Oosight™ Imaging System• General Overview• The Concept• Review of the Biology• Principle Applications• Key Features & Benefits

Seeing Life in a New Light

General Overview• The Oosight™ Imaging System is the next generation

SpindleView™ System, re-engineered to fit the needs of the fast-paced ART lab.

• CRi’s PolScope technology offers real-time, quantitative detection of sub-cellular structures related to oocyte health and may provide a more accurate method for grading sperm and embryos.

• Data support the utility of the system for:– Egg grading– Spindle placement during ICSI– Egg freezing– Lab quality control – In vitro maturation– Nuclear transfer

The ConceptTo visualize and measure the molecular organization of cellular

components without staining.

The Role of the Meiotic Spindle

Microtubules organize into a dense array of aligned filaments and thick bundles forming the MEIOTIC SPINDLE during the first meiotic division.

This structure is responsible for proper chromosomal segregation throughout both meiotic divisions, and activation.

Abnormalities in the spindle have been linked to failed or abnormal fertilization, poor embryo development and failed implantation.

Images courtesy L Rienzi (left) and J LaFontaine (right)

History of the Science• The Oosight is based on technology that is the result of decades of research

pioneered by the scientist Shinya Inoué. • In the 1950s, Inoué was one of the first cytologists to make extensive use of the

polarized light microscope to observe birefringent components of the cell, which led to his landmark discovery in 1951 of the meiotic spindle fibers in living cells.

• In 1957, Inoué added a polarization rectifier to his custom-built microscope, which dramatically decreased distortion and improved the contrast of the image. Rudolf Oldenbourg further improved the polarizing microscope in the mid-1990s, adding liquid crystals with electro-optical controls and software called the LC-PolScope.

• The LC-PolScope was commercialized first as the SpindleView in 1999, later as the Oosight in 2005

• Its technology is being adapted to different application areas by Cambridge Research & Instrumentation CRi and is sold under trademarks such as Oosight. The Oosight has the same optical components and the algorithms as the LC-PolScope, but it is simplified for routine operation.

Current Business Ownership

• CRi were the previous owners of the technology into the IVF market 1999 - 2010.

• CRi and all of their technology have been purchased by the BioTech company Perkin Elmer (2012+)

• Research Instruments Ltd (RI) hold the exclusive distribution rights to all of Europe (including UK and Ireland), Africa, Middle East, South Pacific and most emerging markets.

• RI are the only global value added reseller (VAR) that install and service the system.

Birefringence and The Spindle

Birefringence is an optical effect that is caused by molecular order, such as is found when microtubules align in the meiotic spindle

Birefringence conveys a lot of structural information about the spindle, the multi-laminar zona pellucida and other filamentous structures

Hoffman contrast is entirely insensitive to birefringence

Oosight is capable of measuring birefringence on a pixel-by-pixel basis, reporting quantitative and reproducible measurements that correlate to the structural composition and further to developmental potential

Hoffman Contrast Oosight

How Oosight works: polarization

• Polarized Light:

Schematic A Schematic B

How Oosight works: birefringence• Birefringence: • Some materials are “birefringent”. Birefringence is a physical

property, important in optics.• When a single beam of mixed polarization encounters a

birefringent material it is split into two beams of pure (opposed) polarization. One beam is slowed down (retarded) more than the other and often redirected.

How Oosight works: retardance• Retardance: • “Retardance” is the distance between the faster and slower

beams leaving a birefringent material. Like any distance, it can be measured in kilometres, metres, millimetres, micrometers or nanometers (nm).

retardance

Retardance and Orientation• The Oosight system uniquely measures the magnitude and orientation of

birefringence which are called retardance and slow-axis orientation, respectively

• These two measurements can be thought of as structural density and molecular alignment

Hoffman Contrast

Oosight

Biological relevance• Biological materials with a high degree of linear molecular order

are birefringent. Eg: the spindle and the Zona Pellucida. • Birefringence increases with structural integrity and is therefore

an indicator of health!

Example of a healthy Spindle

Incoming Beam

Conclusion: This spindle has a high-degree of molecular order – an indication of health.

Slow outgoing beam

Fast outgoing beam

Spindle retardance =4nm

Biological relevance• Biological materials with a high degree of linear molecular order

are birefringent. Eg: the spindle and the Zona Pellucida. • Birefringence increases with structural integrity and is therefore

an indicator of health!

Example of an unhealthy Spindle

Incoming Beam

Conclusion: This spindle has a lower-degree of molecular order and thus may be less viable.

Slow outgoing beam

Fast outgoing beam

Spindle retardance =1nm

Normal vs. Dysfunctional Spindle Assembly

metaphaseplate

Centrosomalmatrix?

/-tubulin

Normal Dysfunctional

-tubulin

“Centrosome”

Centrosome Disposition vs. Meiotic Spindle Assembly

Figure courtesy D Battaglia, OHSU.

The Role of the Zona Pellucida

A multi-laminar glycoprotein coat that surrounds growing and mature oocytes as well as the preimplantation embryo

This structure plays a role in sperm-oocyte interactions, direct junctional coupling, cell–cell signalling, and exchange of molecules between the oocyte and the somatic compartment (Albertini and Rider, 1994; Motta et al., 1994)

Abnormalities in the zona have been linked to failed or abnormal fertilization, poor embryo development and implantation.

Image courtesy R Scott, RMA, NJMichelmann HW et al. J of Repro Med and Endorcrinology

Egg Grading and The Zona Pellucida

Mean Inner Zona Retardance = 1.76nm

Conclusion: The magnitude of light retardance by the zona pellucida inner layer appears to present a unique non-invasive marker for oocyte developmental potential.

Egg Grading :: Why Oosight?• Only quantitative method available to measure molecular

changes to critical structures within the egg

• Only method available to measure spindle misalignment

• There are direct correlations of retardance data to outcome

• The system is accurate, reliable and non-invasive

Principle Applications: Identify Optimal Timing for ICSI

Does the polar body accurately determine oocyte maturation?

Conclusion: Spindle imaging may be a better indicator than the presence of the first polar body for the determination of oocyte maturity prior to sperm injection.

Principle Applications: Position the Spindle for ICSI

Rotate spindle to 12 o’clockbefore ICSI

Conclusion: Embryos from spindle-aligned oocytes have an increase in all measured development parameters over control siblings.

ICSI :: Why Oosight?

• Avoid damage to genetic material upon injection

• Ensure full oocyte maturity prior to injection

• Improve overall embryo developmental potential

Principle Applications: Egg Freezing

Temp: 25°CTime: 16:24:02

Temp: 37°CTime: 16:12:35

Spindle IntactSpindle Depolymerized

Conclusion: The current techniques of oocyte freezing and thawing inevitably cause meiotic spindle destruction. All spindles observed in thawed oocytes result from post-thaw reconstruction, and those with spindles have a higher rate of developmental potential.

Egg Freezing :: Why Oosight?

• Only quantitative system to track changes in oocyte quality before freezing and after thawing

• Only method available to ensure complete spindle recovery after thawing