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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
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.
Eshre Bologna, 23-24 January 2009
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
Eshre Bologna, 23-24 January 2009
� 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).
Eshre Bologna, 23-24 January 2009
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
PACS
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
• L´embrione produce durante il
suo sviluppo varie sostanze per
comunicare con l´endometrio
(cross-talk)
• Nei cicli ART, sviluppandosi
• Interleuchine IL-1
• hCG
• Fatori di crescita endoteliale
• Nei cicli ART, sviluppandosi
l´embrione 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.
Eshre Bologna, 23-24 January 2009
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..
Eshre Bologna, 23-24 January 2009
MeaningMeaningvacuole
OrigineOrigine Effect on the Effect on the outcomeoutcome
Eshre Bologna, 23-24 January 2009
• 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
Eshre Bologna, 23-24 January 2009
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
Eshre Bologna, 23-24 January 2009
? 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 ????
Eshre Bologna, 23-24 January 2009
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
Eshre Bologna, 23-24 January 2009
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
Eshre Bologna, 23-24 January 2009
� 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)
Eshre Bologna, 23-24 January 2009
� 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) ?
Eshre Bologna, 23-24 January 2009
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
Eshre Bologna, 23-24 January 2009
Second Second studystudy
Percentage of blastocysts in relation to the method of sperm selectionmethod of sperm selection
ICSI vs. IMSI (sibling study)
Eshre Bologna, 23-24 January 2009
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
Eshre Bologna, 23-24 January 2009
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
Eshre Bologna, 23-24 January 2009
%
ns
ns
Embryo development in relation ICSI or IMSI spermatozoa selection
P<0,05
ns
P<0,05
Eshre Bologna, 23-24 January 2009
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
Eshre Bologna, 23-24 January 2009
[%]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.
Eshre Bologna, 23-24 January 2009
Third studyThird study
% of blastocystesafter IMSI and ICSI in relation to the percentage
of normal sperm formsof normal sperm forms
Eshre Bologna, 23-24 January 2009
% 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