How do external factors affect the morphodynamic behaviour of the human

embryo?

Giles Palmer, Mitera ACU, Athensgpalmer@mitera.gr

There are no commercial relationships or other activities that might be perceived as a

potential conflict of interest

Talk outline

• Review of 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

Review• Continuous viewing of 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

The era of morphokinetics

Chen 2013

Karyokinetics and cytokinetics

Second cell cycle

Third cell cycle

3-4 cell

5-8 cell

Karyokinetics and cytokinetics

Second cell cycle

Third cell cycle

3-4 cell

5-8 cell

*

*

Karyokinetics and cytokinetics

Subject to external factors affecting….•Maturity •Fertilization•Fragmentation•Cleavage rate/Blastocyst rate•Aneuploidy/ multinucleation

In vitro culture of embryos

In vitro culture of embryos

Subject to external factors affecting….•Maturity •Fertilization•Fragmentation•Cleavage rate/Blastocyst rate•Aneuploidy/ multinucleation

External factors affecting human embryoOperator factorsHandling, Media,

ICSI, IMSI, Embryo Biopsy

Clinical Factors

Age, Male, female factor,

protocols

Time-lapse system

LightElectric

fieldDish

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

Are time lapse systems 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

Clinical factors IStimulation ProtocolCan 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 developmentHigher E2 : positive effect on embryo dynamicsProgesterone level no correlation Help to define optimum ranges for stimulation protocols

Clinical factors IIBMI

•Bellver (2013) Similar morphokinetic patterns in embryos derived from obese and normoweight infertile women: a time-lapse studyNo effect on dynamic parameters of embryos but all infertile women had slower development than fertile donors•Lammers (2013) Effect of underweight females on embryo morphokineticsEmbryo development events (4-, 5-, 8-cell) occurred significantly later in underweight women

Smoking•Fréour (2013) Comparison of embryo morphokinetics in smoking and nonsmoking womenSignificantly 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 womenSlower pronuclear breakdown, impact on stages up to 8-Cell•Somava (2013)Peculiarities of early embryo development in women with polycystic ovarian syndromeProlonged timing to 5,8-cell stage & PCO patients had lower top quality embryos.

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 mediaCiray 2012 (2012) Time lapse evaluation of human embryo development in single versus sequential culture media-a sibling oocyte studyNo 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 oocytesNo 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 monitoringCulture 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

Embryo biopsy

• Kirkegaard (2012) Human embryonic development after blastomere removal: a time-lapse studyDuration of the stage at which biopsy occurred prolonged & later reaching t-compaction, t-morula, t-early blastocyst, t-full blastocyst.. but unchanged intervals

External factors affecting human embryoOperator factorsHandling, Media,

ICSI, IMSI, Embryo Biopsy

Clinical Factors

Age, Male, female factor,

protocols

Time-lapse

LightElectric

fieldDish

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

• Using Time lapse 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

Morphokinetics & quality control

CH CH

TX-100TX-100*P<0,05, **P<0,01, ***P<0,001

Optimum timing variables Morphokinetic model

Assessing IVF lab’s quality

• 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)

Use of KPI’s

Precisely defined targets

Minimum standard of proficiencyQuality improvement

Evaluating culture conditions/monitor performance

Time lapse and KPI’s

Time lapse and KPI’s

Take home messages• Time lapse systems archive embryo

development –prospects for selecting embryos with the greatest implantation potentialNew technology,Objective record of embryo development RCT needed

• Valuable tool to assess external /confounding factorsExternal factors may alter % embryos with optimum ranges

• New tool for quality controlAlternative to MEAMonitor drift, Lab conditions using KPI’s

Istanbul consensus workshopAlpha scientists & 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-ICSI28 ± 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