NNuoveuoveTecnichein in MedicinaMedicinadella Riproduzione · Nomarski Interference Contrast 6.600X...

NNuoveuove TecnicheTecniche in in MedicinaMedicina della della RiproduzioneRiproduzioneRiproduzioneRiproduzione

Rimini 23 Marzo 2011

Dott.G.Comploj

„Centri Fivet Prof. Zech“ – International GroupEuBios Italia

ColtureColture BlastocitarieBlastocitarie

AnalisiAnalisi FusoFuso MitoticoMitotico

IMSIIMSI

FollicolometriaFollicolometria 3D AVS3D AVS

SEETSEET

ColtureColture BlasticitarieBlasticitarie

« You can put back as many embryos as you like, but one at a time » (Carl Nygren)

Embryo transfer

Selection of the best embryoSelection of the best embryo

Competent for further development and implantation

Without genetic problem

Oocyte selectionOocyte selection Spermatozoa selectionSpermatozoa selection

Technique

NoNo--invasiveinvasive InvasiveInvasiveSelection of the best embryoSelection of the best embryo

• PGD (Embryo,Polar Body,Sperm)

Oocyte selectionOocyte selection Spermatozoa selectionSpermatozoa selection

Come é Come é possibilepossibileesegiureesegiureunaunaselezioneselezionemofologicamofologicadeldel migliormiglior

embrioneembrione??

SviluppoSviluppo embrionarioembrionarioGiorno 1 Giorno 2 Giorno 3

Ovocita fertilizzati2 pronuclei e2 globuli polari

Embrione a 4 cellule Qualitá A

Embrione a 4 celluleQualitá B Embrione a 8 cellule

Embrione a 4 celluleQualitá C

Embrione a 4 celluleQualitá D

Giorno 4 Giorno 5

Embione in compattazione(morula)

Blastocisti precoce Blastocist G.2

Blastocisti G.3 Blastocisti G.4

Blastocisti G. 5

Blastocisti in Hatching

Età 25a 30a 35a 40

Ovociti (n) 10 10 10 10

Sviluppo Blastocitarioconsiderazioni teoriche

Ovociti (n) 10 10 10 10

Fertilizzazione 7 7 7 7

Sviluppo Blastocitario 50% 40% 30% 20%

Transfer Blastocitario 3-4 2-3 2 1-2

Indici di gravidanza

Qualitá Blastocitaria (eSET)

50

60

70

80

%

0

10

20

30

40

50

5AA 4AA 5(AB, BA, BB) 4 (AB, BA, BB)

%

Indici di gravidanza con „Top Emryo“ in relazione all`

etá femminile (eSET)

40

50

60

%

0

10

20

30

< 30 30 - 35 36 - 39 > 39

% SR

EMBRIONE

di alta qualitá

•Selezione dell` OVOCITA

• Selezione dello SPERMATOZOO

OvocitaOvocita

–– 500 pg RNA500 pg RNA–– 2020--25 pg an Protein25 pg an Protein–– 150 pg an Glykogen150 pg an Glykogen–– 100.000 100.000

MitochondrienMitochondrien–– 1.000.000 1.000.000

MII

–– 1.000.000 1.000.000 RibosomenRibosomen

–– 250 pg Tubulin250 pg Tubulin–– 100 pg Aktin100 pg Aktin–– hohe Level an hohe Level an

EnzymenEnzymen–– 800 pmol ATP800 pmol ATPGV MI

Selezione OvocitariaSelezione Ovocitaria

Valutazione del potenziale di sviluppoValutazione del potenziale di sviluppo

MorfologiaMorfologiaAnomalie citoplasmaticheAnomalie citoplasmaticheAnomalie citoplasmaticheAnomalie citoplasmaticheMorfologia del 1. globulo polareMorfologia del 1. globulo polareSpindelSpindel--Imaging >valutazione fuso mitoticoImaging >valutazione fuso mitoticoRetardance della zona pellucidaRetardance della zona pellucidaBiopsia del globulo polareBiopsia del globulo polare

MORFOLOGIA OVOCITARIA CItoplasma Zona pellucida Vacuoli Globulo polare

Analisi del Fuso Mitotico„Spindel Wiew“

L. Rienzi P. Gassner

“Analisi del Fuso Mitotico”(Spindelwiew)

• Tecnica non invasiva mediante l’ utilizzo di

un microscopio con luce la polarizzata un microscopio con luce la polarizzata

computerizzata che permette di valutare:

• A) Il fuso mitotico

• B) I tre strati della zona pellucida

fuso mitotico

• Il fuso mitotico ha un ruolo importante nella funzionalitá

ovocitaria ed è responsabile di una corretta ripartizione

cromosomica durante la divisione cellulare.cromosomica durante la divisione cellulare.

• Nel 15% - 20% degli ovociti il fuso non è presente

• La presenza del fuso e del primo globulo polare sono indici

di maturazione ovocitaria.

• Con l’ aumentare dell’ età aumentano le anomalie del fuso

e della sua localizzazione.

• L’ assenza del fuso è associata ad una netta riduzione dell’ indice di fertilizzazione e a riduzione della qualità embrionaria.embrionaria.

• La localizzazione del fuso durante le tecniche ICSI è fondamentale per evitare un suo involontario danneggiamento.

�� Analisi del Fuso Mitotico

PolscopioKonc 2004, De Santis 2005, Rienzi 2005

Localizzazione anomala del fuso mitotico rispetto al globulo polare

MII spindle alterations

GroupGroup--11 GroupGroup--22 PP

PatientsPatients 2828 23_____________23_____________

Spindle Spindle LocalisationLocalisation and Embryoand Embryo--DevelopmentDevelopment

PatientsPatients 2828 23_____________23_____________

FertilizationFertilization % (+/% (+/--S.D.S.D.)) 86 %86 % (17)(17) 63%63% (22)(22) <0.05<0.05

ExcellentExcellent EmbryosEmbryos 45.3 %45.3 % 24.0 %24.0 % <0.01<0.01

GoodGood EmbryosEmbryos 30.9 %30.9 % 41.3 %41.3 % <0.05<0.05

PoorPoor EmbryosEmbryos 23.8 %23.8 % 34.7 %34.7 % <0.05<0.05

EmbryosEmbryos TransferredTransferred (+/(+/--S.D.S.D.)) 3.0 (1.0)3.0 (1.0) 3.3 (1.3)3.3 (1.3) NSNS

PregnanciesPregnancies (%)(%) 64%64%(18)(18) 26% (6)26% (6) <0.05<0.05

Spindel and Outcome

AutorAutor SpindelSpindel SpindelSpindel FertilizzazioneFertilizzazionepositivepositive nearnear PbPb SI SI NoNo

Wang et al., 2001 327/533 (61.4)327/533 (61.4) 61 (18.7)61 (18.7) 61.8%61.8% 44.2%44.2%

Wang et al., 2001 1266/1544 (82.0)1266/1544 (82.0) ndnd 69.4%69.4% 62.9%62.9%

Cooke et al., 2003 115/124 (92.7)115/124 (92.7) 35 (30.4)35 (30.4) 70.4%70.4% ndnd

P<0.001P<0.05

Cooke et al., 2003 115/124 (92.7)115/124 (92.7) 35 (30.4)35 (30.4) 70.4%70.4% ndnd

Moon et al., 2003 523/626 (83.6)523/626 (83.6) 252 (48.2)252 (48.2) 84.9%84.9% 75.5%75.5%

Rienzi et al., 2003 484/532 (91.0)484/532 (91.0) 254 (52.5)254 (52.5) 74.8%74.8% 33.3%33.3%

Cohen et al., 2004 585/770 (76.0)585/770 (76.0) ndnd 70.6%70.6% 62.2%62.2%

Konc et al., 2004 320/428 (74.8)320/428 (74.8) 31 (9.7)31 (9.7) 73.4%73.4% ndnd

nd: no datend: no date

IMSI

Intracytoplasmic Morphological Selected Sperm Injection

• World Health Organization• Routine Semen Analysis

• Serial semen samples (at least two) • Kruger s strict criteria

• Morphology• Optional Tests

• Hypo-Osmotic Swelling (HOS) TestINVASIVE TESTSImpossible to use

Sperm DiagnosticsSperm Diagnostics

• Hypo-Osmotic Swelling (HOS) TestCASA

• Sperm Penetration Assay (Hamster-Test)• Hemi-Zona Binding Assay • Acrosome Reaction Assay• Y-Chromosome Microdeletion• Sperm Chromatin (SCSA/TUNEL/Comet Assay)• Kreatinine Kinase Assay

Impossible to use the sperm for IVF or ICSI treatment

• Functional criteria

• Morphological criteria

Which possibility we have to select spermatozoa?

Are there new test available to select spermatozoa for ICSI?

• Morphological criteria

IMSI IMSI Intracytoplasmic Morphological

Selected Sperm Injection

day 0 1 2 3 5

„Early paternal defects“„Late paternal defects“

Impaired development

PaternalPaternal factorsfactors whichwhich influenceinfluence negativelynegatively thethe embryoembryo developmentdevelopment

Impaired development/ abortus

„Early paternal defects“

Sperm-cytoplasm-defects

oocyte-activating factor

centriole

Sperm-nucleus-defect•Meiotic mistakes –

chromosomal aberrations

•cytoplasm-retention

•Persisting histones

•Apoptotic process

•DNA-fragmentation

Delayed cleavage

Emb. fragmentation

�For ICSI:

abnormal sperm shape and aneuploidy

• In semen samples with high incidence of amorphous, round and elongated sperm heads: increased proportion of structural chromosome abnormalities(dicentric and ring chromosomes, chromatids fragments) but no increase in numerical chromosomal aberrations. (Lee 1996)but no increase in numerical chromosomal aberrations. (Lee 1996)

• Association between abnormal sperm with enlarged head and increased frequency of aneuploidies and diploidy. (Bernardini 1998)

• Correlation with different abnormalitiesand significant increase risk of aneuploidy(Colombero1999 Kahraman 1999 Calogero 2001 Rubio 2001 Yakin 2001 Templado 2002)

�For ICSI:

• Injection of abnormal spermatozoa results

– Correlated well with the fertilization outcome 60,7% vs 71,7%

– Did not affect embryo development (day 3)

abnormal sperm shape and pregnancy

– Did not affect embryo development (day 3)

– Reduction in the ongoing pregnancy rates:

20,2% versus 36,7%

– Reduction in the implantation rates:

9,6% versus 18,7%

De Vos et al, 2003

What can we detect under the microscope?

• Abnormal shape in general can be detected

under Hoffman modulation contrast

microscope at magnification x 400microscope at magnification x 400

Current selection of spermatozoa

before ICSI

• Selection of one motile at 400X magnification

We don t know:

Which one is morphologicaly optimal?

Which one has the best maturity?

What can we detect under the microscope?

• Abnormal shape in general can be detected under Hoffman modulation contrast microscope at magnification x 400

• But is it possible to detect other abnormalities such as vacuoles?The existence of big vacuoles in the sperm-nucleus is an index for higher damages in the nuclear DNA-content than the form of the nucleus or differences in size.

Vacuole

Chromatine:

Optimale condensation Bad condensation

Bedford et al,1973 (x 46000)

Big Vacuole

Bad condensation of the chromatin

Normal Nucleus abnormal nucleus

the chromatin

Bisson et David, 1975

Non invasive methods to select

spermatozoa in real timeBartoov developed a system that permits to select in real time

spermatozoa at very high magnification X 6,600

MSOMEMSOME

(motile sperm organelle morphology examination)

Bartoov et al.2001 N Engl J Med,

Bartoov 2002 J.Andrology

MSOME

(Motile Sperm Organelle Morphology Examination)Additional tool to

ICSI

IMSI((II ntracytoplasmic ntracytoplasmic MM orphologically orphologically SSelected elected SSperm perm II njection)njection)

(Bartoov et al., 2001, 2002, 2003)

Eshre Bologna, 23-24 January 2009

Leica 6000

• Inverted microscope, (Leica 6000):

- Nomarski interferential contrast

- Objektives: x20immDIC, x68immDIC, x100immDICx10, x20, x40

• Mikromanipulators Leica AM6000-Eppendorf

Equipments

• Mikroinjektors Eppendorf Cell-Tram

• Variable Zoom (VarioC-mount) Leica= 2.200 - 12.500x

• Analogic Videokamera : Leica DFC280

• Picture processing: Leica Application Suite

• Very thin glass petri dish (170µm) « Wilco »

X 400

X 6.600

Normal sperm with IMSI

Leica 6.000 Bregenz 2005

Teratozoospermie

• Control Experimental

•• previous ICSI previous IMSI• attempts attempts attempts attempts•

• Injected oocytes 10,3 10,2 10,1 10,6

Study on 50 couples after failure of implantation with ICSI,

½ with classical ICSI et ½ with IMSI

•• 2 PN 63,7% 65,5% 63,1% 64,5%

•• Transferred embryos 3,6 3,5 3,6 3,8

•• Clinical pregnancies 0 15 (30%) 0 33 (66%)

•• Implantation rates -- 9,5% -- 27,9%

•

• Bartoov et coll,2003 Fertil.steril

First choice Spermatozoa with morphologically normal shape and nucleus

( 0 or 1 small Vacuole < 4% nuclear area)

Second choice

Abnormal

but oval nuclear shape and

„The morphological normalcy of the sperm nucleus and pregnancy rate of IMSI“ Berkovitz Hum. Reprod. 2005

but oval nuclear shape and

normal nuclear content

Non oval nuclear shape but

normal nuclear content

Abnormal Chromatin

70 cycles FIRST choice 70 cycles SECOND choice

Injected oocytes 9,5 8,9

% 2PN 74,1% 62,3%

Top Emb.% day 3 26,7 16,2

Transferred embryos 3,3 3,2

Pregnancies / cycle 52,8 % 17,1 %

Implantation 26,1 % 8,3 %

Abortion% 9,8 % 33,3 %

4 misc: big vacuoles

Berkovitz et coll,2006. Hum. Reprod.

P< 0,05

Percentage of DNA-Fragmentation<30% 30-40% >40%

Patients 51 11 10

Previous attempts ICSI

Implantation rate 0.9% 0% 0.7%

Birth rate 0% 0% 0%

TUNEL-Assay: 72 Patients

Birth rate 0% 0% 0%

Attempts with IMSI

Implantation rate 23.6% 17.4% 33.3%

Birth rate 18.9% 17.4% 28.6%P< 0.001 P<0.05 P<0.01

Hazout et al RBMonline 2006

Embryo development to day 5 and pregnancy rates aft er ICSI or intracytoplasmic morphologically selected sperm injection (IMSI) in patients with previous failure of

implantation: a sibling studyZech N, Bach M, Neyer T, Stecher A, Zintz M, Petra Baborova, Uher P, Vanderzwalmen P Institute für Reproduktionsmedizin und Endokrinologie. Bregenz, Austria correspondence to: [email protected]

Introduction

With the introduction of a newconcept of ICSI procedure named« IMSI », spermatozoa exhibiting alarge panel of nucleusmalformations in terms of shape,size and presence of vacuoles cannow be observed at magnificationsup tp 12.000x. The existence ofvacuoles in the nuclei ofspermatozoa is associated withreduced blastocysts formation,pregnancy and implantation rates.Such defects can not be detectedduring ICSI at 400x magnification .

Results

Only 7.3% of spermatozoa selected with IMSI were free of anyabnormalities, whereas the majority of the spermatozoapresented small (65.2%) or large (24.3%) nuclear vacuoles a loneor were associated with other abnormalities (3.2%). On day 5 , ascompared to ICSI, IMSI provided a significant higher propor tionof blastocysts (IMSI 39.6% vs ICSI 28.2% P< 0.01) and goodquality blastocysts (IMSI 16.1% vs ICSI 9.0.% P< 0.05).

400X

Hoffman Modulation ContrastNomarski Interference

Contrast6.600X

Materials and methods

39 couples 1-2 previous failures of implantatio n after ICSI.

608 oocytes randomly allocated to:

ICSI 323 IMSI 285

11%

38%

59%

40%

50%

60%

70%

Distribution of transfers Ongoing Pregnancy rates

during ICSI at 400x magnification .As consequence, the pendingquestion to answer is, if IMSI couldbe a better strategy and thussupersede ICSI for selectingspermatozoa. In other word, whatwould have been the percentageselecting spermatozoa of the samequality as can be detected by IMSIby randomly pickingmorphologically normal appearingspermatozoa with conventional ICSIfor the same population ofspermatozoa.

6.600X 12.500X

For IMSI, spermatozoa were selected atmagnifications ranging from 6000x to 12500x under aNomarski interferential inverted microscope (LeicaAM6000 Germany) equipped with a variable zoom lens(HC VarioC-mount Leica). After selecting the bestspermatozoa with IMSI and immobilization, injectionwas performed as described previously forconventional ICSI at 400x magnification. Fertilizedoocytes were cultured to day 5 in 4 well dishescontaining 800µl of non-sequential Global medium(LifeGlobal, Ontario, Canada) supplemented with 7.5%HSA at 37°C in a humidified atmosphere of 6% CO2 inair. The 2 morphologically best blastocysts wereselected for transfers.

33% 56%

25%

0%

10%

20%

30%

40%

IMSI KombinationICSI/IMSI

ICSI

COMBINATION IMSI/ICSI

IMSI

ICSI

Conclusions

Our results demonstrate that if selection had been performe d onthe same sperm population using the conventional ICSI appro achthe likelihood of selecting sperm with a large nuclear vacuo le ormultiple ones with concomitant deleterious effects on embr yodevelopment would have been very high.

As consequence, according to our reports, instead of ICSI, I MSIcan be considered as a useful technique to select normal shap espermatozoa with fewer nuclear defects such as vacuoles

Design

A prospective study, using sibling oocytes was undertaken to compare the outcome after selection of spermatozoa using the conventional ICSI procedure or IMSI.

ASRM, Washington 10-2007

60

70 d5 Embryonen

d5 Top - Embryonen#39

#348

# Nb of injected oocytes

60708090

100 d3 Embryos

d3 Top - Embryos

#39

#17

#348

#130

Blastocyst formation after intracytoplasmic morphol ogically selected sperm injection (IMSI) according to the morphological integrity of human sperm nuclei

Zech N, Bach M, Neyer T, Stecher A, Zintz M, Petra Baborova, Uher P, Vanderzwalmen P Institute für Reproduktionsmedizin und Endokrinologie, Bregenz, A ustria correspondence to: [email protected]

Introduction

Several publications report that theselection of sperm with normalnuclear shapes at a magnification upto 6.600x using Normarski differentialinterference contrast is positivelyassociated with pregnancy rates afterday 3 embryo transfers in coupleswith previous and repeatedimplantation failures and in patientswith elevated degree of DNAfragmented spermatozoa. Insituations where no normalspermatozoa can be found, the onlyalternative consists of choosing the

Results

Out of a total of 81 attempts, 534 MII oocytes were available f orinjection. Only 7.3% of spermatozoa selected for injection werecompletely free of any abnormalities (Grade I) The majority ofthem presented small (Grade II, 65.2%) or large (Grade III, 2 4.3%)nuclear vacuoles alone. The remaining were associated withother abnormalities (Grade IV 3.2%).

Materials and methods

The spermatozoa were selected at magnifications rangingbetween 6.600x and 12500x under a Nomarskiinterferential Leica AM 6000 inverted microscopeequipped with a variable zoom lens (HC VarioC-mountLeica) at 37°°°°C. The primary intention was to selectspermatozoa with absence of vacuole for fertinization.When there was no chance, even after extensive search,to find any normal appearing spermatozoa, the “mostnormal looking” spermatozoon was selected atmagnification up to 12.500x and photo-documented forfurther classification. Following morphological analysis ofeach picture, the spermatozoa were classified in 4 groups.

Spermatozoa classification

Embryo development to day 3 or 5 in relation to the morphological aspect of the sperm

#17

0

1020

3040

50

Grade1

grade2

Grade3

Grade4

d5 Top - Embryonen

#17

#348

#130%

0102030405060

Grade1

Grade2

Grade3

Grade4

%

alternative consists of choosing themorphologically next best. If noapparent early paternal effects onembryo development up to day 3 isobserved when oocytes are fertilizedby spermatozoa with large vacuolespresent in the sperm head, anintriguing question is if the presenceof such nuclear vacuoles, which cannot be detected with conventionalICSI at 200x or 400x magnification,influences the capacity of the embryoto develop to the blastocyst stage.

Conclusions

This study confirms that IMSI is a powerful research tool forinvestigating spermatozoa carrying several abnormalitie s that arenot detectable with conventional ICSI. Such abnormalitiesinfluence embryo development after the third day of culture .Because vacuoles exert a negative effect on embryo developm ent,as shown in our study, it is now time to investigate into theirorigin and under what circumstances the frequency of suchvacuoles increase. In such a way, a treatment may be offered o r astrategy could be established in order to reduce their appea rance.

Design

The aim of our work was to analyseif the existence of vacuoles in thenuclei is associated with the abilityof embryos to develop to theblastocyst stage.

Grade 1

Normal FormNo Vacuole

Grade 2

Normal FormMaximum 2 small Vacuoles<4%

Grade 3

Normal FormAt least one big Vacuole

Grade 4

Abnormal Form, with Vakuole(s)And otherabnormalities

Spermatozoa classification

The embryos were cultured individually to day 5 in Globalmedium (Lifeglobal). On day 5, the quality of the embryoswere recorded and scored according to the degree ofexpansion of the blastocoele, the quality of the inner cellmass (ICM) and of the trophectoderm.

ASRM, Washington 10-2007

Correlation between sperm morphology and embryo quality on day 3

60

70

80

90

100

[%]

#13#66

#33 #15

Day 3 embryos

# injected

oocytes

0

10

20

30

40

50

60[%]

SPZ

normal

SPZ

Vakuole

≤4%

SPZ

Vakuole

> 4%

SPZ with

more than one

vacuole

Day 3 TOP embryos

50

60

70

80

[%]

Correlation between sperm morphology and blastocysts on day 5

Day5 Blastocyst

# injected

oocytes

#66

#13

0

10

20

30

40[%] Top Blastocysts

#15#33

SPZ with

more than

one vacuole

SPZ

Vakuole

> 4%

SPZ

Vakuole

< 4%

SPZ

normal

• Three treatment approaches to the problem of elevated sperm DNA damage have been suggested recently:

– ICSI using surgically-retrieved testicular spermatozoa (TESE) instead of ejaculated ones (Greco et al., 2005b),

– ICSI with ejaculated spermatozoa after two months of – ICSI with ejaculated spermatozoa after two months of oral antioxidant treatment:1g.Vit C + E daily for 2 months. (Greco et al., 2005a),

– ICSI with spermatozoa selected with the use of a high-magnification optical system (IMSI) (Hazout et al., 2006).

Conclusions IMSI

• The morphology of the selected spermatozoa seems to influencethe outcome of embryo development and the pregnancy rate.

• Sperm morphology seems not to influence the number ofembryos on day 3, however there is a tendancy to obtain lessTOP day 3 embryos in case of spermatozoa carrying multipleabnormalitiesTOP day 3 embryos in case of spermatozoa carrying multipleabnormalities

• After IMSI a higher percentage of blastocysts and top blastocystsare obtained as compared to the ICSI technic.

• The size and the number of vacuoles influence the developmentof the embryo especially after day 3. It reflects probably a “latepaternal effect“

IMSI seems a promising technic and

could be offered to couples:

• No implantation

• Idiopathic infertility • Idiopathic infertility

• High degree of DNA Fragmentation

• Absence of fertilization after ICSI

• Severe Teratozoospermia

And in addition:

a new diagnostic and spermocytogram tools

Implementation of IMSI to a large population of ICSI candidate patients Implementation of IMSI to a large population of ICSI candidate patients may be advisable:may be advisable:

if the probability to select a normal spermatozoa using the MSOME(IMSI) approach is higher as compared to the classical ICSI approach.

ifif soso

we hypothezise that negative consequences of vacuolesmayinfluence not only the outcome of embryo developmentbut alsohealth and behaviour of offspring.


Long term effects of mouse ICSI with DNALong term effects of mouse ICSI with DNA--fragmented sperm (DFS) on fragmented sperm (DFS) on health and health and behaviorbehavior of adult offspring.of adult offspring.Fernanderz-Gonzalez Biol. Reprod. 2008

The use of DNA fragmentedspermin ICSIcan generate effectsthat only emerge during later life, such as: aberrant growth, premature aging, abnormal behavior, mesenchymal tumor.Tunnel and comet assay


� At present, there have not been sufficient numbers(or generations) of ICSIchildren to draw any firm conclusionsabout the long-term safety of thisprocedure.

� However, it is important to emphasize that animal dataare absolutelyunequivocal on this point and clearly indicate that DNA damage in the malegermline is potentially damagingfor the embryo and offspring(Anderson,2003; Lewis and Aitken, 2005)

� For the time being, the take-home messageis that DNA damage in themale germline is potentially damaging, and care should be taken whentreating patients exhibiting such damage with ICSI. In light of suchconsiderations, it would seem rational to try to determine the causes of DNAdamage in the male germline and to do everything possible to alleviate thisdamage (antioxidant therapy) and/or use sperm isolation techniques (IMSI)that will select for gametes possessing very low levels of DNA damage(Ainworth et al., 2005, 2007).


news

•Follicolometria 3D

automatizzata (AVS)

•SEET (Stimulation of •SEET (Stimulation of

Endometrium Embryo Transfer)

•Nuove tecniche di

congelamento (vitrificazione)

Follicolometria 3D automatizzata

(AVS)

Right ( Ovary) left ( Ovary)

Which are mature?

Conventional 2D „Follicle Tracking“

Ovaries: longitudinal or tranvers plane

spherical follicle elliptical follicle

+

D 1 + 2 : 2 D 1 + 2 + 3 : 3

e.g. 15 + 17 : 2 = 16mm e.g. 15 + 17 + 22 : 3 = 18mm

V=4/3 ×××× π ×××× radius³

3D Ultrasound – AVC (General Electric)

Automated Antral Follicle Count-Sono AVCFollicolometrie - Sono AVC

Uterine cavity length3D-Scan: FemaleReproductive Organs

Downregulation with GnRH Agonist ›14 days

Bleedingcycle before stimulation

HMG Stimulation

10 000 I.U. HCG OPU

36 h

Embryotransfer d5

Long Protocol

L.P

Standard Workflow

Day of cycle

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Follicles(ml/mm)

+

Pattern Thickness

L.P

Cohort of follicles 16-22 mm

1 2 3 …..10.........21………

Anral Follicle countFertility scan +

Follicle monitoring

Standard Workflow

Oocyte retrivial Follicle monitoring

Standard Workflow

RBMOnline - Vol 19. No 5. 2009 695–699 Reproductive BioMedicine Online

CONCLUSIONI

3D + Sono AVC rappresenta un nuovo standard nella follicolometria.

Impiego clinico in sostituzione della metodica 2D.

Migliore Outcome riguardo la qualitá ovocitaria, gli indici di fertilizzazione, il numero e la qualitá blastocitaria.

Curva di apprendimento rapida.Curva di apprendimento rapida.

Facile integrazione con i sistemi di gestione pazienti informatici per mezzo degli standard DICOM (trasferimento dati in rete).

Miglioramento degli standard qualitativi mediante l´impiego di sistemi di misurazione automatizzati e riproducibili (Direttive EU).

Migliore valutazione della riserva follicolare.

SEETStimulation of Endometrium Embryo Tranfer

SEET

• Lémbrione produce durante il

suo sviluppo varie sostanze per

comunicare con léndometrio

(cross-talk)

• Nei cicli ART, sviluppandosi

• Interleuchine IL-1

• hCG

• Fatori di crescita endoteliale

• Nei cicli ART, sviluppandosi

lémbrione in vitro, questo

meccanismo viene meno

• Ancor piu´ se transfer

blastocitario o su cicli da

crioscongelamento

• Le sostanze rimangono nel

liquido di coltura

Edwards RG et al. 1999, Kapiteijn K. et al. Fert: Steril. 2008,Sakae Goto et al.Fert. Steril. Nov.2007 – Dic.2008

SEET

• Durante cilci con Blastocisti congelate,il tresferimento di

supernatante culturale (ECS) congelato (-20°C) in d2, ha

mostrato un aumento degli indici di impianto e delle

gravidanze cliniche (soprattutto per „Top Blastocysts“) gravidanze cliniche (soprattutto per „Top Blastocysts“)

Outcomes of treatment with SEET and BT (control)

SEET (n=23) BT (control) (n=25) P value

No. of clinical pregnancies 20 12 .006

Single pregnancies 17 10

Twin pregnancies 3 2

Clinical pregnancy rate per transfer (%)a 87,0 48,0 .006

Implantation rate per embryo (%)b 71,9 (23/32) 37,8 (14/37) .007

Serum ß-hCG (IU/mL) on day 30 248 ± 184 138 ± 163 .036

Estradiol (pg/mL) on day 23 370 ± 224 350,5 ± 195 .764

Progesterone (ng/mL) on day 23 6,7 ± 3,6 7,1 ± 2,8 .682

Note: BT, blastocyst transfer; SEET, stimulation of endometrium embryo transfer.

a Clinical pregnancy was identified by development of a gestational sac.

b Implantation rate was determined by dividing the number of gestational sacs by the number of embryos transferred.

Novel method of embryo transfer. Fertil Steril 2007

Thank youThank you

Zona Pellucida

• Con la luce polarizzata si evidenziano i tre strati

(glicoproteici) della zona pellucida.

• In particolare lo strato più interno sembra essere un • In particolare lo strato più interno sembra essere un

marker importante.

• Pazienti over 35 presentano spesso un’ ispessimento dello

strato interno della zona pellucida e le glicoproteine che lo

compongono appaiono disposte in maniera meno

fisiologica.

Lo spermatozoo

Study 1: In all the situations, the probability to select spermatozoa from class 1 – 2 is higher if IMSI is applied.

Study 2:The sibling study shows that higher rate of blastocysts are obtained when IMSI is performed.As consequence, a higher number of transfers are performed with blastocysts that originated from the IMSI group.

Imsi / Blastocysts

that originated from the IMSI group.

Study 3:Independently of the percentages of normal spermatozoa, the rate of blastocysts is higher when IMSI is applied.

The probability to select a normal spermatozoon using the IMSI approach is higher as compared to the classical ICSI approach.


Implementation of IMSI to a large population of Implementation of IMSI to a large population of ICSI candidate patients :ICSI candidate patients :

�� May be advisable,

ifif thethe probabilityprobability toto selectselect aa normalnormal spermatozoaspermatozoa isis higherhigherusingusing thethe IMSIIMSI approachapproach asas comparedcompared toto thethe classicalclassical ICSIICSIapproachapproach..


MeaningMeaningvacuole

OrigineOrigine Effect on the Effect on the outcomeoutcome


• Suggestions:• „Vacuoles may reflect molecular defects responsible for anomalies of sperm

chromatin packaging and abnormal chromatin remodelling during spermmaturation which, in its turn, may render spermatozoa more vulnerable toDNA damage “Berkovitz et al., 2005; Hazout et al., 2006“

• More accurate answer: Isolation and evaluation of single spermatozoon• «Significance of large nuclear vacuoles in human spermatozoa: implications

VACUOLE Meaning ?????

• «Significance of large nuclear vacuoles in human spermatozoa: implicationsfor ICSI » Franco et al, RBMonline 2008

• « High power magnification microscopy and functional status analysis ofsperm in the evaluation and selection before ICSI » Garolla et al RBM online2008

• « Correlation between morphological semen parameters and sperm nucleardamage » Babarova submitted


single sp. Analysis• Sperm DNA integrity - acridine orange staining (Franco, Garolla, Barbarova)

• DNA fragmentation -TUNEL (Franco, Garolla, Barbarova)

• Mitochondrial membrane potential (Garolla)

• alteration seems to be suggestive of an early apoptotic process

• Sperm aneuploidies FISH (Garolla)

VACUOLE Meaning ?????

CONCLUSIONSAssociation between large vacuole in the sperm and DNA damag e.

Advice that the high level of denatured DNA in sperm with larg e nuclear vacuolessuggests: precocious decondensation disaggregation of sp erm chromatinfibers.

Significantly better chromatin status , mitochondrial function, aneuploidy rate (hypospermatogenesis) when nuclear vacuoles were abs ent.

VACUOLEDamage DNAAbnormal DNA packagingChromatin defects

externally or internally produced reactive oxygen species

Default in apoptosis Default in apoptosis processprocess

Origins of DNA damage in the spermatozoa

Aitken 2004, Kelton Tremellen 2008


? Advantage of IMSI ?

Damage DNA

Abnormal DNA packaging

Chromatin defects

Reason

Age

Smoking

Stress

Apoptose

ROS

YES

Selection of sperm

without vacuoles

Signification of vacuoles ?

Consequence

DNA

fragmentation

Impaired embryo development

Impaired implantation

Long term effect ????


Perspectives

Outcome of embryo development according to the morphology of the vacuoles and their localization ?morphology of the vacuoles and their localization ?

Handling and preparation of the sperm in better conditions

Prevent oxydative stress


IMSI has to be considered as a new diagnostic and spermocytogram tools

What are the origins of vacuoles? « In vitro » conditions of the kinetic of vacuoles development


� IMSI is used in very few ART centers.IMSI is used in very few ART centers.Some are reluctant to apply this new approach of selecting Some are reluctant to apply this new approach of selecting spermatozoa before ICSI.spermatozoa before ICSI.

As consequence, we may suggest for those who perform embryo As consequence, we may suggest for those who perform embryo transfer on day 2 or 3 to change their strategy and extend the transfer on day 2 or 3 to change their strategy and extend the culture to day 5: culture to day 5: culture to day 5: culture to day 5:

Extended culture could provide a test by which to select more Extended culture could provide a test by which to select more viable embryos that reflect the quality of the viable embryos that reflect the quality of the gametes from gametes from which they were derivedwhich they were derived

(Spano 2000, Behr 1999, Vanderzwalmen 2008)


� A lot of questions are still in unanswered:

Which attitude to have if only abnormal spermatozoawith large vacuoles are present in the semen sample ?

- If observation before IVF treatment: antioxydant therapy, modify the lifestyle, etc….

- If observation the day of the OPU:

Inject one part of the oocytes ?

Aseptic vitrifcation of oocytes and try to improve the quality of the semen ?

Propose donor (where it is possible) ?


Antinori et al., RBMonline 2008Eshre Bologna, 23-24 January 2009

BENCHMARKING – IVF Zentren Österreich

Fortbildungs-Symposium, Ottobrunn 5.Mai 2007

• Development of news technics with the aim to enhance

the preparation of sperm and to select in a more acccurate

fashion a sperm carrying all the informations for the

future development are mandatory.

• � New sperm preparation technicsNew sperm preparation technics

• � Biochemical markers of human sperm maturity and

function (PICSI)

� Isolation of spermatozoa based on a morphological

approach (MSOE – IMSI)

Sperm separation strategies in

current practice

• Rapid removal of the seminal plasma

• Minimum of physical trauma associated with

centrifugation (ROS)centrifugation (ROS)

• Swim UP motile sp.

• Gradient density centrifugation morphology• Time consumming

• do not avoid the damaging effects of centrifugation

electrophoretic separation of the cells on the basis of their charge and size.

This system is based on two principies:

(i) the highest qualityspermatozoa in the ejaculate are the most electronegative

(ii) spermatozoa can be separated from other contaminating electronegative cells (such as leukocytes and precursor germ cells) by virtue of their small crosssectional size.

outer chambers

polyacrylamide restriction membranes

polycarbonate separation membrane

two inner chambers

outer chambers

outer chambers

polyacrylamide restriction membranes

polycarbonate separation membrane

electrophoretic separation chamber

HR 2006

couple suffering from long-term infertility associated with extensive DNA damage in the male germ linewith extensive DNA damage in the male germ line

Hyaluronidase-Binding-Test

biochemical markers of human sperm maturity and function

Diminished maturity spermatozoa

lack HspA2 expression

• meiotic defects (non disjunction)

• a higher rate of retention of CK and other cytoplasmic enzymes,cytoplasmic enzymes,

• increased level of lipid peroxydation

• DNA fragmentation,

• abnormal sperm morphology,

• deficiency in zona binding and HA binding sites.

• Sperm which bind on immobilised HA:– are mature

– finished the Spermiogenetic process of reorganisation of the sperm-plasma-membrane

– show extrusion of the cytoplasm

– histone-protamine-exchange in the nucleus (mature chromatine)

• HA-bound sperm:

Significance of HA-Binding–Assay

• HA-bound sperm:– show no DNA-degradation and have no active Caspase 3

– show no acrosomal reaction;

– have symmetrical oval heads

– are motile

– have low frequency of chromosomal aneuploidies

Bound sperm can be used for ICSI directly

Based on the above concepts, the team of Huzar Gabor examined the utility of a

diagnostic of sperm binding to HA in a double diagnostic of sperm binding to HA in a double chamber device

PICSI

• Clinical application

PICSI

Selection of HA binding spermatozoa

Incubation, RT, 10 min

Add sperm to the hyaluronan microdot

Gentle aspiration of a bound spermatozoa

The clinical impact associated with the use of PICSI derived embryos

Worrilow ASRM 2006

26 patients

147 oocytes ICSI

126 oocytes PICSI

ICSI only PICSI only PICSI + ICSI

No differences in: fertilization rates, day 3 morphology,blast.morphologyNo differences in: fertilization rates, day 3 morphology,blast.morphology

clinical Pregnancy rates

25% (8) 57% (7) 57% (7)

Higher miscarriage rates in the ICSI group

Patients receiving PICSI derived or a combination of PICSI and ICSI embryos demonstrated greater CPR and lower MR over those receiving only ICSI embryos.

Valutazione del cumulo ooforoValutazione del cumulo ooforo

Grad Definition

11 Gut Gut expandexpandiertiert, , enthält viele Zenthält viele Zellellenen, homogen, homogen

22gutgut expandexpandiieertrt, , enthält „Centhält „Clusterluster“ an Z“ an Zellellen mit en mit intraintrazzellulelluläärren Räumenen Räumen

33Expansion isExpansion istt moderat, moderat, Gruppierungen mit Gruppierungen mit ZZellclusterellclusternn

44 kkompaompakkt, dt, dichticht

(Ng et al., 1999)

Selezione dell’ Ovocita

• Da un modello matematico, per avere tre

ovociti “ideali” dobbiamo avere a

disposizione tra 5 e 14 ovociti. Le nostre disposizione tra 5 e 14 ovociti. Le nostre

strategie di stimolazione non devono

cambiare dopo la legge 40.(R.Palermo 2004)

Conclusions IMSI

• Microinjection of selected spermatozoa withstrictly defined morphologically normal nucleiimproves the incidence of ongoing pregnancy incouples with recurrent negative conventional ICSI

• Improvement of results for patients with elevated levels of Sperm-DNA-fragmentation (Tunel)

Study1: In all the situations, the probability to select spermatozoa from class 1 – 2 is higher if IMSI is applied

Conclusions


Second Second studystudy

Percentage of blastocysts in relation to the method of sperm selectionmethod of sperm selection

ICSI vs. IMSI (sibling study)


ICSI vs. IMSI (sibling study)53 Patients with at least 1-2 previous failure of implantation , > 3 oocytes

Woman age: 38 Man: OAT (WHO)

Nb oocytes 833

IMSIICSI

403430Nb injected oocytes


First studyFirst study

Probability to select a normal spermatozoa in relation to the method of observation: in relation to the method of observation:

Nomarski or Hoffman


%

ns

ns

Embryo development in relation ICSI or IMSI spermatozoa selection

P<0,05

ns

P<0,05


65%59%

50%60%70%

% of deliveries in relation tothe method of sperm

selection

% of transfers in relation to the origin of the embryo

selected (max 2 embryo transferred)

Percentages of transfer and deliveries in relation to ICSI or IMSI spermatozoa

selection

38%

0%10%20%30%40%50%60%

IMSI Kombination

ICSI/IMSI

ICSI

IMSI

KombinationICSI / IMSI

ICSI


[%]Normal

Probability to select a normal spermatozoa after ICSI and IMSI in relation to the percentage of spermatozoa from class 1-2

SpermocytogrameAfter ICSI selectionAfter IMSI selection

NormalSp

Groups of normal form (class 1 – 2)

Study 1: In all the situations, the probability to select spermatozoa from class 1 – 2 is higher if IMSI is applied.

Study 2:The sibling study shows that higher rate of blastocysts are obtained when IMSI is performed.As consequence, a higher number of transfers are performed with blastocysts

Conclusions

As consequence, a higher number of transfers are performed with blastocysts that originated from the IMSI group.


Third studyThird study

% of blastocystesafter IMSI and ICSI in relation to the percentage

of normal sperm formsof normal sperm forms


% of blastocysts after IMSI and ICSI in relation to the percentage of class 1-2 spermatozoa in the semen sample

%blastocystsblastocysts

Percentages normal spermatozoa

Nb of cases 7 5 3 9 6 3 1Eshre Bologna, 23-24 January 2009

% of top quality blastocysts after IMSI and ICSI in relation to the percentage of class 1-2 spermatozoa in the semen sample

% of TOPblastocystsblastocysts

Percentages normal spermatozoa

Nb of cases 7 5 3 9 6 3 1Eshre Bologna, 23-24 January 2009

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