OOCYTE SELECTION BASED ON MORPHOLOGICAL CRITERIA AND POLOSCOPE DAL CANTO MARIABEATRICE BIOGENESI REPRODUCTIVE MEDICINE CENTER, ISTITUTI CLINICI ZUCCHI V. Zucchi, 24-Monza (Italy) tel/fax +39.39.8383314 First of all, I wish to thank everybody for being here today. Since March 2004, a new law has been introduced in Italy to regulate the assisted reproduction. The prohibitions introduced by the new law have on one side reduced the expectation of success of the current techniques, and on the other side stimulated the doctors and the embryologists to perfect new therapeutic strategies in order to offer the highest chances of success with the lowest risks. Today in Italy we are not any more allowed to crio-preserve embryos, and we cannot use more than 3 oocytes per IVF cycle.

Performing a right evaluation of nuclear and cytoplasmic maturation of human oocytes is extremely important for the result of In Vitro Fertilization: low quality oocytes are hardly going to fertilize or won’t be competent to produce good embryos. More than half of the collected oocytes during IVF show at least one morphologic anomaly that could negatively influence the embryo development.

Morphologic evaluation of oocytes with cumulus-corona complex It’s difficult! It’s necessary “the spreading” of the cumulus-complex (Veeck)

Metaphase II Oocytes without cumulus can be accurately evaluated analyzing the nuclear maturity, the characteristics of the cytoplasm, the aspect of the polar body, the perivitelline space and the ZP.

Two processes need to be synchronize to produce a competent oocyte to use in IVF: Nuclear Maturation can be verified analyzing the presence of the first polar body and the Meiotic spindle signal Cytoplasmic Maturation can be only hypnotized using several morphological criteria based on cytoplasm characteristic

Polar body The first polar body is the real signal of nuclear maturation: Oocyte with 1°polar body is arrested at Metaphase II The first polar body in human remains intact more than 20 hr after ovulation, while in mammals it has a shorter life. We can postulate that the first polar body morphology gives informations not only on the nuclear maturation of the oocytes but on the “AGE” of the oocyte also.

Cytoplasmic Maturation More than a half of the collected oocytes show at least one anomaly. Ebner suggests to divided these anomalies in intracytoplasmic and extracytoplasmic (Ebner T. 2001)

Cytoplasmic Maturation Intracytoplasmic anomalies: variations in colour or granularity of the cytoplasm presence of inclusions, vacuoles or retractable bodies (Ebner et al. 2003, 2005) aggregations of the smooth endoplasmic reticulum (Otsuki J. 2004)

Cytoplasmic Maturation Extracytoplasmic anomalies: include irregularities in the shape of the oocyte, presence of detritus or increment in the perivitelline space,, abnormal consistency of ZP and fragmentation of the first polar body

Oocyte morphology can impact IVF outcome?

Alikani’s group (Alikani 1995) did not find any reduction in the fertilization rate after ICSI on dysmorphic oocytes versus to normal oocytes

Some groups (Balaban et al. , 1998; De Sutter et al Some groups (Balaban et al., 1998; De Sutter et al., 1996) have not found correlation between the oocytes’ morphology and the fertilization rate or the embryo quality after ICSI.

the Serhal’s group (Serhal et al., 1997) and Loutradis’ group (Loutradis et al., 1999), reported higher pregnancy rate in patients in which had been transferred embryos that derived exclusively from oocytes without anomalies

Xia et al. in 1997 correleted the first polar body apparence and periviteline space size with the oocyte’s potenziality to fertilize. Ebner et al. correlated the cytoplasm and polar body morphology with embryo quality and embryo capacity to reach blastocyst stage (Ebner 1999, Ebner 2001, Ebner 2005)

Influence of first polar body morphology The post-maturity of the oocytes seems to be associated to the polar body degeneration Grade 1° PB N° oocytes % 2PN <25% fragm. 1 174 89.1 a 89.0 d 2 187 85.6 b 56.3 e 3 159 70.4 c 47.3 f 4 18 50.0 22.2 a,b,c p< 0.0025 d,e,f p < 0.001 Ebner et al. 2000 It has been found that an integral first polar body is a favourable prognostic factor in terms of fertilization, embryo quality and pregnancy rate.

Influence of first polar body morphology On the contrary Ciotti (Ciotti et al. 2004) published results against a predictive value of PBI morphology, confirmed by De Santis (De Santis et al. 2005) on fertilization rate and embryo quality. De Santis L. 2005

The introduction of polarization light microscopy favoured a new approch to analize the oocyte (Wang et al. 2001). At the stage of metaphase II (second meiotic division) the signal of chromosomes aligned at the equator of the meiotic spindle (MS) can be capture preserving the oocyte’s funzionality. The presence of spindle-signal could be consider a prognostic factor of good quality.

The signal is produced by microtubules located at the oocyte periphery and it is very dynamic structure (Rienzi et al. 2003).

The percentage of oocytes showing a detectable MS varied between 60% and 90%. This difference seems to be related to two important parameters: the high sensitiveness to temperature and the technique of spindle visualization (Wang et al. 2002, Rienzi et al.2003; Cooke et al. 2003).

The position of the signal could be correlate with the “age of oocyte”. Some oocytes showing a first polar body may not have finished nuclear maturation and could be at telophase I stage.

Sometimes oocytes show the spindle far from the polar body. 5% of oocytes show an angle of 45° or 90° between polar body and meiotic spindle (Moon et al., 2003; Rienzi et al., 2003, Rienzi et al.2005)

Rienzi et al. reported that oocytes with visible meiotic spindle have higher fertilization rate than oocytes where it isn’t observable, and the number of high quality embryos and blastocysts increase (Rienzi et al. 2003) There are no difference in fertilization rate and embryo quality if the angle of displacement does not exceed 90°; if the angle is >90°, lower fertilization rate is observed. (Moon et al., 2003; Rienzi et al., 2003, Rienzi et al.2005) Oocyte at telophase I stage failed the fertilization process. (De Santis et al. 2005)

Our study According to the Italian law we cannot select embryos, we cannot use more than three oocytes per cycle. Since April 2005 we’ve started to select oocytes before ICSI in order to obtain the best fertilization and high quality embryos to transfer. Our STRICT criteria have been: Meiotic spindle: present absent or irregular (45°, 90°) Polar body: regular anomalous Cytoplasmic: regular anomalous (intra+extra anomalies)

Our study 2286 oocytes regular anomalous Spindle 1680 73.5% 606 26.5 % Polar body 874 38.2% 1412 61.8% Cytoplasm 542 23.7% 1744 76.3%

Oocyte morphology and fertilization

p<0.0001 Meiotic spindle 2286 visible not visible n° MII Inseminated 1679 606 No fertilization 192 11.4% 96 15.8% 2 PN 1303 77.6% 425 70.1% 1 PN or 3 PN 64 3.8% 38 6.3% degenerated 120 7.2% 47 7.8% p<0.0001

P= ns Polar Body 2285 regular irregular n° MII Inseminated 874 1412 No fertilization 110 12.6% 179 12.7% 2 PN 660 75.5% 1068 75.6% 1 PN or 3 PN 40 4.6% 62 4.4% degenerated 64 7.3% 103 P= ns

P<0.0001 Cytoplasm 2285 regular irregular n° MII Inseminated 542 1744 No fertilization 60 11.0% 229 13.1% 2 PN 436 80.4% 1292 74.1% 1 PN or 3 PN 25 4.6% 77 4.4% degenerated 21 3.9% 146 8.4% P<0.0001

Multiple linear regression Logistic Regression Analysis : Meiotic spindle and cytoplasm morphology play a SIGNIFICANT INDIPENDENT role on Fertilization Parameter Multiple linear regression Fertilization rate Coefficient (95% CI) p-value Meiotic Spindle Polar Body Cytoplasm 1.61 (1.37-1.89) 0.93 (0.79 -1.10) 1.42 (1.17-1.73) < 0.0001 0.390

Oocyte morphology and embryo quality

Results MS + - PB + - CYTO + - P= ns 0% 10% 11-20% 21-30% >30% Embryo quality (% of fragmentation) MS + - 0% 464 35.9% 162 38.7% 10% 525 40.6% 155 37.1% 11-20% 140 10.8% 51 12.2% 21-30% 104 8.0% 29 6.9% >30% 61 4.7% 21 5.2% PB + - 239 36.6% 387 36.5% 262 40.1% 418 39.4% 77 11.8% 114 10.7% 44 6.7% 89 8.4% 30 4.6% 52 4.9% CYTO + - 157 36.3% 469 36.7% 159 521 40.7% 52 12.0% 139 10.9% 30 6.9% 103 8.1% 35 47 3.7% P= ns At the moment of the transfer, 48 hrs after ICSI, we transferred embryo to two, three and four cells No particularly cleavage delay was observed

Results ? 1° step 2° step

Results 2° step 3° step 1° step Women ??? 4° step

Thank you very much for you attention