iogenesi centro di medicina della riproduzione b oocyte selection based on morphological criteria...

IOGENESI

Centro di Medicinadella RiproduzioneB

OOCYTE SELECTION OOCYTE SELECTION BASED ON BASED ON

MORPHOLOGICAL MORPHOLOGICAL CRITERIA AND CRITERIA AND POLOSCOPEPOLOSCOPE

DAL CANTO MARIABEATRICE

BIOGENESI REPRODUCTIVE MEDICINE CENTER,

ISTITUTI CLINICI ZUCCHIV. Zucchi, 24-Monza (Italy) tel/fax +39.39.8383314

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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.

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Morphologic evaluation of oocytes with Morphologic evaluation of oocytes with

cumulus-corona complexcumulus-corona complex

It’s difficult!

It’s necessary “the spreading” of the cumulus-complex (Veeck)

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Metaphase IIMetaphase IIOocytes 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.

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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

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Polar bodyPolar bodyThe 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.

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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 Cytoplasmic Maturation

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

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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

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Oocyte morphology can Oocyte morphology can impact IVF outcome?impact IVF outcome?

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

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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.

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the Serhal’s group (Serhal et al., 1997)and Loutradis’ group (Loutradis et al.,

1999), reported higher pregnancy rate in patientsin which had been transferred embryos thatderived exclusively from oocytes withoutanomalies

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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)

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Grade 1° Grade 1° PBPB

N° N° oocytesoocytes

% 2PN% 2PN <25% <25% fragm.fragm.

11 174174 89.1 89.1 aa 89.0 89.0 dd

22 187187 85.6 85.6 bb 56.3 56.3 ee

33 159159 70.4 70.4 cc 47.3 47.3 ff

44 1818 50.050.0 22.222.2

Influence of first polar body morphology

a,b,c p< 0.0025 d,e,f p < 0.001

Ebner et al. 2000

The post-maturity of the oocytes seems to be associated to the polar body degeneration

It has been found that an integral first polar body is a favourable prognostic factor in terms of fertilization, embryo quality and pregnancy rate.

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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.

Influence of first polar body morphology

De Santis L. 2005

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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 introduction of polarization light microscopy favoured a new approch to analize the oocyte (Wang et al. 2001).

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

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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).

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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.

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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)

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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)

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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

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Our study

2286 oocytes

regular anomalous

Spindle 1680 73.5%

606 26.5 %

Polar body 87438.2%

141261.8%

Cytoplasm 54223.7%

174476.3%

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Oocyte morphology and Oocyte morphology and fertilizationfertilization

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1 PN or 3 PN 643.8%

386.3%

degenerated 1207.2%

477.8%

p<0.0001

Meiotic spindle

2286 visible not visible

n° MII Inseminated

1679 606

No fertilization

19211.4%

9615.8%

2 PN 1303 77.6%

42570.1%

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Centro di Medicinadella RiproduzioneBP= ns

1 PN or 3 PN

404.6%

624.4%

degenerated

647.3%

1037.3%

Polar Body

2285 regular irregular

n° MII Inseminate

d

874 1412

No fertilizatio

n

11012.6%

17912.7%

2 PN 660 75.5%

106875.6%

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P<0.0001

1 PN or 3 PN

254.6%

774.4%

degenerated

213.9%

1468.4%

Cytoplasm

2285 regular irregular

n° MII Inseminate

d

542 1744

No fertilizatio

n

6011.0%

22913.1%

2 PN 436 80.4%

129274.1%

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Parameter Multiple linear regressionFertilization rate

Coefficient (95% CI)

p-value

Meiotic SpindlePolar BodyCytoplasm

1.61 (1.37-1.89)0.93 (0.79 -1.10)1.42 (1.17-1.73)

< 0.00010.390

< 0.0001

Logistic Regression Analysis : Meiotic spindle and cytoplasm morphology play a SIGNIFICANT INDIPENDENT role on Fertilization

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Oocyte morphology and Oocyte morphology and embryo qualityembryo quality

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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%

Results

P= ns

CYTO

+ -157

36.3%

469

36.7%

159

36.7%

521

40.7%

52

12.0%

139

10.9%

30

6.9%

103

8.1%

35

8.1%

47

3.7%

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Results

1° step

2° step

?

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Results

1° step

2° step

4° step

3° step

Women ???

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Thank you very much for you attention

iogenesi centro di medicina della riproduzione b oocyte selection based on morphological criteria...

Documents

polar body slide

pn iogenesi centro

oocytes morphology

cytoplasmic maturation

collected oocytes

dysmorphic oocytes

normal oocytes

oocytes potenziality