Morphological aspects at zygote stage Markus Montag, Prof., Ph.D. University of Bonn Germany Imaging zygotes Stereo microscope - Standard contrast - Stereo microscope - Relief contrast - Inverted microscope - Hoffman contrast - Zygotes - what can we see? Pronuclei Halo Nucleoli N l li Precursor Bodies Number of pronuclei Mechanism of 1 PN formation - Fusion of 2PN - 11.4h 11.7h 16.1h post ICSI Mechanism of 3PN formation in ICSI cycles I • 3 PN from oocytes in a transition from telophase to metaphase Mechanism of 3PN formation in ICSI cycles II • 3 PN ffrom di diploid l id ((giant) i ) oocytes 240 µm µ Micro pronuclei Micro-pronuclei • M May arise i b by chromatid/chromosome h tid/ h llagging i during division – High risk for aneuploidy Shape and size of pronuclei • Equal E l - good d / unequall - bad b d Zollner et al., 2002 Position of pronuclei • PN in i the th periphery i h or apartt from f each h other th are considered developmentally abnormal Garello et al., 1999 Scott & Smith, 1998 Polar bodies: 1st and 2nd PB 1 Videos of polar body formation PB2 Position of pronuclei versus polar bodies • Alignment Ali t (Garello (G ll ett al., l 1999) • Th The possible ibl benefit b fit off this thi criterion it i iis questionned ti d due to a possible impact of denudation on polar b d position body iti (T (Taylor l ett al., l 2008) • Polar bodies move (Scott et al., 2008) The HALO • Th The Halo H l iis primarily i il a sign i th thatt th the cytoskeleton t k l t off th the developping zygote is functionally active • The Halo is not correlated to babies born (Scott, 2006) The nature of the Nucleoplar Precursor Bodies (NPBs) Distribution of DNA in relation to the NPBs Morphological p g aspects p and zygote scoring Zygote scoring systems • Scott & Smith, Smith 1998 • Tesarik & Greco, 1999 • Garello G et al., 1999 • Balaban et al., 2001 • Montag et al., 2001 • Scott et al., 2001 • Zollner et al., 2002 • Ebner et al., 2003 • Senn et al., 2006 PN, NPB, PN NPB Halo PN, NPB PN, PB PN, NPB PN, NPB PN, NPB Multi-factorial, Score PN, Halo Multi-factorial, Score Widely used scores Tesarik & Greco Greco, 1999 Scott et al., al 2002 Automatic scores 1-3 points per criterion: 1. Distance of PN 2. Orientation of PN versus PB 5 1 4 6 3. Position of PN 3 4. Cytoplasmic Halo 2 5. Number of Nucleoli 6. Polarisation of Nucleoli Senn et al., al 2006 R l Relevant t ffor pregnancy rate t Criticism of PN score Limited value of zygote (PN) scoring systems • Payne et al., 2005 • James et al., 2006 • Nicoli et al., 2007 • Brezinova et al al., 2009 • Nicoli et al., 2010 The time-point time point • Z Zygote t assessmentt is i usually ll performed f db by a static observation • Difference between IVF and ICSI cycles y in timing (Montag et al., 2001) • In ICSI cycles: strict timing required post-ICSI for studies on inter inter-cycle cycle comparison Imaging zygote development Payne ett al., P l 1997 • The male pronucleus is shortly formed prior to th ffemale the l pronucleus l • The female pronucleus is closer to the 2nd polar body and contains less NOR-precursor bodies compared d tto the th male l pronucleus l Imaging of embryo development • Time-lapse p studies – 1st cleavage cycle – Embryo y development p – Blastocyst formation • Adequate cleavage timing – Constant cleavage intervals • Being fast is not always good – Too fast development: low IR Imaging zygote development Videos showing zygote formation Imaging g g NPB variation - Symetric from the beginning- Post-ICSI: 11.7h 15.4h 18.3h 23.8h Imaging NPB variation Post-ICSI: 12.7h 14.3h 13.0h 18.3h 13.7h 21.0h Timing of PN formation Correlation appearance 2nd PB and appearance pronuclei 800 PB formation in relation to spindle PN appearan nce [min.] 300 200 100 400 200 en nt es Linear regression analysis: P = 0.0008 Pr se 600 t P = 0.0395 0 Ab Time of PB formation 400 0 0 100 200 300 PB2 appe arance [min.] 400 PN formation and PN score Time of formation of pronuclei 700 P < 0.05 0 05 P < 0.05 0 05 600 500 400 300 200 100 0 Muster Z1/Z2 Pattern Z1/Z2 Muster Z3/Z4 Z3/Z4 4 Zeller Tg2 4 Cell d2 <4 Zeller Tg2 <4cell d2 Variations in PN formation Variations in the first cell cycle of human oocytes 800 700 600 500 PB2 appearance 400 PN appearance 300 200 100 0 30 32 34 36 38 40 Conclusions • Certain zygote parameters are an indicator for the progression through the cell cycle • Pronuclear morphology, • Orientation of the polar bodies • Changes in the cytoplasm (e.g. halo) • S Scoring i systems ffor these h parameters are iin place, but their absolute benefit is somehow questionned in the era of blastocyst culture • Imaging zygote development may proof as a new parameter however parameter, however, it´s it s relevance is under investigation
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