Analysis of optical properties of paracrystalline ultrastructures in human oocytes by PolScope microscopy correlated to embryo quality and viability [Elektronische Ressource] / vorgelegt von Ying Shen

Sujets

• Gesundheit

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2007
• Nombre de lectures: 72
• Poids de l'ouvrage: 3 Mo

Extrait

ANALYSIS OF OPTICAL PROPERTIES OF
PARACRYSTALLINE ULTRASTRUCTURES IN
HUMAN OOCYTES BY POLSCOPE
MICROSCOPY CORRELATED TO EMBRYO
QUALITY AND VIABILITY
YING SHEN
INAUGURALDISSERTATION
zur Erlangung des Grades eines
Doktors der Humanbiologie
des Fachbereichs Medizin der
Justus-Liebig-Universität Giessen

édition scientifique
VVB LAUFERSWEILER VERLAG
VVB LAUFERSWEILER VERLAG ISBN 3-8359-5180-7
STAUFENBERGRING 15
D-35396 GIESSEN
Tel: [phone] Fax: -5599890
[e-mail]
www.doktorverlag.de 9 7 8 3 8 3 5 9 5 1 8 0 8 édition scientifique
VVB VVB LAUFERSWEILER VERLAG
SHEN YING SPINDEL, ZONA PELLUCIDA & OOCYTEQUALITYDas Werk ist in allen seinen Teilen urheberrechtlich geschützt.
Jede Verwertung ist ohne schriftliche Zustimmung des Autors
oder des Verlages unzulässig. Das gilt insbesondere für
Vervielfältigungen, Übersetzungen, Mikroverfilmungen
und die Einspeicherung in und Verarbeitung durch
elektronische Systeme.
1. Auflage 2007
All rights reserved. No part of this publication may be
reproduced, stored in a retrieval system, or transmitted,
in any form or by any means, electronic, mechanical,
photocopying, recording, or otherwise, without the prior
written permission of the Author or the Publishers.
st
1 Edition 2007
© 2007 by VVB LAUFERSWEILER VERLAG, Giessen
Printed in Germany
VVB LAUFERSWEILER VERLAG
édition scientifique
STAUFENBERGRING 15, D-35396 GIESSEN
Tel: [phone] Fax: [phone]
email: [e-mail]
www.doktorverlag.de
Analysis of Optical Properties of Paracrystalline
Ultrastructures in human oocytes by PolScope
Microscopy Correlated to Embryo Quality and Viability
INAUGURALDISSERTATION
zur Erlangung des Grades eines
Doktors der Humanbiologie
des Fachbereichs Medizin der
Justus-Liebig-Universität Giessen





vorgelegt von




YING SHEN

aus Beijing, China




Gießen, 2006
Aus dem Medizinischen Zentrum für Frauenheilkunde und Geburtshilfe
des Fachbereichs Medizin der Justus-Liebig-Universität Gießen

Leiter: Prof. Dr. Dr. h.c. Hans-Rudolf Tinneberg




























Gutachter: Prof. Dr. Dr. h.c. H.-R. Tinneberg rof. Dr. K. Steger

Tag der Disputation: 14. 05. 2007




Content I

1. Abstract …………………………………………………………………………… 1
2. Introduction ………………………………………………………………………. 4
2.1 Oocyte growth, maturation, fertilization, implantation and
developmental potential after fertilization ………………………………… 4
2.1.1 Oogenesis and female meiosis …………….…………………………… 4
2.1.2 Oocyte arrest and growth ………………………………………………. 9
2.1.3 Folliculogenesis ……………………………………................................ 10
2.1.4 Spindle formation and cell divisions …………………………………… 14
2.1.5 Aged oocytes …………………………………………………………… 18
2.1.6 Zona Pellucida formation ……………………………………………..... 19
2.1.7 Organisation of spindle apparatus and zona pellucida may reflect the
true state of oocyte healthy and their developmental competence ……... 21
2.2 Methodologies to evaluate the sub-microscopic structures (spindle and
zona pellucida) in human oocytes ………………………………………….. 23
2.2.1 Invasive analysis procedures for illumination of spindle apparatus and
zona pellucida …………………………………………………………... 23
2.2.1.1 Indirect anti-tubulin immunofluorescence and laser scanning
confocal microscopy ……………………………………………… 23
2.2.1.2 Electron-Microscopy …………………………………………… 24
2.2.2 Non-invasive analysis of ultra-structures in mammalian oocytes ……… 25
2.2.2.1 Development history of polarized light microscopy …………… 25
2.2.2.2 Quantitative analysis of birefringence property using polarized
light microscopy …………………………………………………. 28
2.2.2.3 Non-invasive nature of PolScope microscopy …………………. 29
2.3 Human infertility and current practices in assisted reproduction ……….. 30
2.3.1 Current practice in assisted human reproduction ……………………….. 30
2.3.2 In vitro maturation of human oocytes is becoming a novel method for
some infertile patients …………………………………………………... 32
2.3.3 Assessing oocyte Quality as a way to improve ART outcomes ………... 33
3. Aims of the study and experimental design ……………………………………… 35
3.1 Requirement of identifying the oocyte quality to improve the success rate of
ART …………………………………………………………………………... 35
3.2 Aims of the study ……………………………………………………………... 35
3.3 Experimental design ………………………………………………………….. 37
4. Materials and methods …………………………………………………………….. 39
4.1 Source of human oocytes ……………………………………………………... 39
4.2 Retrieval and culture of oocytes ……………………………………………… 39
4.3 Analysis of maturation kinetics of immature GV-stage human oocytes in vitro 40
4.4 Invasive analysis of spindle apparatus in fixed oocytes by indirect anti-
tubulin immunofluorescence ………………………………………………..... 40
4.4.1 Solutions and chemicals ………………………………………………... 40
4.4.1.1 Ring-solution and fibrinogen preparation for use in fibrin clots. 40
4.4.1.2 PBS solution ……………………………………………………. 40
4.4.1.3 Thrombin-solution ……………………………………………..... 42
4.4.1.4 NGS wash solution …………………………………………….. 42



Content II
4.4.1.5 5x SB-stock ……………………………………………………. 42
4.4.1.6 Simple-fix ……………………………………………………… 42
4.4.1.7 Other chemicals ………………………………………………... 42
4.4.2 Procedure ………………………………………………………………. 43
4.4.2.1 Preparation of slides …………………………………………… 43
4.4.2.2 Preparation of clot ……………………………………………… 43
4.4.2.3 Placement of oocytes in a fibrinogen/thrombin clot …………… 43
4.4.2.4 Fixation of oocytes …………………………………………….. 44
4.4.2.5 Indirect anti-tubulin immunofluorescence …………………….. 44
4.4.3 Analysis of spindle morphology and chromosomal behaviour ………… 44
4.5 Non-invasive analysis of birefringent structures in living oocytes by
PolScope microscopy ………………………………………………………... 45
4.5.1 Installation of the hardware ……………………………………….......... 45
4.5.2 The SpindleView software and oocyte imaging ……………………….. 46
4.5.3 Imaging of birefringent structures in living human oocytes …………… 46
4.5.3.1 Quantitative assessment of the birefringent property of zona
pellucida ………………………………………………………… 47
4.5.3.2 Quantitative assessmrefringent property of spindle
apparatus ……………………………………………………….. 50
4.6 Assessment of PN score for embryo selection, development of embryos at
day 2 and pregnancy ……………………………………………………........ 53
4.7 Statistical analysis …………………………………………………………… 55
5. Results ………………………………………………………………………………. 56
5.1 Assessment of spindle formation in immature oocytes from ICSI cycles... 56
5.2 Quantitative analysis of expression of a birefringent spindle apparatus in
human in vivo maturing oocytes using PolScope microscopy ……………. 59
5.2.1 Non-invasive nature of PolScope microscopy …………………………. 59
5.2.2 Presence of a visible birefringent spindles in human living oocytes
matured in vivo …………………………………………………………. 60
5.2.3 Fertilization rate of oocytes with and without birefringent spindle …….. 61
5.2.4 Fate of oocytes with and without a birefringent spindle ………………... 62
5.2.5 Fate of oocytes with and without a displaced spindle …………………... 63
5.3 Quantitative assessment of spindles in oocytes with respect to
developmental potential/PN score ………………………………………….. 64
5.3.1 Oocytes included to the quantitative assessment ……………………….. 64
5.3.2 Retardance magnitude and Pole-to-pole distance of the meiotic spindle
of human oocytes ……………………………………………………….. 65
5.3.3 Maternal age and spindle organization …………………………………. 68
5.3.4 Biochemical pregnancy in oocytes with lower or higher mean
retardance of the spindle ………………………………………………... 68
5.3.5 Mean retardance in relation to transfers with good or mediocre/low PN
score and pregnancy rate ……………………………………………….. 70
5.4 Quantitative analysis of the zona pellucida of human oocytes using
PolScope microscopy and developmental potential ……………………….. 72
5.4.1 Characteristic morphology of the human zona pellucida in oocytes of
the CC and NCC group …………………………………………………. 72
5.4.2 Quantitative analysis of retardance magnitude and thickness of the zona



Content III
layers in CC and NCC groups ………………………………………….. 75
5.4.3 Mean retardance as predictive parameter for conception ……………… 76
5.4.4 Relationship between thickness and ratardance ……………………….. 79
5.4.5 Influence of maternal age and differences in zona thickness and
retardance between transfer and non-transfer oocytes ………………….. 80
6. Discussion …………………………………………………………………………... 83
6.1 Analysis of spindle formation by PolScope in improvement of in vitro
maturation and selection of human oocytes ………………………………. 83
6.2 PolScope analysis of birefringent spindles in in vivo maturing oocytes ….. 85
6.2.1 No