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Final external factors
External factors such as culture conditions, stimulation protocols, and patient characteristics can affect the morphokinetic development of human embryos observed through time-lapse monitoring. Specifically, higher FSH doses, higher estrogen levels, underweight BMI, smoking, and PCOS are associated with slower embryo development. Differences in culture media brands and oxygen concentration can also influence timing of developmental stages. While time-lapse monitoring provides more detailed data on embryo development and a tool for quality control, further randomized clinical trials are still needed to determine the impact on pregnancy outcomes.
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Final external factors
- 1. How do externalfactors affect the morphodynamic behaviour of the human embryo? Giles Palmer, Mitera ACU, Athens gpalmer@mitera.gr
- 2. There are nocommercial relationships or other activities that might be perceived as a potential conflict of interest
- 3. Talk outline • Reviewof time lapse embryo monitoring • External factors & how they affect the in vitro embryo • Utilization of time lapse monitoring to assess external factors • Time lapse monitoring as a tool for quality control
- 4. Review • Continuous viewingof in vitro human embryos development • More observations/less disturbance • Archiving the “history” of embryos • Greater implantation potential? choosing optimum morphokinetic patterns • Selecting out less viable embryos • Teaching, communication tool…..research tool Conventional incubator Time lapse microscope Microwell embryo culture dish with the developing embryos
- 5. The era ofmorphokinetics Chen 2013
- 6. Karyokinetics and cytokinetics Secondcell cycle Third cell cycle 3-4 cell 5-8 cell
- 7. Karyokinetics and cytokinetics Secondcell cycle Third cell cycle 3-4 cell 5-8 cell * *
- 8.
- 9. Subject to externalfactors affecting…. •Maturity •Fertilization •Fragmentation •Cleavage rate/Blastocyst rate •Aneuploidy/ multinucleation In vitro culture of embryos
- 10. In vitro cultureof embryos Subject to external factors affecting…. •Maturity •Fertilization •Fragmentation •Cleavage rate/Blastocyst rate •Aneuploidy/ multinucleation
- 11. External factors affectinghuman embryo Operator factors Handling, Media, ICSI, IMSI, Embryo Biopsy Clinical Factors Age, Male, female factor, protocols Time-lapse system Light Electric field Dish incubation Laboratory (direct and indirect) Culture conditions, Temp °C, pH, %CO2, %O2, air quality (VOC’s) t2 t3 t4 t5 t8 cc1 cc2 cc3 s2 s3
- 12. Are time lapsesystems safe? • Cruz et al. (2011) Embryo quality, blastocyst and ongoing pregnancy rates in oocyte donation patients whose embryos were monitored by time lapse imaging • Nakahara et al. (2010) Evaluation of the safety of time lapse observations for human embryos • Kirkegaard et al. (2012) A randomized clinical trial comparing embryo culture in a conventional incubator with a time-lapse incubator • Meseguer et al. (2013) Embryo incubation and selection in a time-lapse monitoring system improves pregnancy outcome compared with standard incubator: a retrospective cohort study • Vajta (2008), Hoelker (2009), Dai (2012), Francovits (2013) WOW dish group culture in micro well improving embryo development
- 13. Clinical factors I StimulationProtocol Can the type of protocol used in COH influence the kinetics of embryo development ? •Muñoz (2012) Dose of recombinant FSH and oestradiol concentration on day of HCG affect embryo developmental kinetics Examined the use of rFSH only, HMG and FSH and HMG in oocyte donors: No difference in embryo kinetics between the groups Embryos obtained by rFSH showed increase in the proportion of optimum timing embryos but not significant. Dose of gonadotrophin and E2 concentration showed differences in embryo development: Higher FSH dose: slower embryo development Higher E2 : positive effect on embryo dynamics Progesterone level no correlation Help to define optimum ranges for stimulation protocols
- 14. Clinical factors II BMI •Bellver(2013) Similar morphokinetic patterns in embryos derived from obese and normoweight infertile women: a time-lapse study No effect on dynamic parameters of embryos but all infertile women had slower development than fertile donors •Lammers (2013) Effect of underweight females on embryo morphokinetics Embryo development events (4-, 5-, 8-cell) occurred significantly later in underweight women Smoking •Fréour (2013) Comparison of embryo morphokinetics in smoking and nonsmoking women Significantly later cleavage in most embryo development events Polycystic ovary syndrome •Hoest (2012) Slower early embryo development in women with Polycystic ovary syndrome (PCOS) compared to regularly cycling women Slower pronuclear breakdown, impact on stages up to 8-Cell •Somava (2013)Peculiarities of early embryo development in women with polycystic ovarian syndrome Prolonged timing to 5,8-cell stage & PCO patients had lower top quality embryos.
- 15. Culture conditions • Pribenszky(2012) Effect of maternal factors and culture conditions on in vitro development Differences (mixed effect model) reported in 5-8-Cell interval with different brands of media Ciray 2012 (2012) Time lapse evaluation of human embryo development in single versus sequential culture media-a sibling oocyte study No differences between the durations for second cell cycle cc2 (t3-t2) and s2(t4-t3) but embryos cultured in single media were advanced from the first mitosis cycle and reached 2- to 5-cell stages earlier • Basile N (2013). Type of culture does not affect embryo kinetics: a time lapse analysis of sibling oocytes No differences comparing two different types of concept media in variables studies such as t3, t4 etc. or % embryos falling out of the optimum time ranges • Kirkegaard (2012) Effect of oxygen concentration on human embryo development evaluated by time lapse monitoring Culture in 20% O2 reduces development rates and delays completion of 3rd cell cycle. More embryos in 20% and 5%, and 5% only reached 8-Cell, EBC, BC than 20% O2 only
- 16. Embryo biopsy • Kirkegaard(2012) Human embryonic development after blastomere removal: a time-lapse study Duration of the stage at which biopsy occurred prolonged & later reaching t-compaction, t- morula, t-early blastocyst, t-full blastocyst.. but unchanged intervals
- 17. External factors affectinghuman embryo Operator factors Handling, Media, ICSI, IMSI, Embryo Biopsy Clinical Factors Age, Male, female factor, protocols Time-lapse Light Electric field Dish incubation Laboratory (direct and indirect) Culture conditions, Temp °C, pH, %CO2, %O2, air quality (VOC’s) t2 t3 t4 t5 t8 cc1 cc2 cc3 s2 s3
- 18. • Using Timelapse as a QC tool • Observations of timing & patterns of cell divisions of mouse embryos with exposure to toxins (Cumene Hydroperoxide and Triton X-100) • Challenge to MEA test vs. continuous objective records • Morphokinetic patterns more sensitive than BC formation • Enhanced assay system detection 24-48hrs sooner than MEA test
- 19. Morphokinetics & qualitycontrol CH CH TX-100TX-100 *P<0,05, **P<0,01, ***P<0,001 Optimum timing variables Morphokinetic model
- 20. Assessing IVF lab’squality • Implantation /Clinical pregnancy/Delivery rate • Fertilization rate/Cleavage rate • “Good Quality” Blastocysts • Embryo utilization • Use of Key Performance Indicators (KPI’s) Use of Key Performance Indicators (KPI’s)
- 21. Use of KPI’s Preciselydefined targets Minimum standard of proficiency Quality improvement Evaluating culture conditions/monitor performance
- 22. Time lapse andKPI’s
- 23. Time lapse andKPI’s
- 24. Take home messages •Time lapse systems archive embryo development –prospects for selecting embryos with the greatest implantation potential New technology, Objective record of embryo development RCT needed • Valuable tool to assess external / confounding factors External factors may alter % embryos with optimum ranges • New tool for quality control Alternative to MEA Monitor drift, Lab conditions using KPI’s
- 26. Istanbul consensus workshopAlphascientists & ESHRE SIG embryology, 2011 Type of observation Timing (hrs. post insemination Expected stage of development Fertilization Check 17 ± 1 Pronuclear stage Syngamy check 23 ± 1 Expect 50% to be in syngamy (up to 20% may be at the 2-cell stage) Early cleavage check 26 ± 1 post-ICSI 28 ± 1 post-IVF 2-cell stage Day-2 embryo assessment 44 ± 1 4-cell stage Day-3 embryo assessment 68 ± 1 8-cell stage Day-4 embryo assessment 92 ± 2 Morula Day-5 embryo assessment 116 ± 2 Blastocyst
Editor's Notes
- #4 Apologise if heard first talk immerging technology How precisely tl can be used to study external factors Time lapse as a means to study external factors
- #5 Continuous viewing of in vitro human embryos development- picture of embryo from within the incubator approx ev. 10 mins More observations/less disturbance-most evaluate embryo by removal from incubator Archiving of “history” of embryos- review each embryo before embryo transfer Embryos chosen by time lapse parameters- according to Morphokinetic pattern and timing of cell division it’s a non invasive tool for embryo selection Prospects of greater implantation potential- has been reported good prediction of viability, blastocyst formation and implantation potential algoriths-search for algorith Teaching, communication, aid to colleagues and clinicians Selecting out less viable embryos-exclusion criteria, abnormal cell division, 1-3 cells, slow growth, some parameters assoc with aneuploidy
- #6 Overall time lapse markers have exhibited remarkable correlation wth human embryo devstudies shoe remarkable reproducibility across different clinics and labs..clustering on early stages before 5 cell stage Timing from later stages a bit more subject/diff to define but may give us addition help Dark red =at least three publications
- #10 All embryos in culture subject to external factors No doubt extrernal factors effect embryo development either pos or neg effect. Done with static observations but now more precisely with TL Maturity- related to stimulation protocol-syngamy which is a sensitive indicator of maturity (Lawler 2007), can effect timing of embryo development (Lechniak 2008) Fertilization Fragmentation-sign perhaps of embryo stress, ,10% negligible (van royen 2001), trend between frags and aneuploidy (ziebe 2003, 2006) We have seen fragmentation can be a dynamic event Cleavage arrest/rate-single most important indicator of viability..numerous authors. And reported too fast or too slow embryo cleavage has negative impact on implantation ( edwards 1980, Giorgetti 1995, Ziebe 1997, Van Royen 1999) Blastocyst rate-end product of many studies showing good embryo culture conditions Euploidy/ multinucleiation- report vary as too extent but …….Munne ????, multinuclietion may effect embryos in over 44% of patients and may be efferted by sub optimun culture conditions ( winston 1991, Pickering 1990) We states earlier that link between cleavage patterns and aneuploidy (Davies 2012, Basile 2013) See stress in notes
- #11 All embryos in culture subject to external factors No doubt extrernal factors effect embryo development either pos or neg effect. Done with static observations but now more precisely with TL Maturity- related to stimulation protocol-syngamy which is a sensitive indicator of maturity (Lawler 2007), can effect timing of embryo development (Lechniak 2008) Fertilization Fragmentation-sign perhaps of embryo stress, ,10% negligible (van royen 2001), trend between frags and aneuploidy (ziebe 2003, 2006) We have seen fragmentation can be a dynamic event Cleavage arrest/rate-single most important indicator of viability..numerous authors. And reported too fast or too slow embryo cleavage has negative impact on implantation ( edwards 1980, Giorgetti 1995, Ziebe 1997, Van Royen 1999) Blastocyst rate-end product of many studies showing good embryo culture conditions Euploidy/ multinucleiation- report vary as too extent but …….Munne ????, multinuclietion may effect embryos in over 44% of patients and may be efferted by sub optimun culture conditions ( winston 1991, Pickering 1990) We states earlier that link between cleavage patterns and aneuploidy (Davies 2012, Basile 2013) See stress in notes
- #12 External factors…..Analyse factors that may effect the accuracy of timelapse If external factors do something then we can not have a model Chrcvk diagram Time lapse less intervention but concerns Conclusion????Comprehensive assessment of human embryos has been unachievable with trad. Static observations without comprimizing development….are there stage specific temporal effects
- #13 Say concerns If not better embryo culture less disturbance See kirkaard Technology driven vs rct Implimant new technology we must…….. Mess even better than convent incubators Now long term study
- #14 Dose of gonadotrophin and E2 concentration showed differences in embryo development allowing for them to say an optimum dose of FSH and E2 …………….this probably echos a study showing overstim = less eupoidy embryos FINF These kinetcic difeference did not translate to increased implantaion potential Although ovarian stimulation has an important role in assisted reproduction, it may also have detrimental effects on oogenesis, embryo quality, endometrial receptivity and perinatal outcomes. Sertyel 2013 overstim effect eupoidy Allows for the first time to assess thing..like..that might impair embryo qual & development Obese females reprduction wise have lower fecundity, and miscarrieage rates alos in IVF have lower preg rates as BMI increase (Bellver 2010).. Related to reduction in oocyte competance embryo quality and uterine recptivity (Bellver 2007) No different but obese or infert bith slower than fertile donors at t2, t4, t5 and more embryos from o.d were in the optimum range (not signif) High glucose may effect metabolic pathways but studies vary in if effects or not Interstingly a poster on underweight infert patients showed a delaying of cellular events in all catorgories (Lammers) Slower development of PCOS woemen compared to regulary cycling women but the morphokinetic profile of embryos from Pregnnant PCO patients were similar to the reg cycling ones
- #15 Dose of gonadotrophin and E2 concentration showed differences in embryo development allowing for them to say an optimum dose of FSH and E2 …………….this probably echos a study showing overstim = less eupoidy embryos FINF These kinetcic difeference did not translate to increased implantaion potential Although ovarian stimulation has an important role in assisted reproduction, it may also have detrimental effects on oogenesis, embryo quality, endometrial receptivity and perinatal outcomes. Sertyel 2013 overstim effect eupoidy Allows for the first time to assess thing..like..that might impair embryo qual & development Obese females reprduction wise have lower fecundity, and miscarrieage rates alos in IVF have lower preg rates as BMI increase (Bellver 2010).. Related to reduction in oocyte competance embryo quality and uterine recptivity (Bellver 2007) No different but obese or infert bith slower than fertile donors at t2, t4, t5 and more embryos from o.d were in the optimum range (not signif) High glucose may effect metabolic pathways but studies vary in if effects or not Interstingly a poster on underweight infert patients showed a delaying of cellular events in all catorgories (Lammers) Slower development of PCOS woemen compared to regulary cycling women but the morphokinetic profile of embryos from Pregnnant PCO patients were similar to the reg cycling ones
- #16 Media comaprisons have focussed in the past by static observations with possibly poorely controlled time points an often did not supply specific cell numbers Mauri 2001, xella 2010 Morphokinetics differences may be attributed to media ingrediates that may effect metaboloc pathways Basile…..may validate the algorith for embryo selection in diverse culture conditions To test the media is to test the accuracy of time lapse ------------- Traditionally at 20% o2. Animal studies show oviduct 7% with furher decrease in uterine enviromenty SUDIES SHOWING IMPROVEMENT????? All media = preg and implantation were similar
- #17 Blastomere STILL the most comon form of biopsy Earlier embryo bipsy papers show BC rate is lower when 2 cells removed compared with one (Goosens 2008) De vos indicating impairs development but ……………………..think to add Biopsy also alters hatching process..maybe dekayed or abnormal hatching (Cohen 1991, Duncan 2009) Can use tl to measure BC size!!!!! Which is good and shed light on hatching process ( poss expandion causes thinning of zona) Now change of course in time events before biopsy but then duration at which the stage whem they were biopsied was longer.And reaching tcompaction, tmorula, tearly blastocyst, tfullblastocyst later..but unchanged intervals BUT there was no zona thinning and hatched at the same time but because of delay were signif smaler in size Spending shorter time in the Full blastomere stage!!!!!
- #18 External factors…..Analyse factors that may effect the accuracy of timelapse If external factors do something then we can not have a model Chrcvk diagram Time lapse less intervention but concerns
- #19 First study to use TL as QC for Mouse embryos..to detect toxins in culture media: from MAYO CLINIC In vestigated Cumene Hydroperoxide ( which can accumulate naturally in oil overlays) and Triton X-100 (a frequent contaminate of oil) And their affect on mouse embryo morphokineticas Cryo preserved 1 cell mouse embryos F1 bred Usually bioassy such as MEA and sperm survival test used by labs and manufactures to test IVF products. In the case of MEA the end test is blastocyst formation but this study shows may not be as sensitive as we think because toxins were detected by time lapse at concentrations that did not effect the BC rate??? And bc not always assoc with viability..if test as end point bc it may not always pick up clin relevant toxins
- #20 See % optimum timing embryos with relation to certain developmental events:such as cc2 the 2nd cell cycle ie the time the embryo is at 2cell, t2 the time to 5 cell and tb the time to see the blastocoel With time lapse monitoring, all but lowest concentration of hydroperoxide was detected Early delay in development with CH of 0,4 conc with more profound effect at t5 ??????? b)With Triton X a formation of blastocoel cavity was very sensitive with delay of 5 hours ---------------- Interesting thing with tl isd that swe can combine several objective measurements into one analysis model ie cc2, t2, tb combined to 1 mophokinetic model….in thus model the lowest conc of both toxins were detected. While looking at bc alone only saw a signif differnce at 6um for CH, and highest conc for triton So TK with its objective recording of embryo development couldd be arobust test in future
- #21 Tl surpasse the phase of embryo selection and can be used for quality control setting. Optimum culture conditions are a product of good QC sytem TL to provide monitoring of standard conditions Especially if difference in media and lab time frames
- #22 Essential for evaluating things in the lab Benchmark=aspiration value..best practice goal Alpha set minimum criteria for oocyte and embryo assessment Detect changes in Lab Environment (air) Track Lot number variation (cm, stim, lab tech) Develop KPI to compare laboratories Improve QC of suppliers
- #23 Essential for evaluating things in the lab Benchmark=aspiration value..best practice goal Alpha set minimum criteria for oocyte and embryo assessment Detect changes in Lab Environment (air) Track Lot number variation (cm, stim, lab tech) Develop KPI to compare laboratories Improve QC of suppliers
- #24 Essential for evaluating things in the lab Benchmark=aspiration value..best practice goal Alpha set minimum criteria for oocyte and embryo assessment Detect changes in Lab Environment (air) Track Lot number variation (cm, stim, lab tech) Develop KPI to compare laboratories Improve QC of suppliers
- #25 Future greater numbers Quality management should utilize metrics that are sensitive and easy to obtain Customixed TL patterns: Profiling of embryo development containg subgroups for subfertilie couples May be diff due to ex factors (x factor!) May see import of interphases than timing events? To test accuracy of time lapse Large no. confounding papers, [posters inheret in clinical embryology must be concidered when analsising embryo development Investigation of ex facter fact must .. Choose Best fit pattern or %optimum morphokinetic Lab use qc to monitor periods of poor performance
- #27 Difficult to do in a lab where insem times difdfer and observations restricted to work load Concencus important for uniformitry and critical for coparison