-implantation human embryos. Proteomic assessment meanwhile remains in its infancy. Unlike DNA and RNA, proteins are not subject to amplification, and conventional analysis of this vast repertoire of macromolecules is technically challenging and labor intensive98 . However, recent developments in mass spectrometry have for the first time allowed us to identify protein expression related to morphology, and degenerating embryos exhibiting significant up-regulation of potential markers can be identified99.While pre-implantation genetic diagnosis (PGD) has been of benefit to countless couples at risk of transmitting a genetic disease since 199079, its cousin, pre-implantation genetic screening (PGS), a method for selecting euploid embryos for transfer in an attempt to increase the success rate of IVF, has not fulfilled its expectations and is now under scrutiny. To select the most viable embryos, many centers have screened for numerical chromosomal abnormalities using fluorescence in situ hybridization (FISH) 80, 81, although in the wake of several randomized controlled trials are not encouraging82. The inherent problems within the embryo, such as mosacism, coupled to the technical problems associated with FISH have been blamed for any positive influence on pregnancy outcomes83.The progress in the genetic analysis of embryos has relied upon joint developments within IVF and genetic laboratories. The use of lasers for embryo biopsy84, the success of blastocyst culture85, and the beneficial outcomes of cryopreservation by vitrification86 have extended the viable window for diagnosis. Simultaneously, advances in genetics have also fuelled the genetic testing of preimplantation embryos: two colored FISH evolved to multi-probe FISH87 and amplification of DNA by conventional PCR has since been replaced by fluorescent and multiplex PCR, becoming more reliable and more informative88.  The future of aneuploidy screening perhaps now rests upon the success of a new genetic screening tool that offers a more “global view” of the developing embryos. Whole genome amplifications have become very efficient, incorporating comparative genomic hybridization (arrayCGH) or single nucleotide polymorphism (SNP) arrays to yield high resolution molecular and cytogenetic analysis at the single cell level 89, 90 .     Applied to PGS, array technology offers the opportunity for a genome wide analysis, eliminating the necessity to generate patient specific DNA probes. Together with the renewed interest in blastocyst stage biopsy these techniques may provide a more favorable outcome, overcoming diagnostic problems such as moscaism91,92. Array technology also affords the ART community a first glimpse of the possibilities offered by genetic profiling. Complementary DNA (cDNA) microarray technology combined with cDNA amplification means that it is now possible to analyze the entire transcriptome within a single cell as a parallel to aneuploidy screening. Improved embryo selection could be accomplished by selecting favorable gene expression patterns in biopsied cells. DNA fingerprinting, namely matching gene expression profiles of blastocysts transferred to the resulting offspring, has provided us with the ultimate system for biometric identification, and could be used to predict positive diagnostic biomarkers for developmentally competent embryos93.
  • METANon invasiveNone of all these emerging technologies are as close to delivering a rapid, reliable non-invasive screening test as “Metabolomics” –the study of the metabolite profile of the embryo in culture. The changing metabolic and nutrient requirements reflect the embryos well being, and such measurements may help to determine embryo viability prior to transfer105,106. Metabolites can be perceived as the final end products of gene expression and, unlike genes or proteins, the number of prospective candidates to screen are significantly fewer. A great body of work is being accumulated detecting differences between viable and nonviable embryos grown in culture through measurements of energy substrates, proteins and amino acids107. Early work by Leese and Conaghan108 measuring the energy requirements of the embryo in-vitro provided a scientific foundation for subsequent spent media analysis, although its value was limited in routine IVF. The use of optic spectroscopy, namely Raman and NIR (near
  • METANon invasiveNone of all these emerging technologies are as close to delivering a rapid, reliable non-invasive screening test as “Metabolomics” –the study of the metabolite profile of the embryo in culture. The changing metabolic and nutrient requirements reflect the embryos well being, and such measurements may help to determine embryo viability prior to transfer105,106. Metabolites can be perceived as the final end products of gene expression and, unlike genes or proteins, the number of prospective candidates to screen are significantly fewer. A great body of work is being accumulated detecting differences between viable and nonviable embryos grown in culture through measurements of energy substrates, proteins and amino acids107. Early work by Leese and Conaghan108 measuring the energy requirements of the embryo in-vitro provided a scientific foundation for subsequent spent media analysis, although its value was limited in routine IVF. The use of optic spectroscopy, namely Raman and NIR (near infrared) spectroscopy has reduced both the instrumentation and complexity associated with fluid analysis, as compared with non-optical spectroscopy methods109, and produced a rapid method for the analysis of biofluids. Spectral analysis of spent culture media using these methods has yielded a unique metabolic profiles which, when combined with bioinformatics, resulted in viability scores highly correlated with reproductive potential110-112. Only time will tell if this technology can differentiate the most competent embryos from a cohort of Leese’s “quiet” embryos113. Nethertheless, metabolomics is stirring great commercial interest, and we may soon see a bench top model in the IVF laboratories of the future (Fig. 8).It has even been suggested that there may be yet another role for microfluidics in the non-invasive assessment of the in vitro embryo, allowing real-time biomarker analysis by integrating an ELISA (enzyme-linked immunosorbent assay) on a chip44 or parallel measurements of pyruvate, glucose and lactose levels in a novel microfluidic system114. Open label one armed observational, multi centre pilot study with retrospective amino acid analysisand explorative statistical analysis5 study sites in the UK participating400 patients to be includedInterim analysis after 200 patients None of all these emerging technologies are as close to delivering a rapid, reliable non-invasive screening test as “Metabolomics” –the study of the metabolite profile of the embryo in culture. The changing metabolic and nutrient requirements reflect the embryos well being, and such measurements may help to determine embryo viability prior to transfer105,106. Metabolites can be perceived as the final end products of gene expression and, unlike genes or proteins, the number of prospective candidates to screen are significantly fewer. A great body of work is being accumulated detecting differences between viable and nonviable embryos grown in culture through measurements of energy substrates, proteins and amino acids107.    Early work by Leese and Conaghan108 measuring the energy requirements of the embryo in-vitro provided a scientific foundation for subsequent spent media analysis, although its value was limited in routine IVF. The use of optic spectroscopy, namely Raman and NIR (near infrared) spectroscopy has reduced both the instrumentation and complexity associated with fluid analysis, as compared with non-optical spectroscopy methods109, and produced a rapid method for the analysis of biofluids. Spectral analysis of spent culture media using these methods has yielded a unique metabolic profiles which, when combined with bioinformatics, resulted in viability scores highly correlated with reproductive potential110-112. Only time will tell if this technology can differentiate the most competent embryos from a cohort of Leese’s “quiet” embryos113. Nethertheless, metabolomics is stirring great commercial interest, and we may soon see a bench top model in the IVF laboratories of the future (Fig. 8).
  • First one I want to talk about is tlModern time-lapse imaging offers an insight into the developing embryo (Fig7), allowing morphological and temporal analysis that have been shown to not impair embryo quality and viability69. Monitoring can aid the embryologist in studying kinetic markers for embryo quality and such as synchrony of appearance of nuclei70, cell division71, and fragmentation events72. A static observation may not help embryo selection. Early cleavage has been reported to be an important parameter in embryo quality scoring73 and implantation potential74. Moreover, a more accurate assessment of timing of first cleavage reveals selection of embryos with higher implantation potential 75. Adaptations and enhanced software may present further diagnostic value. Wong et al., observing membrane ruffling using automatic cell outline tracking imagery was able to predict zygote progression to blastocyst with over 90% sensitivity76. While, using a nanospirometer77 in combination with time lapse ima
  • First one I want to talk about is tlModern time-lapse imaging offers an insight into the developing embryo (Fig7), allowing morphological and temporal analysis that have been shown to not impair embryo quality and viability69. Monitoring can aid the embryologist in studying kinetic markers for embryo quality and such as synchrony of appearance of nuclei70, cell division71, and fragmentation events72. A static observation may not help embryo selection. Early cleavage has been reported to be an important parameter in embryo quality scoring73 and implantation potential74. Moreover, a more accurate assessment of timing of first cleavage reveals selection of embryos with higher implantation potential 75. Adaptations and enhanced software may present further diagnostic value. Wong et al., observing membrane ruffling using automatic cell outline tracking imagery was able to predict zygote progression to blastocyst with over 90% sensitivity76. While, using a nanospirometer77 in combination with time lapse imagery demonstrated for the first time a correlation between oxygen consumption and developmental events. A peak of oxygen consumption at time of fertilization and prior to first cell division suggest this can be used as an indicator of embryo competence78. Used in my lab…gfreta success, 10 % world wide uses it.Lot of data coming out
  • The emergence of the so-called “omic” sciences, including genomics, transcriptomics, proteomics and metabolomics, are improving our collective understanding of cellular processes in addition to providing diagnostic tools for the evaluation of pre-implantation human embryos. While pre-implantation genetic diagnosis (PGD) has been of benefit to countless couples at risk of transmitting a genetic disease since 199079, its cousin, pre-implantation genetic screening (PGS), a method for selecting euploid embryos for transfer in an attempt to increase the success rate of IVF, has not fulfilled its expectations and is now under scrutiny. To select the most viable embryos, many centers have screened for numerical chromosomal abnormalities using fluorescence in situ hybridization (FISH) 80, 81, although in the wake of several randomized controlled trials are not encouraging82. The inherent problems within the embryo, such as mosacism, coupled to the technical problems associated with FISH have been blamed for an
  • The emergence of the so-called “omic” sciences, including genomics, transcriptomics, proteomics and metabolomics, are improving our collective understanding of cellular processes in addition to providing diagnostic tools for the evaluation of pre-implantation human embryos. While pre-implantation genetic diagnosis (PGD) has been of benefit to countless couples at risk of transmitting a genetic disease since 199079, its cousin, pre-implantation genetic screening (PGS), a method for selecting euploid embryos for transfer in an attempt to increase the success rate of IVF, has not fulfilled its expectations and is now under scrutiny. To select the most viable embryos, many centers have screened for numerical chromosomal abnormalities using fluorescence in situ hybridization (FISH) 80, 81, although in the wake of several randomized controlled trials are not encouraging82. The inherent problems within the embryo, such as mosacism, coupled to the technical problems associated with FISH have been blamed for any positive influence on pregnancy outcomes83.The progress in the genetic analysis of embryos has relied upon joint developments within IVF and genetic laboratories. The use of lasers for embryo biopsy84, the success of blastocyst culture85, and the beneficial outcomes of cryopreservation by vitrification86 have extended the viable window for diagnosis. Simultaneously, advances in genetics have also fuelled the genetic testing of preimplantation embryos: two colored FISH evolved to multi-probe FISH87 and amplification of DNA by conventional PCR has since been replaced by fluorescent and multiplex PCR, becoming more reliable and more informative88.  The future of aneuploidy screening perhaps now rests upon the success of a new genetic screening tool that offers a more “global view” of the developing embryos. Whole genome amplifications have become very efficient, incorporating comparative genomic hybridization (arrayCGH) or single nucleotide polymorphism (SNP) arrays to yield high resolution molecular and cytogenetic analysis at the single cell level 89, 90 .     Applied to PGS, array technology offers the opportunity for a genome wide analysis, eliminating the necessity to generate patient specific DNA probes. Together with the renewed interest in blastocyst stage biopsy these techniques may provide a more favorable outcome, overcoming diagnostic problems such as moscaism91,92. Array technology also affords the ART community a first glimpse of the possibilities offered by genetic profiling. Complementary DNA (cDNA) microarray technology combined with cDNA amplification means that it is now possible to analyze the entire transcriptome within a single cell as a parallel to aneuploidy screening. Improved embryo selection could be accomplished by selecting favorable gene expression patterns in biopsied cells. DNA fingerprinting, namely matching gene expression profiles of blastocysts transferred to the resulting offspring, has provided us with the ultimate system for biometric identification, and could be used to predict positive diagnostic biomarkers for developmentally competent embryos93.
  • the embryologists’ of tomorrow will require not only a broad scientific foundation, but the ability to assimilate innovations from multiple scientific disciplines quickly and effectively.
  • Aesop used already in 50 gynaecolproceedues by voice control/no tremourPGS better results if standardised and not subjection, sperm selection ..maccs,oocytes polarized light/denudation/ nmicrofluidics and time lapse, metaboloismFuture devices could facilitate the automation of embryonic culture, providing an enclosed, multi-step culture system, without the need for manual manipulation of the embryo. Already models have been designed which enable the physical manipulation of the embryo through changes in the flow of media. Utilizing peristaltic pressure changes and electro-rotation, the oocyte can be immobilized by suction through a microholein the same way a conventional holding pipette holds an oocyte during ICSI48. Another device serves to simultaneously immobilize and inseminate the oocyte, remove cumulus and track the resulting development through to the blastocyst stage49.
  • Surely the quest for the preservation of fertility remains the last great challenge for assisted reproduction. Fertility preservation is an emerging medical discipline, designed to treat the increasing number of young women surviving cancer115 . Owing to a heightened public awareness of ART, greater emphasis is being placed upon restoring fertility after the effects of damaging cancer treatment116.The prospect of IFM holds tremendous potential, and further developments in tissue engineering could provide us with a better understanding of human folliculogenesis and allow us to enter a new age of ovarian stimulation. A solution to maintain and mature ovarian tissue in vitro, not only for cancer patients, but also for the purposes of ovarian banking, for women who wish to postpone childbearing for social or professional reasons.      Ovarian stimulation and cryopreservation of oocytes or embryos before cancer treatment may not always be the most appropriate solution117. The cryopreservation
  • Surely the quest for the preservation of fertility remains the last great challenge for assisted reproduction. Fertility preservation is an emerging medical discipline, designed to treat the increasing number of young women surviving cancer115 . Owing to a heightened public awareness of ART, greater emphasis is being placed upon restoring fertility after the effects of damaging cancer treatment116.The prospect of IFM holds tremendous potential, and further developments in tissue engineering could provide us with a better understanding of human folliculogenesis and allow us to enter a new age of ovarian stimulation. A solution to maintain and mature ovarian tissue in vitro, not only for cancer patients, but also for the purposes of ovarian banking, for women who wish to postpone childbearing for social or professional reasons.      Ovarian stimulation and cryopreservation of oocytes or embryos before cancer treatment may not always be the most appropriate solution117. The cryopreservation
  • In 1978 when Robert Edwards and Patrick Steptoe announced the birth of the first human fertilized in vitro from a small cottage hospital in Oldham a worldwide industry was born. Once recommended for the treatment of fallopian tube pathologies, the field of in vitro fertilization (IVF) has enveloped a spectrum of reproductive and genetic disorders creating social, moral, legal and religious issues in its wake. The nascent field of embryology has evolved, Assisted Reproductive Technologies (ART) have progressed, experience gained and techniques refined.We live in a society where never before has scientific data been so readily accessible and so quickly disseminated. Future developments will be due not only to the number of dedicated scientists involved in research but to the merging of multiple scientific fields. The advances of biological sciences, biotechnology, bio-engineering and informatics converge to create new innovations: many of which are of immediate relevance to the field of ART, and some of which
  • 5 centers in UK, 400 patientsChallenge Can we create a nice logarithm ?Problems in development
  • The emergence of the so-called “omic” sciences, including genomics, transcriptomics, proteomics and metabolomics, are improving our collective understanding of cellular processes in addition to providing diagnostic tools for the evaluation of pre-implantation human embryos. Proteomic assessment meanwhile remains in its infancy. Unlike DNA and RNA, proteins are not subject to amplification, and conventional analysis of this vast repertoire of macromolecules is technically challenging and labor intensive98 . However, recent developments in mass spectrometry have for the first time allowed us to identify protein expression related to morphology, and degenerating embryos exhibiting significant up-regulation of potential markers can be identified99.While pre-implantation genetic diagnosis (PGD) has been of benefit to countless couples at risk of transmitting a genetic disease since 199079, its cousin, pre-implantation genetic screening (PGS), a method for selecting euploid embryos for transfer in an atte
  • The emergence of the so-called “omic” sciences, including genomics, transcriptomics, proteomics and metabolomics, are improving our collective understanding of cellular processes in addition to providing diagnostic tools for the evaluation of pre-implantation human embryos. Proteomic assessment meanwhile remains in its infancy. Unlike DNA and RNA, proteins are not subject to amplification, and conventional analysis of this vast repertoire of macromolecules is technically challenging and labor intensive98 . However, recent developments in mass spectrometry have for the first time allowed us to identify protein expression related to morphology, and degenerating embryos exhibiting significant up-regulation of potential markers can be identified99.While pre-implantation genetic diagnosis (PGD) has been of benefit to countless couples at risk of transmitting a genetic disease since 199079, its cousin, pre-implantation genetic screening (PGS), a method for selecting euploid embryos for transfer in an attempt to increase the success rate of IVF, has not fulfilled its expectations and is now under scrutiny. To select the most viable embryos, many centers have screened for numerical chromosomal abnormalities using fluorescence in situ hybridization (FISH) 80, 81, although in the wake of several randomized controlled trials are not encouraging82. The inherent problems within the embryo, such as mosacism, coupled to the technical problems associated with FISH have been blamed for any positive influence on pregnancy outcomes83.The progress in the genetic analysis of embryos has relied upon joint developments within IVF and genetic laboratories. The use of lasers for embryo biopsy84, the success of blastocyst culture85, and the beneficial outcomes of cryopreservation by vitrification86 have extended the viable window for diagnosis. Simultaneously, advances in genetics have also fuelled the genetic testing of preimplantation embryos: two colored FISH evolved to multi-probe FISH87 and amplification of DNA by conventional PCR has since been replaced by fluorescent and multiplex PCR, becoming more reliable and more informative88.  The future of aneuploidy screening perhaps now rests upon the success of a new genetic screening tool that offers a more “global view” of the developing embryos. Whole genome amplifications have become very efficient, incorporating comparative genomic hybridization (arrayCGH) or single nucleotide polymorphism (SNP) arrays to yield high resolution molecular and cytogenetic analysis at the single cell level 89, 90 .     Applied to PGS, array technology offers the opportunity for a genome wide analysis, eliminating the necessity to generate patient specific DNA probes. Together with the renewed interest in blastocyst stage biopsy these techniques may provide a more favorable outcome, overcoming diagnostic problems such as moscaism91,92. Array technology also affords the ART community a first glimpse of the possibilities offered by genetic profiling. Complementary DNA (cDNA) microarray technology combined with cDNA amplification means that it is now possible to analyze the entire transcriptome within a single cell as a parallel to aneuploidy screening. Improved embryo selection could be accomplished by selecting favorable gene expression patterns in biopsied cells. DNA fingerprinting, namely matching gene expression profiles of blastocysts transferred to the resulting offspring, has provided us with the ultimate system for biometric identification, and could be used to predict positive diagnostic biomarkers for developmentally competent embryos93.
  • The emergence of the so-called “omic” sciences, including genomics, transcriptomics, proteomics and metabolomics, are improving our collective understanding of cellular processes in addition to providing diagnostic tools for the evaluation of pre-implantation human embryos. Proteomic assessment meanwhile remains in its infancy. Unlike DNA and RNA, proteins are not subject to amplification, and conventional analysis of this vast repertoire of macromolecules is technically challenging and labor intensive98 . However, recent developments in mass spectrometry have for the first time allowed us to identify protein expression related to morphology, and degenerating embryos exhibiting significant up-regulation of potential markers can be identified99.While pre-implantation genetic diagnosis (PGD) has been of benefit to countless couples at risk of transmitting a genetic disease since 199079, its cousin, pre-implantation genetic screening (PGS), a method for selecting euploid embryos for transfer in an atte
  • Implementation of new technologies is essential to ensure safety and consistency in future laboratory practice. Although reported ART processing errors are rare14, several technological solutions currently exist to safeguard against potential mismatches of patients’ gametes. Methods of witnessing are debatable but incorporation of passive recognition and verification systems are paramount for patient confidence. Radio frequency identification (RFID) is gathering popularity and with further miniaturization of tags15 their use may increase further. One ingenious line of research is the use of silicon based microscopic barcodes, originally designed for use with cultured human macrophages16. These intracellular tags have demonstrated their biocompatibility, having been successfully micro-injected into the perivitelline space and used to track individual embryos17.

Transcript

  • 1. Future developments in the IVF lab Giles Palmer Mitera IVF, Athens
  • 2. Future developments in the IVF lab Areas of interest • The laboratory • Embryo culture • Embryo selection • Fertility preservation Tomorrow • Biotech Arena • Integration • Automation IVF labs generally resist change - maintain consistency Cost/benefit Research grants
  • 3. Future developments in the IVF lab The story so far….. • Change from apprentice system • Accreditation/documentation • Commercially available media and instruments • QMS/QC cornerstone of todays IVF labs • Purpose built labs ?
  • 4. Future developments in the IVF lab Regulations and the search llence • EUTCD 2004- specific quality and safety requirements with a critical point in the doctrine being clean air • Air quality and VOCs (Cohen 1997, Legro 2010) • Clean room environment- ”protect the manufactured product”
  • 5. Future developments in the IVF lab Regulations and the search flence • EUTCD 2004- specific quality and safety requirements with a critical point in the doctrine being clean air • Air quality and VOCs (Cohen 1997, Legro 2010) • Clean room environment- ”protect the manufactured product”
  • 6. Future developments in the IVF lab Laboratory equipment
  • 7. Future developments in the IVF lab Laboratory equipment
  • 8. Future developments in the IVF lab Laboratory equipment
  • 9. Future developments in the IVF lab Heated glass worktop, integrated RFID antenna, touch screen interface
  • 10. Future developments in the IVF lab Automation of ICSI • Since 1992 : male infertility • Experience operator, highly skilled • Auto injection – transgenics • Use of MEMS and micro-robotics, Nano-newton force sensors, Servo optical control • RICSI : 7200 per second with an accuracy of 0.240, Immobilization of sperm in 6-7 seconds (CASA adapted) • Survival rate 90% (Sun 2011) • Human trials on going
  • 11. Future developments in the IVF lab Automation of ICSI • Since 1992 : male infertility • Experience operator, highly skilled • Auto injection – transgenics • Use of MEMS and micro-robotics, Nano-newton force sensors, Servo optical control • RICSI : 7200 per second with an accuracy of 0.240, Immobilization of sperm in 6-7 seconds (CASA adapted) • Survival rate 90% (Sun 2011) • Human trials on going
  • 12. Future developments in the IVF lab Automation of ICSI • Since 1992 : male infertility • Experience operator, highly skilled • Auto injection – transgenics • Use of MEMS and micro-robotics, Nano-newton force sensors, Servo optical control • RICSI : 7200 per second with an accuracy of 0.240, Immobilization of sperm in 6-7 seconds (CASA adapted) • Survival rate 90% (Sun 2011) • Human trials on going • Robot assisted oocyte retrieval!!
  • 13. Future developments in the IVF lab Further developments of in vitro culture • 40 years on…Biggers, Brinster • Basic physiological principles of Leese, Quinn and Gardner • In vitro culture sub-optimum (Schieve 2002, Richter 2008) • Call for “new generation media” with bio active factors
  • 14. GM-CSF Future developments in the IVF lab • GM-CSF in IVF medium improves success rates in Recurrent Implantation Failure patients (Spandorfer et al., Am J ReprodImmunol 2008) • GM-CSF is reduced in recurrent miscarriage patients (Perricone et al Am J Reprod Immunol 2003) • Follicular fluid GM-CSF is reduced in women experiencing unexplained infertility (Calogero et al., Cytokine 1998) Positive effect on cell number, implantation, blastocyst formation, regulates apoptosis Robertson 2011, Sjoblom 1999
  • 15. GM-CSF LIF PAF GH IGF-I IGF-II EGF TGFβ TNFα IFNγ Future developments in the IVF lab Many other compounds possible inclusion culture media ? lipids/ prostoglandins
  • 16. Future developments in the IVF lab Static culture methods • Polystyrene tubes and dishes • Accumulation of toxins, free radicles Systems to mimic reproductive tract • Microfluidics- technology of handling small volumes of liquids “Lab on a chip” • Application in co-culture (Mizuno 2007) • Sperm sorting, fertilization (Suh 2006) • Cumulus removal (Zeringue 2004) • Dynamic micro-funnel device enhancing mouse embryo development (Heo 2010) • Possibility of real time measurements?
  • 17. Future developments in the IVF lab • Further developments of in vitro culture
  • 18. Future developments in the IVF lab • Further developments of in vitro culture m
  • 19. Future developments in the IVF lab Selecting for success • How to predict viability? • Cell morphology • Early cleavage, polar body orientation and pronuclei morphology
  • 20. Future developments in the IVF lab Selecting for success Invasive Genetic analysis has evolved with IVF techniques. • Euploid selection (PGS) not fulfilled expectations • Global view- array CGH, SNP, New generation sequencing • RCT underway….promising (Yang 2011) • Search for non invasive molecular techniques • DNA array –cumulus cells (Assou 2008), Follicular fluid (Hamel 2010), Proteomic approach to sex selection (Picton 2010) • Closest to implimentation –Systematic study of the unique chemical fingerprint that in vitro embryo leave behind • Compulsory embryo biopsy & PGS????
  • 21. Future developments in the IVF lab Selecting for success Invasive Genetic analysis has evolved with IVF techniques. • Euploid selection (PGS) not fulfilled expectations • Global view- array CGH, SNP, New generation sequencing • RCT underway….promising (Yang 2011) • Search for non invasive molecular techniques • DNA array –cumulus cells (Assou 2008), Follicular fluid (Hamel 2010), Proteomic approach to sex selection (Picton 2010) • Closest to implimentation –Systematic study of the unique chemical fingerprint that in vitro embryo leave behind • Compulsory embryo biopsy & PGS????
  • 22. Future developments in the IVF lab Selecting for success Invasive Genetic analysis has evolved with IVF techniques. • Euploid selection (PGS) not fulfilled expectations • Global view- array CGH, SNP, New generation sequencing • RCT underway….promising (Yang 2011) • Search for non invasive molecular techniques • DNA array –cumulus cells (Assou 2008), Follicular fluid (Hamel 2010), Proteomic approach to sex selection (Picton 2010) • Closest to implimentation –Systematic study of the unique chemical fingerprint that in vitro embryo leave behind • Compulsory embryo biopsy & PGS????
  • 23. Future developments in the IVF lab Selecting for success Invasive Genetic analysis has evolved with IVF techniques. • Euploid selection (PGS) not fulfilled expectations • Global view- array CGH, SNP, New generation sequencing • RCT underway….promising (Yang 2011) • Search for non invasive molecular techniques • DNA array –cumulus cells (Assou 2008), Follicular fluid (Hamel 2010), Proteomic approach to sex selection (Picton 2010) • Closest to implimentation –Systematic study of the unique chemical fingerprint that in vitro embryo leave behind • Compulsory embryo biopsy & PGS????
  • 24. Future developments in the IVF lab Selecting for success Invasive Genetic analysis has evolved with IVF techniques. • Euploid selection (PGS) not fulfilled expectations • Global view- array CGH, SNP, New generation sequencing • RCT underway….promising (Yang 2011) Search for non invasive molecular techniques • DNA array –cumulus cells (Assou 2008), Follicular fluid (Hamel 2010), Proteomic approach to sex selection (Picton 2010) • Closest to implementation –Systematic study of the unique chemical fingerprint that in vitro embryo leave behind in culture media • Compulsory embryo biopsy & PGS????
  • 25. Future developments in the IVF lab • Leese & Conaghan- late 1980’s- pyruvate uptake and embryo viability • Bio-spectroscopy-applied to spent media (NMR/MS & HPLC) Houghton 2002, Brison 2004, Katz-Jaffe 2006
  • 26. Future developments in the IVF lab • Sakkas- biochemical factors representative of embryos that give pregnancy or no pregnancy • 2009: Reduced complexity-Raman/NIR spectroscopy - rapid metabolic profile=Viability scores • Automative system of embryo metabolisms – problems in its development • Pipeline “Embryosure “- Amino acid profiling (Origio/Leese) • Incorporation in microfluidic devices? (Swain 2009)
  • 27. Future developments in the IVF lab Time lapse evaluation Morphological selection criteria in literature: Cell division Cell symmetry Synchronicity Multi-nucleation Fragmentation Abnormal cell division
  • 28. Future developments in the IVF lab Time lapse evaluation Morphological selection criteria in literature: Cell division Cell symmetry Synchronicity Multi-nucleation Fragmentation Abnormal cell division m
  • 29. Future developments in the IVF lab Time lapse evaluation • Use of morphokinetics as a predictor of embryo implantation (Meseguer 2011) • Search for algorithm for implantation potential • Morphokinetics link to aneuploidy detection (Davies 2012) • Classification system based on time parameters relates to selection of euploidy embryo ( Basile 2013) • Applications: Oxygen consumption (lopes 2005), cell membrane tracking ( Wong 2010) t2 t3 t4 t5 cc2 cc3s2 Cc: cell cycle cc2 = t3-t2 cc3 = t5-t4 s2 = t4-t3 cc2=11,8h s2 = <0.76h t2=25,6h (24,3-25,8h) t3=37,4h (35,4-37,8h) t4=38h (36,4-38,9h) t5=52,3h (48,8-56-6,h)
  • 30. Future developments in the IVF lab Time lapse evaluation • Use of morphokinetics as a predictor of embryo implantation (Meseguer 2011) • Search for algorithm for implantation potential • Morphokinetics link to aneuploidy detection (Davies 2012) • Classification system based on time parameters relates to selection of euploidy embryo ( Basile 2013) • Applications: Oxygen consumption (lopes 2005), cell membrane tracking ( Wong 2010) t2 t3 t4 t5 cc2 cc3s2 Cc: cell cycle cc2 = t3-t2 cc3 = t5-t4 s2 = t4-t3 cc2=11,8h s2 = <0.76h t2=25,6h (24,3-25,8h) t3=37,4h (35,4-37,8h) t4=38h (36,4-38,9h) t5=52,3h (48,8-56-6,h)
  • 31. “Lab on a chip” Sperm sorting Imaging Co-culture Oxygen consumption Biomarker analysis
  • 32. Future developments in the IVF lab
  • 33. Extra slides
  • 34. The IVF lab of the future Automation in the ART. (mes) • Mostly manual/sperm selection/oocyte selection/denudation/ICSI/monitor ing/vitrification • Emerging technology-alternative in all fields of medicine/ telepresence/education • Da Vinci/AESOP/Zeus • Robotic assisted follicle aspiration!
  • 35. The IVF lab of the future Fertility Preservation • Emerging medical discipline • Ovarian stimulation/Oocyte freezing may not be always appropriate Options for cancer patients • IVM of primordial follicles • Xenographing • Cryopreservation/transplantation of ovarian tissue (Donnez 2004) Ovarian tissue culture, IVG, folliculogenesis • Live births with pre-antral primordial follicles in mice (Eppig 1996) • Advances in tissue/biomaterial engineering • 2D vs 3D models (alginate, poly ethylene glycol PEG) • Promoted secondary follicles to antral and MII in primates (Xu 2011) • Artificial ovary-thecal and granulosa cell self assemble (Krotz 2010) Folliculagenesis • Complex process-endocrine & paracrine interactions
  • 36. The IVF lab of the future Fertility Preservation Perfusion system –pulsatile gonadotrophin administration mimic pituitary-enhances follicular development Winkler 2009,2013
  • 37. The IVF lab of the future Moore’s law • Intel based business strategy • Exponential growth every 2 years • Generally difficult in medicine • ART under utilized • IVF labs generally resist change - maintain consistency
  • 38. • Sakkas- biochemical factors representative of embryos that give pregnancy or no pregnancy • Bio-spectroscopy- metabolic profile/ with bio-informatic analysis=Viability scores • 2009-Automative system of embryo metabolisms – problems in its development • Pipeline “Embryosure “- Amino acid profiling (Origio/Leese) • Sakkas. Fertil Steril 90.6.(2008 ) The IVF lab of the future
  • 39. The IVF lab of the future The “omic” approach • Genomics, transciptomics, proteomics etc • Genetic analysis has evolved with IVF techniques • Euploid selection (PGS) not fulfilled expectations • Fgf • Bvv • Vcvc • Vcv • Cxcc • N
  • 40. The IVF lab of the future The “omic” approach • Genomics, transciptomics, proteomics etc • Genetic analysis has evolved with IVF techniques • Euploid selection (PGS) not fulfilled expectations • Fgf • Bvv • Vcvc • Vcv • cxcc
  • 41. The IVF lab of the future The “omic” approach • Genomics, transciptomics, proteomics etc • Genetic analysis has evolved with IVF techniques • Euploid selection (PGS) not fulfilled expectations • Fgf • Bvv • Vcvc • Vcv • cxcc
  • 42. The IVF lab of the future Laboratory equipment • Witness system, RFID • Microscopic silicon based barcoding (Novo 2010)