laura rienzi g.en.e.r.a centre for reproductive medicine ... · declared no potential conflict of...
TRANSCRIPT
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Declared no potential conflict of interest
Laura Rienzi G.EN.E.R.A Centre for Reproductive Medicine Valle Giulia Clinic Rome, Italy
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Laura Rienzi, Rome, Italy Senior Clinical Embryologist
New culture devices in ART
CLINICA VALLE GIULIA, Rome
www.generaroma.it
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New culture devices in ART
The majority of research have for many years focused on manipulation of
chemical components (salts, amino acids, energy substrate, grow factors, …).
Little has be done examining the physical requirements of the preimplantation
embryos and the role of platforms, devices, that can influence embryo
development:
- reduced media volume
- reduced embryo spacing
- dynamic culture
-embryo movement
-special culture surface
Swain and Smith, 2011
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Current devices for embryo culture: limitations
• Artificial surface
• Oil coverage
• Dilution of autocrine factors
• No dynamic movements
• Drastically interruption of darkness
• Narrow tube lined by the dynamic layer of
microvilli
• Dynamic mechanical and biochemical conditions
• Mild exposure to constituents of oviductal fluid
• Gravity position change
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Current devices for embryo culture
Static culture platforms:
Microdrops
Ultramicrodrops
Submicroliter platforms
Microwells
Micro channels
Swain and Smith, 2011
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Specialized microdrop dishes
This approach affects embryos spacing and prevents flattening or coalescing of traditional microdrops.
Collection of embryos at the bottom of a concave depression
Specialized polystyrene dishes
Swain and Smith, 2011
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Microwells
Small microenvironment for individual or small groups of
embryos larger common culture
media reservoir
Microwell approaches are often termed the well-of-the-well (WOW) approach, as first described by Vajta
Vajta et al., 2000, 2008 , Swain and Smith, 2011
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The WOW approach
Swain and Smith, 2011
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The WOW approach
Non humidified environment
Unique identification of each embryo
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Automatic time-lapse system
Retrospective analysis of 247 embryo transfers, with known destiny of the embryos (no implantation or all implanted).
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Morphodynamic evaluation: new parameters
Meseguer et al., 2011
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Hierarchical classification
Meseguer et al., 2011
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Microchannels
Increase surface area
The microenvironment of a micro channel more closely resembles in vivo
fertilization conditions than a culture dish or microdrop.
Glass microscopic slide base
Connections to mechanical or
pneumatic pumps
Plastic layer with the channels and valves
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The GO system
Culture in microliter volumes in glass capillary tubes
Lane and Gardner, 1992; Thouas et al., 2003, Vajta, unpublished
Increased cell contact Improvement of the qualitative parameters of mouse embryos (total cell number, hatching rates)
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Microfluidic technology
Microfluidic technology may provide an automated platform for
performing multiple steps of IVF:
1. Selection of normal oocytes Choi et al., 2008
2. Isolation motile sperm from non-motile sperm, debris and
seminal plasma Cho et al., 2003; Schuster et al.,2003
3. Control of embryo positioning, movement and zona pellucida
removal for chimera and transgenic production.
Zeringue et al. ,2005
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Advances in embryo culture platforms
Specialized surfaces
•Agarose •Matrigel •Hyaluronic acid •Co-culture •3-Dimensional matrix
Dynamic culture platforms
•Shaking/rotation •Tilting •Vibration •Controlled fluid flow
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Advances in embryo culture platforms
Dynamic culture platforms
Swain and Smith, 2011
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Two promising dynamic culture approaches
Swain and Smith, 2011
Tilting embryo culture system
Dynamic microfunnel culture
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Controlled fluid flow
Cabrera et al., 2006; Heo et al., 2010, Swain and Smith, 2011
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Dynamic microfunnel culture
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Dynamic microfunnel culture
Heo et al., 2010
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Dynamic microfunnel culture
Heo et al., 2010
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Dynamic microfunnel culture
Heo et al., 2010
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Advances in embryo culture platforms
Use of specialized surface coatings in combination with approaches that maximize cell-surface contact may allow for embedding and orientation of macromolecules that could convey benefit to embryo development in vitro
Specialized surfaces
•Agarose •Matrigel •Hyaluronic acid •Co-culture •3-Dimensional matrix
Swain and Smith, 2011
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Robotic ICSI
• Integration of key robotic functions:
– Non-invasive manipulation of gametes
– High precision positioning of multiple oocytes
– Specific cellular orientation control
– Sperm tracking, immobilization and capture
– Highly consistent cellular penetration and injection of biomaterial
– Real-time visual surveillance by computer vision microscopy
– Integration of multivariate functions by proprietary algorithms
– Fast, reproducible, skill-independent
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Key Components of RICSI™System
• Single Use Gamete Slide (SUGS; cell holding device)
• Precision vacuum pump for immobilizing oocytes
• Motorized precision micro-syringe for sperm aspiration and deposition
• Motorized rotational stage
• Integrative software
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Proprietary Components of the
Marksman RICSI™ System
Motorized
Rotational Stage
Single Use Gamete Slide
(SUGS)
Oocyte immobilization platform
Sperm well
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Marksman RICSI Technology
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Electronic Witnessing
Since the first known case of an ART mix-up in 1987 in Manhattan, USA), the
accidental use of incorrect gametes or embryos during ART procedures has
been reported in several centers around the world Liebler, 2002, Spriggs, 2003; Bender, 2006
Barcodes (MatcherTM, FertilityQMS Ltd, UK)
RFID reader Scanner
Electronic witnessing
Radio frequency identification labels (IVFWitnessTM, UK)
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Silicon-based barcodes
To further minimize this risk, a method of labeling the gametes or embryos directly has been proposed
Required proprieties of a direct embryo labelling system
Made of a biocompatible material
Small enough not to compromise gamete fertilization and
embryo developmental potential
Large enough to hold a sufficient amount of information for
sample identification purposes
Readable under a standard inverted microscope
Electronic Witnessing
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Silicone-based barcodes
To provide a proof of concept for a direct oocyte/embryo labeling system
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Silicone-based barcodes
Novo et al., 2011
Microinjected embryos were maintained in culture until the blastocyst stage
Barcodes are microinjected into the perivitelline space of mouse embryos
Evaluated outcomes:
•Rates of development •Embryo identification •Retention of barcodes in the perivitelline space during culture •Release of barcodes after blastocyst hatching •Effectiveness of the labeling system after embryo cryopreservation
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Silicone-based barcodes
Type C: 2D- polysilicon barcodes
Novo et al., 2011
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Silicone-based barcodes
Remaining barcodes
In vitro development of embryos microinjected with different types of polysilicon barcodes into their perivitelline space.
Magnified images of the barcodes Novo et al., 2011
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Vision of a complex automated embryo production system
Camera
Dinamic morphological evaluation
(light and polarized microscopy)
Media sampling: Metabolomic,
Respiration, Amino acid and
Sugar uptake, Gene expression,
…..
Denudation Robotic ICSI Robotic
biopsy
Bar code for identification of the samples
Rienzi et al., 2011
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Vision of a complex automated embryo production system
Sakkas, personal comunication
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• The physical environment during in vitro embryo culture can affect resulting embryo quality.
• Examination of these physical requirements through development of novel culture platforms may lead to new approaches to future help improve ART
Conclusions
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• The automation of the whole IVF procedure based on microchannel systems is a realistic perspective, althouth 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.
Conclusions
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CLINICA VALLE GIULIA, Roma
www.generaroma.it
Gynecology:
Filippo Ubaldi
Elena Baroni
Silvia Colamaria
Maddalena Giuliani
Fabio Sapienza
Embryology:
Laura Rienzi
Stefania Romano
Laura Albricci
Antonio Capalbo
Roberta Maggiulli
Benedetta Iussig
Nicoletta Barnocchi
SALUS – ASI MEDICAL, Marostica GENERA UMBERTIDE, Perugia