The future ivf lab

Future developments in the IVF lab

Giles Palmer Mitera IVF, Athens


Areas of interest• The laboratory• Embryo culture • Embryo selection

• Fertility preservation

Tomorrow• Biotech Arena• Integration • Automation

IVF labs generally resist change - maintain consistencyCost/benefit

Research grants


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 ?


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”


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”


Laboratory equipment






Heated glass worktop, integrated RFID antenna, touch screen interface

Future developments in the IVF labAutomation 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


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



Future developments in the IVF labAutomation 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



• Robot assisted oocyte retrieval!!


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

GM-CSF


• 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 apoptosisRobertson 2011, Sjoblom 1999

GM-CSFLIFPAFGHIGF-IIGF-IIEGFTGFβ

TNFαIFNγ


Many other compounds possible inclusion culture media? lipids/ prostoglandins

Future developments in the IVF labStatic 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?


• Further developments of in vitro culture


• Further developments of in vitro culture

m


Selecting for success

• How to predict viability?• Cell morphology• Early cleavage, polar body

orientation and pronuclei morphology


Selecting for successInvasive 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????



• Search for non invasive molecular techniques• DNA array –cumulus cells (Assou 2008), Follicular fluid (Hamel 2010),

Proteomic approach to sex selection (Picton 2010)

















Search for non invasive molecular techniques• DNA array –cumulus cells (Assou 2008), Follicular fluid (Hamel 2010), Proteomic


• Closest to implementation –Systematic study of the unique chemical fingerprint that in vitro embryo leave behind in culture media



• 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


• 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)


Time lapse evaluation

Morphological selection criteria in literature:

Cell divisionCell symmetry SynchronicityMulti-nucleationFragmentation

Abnormal cell division


Time lapse evaluation

Morphological selection criteria in literature:

Cell divisionCell symmetry SynchronicityMulti-nucleationFragmentation

Abnormal cell division

m


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 cyclecc2 = t3-t2cc3 = t5-t4s2 = 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)


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 cyclecc2 = t3-t2cc3 = t5-t4s2 = 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)

“Lab on a chip”

Sperm sorting

Imaging

Co-culture

Oxygen consumption

Biomarker analysis


Extra slides

The IVF lab of the futureAutomation in the ART. (mes)

• Mostly manual/sperm selection/oocyte selection/denudation/ICSI/monitoring/vitrification

• Emerging technology-alternative in all fields of medicine/ telepresence/education

• Da Vinci/AESOP/Zeus

• Robotic assisted follicle aspiration!

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


Fertility Preservation

Perfusion system –pulsatile gonadotrophin administration mimic pituitary-enhances follicular developmentWinkler 2009,2013


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

• 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 “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


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


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


Laboratory equipment• Witness system, RFID• Microscopic silicon based barcoding (Novo 2010)

Health & Medicine

The future ivf lab