The ART-laboratory in the future; from Ego-based to Evidence-based?

1. The ART-laboratory in the future; from Ego-based toEvidence-based? Arne Sunde Head, The Fertility Clinic St. Olav’s University Hospital in Trondheim, Norway Professor, Norwegian University of Science and Technology in Trondheim, Norway Declaration of interest: Consultant to CellCura of Norway

2. Trondheim, Norway

3. The lecture • The ART laboratory • Procedures and strategies • Gametes and embryos • Culture • Scoring and selection • Safety… • Quality management

4. Ego-based ? • When I started in IVF in the early 80-ties, everything was ego-based • I think.., I have heard.., I believe..I suggest… • This was especially true for the clinical part • Now, a lot of the clinical procedures are evidence-based… • while the laboratory practise to a large extent still • is ego-based • We need to change this 

5. The black box in the middle The ART laboratory Evidence or Ego based? Luteal phase Endometrium Poorly understood Ovarian stimulation Regulated Evidence based

6. Microscopes Micromanipulators Workbenches LAF benches Centrifuges Incubators Monitoring systems Temperature, gas, humidity …. Culture dishes Tubes Pipettes Pipette-tips ICSI needles Micropipettes ……. Laboratory hardware, utensils and consumables for the ART lab

7. Some of it general purpose equipment Some of it adapted to ART Not necessarily well adapted Some of it designed for the ART lab Optimal design? Objectives? Validated design? ? The ART lab needs to be totally redesigned: Purpose built Validated No touch – closed systems Automatic Traceability QC/QA Laboratory hardware, utensils and consumables for the ART lab

8. Modern Lab installed at University of Leuven, Belgium

9. Culture media • Formulations differ substantially between media types/producers • Why? • Not often a lot of science backing a change of formulation • Clinical trials are expensive • Producers tend to keep formulations secret • Patent protection difficult • They add components that perhaps should not be there • Why?

10. Culture media – a warning ! • Culture media will influence many of the variables we use for monitoring the embryo • Growth kinetics • Morphology • Metabolism • Genetics ? • Your scoring system might not work independently of the culture system/media • Don’t forget that… !!!

11. Culture media – a warning ! • Some of the culture media have been designed to give embryos that we “like” • Fast growing, good looking • This may not necessarily select for the “best” embryos • Likely to contribute to the skewed sex ratio observed after blastocyst transfer (?)

12. Culture media – a warning ! • It is well established that IVF-culture media may introduce epigenetic changes and affect the offspring in animal models • (and in humans?.. Later in the talk..) • I am reluctant to use media: • that have been devised to increase growth rate • that contains hormones and growth factors • this may introduce gender bias or epigenetic changes

13. Culture Strategies and protocols? • Day 1/2/3 or blastocyst? • Fixed or adaptive? • Indications and protocols? • Cryopreservation • Slow freeze or vitrification? • When and how? • Culture media/protocols • Sequential or mono? • Complex or simple? • Protein supplements ? • Selection of gametes and embryos • Algorithms and methods? • PGS?

14. Strategies and protocols? • What are your success criteria: • Pregnancy rate from fresh transfers? • Cumulative delivery rate (fresh + frozen)? • Multiple pregnancy rate? • Or…? • Your success criteria will to a large extent: • Define your selection parameters • Have a strong influence on your culture strategy

15. St. Olav’s Hospital • Governmental - regulated • 1) Multiple pregnancy rate below 5% • >80% eSET • 2) Cumulative delivery rate above 55% • 3) Delivery rate from fresh transfers above 30% • Fertility INC • Private - competitive • 1) Delivery rate from fresh transfers above 45% • 2) Cumulative delivery rate high • 3) Multiple pregnancy rate? Success criteria defines: culture and selection strategy

16. Patients starting their first treatment in a given year Cumulative delivery rate including FER (including only first delivery in a couple) University Hospital Patients allocated up to 3 OPU’s 270 – 350 new patients each year

17. Cleavage stage or blastocyst? • Not many good studies done • a nightmare of bad design and confounders

18. Day 2/3 versus BlastocystCohrane 2007

19. Day 2/3 versus BlastocystCohrane 2007 Cumulative pregnancy rate including FER cycles

20. A good study:eSET cleavage stage or Blastocyst? Good prognosis patients < 36 years of age (mean 30,5) 1st. or 2nd. Trial FSH at Day 3 <12 IU/l Candidate for eSET Antagonist cycle Puregon on day 2 0,25 mg Ganirelix from day 6 hCG > 3 follicles > 17mm Papanikolaou, et al. ,N Engl J Med,354, 1139, 2006.

21. Cleavage stage or blastocyst? Papanikolaou et al. N Engl J Med. 2006;354:1139.

22. BUT what about cumulative delivery ratefresh + frozen? 4.2 vs. 2.2 P=0.001 Papanikolaou et al. N Engl J Med. 2006;354:1139.

23. Blastocyst transfer • Works fine (better) in good prognosis patients • What about the others ? • Swedish epidemiological data suggests a less favourable outcome (?) “the risk of preterm birth among singletons was significantly greater after blastocyst-stage transfer than after cleavage-stage transfer. The risk of congenital malformations was also significantly higher.” FertilSteril. 2010;94:1680–3.

24. Basis for selection of gametes and embryos General points in evaluating a selection parameter • Dependent or independent variable (s)? • Relative weight? • Predictive power? • Robustness? • Objective/subjective? • Practical in a routine setting? • Expensive? • Laborious? • Time consuming? • Useful in the general population? • Does it give the answer you are looking for ?

25. Dependent or independent variables?Spruce or pine ?

26. Dependent or independent variables?Spruce or pine ?

27. Dependent or independent variables?Spruce or pine ?

28. Dependent or independent variables?Spruce or pine ?

29. Dependent or independent variables?Spruce or pine ?

30. Dependent or independent variables?Spruce or pine ? Good correlates The three itself The needles The cones The smell The wood Appearance Microstructure The bark Colour, Appearance The seeds Shape, colour Associated micro fauna Fungi and mushrooms Chemical analysis The resin The seed oils Genotyping Weaker correlates Associated macro fauna Beetles, birds and squirrels Associated flora Flowers and plants Closest three a pine or a spuce? Frequency of emitted /absorbed light Greenness Elevation above sea level Not a true correlation Has a trunk Has branches Has roots Has needles Always next to a road Not in a city

31. Dependent or independent variables?Spruce or pine ? Choose independent variables that has predictive power and is easy to measure

32. Basis for selection of gametes and embryos • Dependent or independent variable (s)? • Do NOT waste time, money and energy on collecting redundant information. • The selection variable should ideally provide independent predictive power

33. Sperm • Spermiogram • Real-time morphology • Karyotyping • Aneuploidy • DNA integrity • DNA fragmentation • Binding to Hyaluronic acid • Normal chromatin (?) • Birefringence • Normal chromatin (?) • Oocytes • Transcriptome in cumulus cells (?) • Morphology • Zonapellucida, • Perivitelline space • 1st polar body appearance/karyotype • Granulated cytoplasm • Centrally located granular area • Meiotic spindle • location/appearance • Polar body karyotype • Aneuploidy • Mitochondria • polarity, number Selection of gametes correlates to fertilization and embryo development

34. Zona pellucida Polar bodies Perivitelline space Pronuclei Shape of embryo Shape of blastomeres Relative size of blastomeres Number of blastomeres Fragmentation Multinucleated Granulation Vacuoles Compaction Number of cells in ICM Shape of TE cells …. .. Morphological selection of embryos?

35. Embryo prediction model. J. Holte et al. Human Reproduction (22), 548–557, 2007 Univariate analysis

36. Embryo variables - logistic regression model J. Holte et al. Human Reproduction (22), 548–557, 2007 Variable P 1. Number of blastomeres <0.0001 2. Even-sized blastomeres <0.0036 3. Single nuclei <0.0004 Fragments NS Symmetry NS

37. Embryo scoring based on morphology • Embryo scoring has until now been done subjectively following various scoring systems • Negative • Very operator dependent • Positive • Fast, and powerful (parallel processing)

38. The new incubator generation –Not only an incubator… Incubator with: Non-invasive O2 sensors Automatic time-lapse recording Real-time computer analysis

39. Embryo physiology as basis for selection “Metabolic profiling” • It is possible to measure embryo metabolism non-invasively • Metabolic profiling could then be used to select the most viable embryos? Pioneered by Prof. Henry Leese, York

40. Near infrared and Raman spectra? • Spent culture media analysed by infrared spectroscopy. • Unclear why it seems to work ! Seli E, et al. Fertil Steril.2007;88:1350–1357.

41. FISH analysis for chromosomes 13, 16, 18, 21, 22, X and Y in all blastomeres of IVF pre-embryos from 144 randomly selected donated human oocytes Ziebe et al. Human Reproduction. 2003;18:2575. Aneuploidies and mosaicism in early embryos are physiological in humans(?)

42. Is PGS using FISH dead? Mastenboek et al, New Engl. J.Med. 357, 2007,

43. Lets’ not be too confused • CGH can be used to identify embryos that are euploid for all chromosomes. • Almost 80% of all embryos contains cells that are aneuploid • Embryos that are euploid for all chromosomes implant more frequent than abnormal embryos. • Then is should be easy • Just find the euploid embryos and transfer those  ??

44. Just for the sake of the argument • 100 patients • 20 with at least one uniformly euploid embryo • 16 pregnant (80%) • Great  • “It just to screen all embryos in order to find the right one, then most of the patients will become pregnant.. ..”

45. Just for the sake of the argument • In the real world: • 80% of the patients does not have an uniformly euploid embryo • What do you do then ? • Do we know which one (s) of the abnormal embryos we should transfer/freeze??

46. VOLUME 15 [ NUMBER 5 [ MAY 2009 Black: normal Purple: homozygous deletion Red: hemizygous deletion Green: duplication Dark green: amplification Yellow: UPID

47. VOLUME 15 NUMBER 5 MAY 2009 “….analysis unexpectedly revealed a high frequency of chromosome instability in cleavage-stage embryos involving complex patterns of segmental chromosomal imbalances and mosaicism for whole chromosomes and uniparental isodisomies. These patterns are reminiscent of the chromosomal instabilities observed in human cancers and correspond with the complex chromosomal aberrations observed in individuals with birth defects…”

48. I have this sinking feeling that we haven't understood it at all…

49. SummaryMarkers of oocyte/embryo competence • Morphology and growth kinetics can be used to select the “best” embryos in a cohort • Measurements of morphology and growth kinetics may be less operator-dependent in the future • Objective measurement • Decision algorithms • Genome • Karyotyping using FISH seems to be of limited clinical value • Why…? • Do we have the wrong approach ? • Transcriptome • Still unclear whether it will prove useful in the clinical routine • Metabolome • Secretion/uptake of metabolites may prove useful in the future • Near infrared spectroscopy may be useful – unclear why?

50. Safety..? Is the clinical embryology lab a safe place to be for gametes and embryos.? Random events Accidents, equipment failure, loss of biological traceability….. Systematic effects Systematic effect of In Vitro Culture? Epigenetic effects ?