1. Assisted Reproduction Unit, Istanbul, Turkey
Oocyte quality and It’s influence on Embryo Cryopreservation Survival and Outcome
Başak Balaban, BSc
VKV American Hospital
Assisted Reproduction Unit, Istanbul, Turkey
AMERICAN
HOSPITAL

2. OOGENESIS
The ability of an embryo to implant, sustain and give rise to a viable offspring is unquestionably rooted to oogenesis
During oogenesis significant variations are known to exist among oocytes
**Meiotic competence (ability to undergo meiotic maturation)
**Developmental competence (support later embryonic development

3. GOOD QUALITY OOCYTE?
Mature oocyte that gives rise to the best quality embryo with the highest implantation potential
Viable, genetically normal oocyte that gives rise to an healthy embryo and healthy offspring

4. FACTORS AFFECTING OOCYTE QUALITY
Intrinsic factors
**- Chromosomal abnormalities, genetic defects,suboptimal characteristics of the oocyte cytoplasm
Extrinsic factors
**- Stimulation protocols
- Culture conditions (Ebner et al.2005, protocols and factors spesific to the laboratory
- Impact of nutrition??(Moderately fat animals on high level of feeding were found to be hyperinsulinaemic,and ruminants with this condition are associated with impaired oocyte quality, Adamiak et.al.2005)

5. GOOD QUALITY OOCYTE MORPHOLOGY
Perfectly spherical shape
Small Perivitelline Space(PVS)
Regular Zona Pellucida (ZP)
Intact 1st. Polar Body (PB)
Translucent, homogeneously coloured cytoplasm without inclusions
Veeck et al. Ann.N.Y.Acad.Sci, 1988
**Only 34% of all oocytes retrieved are of ideal morphology

6. Markers of oocyte quality /viability in relation to morphology
Cumulus-corona morphology
1st.Polar body morphology
Metaphase II (MII) OOCYTE MORPHOLOGY

7. Cumulus-corona morphologyB C D
Ng, Fertil Steril, 1999

8. Cumulus-corona morphology
Pro:
Ng, Fertil Steril, 1999 (IVF)
Lin et al.,JARG 2003 (IVF)
Con:
Rattanachaiyanont, Fertil Steril, 1999 (ICSI)
Hammitt, JARG, 1992 (IVF)
Hammitt, Fertil Steril, 1993 (IVF)
Highly dependant on embryologist’s experience
Asynchrony in 28%

9. Polar body morphology
Oocyte grading according to first polar body morphology
Grade
Shape of first polar body 1
Ovoid or round, smooth surface 2
Ovoid or round, rough surface 3
Fragmented 4
Broken into two parts 5
Large
Ebner, Fertil Steril, 1999
Ebner, Hum Reprod, 2000
Ebner, Hum Reprod, 2002

10. Predictive value of 1st.PB morphology evaluation??
Formation of the 1st.PB is a dynamic procedure and morphological characteristics (fragmentation) of the PBI of human oocytes can significantly change before ICSI and after 16 h in culture. Therefore cannot be used as a diagnostic criteria determining the quality of the subsequent embryo and it’s implantation potential (Verlinsky et al., RBM Online 2003 )
Predictive value of 1st.PB morphology is limited,and could not be used as a prognostic factor of embryo quality& PR & IR (Ciotti et al., RBM Online 2004)

11. MORPHOLOGICAL ABNORMALITIES OF MII OOCYTE
Extracytoplasmic abnormalities
**Shape abnormalities (irregular shape of MII oocyte)
**ZP abnormalities (dark or thick ZP)
**PVS abnormalities (large PVS, PVS granulation)
Cytoplasmic abnormalities
**Dark cytoplasmic colour (slight granulation)
** Excessive whole or centrally located granulation
**Refractile bodies,sERC or vacuoles in the ooplasm

12. Effect of extracytoplasmic abnormalities on embryo development&CPR&IR
**CON:
De Sutter et al.,Hum.Reprod. 1996
Balaban et al.,Hum.Reprod. 1998
H.Hassan-Ali et al.,Hum.Reprod.1998
Loutradis et al.,F&S 1999
Ebner et al.,JARG 2001
Plachot et al.,Gyn. Obst. Fertil 2002
**PRO:
Xia et al.,Hum.Reprod. 1997(CPR not mentioned)

13. Effect of cytoplasmic abnormalities on embryo development&CPR&IR
**PRO
Serhal et al., Hum.Reprod. 1997
Xia et al.,Hum.Reprod. 1997
Loutradis et al.,F&S 1999
Ebner et al.,HumReprod. 2003
Otsuki et al.,Hum.Reprod. 2004
Ebner et al.,Hum.Reprod. 2005
**CON
De Sutter et al.,Hum.Reprod. 1996
Balaban et al.,Hum.Reprod. 1998(severe defects excluded)

14. Severe cytoplasmic defects

15. Smooth Endoplasmic Reticulum Clusters (sERC)
and vacuolization appearance in MII oocytes
Otsuki et al.Hum.Reprod.,2004

16. The presence of sERC is associated
with lower chances of successful pregnancy
sERC(-)
sERC(+)
p value
No.of patients
174 18
Fertlilization by ICSI(%)
85.2
81.6
Fertlilization by IVF(%)
77.8
80.0
Cleavage on day 3(%)-ICSI
95.5
96.8
Cleavage on day 3(%)-IVF
96.1
97.2
Mean no.of ET
2.6
2.7
CPR/ET(%)
28.2
5.6
<0.05
IR/ET(%)
13.0
2.1
Otsuki et al.Hum.Reprod.,2004

17. Both normal and CLCG oocytes
Very low OPR% are obtained with the transfer
of embryos obtained from CLCG oocytes
Both normal and CLCG oocytes
Only CLCG oocytes
Total
No.of cycles 21 18 39
No.of pregnancies
6(28.6)
5(27.8)
11(28.2)
Abortion
4(19.0)
2(11.1)
6(15.4)
Ongoing pregnancies
2(9.5)
3(16.7)
5(12.8)
Kahraman&Yakin et al.Hum.Reprod.,2000
CLCG-Centrally located granular cytoplasm
Fert%+embryo quality: no sig.

18. Correlation of oocyte morphology with aneuploidy rates
Normal morphology
Abnormalities
Single
Multiple
Ext.cytop
Cytop.
Shape
Double
Triple
Diagnosed embryos 43
(93.4) 62
(89.8) 55
(93.2) 54
(94.7) 19
(95.0) 15
(100)
Euploid embryos 25
(58.1) 33
(53.2) 22
(40.0) 31
(57.4) 7
(36.8) 6
(40)
Aneuploid embryos 18
(41.8) 28
(46.7)
(60.0) 23
(42.5) 12
(63.1) 9
Yakin&Balaban et al.,F&S 2007

19. Oocytes without vacuoles (n=1151)
Effect of cytoplasmic vacuoles throughout embryo development
Oocytes without vacuoles (n=1151)
Vacuolar
Oocytes (n=47)
(3.9%)
p value
Single vacuole
Two vacuoles
Multiple vacuoles
31(66)
10 (21.3)
6 (12.7)
Fert. (%)
51.6%
43.8%
65.3%
48.9%
<.05
Mean diameter of vaculoes with fertilization :9.8m
Mean diameter of vaculoes with FF :17.6 m (p<.05)
Cut off value :14 m
Patients variables similar between
vacuol (+) & (-) cycles
Ebner et al. F&S, 2005

20. Markers of Oocyte Quality/Viability
Examination of the structure of miotic spindle, and zona pellucida of the oocyte by using polirized microscopy

21. Spindle imaging of the MII oocyte
De Santis et al., RBM Online 2005
At the time of ovulation mammalian oocyte is arrested at MII of the meiotic
Cell cycle, when chromosomes are tethered by microtubule fibres of the meiotic
Spindle. During meiosis and fertilization meiotic spindles are responsible for proper
segregation of the nuclear material, and abnormalities in this fragile structure can lead to
İnfertility, miscarriage and genetic diseases, such as Down syndrome

22. 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)

23. Abnormalities of oocyte morphology do effect embryo quality and viability in certain cases
Whether morphological abnormalities of the oocyte influence cryosurvival and further development of derived embryos in not known

25. Aim of the study
To compare survival, blastocyst formation and hatching rates of frozen-thawed day 3 cleavage stage embryos derived from morphologically abnormal or normal metaphase II (MII) oocytes

26. Material and Methods
5292 day 3 excess embryos of good quality from 964 patients (mean age: 32.4) were frozen and subsequently cultured up to the blastocyst stage after thawing
Fertilization was affected by ICSI (cycles with testicular/epididymal sperm were excluded)
GIII series (Vitrolife-Sweden) for fresh embryo culture, and Freeze/Thaw Kıt-1(1-2 propandiol based- Vitrolife)/with a slow freezing program was used for freezing

27. Balaban et al.,HR 2008

28. Blastocyst development of cryo-thawed top quality day 3 embryos
(Single morphological abnormality)
Normal MII
Irregular
shape
Large
PVS
Dark ZP
Dark cytoplasm with slight granulation
Vacuolar
cytoplasm
Central
Granulation
G1 EMBRYOS
352
117
122
109
100 12 14
Cryosurvival (%)
317
(90.0) 99
(84.6)
(81.9) 93
(85.3)
80 *
(80)
6 *
(50)
7 *
Blastocyst development
(%)
170
(53.6) 50
(50.5) 49
(49) 46
(49.4) 39
(48.8) 2
(33.3)
(28.5)
Good quality blastocyst
106
(62.3) 30
(60) 29
(59.1)
(63.0) 22
(56.4)
Hatching blastocyst 48
(28.2)
(28.0) 13
(26.5)
(26) 10
(25.6)
Balaban et al.HR 2008
* Sig. diff. from normal MII

29. Blastocyst development of cryo-thawed good quality day 3 embryos
(Single morphological abnormality)
Normal MII
Irregular
shape
Large
PVS
Dark ZP
Dark cytoplasm with slight granulation
Vacuolar
cytoplasm
Central
Granulation
G2 EMBRYOS
1047
363
393
318
321 96 68
Cryosurvival (%)
914
(87.2)
297
(81.8) *
(80.9) *
266
(83.6)
258
(80.3) * 43
(44.7) * 29
(42.6) *
Blastocyst development
(%)
482
(52.7)
151
(50.8)
155
(48.7)
133
(50)
126
(48.8) 10
(23.2) * 6
(20.6) *
Good quality blastocyst
296
(61.4) 89
(58.9) 93
(60.0) 81
(60.9) 72
(57.1) 1
(10.0)
Hatching blastocyst
137
(28.4) 40
(25.8) 35
(26.3) 30
(23.8)
Balaban et al.,HR 2008
*Sig.diff. from normal MII

30. Blastocyst development of cryo-thawed 8 cell embryos
(Single morphological abnormality)
Normal MII
Irregular
shape
Large
PVS
Dark ZP
Dark cytoplasm with slight granulation
Vacuolar
cytoplasm
Central
Granulation
8 CELL
EMBRYOS
570
180
199
166
168 21 22
Cryosurvival (%)
514
(90.1)
152
(84.4)
142
(85.5)
138
(82.1) * 10
(47.6) *
(45.4) *
Blastocyst development
(%)
280
(54.4) 80
(52.6) 84
(50) 73
(51.4) 68
(49.2) 3
(30)
Good quality blastocyst
(64.2) 48
(60) 51
(60.7) 46
(63) 39
(57.3) 1
(31.3)
Hatching blastocyst 82
(29.2) 24
(26.1) 20
(27.3) 17
(25)
*Sig.diff. from normal MII
Balaban et al.,HR 2008

31. Blastocyst development of cryo-thawed top quality day 3 embryos
(Multiple morphological abnormality)
Normal
MII
Double
extracytop. abn.
cytop.
abn.
Double combined abn.
Triple extracytop.
Triple combined G1
EMBRYOS
352
153 16 84 79 48
Cryosurvival (%)
317
(90.0)
121
(79) * 5
(31.2) * 39
(46.4) * 56
(70.8) * 18
(37.5) *
Blastocyst development
(%)
170
(53.6) 59
(48.7) 1
(20.0) 11
(28.2) * 26
(46.4) 4
(22.2) *
Good quality blastocyst
106
(62.3) 33
(56.0) 2
(18.1) 14
(53.8)
Hatching blastocyst
(28.2)
(23.7) 6
(23)
*Sig.diff. from normal MII
Balaban et al.,HR 2008

32. Blastocyst development of cryo-thawed good quality day 3 embryos
(Multiple morphological abnormality)
Normal
MII
Double
extracytop. abn.
cytop.
abn.
Double combined abn.
Triple extracytop.
Triple combined G2
EMBRYOS
1047
477
161
334
315
193
Cryosurvival (%)
914
(87.2)
375
(78.6) * 50
(31.0) *
155
(46.4) *
226
(71.7) * 72
(37.3) *
Blastocyst development
(%)
482
(52.7)
182
(49.5) 7
(14) * 41
(26.4) *
101
(44.6) * 12
(16.6) *
Good quality blastocyst
296
(61.4)
102
(56.0) 8
(19.5) * 55
(54.4)
Hatching blastocyst
137
(28.4)
4(23) 22
(21.7)
*Sig.diff. from normal MII
Balaban et al.,HR 2008

33. Blastocyst development of cryo-thawed 8 cell embryos
(Multiple morphological abnormality)
Normal
MII
Double
extracytop. abn.
cytop.
abn.
Double combined abn.
Triple extracytop.
Triple combined 8C
EMBRYOS
570
237 20
134
138 72
Cryosurvival (%)
514
(90.1)
188
(79.3) * 7
(35) * 64
(47.7) *
101
(73.1) * 29
(40.2) *
Blastocyst development
(%)
280
(54.4) 4
(50) 2
(28.3) 18
(28.1) * 47
(46.5) 6
(20.6)
Good quality blastocyst
180
(64.2) 54
(57.4)
(22.2) * 26
(55.3)
Hatching blastocyst 82
(29.2) 23
(24.5) 11
(23.4)
*Sig.diff. from normal MII
Balaban et al.,HR 2008

34. Conclusions- I
Presence of extracytoplasmic abnormalities alone does not affect blastocyst development despite decreasing cryosurvival
However, embryos derived from oocytes with vacuolar cytoplasm or central granulation do not seem to bear the potential to develop into good quality blastocyst or to reach hatching stage after cryopreservation
These cytoplasmic abnormalities may be reflections of genetic, epigenetic or metabolic defects in the oocyte

35. Conclusions-II
Embryos with severe cytoplasmic abnormalities comprise around 5% of all embryos suitable for cryopreservation. The formation of a good quality blastocyst, or a blastocyst with the capibility of completing the hatching procedure after thawing of such embryos is ≤1%
Although transfer of such embryos may not affect overall success rate of a cryopreservation program, priority should be given to the transfer of embryos derived from oocytes without cytoplasmic abnormalities whenever possible

36. Conclusions-III
Although it’s difficult to estimate the impact of oocyte morphology on the implantation potential of the derived embryo when multiple embryos are transferred, and when considering the fact that the transition from the gamete to the embryo is a continuum with the difficulty of evaluating the contribution of the oocyte on implantion potential of the derived embryo, it’s shown that the developmental behaviour of frozen-thawed embryos derived from dysmorphic oocytes are similar when compared to results from the literature reported for fresh embryos (Balaban&Urman 2006, Ebner et al., 2006)

39. Fertilization&embryo quality outcome in relation to the
presence/absence of visible spindle in M2 oocytes
Spindle visible
No.of oocytes (%)
% Fertilization
% High quality embryos
Wang et al., 2001b +
327 (61.4)
61.8a NA –
206 (38.6)
44.2
Wang et al., 2001a
1266 (82.0)
69.4a
66.3a
278 (18.0)
62.9
55.4
Moon et al., 2003
523 (83.5)
82.2
64.2a
103 (16.5)
75.7
35.9
Rienzi et al., 2003
484 (91.0)
74.8a
48 (9.0)
33.3
Cohen et al., 2004
585 (76.0)
70.4a
47.2
62.2
35.6
NA:not applicable,
a: p<0.05
Borini et al RBM Online 2005

40. Why are the results contradictory??
The dynamics of spindle formation during oocyte maturation were not considered:
*Spindle visualization might change within the maturation stages: MI-1st.PB extrution- telaphase I (MS dissepear for minutes!!)- MII. Ideal time at least 38 hours after the HCG(Cohen 2004)
*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 Onilne 2005,
Montag et al.,RBM Online 2006
Not only the presence of the spindle but also the angle to the 1st.PB,
retardance and the length can be evaluated to determine the quality of the OOCYTE
Rienzi 2003, Rama Raju 2007

41. M.Spindle visualization and ICSI outcome: Meta-Analysis
Petersen C et al.,RBM Online 2009
PN morphology p:0.003, Cleavage: p<0.0001, Blastocysts:p<0.0001

42. Transfer of the embryos derived from oocytes
with HZB reveals higher IR&OPR
Montag et al.,RBM Online 2008
Rama Raju( 2007) had also shown sig. higher blastocyst formation from oocytes with
higher zona inner layer retardance measurements

43. fewer oocytes not detected
35.6%
No.of patients: 69
No.of day 5 BT: 49
No.of day 3 ET: 20
LBR: 34.8%
IR:29.1%
No.dif in PR for day3&5ET
Patients with + PR had sig.
fewer oocytes not detected
(22.6% vs %)
Ebner &Balaban
F&S 2009 in press

44. Ebner &Balaban F&S 2009 in press