30
Giovanni Coticchio Oocyte quality: dysmorphic patterns and cytoskeletal alterations in human oocytes Building a bridge between science and clinical practice 15 – 16 September, Barcelona
Giovanni Coticchio
Oocyte quality: dysmorphic patterns and cytoskeletal alterations in human oocytes
Building a bridge between science and clinical practice 15 – 16 September, Barcelona
Physicians R. Fadini M. Mignini Renzini C. Brigante F. Benedetti A. Caiazzo I. Caliari R. Comi S. Gippone A. Epis R. Iemmello L. Maragno A. Villa
Embryologists M. Dal Canto F. Brambillasca M. Crippa M. Lain M. Merola C. Moutier C. Guglielmo P. Novara M.Sottocornola D. Turchi A. Longoni
Acknowledgements David Albertini Center for Human Reproduction New York
Stefania Nottola Università “La Sapienza” Rome
Disclosure
No interests shared
with companies or commercial organizations
active in the subject covered by this talk
Heterogeneity of human oocytes
Size / Shape
Source: ESHRE Atlas of human embryology — http://atlas.eshre.eu
Diploid and often with two spindles
Size / Shape Cytoplasm
Source: ESHRE Atlas of human embryology — http://atlas.eshre.eu
Not only “what” but also “how much” is problably an important question
Size / Shape Cytoplasm Zona pellucida / PBI /PVS
Source: ESHRE Atlas of human embryology — http://atlas.eshre.eu
Large PV space …. or small oocyte?
The extent of the phenomenon
Oocytes No. %
MII oocytes analysed 1191 100
Cytoplasmic abnormalities 739 62.0
— Cytoplasmic granularity 441 37.0
— Vacuoles 37 3.1
— Smooth endopl. ret. (SER) clusters 60 0.5
— Refractile bodies 255 21.4
Abnormal PBI 52 4.4
Abnormal zona pellucida 51 4.3
Abnormal oocyte shape 23 1.9
Rienzi et al., 2008 Balaban et al., 1998
“In 92.6% of cycles at least one oocyte was recorded as morphologically abnormal.’’
Female condition/treatment and oocyte morphology
Study No. of patients in study group
Condition or treatment Outcome
Xia et al., 2001 80 High E2/P or E2/T rate Increased overall rate of dysmorphic oocytes
Rienzi et al., 2008 516 Higher maternal age and FSH Increased overall rate of dysmorphic oocytes
Valeri et al., 2011 36 Maternal age Decreased oocyte size and increased ZP thickness in older women
Cota et al., 2012 32 GnRH agonist/antagonist
suppression No impact on the rate of three major dysmorphisms
Setti et al., 2012 1105 High BMI No impact on the rate of all observed dysmorphisms
Santos et al., 2015 97 High FSH Increased rate of oocytes with granular cytoplasm and vacuoles
Sigala et al., 2015 48 PCO No impact on the overall rate of dysmorphic oocytes
Which endpoints to appraise the impact of oocyte dysmorphism?
Cryosurvival
Fertilization
Implantation
Biases
Cryopreservation approach and skills
IVF vs. ICSI (time of observation)
Embryo selection strategies and clinical factors
Embryo quality Assessment criteria (stages, parameters) and culture system
Which endpoints:
In many laboratories, it is common practice to cryopreserve only morphologically normal oocytes. Is this attitude justified?
Dysmorphisms RC No. (%) P value
Cryosurvival
No association between oocyte morphology and post-warming survival rate (donor oocytes)
Setti et al., 2011
Dysmorphism De Sutter,
1996 Balaban,
1998 Ebner, 2000
Otsuki, 2004
Ebner, 2005, 2008
Ten, 2007
Rienzi, 2008
Setti. 2011
Dark cytoplasm — — — n.a. n.a. — — —
Central granularity — — — n.a. n.a. — — —
SER clusters n.a. n.a. n.a. — ✔︎ n.a. — n.a.
Refractile bodies — — — n.a. n.a. n.a. — ✔︎
Vacuoles — n.a. n.a. n.a. ✔︎ — ✔︎ ✔︎
Abnormal shape — — n.a. n.a. n.a. — — n.a.
Abnormal zona — — — n.a. n.a. — — n.a.
Large PV space n.a. — n.a. n.a. n.a. — — ✔︎
Large polar body n.a. n.a. ✔︎ n.a. n.a. n.a. n.a. ✔︎
Legend: n.a. = not assessed; — = unaffected; ✔︎ = affected
Which endpoints: Fertilization
Dysmorphism De Sutter,
1996 Serhal, 1997
Balaban, 1998
Ebner, 2000
Ebner, 2008
Ten, 2007
Rienzi, 2008
Lazzaroni Tealdi, 2015
Dark cytoplasm — n.a. — — n.a. ✔︎ — n.a.
Central granularity — — — — n.a. — ✔︎ n.a.
SER clusters n.a. n.a. n.a. n.a. ✔︎ n.a. — n.a.
Refractile bodies — — — — n.a. n.a. — n.a.
Vacuoles — — n.a. n.a. — — — n.a.
Abnormal shape — n.a. — n.a. n.a. — — n.a.
Abnormal zona — n.a. — — n.a. — — n.a.
Large PV space n.a. n.a. — n.a. n.a. — — n.a.
Overall oocyte score n.a. n.a. n.a. n.a. n.a. n.a. n.a. ✔︎
Legend: n.a = not assessed; — = unaffected; ✔︎ = affected
Which endpoints: Embryo quality
Dysmorphism Serhal, 1997
Balaban, 1998
Otsuki, 2004
Ebner, 2008
Chamaiou, 2006
Sauerbrun-Cutler, 2015
Rienzi, 2008
Lazzaroni Tealdi, 2015
Dark cytoplasm n.a. — n.a. n.a. — n.a. n.a. n.a.
Central granularity ✔︎ — n.a. n.a. — n.a. n.a. n.a.
SER clusters n.a. n.a. ✔︎ — n.a. n.a. n.a. n.a.
Refractile bodies ✔︎ — n.a. n.a. n.a. n.a. n.a. n.a.
Vacuoles ✔︎ n.a. n.a. — n.a. n.a. n.a. n.a.
Abnormal shape n.a. — n.a. n.a. n.a. n.a. n.a. n.a.
Abnormal zona n.a. — n.a. n.a. n.a. ✔︎ n.a. n.a.
Large PV space n.a. — n.a. n.a. — n.a. n.a. n.a.
Overall oocyte score n.a. n.a. n.a. n.a. n.a. n.a. ✔︎ ✔︎
Legend: n.a = not assessed; — = unaffected; ✔︎ = affected
Which endpoints: Implantation
Then, why bother with oocyte dysmorphisms
Otsuki et al., 2004
• One baby diagnosed with Beckwith-Wiedemann syndrome
Ebner et al., 2008
• Increased “obstetric problems” and decreased birth weight
Akarsu et al., 2009
• One bay with multiple marformation
Alpha-ESHRE consensus on embryo assessment, 2011
• “Oocytes with SER aggregates should not be inseminated”
Alert raised by several studies on health risks associated with oocytes displaying SER aggregates
Ultrastructure of SER aggregates in dysmorphic oocytes is abnormal
Sa et al., 2011
18 mm 4 mm
SER aggregates in dysmorphic oocytes
5 mm 5 mm
Coticchio et al., 2010; 2016
Normal SER formations
Evidence on oocyte SER aggregates and baby health remains controversial
Embryological outcome
of SER⊕ and SER⊖ oocytes Neonatal outcome
of SER⊕ and SER⊖ oocytes Mateizel et al., 2013
Similar conclusions drawn in a more recent study (Shaw-Jackson et al., 2016)
Study performed by the Biogenesi team on possible associations between
specific dysmorphic patterns and cytoskeletal alterations
Coticchio et al., 2013; 2014
Object of study
SER aggregates
Granular Cytoplasm
Choice of dysmorphisms based on • High prevalence (cytoplasmic granulation) • Specific health concern (SER aggregates) • Evidence from animal model (large PBI)
Outcome measures
Cortical actin integrity Spindle morphometry and chromosome configuration
Control Large PBI
Oocytes with large PBI are rare (approx. 2%) but have multiple cytoskeletal and chromosome anomalies
In mouse oocytes with an abnormally large polar body, the spindle shape is altered and the spindle fails to translocate to the cortex, leading to the establishment of an altered cleavage plane.
Choi et al., 1996
Abnormal
In the mouse, extrusion of a large PBI is caused by abnormal maturation
Sanfins et al., 2003 Coticchio et al., 2015
Normal
In oocytes with SER aggregates and CG spindle and chromosome configurations are altered
Spindle length is larger in dysmorphic oocytes
The metaphase plate of dysmorphic oocytes tends to be localized equatorially, while that of control oocytes is closer to the pole proximal to the oolemma
Dal Canto et al., 2017
Chromosomes are less aligned on the metaphase plate in oocytes with cytoplasmic granularity
Dal Canto et al., 2017
Chromosomes are less aligned on the metaphase plate in oocytes with cytoplasmic granularity
Dal Canto et al., 2017
From a polar angle, chromosomes are less organized radially in oocytes with cytoplasmic granularity
Dal Canto et al., 2017
Dal Canto et al., 2017
From a polar angle, chromosomes are less organized radially in oocytes with cytoplasmic granularity
Sub-oolemmal actin organization is disrupted in oocytes displaying SER and CG
Dashed
Normal (continuous)
Sub-oolemmal actin pattern
Dal Canto et al., 2017
The cortex is emerging as a crucial oocyte domain
- The subcortical maternal complex (SCMC) -
FLOPED
Li et al., 2008
TLE6 is the earliest known human embryonic lethal phenotype
Maternal-effect gene products
Proven (continuous line) and hypothesized (dotted line)
functions of the SCMC
Bebbere et al., 2016
Final considerations
Possible biases in the classification and detection of oocyte dysmorphisms
Most studies are small and not recent. Quality of data is therefore limited
Evidence on effects of specific oocyte dysmorphisms on fertilization, development and implantion is contradictory
Early reports on obstetrics risks or congenital malformations associated with SER⊕ oocytes are not confirmed by recent studies
Oocytes with SER or especially cytoplasmic granulations show anomalies of the cytoskeleton and chromosome configuration. This may expose them to perturbations of cell cleavage and chromosome segregation.
Grazie per l’attenzione
