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

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

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

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)

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

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

Cumulus-corona morphology B C D Ng, Fertil Steril, 1999

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%

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

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)

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

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)

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)

Severe cytoplasmic defects

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

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

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.

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

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

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

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

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)

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

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

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

Balaban et al.,HR 2008

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

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

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

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

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

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

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

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

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)

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

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 40-60 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

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

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

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 35.5.%) Ebner &Balaban F&S 2009 in press

Ebner &Balaban F&S 2009 in press