Implementation of Novel Technologies in the IVF lab.

Basak BalabanHead of IVF Laboratory

VKF American Hospital, Istanbul-Turkey

Rapid inclusion of new technologies in IVF lab. 1994- 2014

• 1994 Intra-uterin insemination, classical in-vitro fertilization

• 1995-1996 ICSI, PESA, TESE/A• 1998 Embryo freezing• 2000 Novel technologies, introduction of objective

biomarkers;• OMICS (Metabolomics..), PGD, IMSI, polarized

microscopy (Meiotic spindle, birefrigence..) PICSI...• Late 2000s Time-lapse• 2014 RI Witnessing System

The phases of new techniques

Phase 1: Introduction (e.g. Initial publication)

Phase 2: Validating (e.g. RCT)

Phase 3: Long term use and validation (longditutional studies)

Vergouw et al., HR 2012

Lack of benefit from PGS with FISH;

*Injury to the embryo from blastomere biopsy*Mosaicism of the embryo for aneuploidy(potentially leading to false + diagnosis)*Limited no.of chromosomes studied (potentially leading to false – diagnosis)*FISH only detects the presence or the absence of the chromosomal region targeted by the probe(usually at the centromere), and does not provide information about the remainder of the chromosome

*Day 5 biopsy & 6 ET can only be used for good prognosis patients

*TB can only be applied for exp.blasts.

*Risk of decreasing the implantation potential of a poor quality blast. by day6 ET

Scott R et al., F&S 2013 in press

Spindle imaging of the MII oocyte

De Santis et al., RBM Online 2005

During meiosis and fertilization meiotic spindles are responsible for propersegregation of the nuclear material, and abnormalities in this fragile structure can lead to infertility, miscarriage and genetic diseases, such as Down syndrome

Viability assessment by birefringence imaging with polarization microscopy

Why are results contradictory?

• The dynamics of spindle formation during oocyte maturation were not considered

1. Spindle visualization might change within the maturation stages: MI-1st.PB extrution- telaphase I (MS dissapear for 40-60 minutes!!)- MII

2. The microtubules of the MS are highly sensitive to chemical (hyaluronidase), and physical changes ( temperature& pH variations) that may occur during oocyte handling. Shift of the PB1 position may also be related to physical displacement during denudation.

• A precise classification spindle imaging should be performed repeatedly: after hyaluronidase treatment and immediately prior to ICSI

Rienzi et al., RBM Online 2005Montag et al.,RBM Online 2006

Oocyte zona birefringence intensity

High zona birefringence Low zona birefringence

Montag et al.,RBM Online 2008

Polarization microscopy allows the distinction of three layers within the ZP. Inner layer exhibits the highest birefringence(Pelletier 2004). Zona birefringence intensity is higher in conception cycles(Shen 2005)

The multilaminar structure of the ZP revealed by polarization microscopy is directly linked to the paracrystalline network structure of the zona which is formed during the follicular maturation by the oocyte.

So a high birefringence of the inner zona layer might indicate an optimal formation of the ordered structure during oocyte maturation. HZB oocytes can have better conditions during follicular growth and maturation compared with a LZB oocyte with unordered zona structure. Regular structural integrity of ZP may reflect an optimal cytoplasmic potential of an oocyte and its various cellular and molecular structures

Ebner-Balaban F&S 2010

undetectable

Ebner - Balaban F&S 2010

Time-lapse imaging; morphokinetics • One of the fundamental problems of the current embryo quality assessment by

morphology is the static evaluation of a highly dynamic developing entity. Current systems analyze the morphology at a few predefined time points during embryo development preimplantation, with the consequent lack of information about what happened between the analyzed points. Thus continual monitoring might provide one strategy to collect a complete picture of embryo developmental kinetics and improve the success rate of viable embryo selection

• So far 2 algorithm have been proposed for morphokinetic embryo selection to improve success rates based on parameters derived from time-lapse measurements

** Wong Nat Biotechnology 2010; Analyzed time-lapse image series of embryo development to identify predictive markers of blastocyst development and the underlying gene expression profile (arrested /normal developing) Sample was composed of supernumerary embryos cryopreserved at the 2PN stage and donated to research. None of the embryos were transferred. No data on IR were obtained. Hierarchy of morphokinetic markers were not used for decision making

** Meseguer HR 2010, F&S 2012

Time-lapse markers used for clinical outcome predictions

Chen et al.,F&S 2013

CGH from TE biopsy

RBM Online 2013 in press

**Morphokinetics and embryo aneoploidy: has time come or not yet?**Will it be possible to define exact boundries for the timing of the different risk classification

groups in the proposed model, as variations/confounding parameters (different patient profiles, lab. conditions....)should be expected for individual labs and clinics

Montag M. RBM Online 2013

MSOME: Motile sperm organelle morphology examination IMSI: Intracytoplasmic morphologically selected sperm injection

Bartoov et al.,HR 2006

Selection of the motile spermatozoa with normal nucleus at 6300X objectiveElimination of the sperm cells with vacuoles within the nucleus

Bartoov 2001,2002,2003; Berkovitz 2005IMSI range:X6000-X12.500

**Selected group of male factor infertility

Significantly improvedCPR & IR,

Significantly reducedmiscarriage rates

Vanderzwalmen 2008 had shown that only sperm with ≥1 or large vacuoles with other head abnormalities do effect embryo development. Perhaps the group of patients who do contain a high % of such spermatozoa are the target group who would most likely benefit IMSI

Balaban et al., RBM Online 2011

Increased workload??

Ratio had only been suggested for no.of cycles. Details of the procedures, inclusion of the novel technologies are not included!!!! RISK MANAGEMENT should carefully be evaluated!

How often do you make mistakes?We never make mistakes!

Mistakes are very rare in our lab...

Scientific TRUTH!! is sample identification and mismatching errors may occur, and their incidence can increase with the increased workload in the lab.!! (1987, Liebler 2002, Spriggs 2003, Bender 2006)

**Even ART mix-ups is rare, their consequences can be devastating for both couples and fertility centers, leading to complex paternity suit and legal actions against the clinics

We think it is low...but we don’t know

Mandatory guidelines of medical-scientific societies

• ESHRE, FLASEF(Latin America), HFEA mandates in their guidelines and codes of practice the permanent labeling of all labware to identify the source of the biological material inside the tube or dish, and the application of witnessing protocols to double check the identification of samples and the patients or donors whom they relate, at all aforementioned critical points of the clinical and lab. procedures

Witnessing protocols should be followed when any of the following clinical or laboratory procedures take place:

• Collecting eggs• Collecting sperm• Preparing sperm• Mixing sperm and eggs or injecting sperm into eggs• Transferring gametes or embryos between tubes or dishes• Transferring embryos into a woman• Inseminating a woman with sperm prepared in the laboratory• Placing gametes or embryos into cryopreservation• Removing gametes or embryos from cryopreservation• Disposing of gametes or embryos• Transporting gametes or embryos

**Inclusion of novel technologies

BarcodesMatcherTM

RFID TagsRI WitnessTM

Silicon-based barcodes

Labelling Systems; Available Alternatives

Double manual witnessing is time consuming and distracting, and sufficient no.of lab. stuff is needed!!

Direct labeling system: Silicone-based barcodesTo provide a proof of concept for a direct oocyte/embryo labeling system

• Radio Frequency Identification (RFID)- Consumables are ‘tagged’ with an adhesive RFID label which when

detected by a reader generates a signal- This detected signal is used to uniquely assign the item- The allocation is permanently and irreversibly stored- Every time an item enters the read range it is detected and identified

in the database

• RI Witness™ relies on three components:- RFID tags- RFID tag reader hardware- Controlling software

The hardware – RFID tags

1. Circular tag1 2

3 4

2. Standard tag

3. Square tag

4. ID card

The hardware – tag readers

Real time process protection

Is it easy to use??...

1. Log in

2. Enter PIN

3. Select

4. Allocate

5. Confirm

6. Complete

Is it effective?

• Number of IVF Mix-up events in IVF centres using RI Witness worldwide =

0• ALL potential incidents prevented as ‘Near

Misses’

Is it safe?• Independently tested in Belgium in a large testing

facility• Tests on mouse embryos exposed to an RFID field

700 times stronger and for 48 times the duration of normal working practice

• No differences in embryo development rate, quality of embryos or implantation rate following these tests

• The system has been in use clinically since 2007;– No reported no change in fertilisation rate, cleavage rate,

embryo & blastocyst quality, pregnancy, implantation or live birth rates.

RI Witness records all laboratory proceduresType of procedure + Patient + Operator + Time

Identification of the true mismatch rate (0.11%) (Guy’s London)

Error Rate Type of study Author

6% Mismatches using barcode based system (MatcherTM) Schnauffer et al.

0.2% 1000 drugs administration (by 2 nurses) Krause et al.

7 - 10 % Pathology studies Elson D.

0.3 % Mistakes in 4 hospital departments Carraro et al.

0.8 % Data input: keyboard vs. Barcode system Shaw et al.

0.1% Wristband mix-ups involving 2 patients Valenstein P.

What is the mismatch rate??

Implementing an electronic witnessing system into a busy IVF clinic – one clinic’s experience.

Sanges, et al.,Genera Center for Reproductive Medicine, Rome, Italy ESHRE 2013 London , Poster presentation

Study question: The aim of the study was to evaluate the time required to install an ex-novo electronic witnessing system (RI Witness™) into a busy IVF clinic.

Participants/materials, setting, methods: The simultaneous presence of two different patient samples in the working area was defined as “true mismatches”, whilst mismatches derived from acceptable common errors were defined as “secondary mismatches”. Finally, we estimated the level of satisfaction to laboratory staff (5= very,4= fair,3= adequate,2= poor, 1= not at all).

Main results and the role of chance: The installation and integration of the system required about 4 working days whilst the training of all the laboratory staff was carried out the week after. The validation period took one month and a total of 2099 witnessing steps were carried out. During this period, a total of 17 mismatches (0.81%) were recorded, 2 of which were true human errors (0.09%) and required additional intervention. During the post-validation period, a total of 5921 witnessing steps we carried out with 852 patients. The total mismatch rate was 0.66%(39/5921). Excluding secondary errors, we recorded a true human error rate equal to 0.10%(6/5921). Errors due to system configuration were recorded during and after the validation period (0.47 and 0.05, respectively). The user satisfaction index was 4.8.

Summary answer: The integration of the RI Witness™ system in the daily routine is simple, fast and safeguards the reliability of the entire IVF process.

Wider implications of the findings : Our experience suggests that the integration of RI Witness™ in the daily routine is simple and fast and allows for an improvement in system usage after a short time. RI Witness™recorded a constant true human error rate that is within the acceptability range. Moreover, the warning of mismatches permits immediate corrective intervention, safeguarding the reliability of the entire IVF process. Other benefits include the traceability of each step performed and a reduction of staff workload and distractions, thus increasing operator satisfaction.

Patient’s views

– Impressed with deployment of a high-tech approach to prevent laboratory errors

• Overwhelming support for system implementation

– Confident the embryos created and transferred were created with their gametes

– Feel that the system affords a sense of ownership and control throughout the treatment process

ELECTRONIC WITNESSING: Benefits

• Easy to implement

• Safeguards the reliability of the entire IVF process

• Traceability of each step performed

• Reduction of staff workload and distractions

• Patients reassurance

Special Thanks to...

• Laura Rienzi, Rome ITALY• Alan Thornhill, London UK• Sally Lloyd, Research Instruments UK

Configuration of the working flow chart

Implementing An Electronic Witnessing System Into A Busy IVF Clinic – MUST for a 21th.century IVF Lab!!

GOOD MANAGEMENT IN THE IVF LABORATORY

LAWS -EU

DIRECTIVES

SCIENTIFIC BACKGROUND

-UPDATE

QUALITY SYSTEM

RISK MANAGEMENT

MINIMIZE RISK MAXIMIZE TRACEABILITY

Each lab must put in place a quality management system and implement this system to continually

improve the quality and effectiveness of the service provided in accordance with the guidance on good

practice