Zusammenfassung
Fragestellung: Um prädiktive Aussagen über einen Erfolg in der assistierten Reproduktion machen
zu können, ist man auf der Suche nach reliablen Tests, um die Qualität der verwendeten
Oozyten und Spermatozoen überprüfen zu können. Beispielsweise ist eine direkte Beurteilung
der Ooyzte neben einer groben morphologischen Abschätzung nicht möglich. Aufgrund
der engen Beziehung zwischen der Oozyte und den sie umgebenden Cumuluszellen könnte
eine nähere Untersuchung der Cumuluszellen als Prädiktor für die Eizellqualität geeignet
sein. In dieser Arbeit soll die apoptotische Aktivität der Cumuluszellen mit der Eizellqualität
und dem Erfolg der IVF-Behandlung verglichen werden.
Material und Methode: Mithilfe des Comet Assay wurde im Rahmen der Sterilitätsbehandlung mit intrazytoplasmatischer
Spermieninjektion (ICSI) an insgesamt 246 gewonnenen und vereinzelten Cumulus-Eizell-Komplexen
der Anteil apoptotischer Cumuluszellen jedes einzelnen Komplexes bestimmt und mit
der Qualität der jeweiligen Eizelle verglichen. Bei insgesamt 86 Patientinnen wurde
die Gesamtrate an apoptotischer Aktivität der Cumuluszellen mit dem Erfolg der IVF-Behandlung
verglichen.
Ergebnisse: Unsere Ergebnisse zeigen tendenziell, dass Oozyten aus Cumuli mit geringem Anteil
apoptotischer Zellen zu qualitativ besseren Embryonen heranreifen. Die detektierten
Unterschiede hinsichtlich des Schwangerschaftsoutcomes waren statistisch nicht signifikant.
Schlussfolgerungen: Die bisher erhaltenen tendenziellen Unterschiede stehen in Einklang mit anderen bisher
veröffentlichten Studien. Um statistisch gesicherte Parameter für die Vorhersage der
Entwicklungschance einzelner Eizellen zu bekommen, bedarf es allerdings noch einer
Erweiterung der Patientenzahl.
Abstract
Objective: To be able to predict the success of ART reliable tests for determining the quality
of the oocytes are necessary. Apart from a vague morphologic assessment via microscopy
a direct analysis of the oocyte quality is not possible. Because of the very close
relation between the oocyte and the cumulus cells the analysis of the cumulus cells
might give sufficient information on the oocyte quality. In this study we correlate
the apoptotic activity of cumulus cells to the outcome of fertilized oocytes after
Intracytoplasmic Sperm Injection (ICSI).
Material and Methods: 246 cumulus-oocyte-complexes from patients undergoing infertility treatment with
the ICSI procedure were individually collected. The comet assay was used to determine
the proportion of apoptotic cells within the cumulus population of each oocyte and
correlated with oocyte fertilization and oocyte quality as well as with pregnancy
outcome in 86 patients.
Results: We were able to show that high quality embryos correlate to a low rate of apoptotic
cells in their corresponding cumuli. Differences regarding the pregnancy outcome were
statistically not significant.
Conclusions: Our results on cumulus cell apoptosis and embryo quality confirm other publications.
To arrive at statistically proven criteria for the further development of single oocytes
an increase in the number of analyzed patients is necessary.
Schlüsselwörter
Comet Assay - ART - Cumuluszellen - Eizellqualität
Key words
Comet Assay - ART - Cumulus cells - Oocyte quality
Literatur
1
Al-Hasani S, Ludwig M, Palermo I, Kupker W, Sandmann J, Johannisson R, Fornara P,
Sturm R, Bals-Pratsch M, Bauer O, Diedrich K.
Intracytoplasmic injection of round and elongated spermatids from azoospermic patients:
results and review.
Hum Reprod.
1999;
14 (Suppl 1)
97-107
2
Billig H, Chun S Y, Eisenhauer K, Hsueh A J.
Gonadal cell apoptosis: hormone-regulated cell demise.
Hum Reprod Update.
1996;
2
103-117
3
Billig H, Futura I, Hsueh A JW.
Gonadotropin-releasing hormone directly induces apoptotic cell death in the rat ovary:
Biochemical and in situ detection of deoxyribonucleic acid fragmentation in granulosa
cells.
Endocrinology.
1994;
134
245-252
4 Bortz J, Lienert G, Boehnke K. Verteilungsfreie Methoden in der Biostatistik. 2nd
ed. Springer-Verlag, Heidelberg 2000
5
Diedrich K, Felberbaum R, Kupker W, al-Hasani S.
New approaches to male infertility: IVF and microinjection.
Int J Androl.
1995;
18 (Suppl 2)
78-80
6
Driancourt M A, Thuel B.
Control of oocyte growth and maturation by follicular cells and molecules present
in follicular fluid. A review.
Reprod Nutr Dev.
1998;
38
345-362
7
Fairbairn D W, Olive P L, O'Neill K L.
The comet assay: a comprehensive review.
Mutation Research.
1995;
339
37-59
8
Felberbaum R, Diedrich K.
Ovarian stimulation for in-vitro fertilization/intracytoplasmic sperm injection with
gonadotrophins and gonadotrophin-releasing hormone analogues: agonists and antagonists.
Hum Reprod.
1999;
14 (Suppl 1)
207-221
9
Host E, Mikkelsen A L, Lindenberg S, Smidt-Jensen S.
Apoptosis in human cumulus cells in relation to maturation stage and cleavage of the
corresponding oocyte.
Acta Obstet Gynecol Scand.
2000;
79
936-940
10
Jose de los Santos M, Anderson D J, Racowsky C, Hill J A.
Presence of Fas-Fas ligand system and bcl-2 gene products in cells and fluids from
gonadotropin-stimulated human ovaries.
Biol Reprod.
2000;
63
1811-1816
11
Kerr J F, Wyllie A H, Currie A R.
Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue
kinetics.
Br J Cancer.
1972;
26
239-257
12
Kupker W, Schlegel P N, Al-Hasani S, Fornara P, Johannisson R, Sandmann J, Schill T,
Bals-Pratsch M, Ludwig M, Diedrich K.
Use of frozen-thawed testicular sperm for intracytoplasmic sperm injection.
Fertil Steril.
2000;
73
453-458
13
Lee K S, Joo B S, Na Y J, Yoon M S, Choi O H, Kim W W.
Cumulus cells apoptosis as an indicator to predict the quality of oocytes and the
outcome of IVF-ET.
J Assist Reprod Genet.
2001;
18
490-498
14
Ludwig M, Schopper B, Katalinic A, Sturm R, Al-Hasani S, Diedrich K.
Experience with the elective transfer of two embryos under the conditions of the german
embryo protection law: results of a retrospective data analysis of 2 573 transfer
cycles.
Hum Reprod.
2000;
15
319-324
15
McKelvey-Martin V J, Green M HL, Schmezer P, Pool-Zobel B L, De Meo M P, Collins A.
The single cell gel electrophoresis assay (comet assay): A European review.
Mutation Research.
1993;
288
47-63
16
Nader S, Berkowitz A S, Maklad N, Wolf D P, Held B.
Characteristics of patients with and without gonadotropin surges during follicular
recruitment in an in vitro fertilzation/embryo transfer program.
Fertil Steril.
1986;
45
75-78
17
Olive P L, Banath J P, Durand R E.
Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells
measured using the comet assay. Radiat.
Res.
1990;
122
86-94
18
Ostling O, Johanson K J.
Microelectrophoretic study of radiation-induced DNA damages in individual mammalian
cells.
Biochem Biophys Res Commun.
1984;
123
291-298
19
Palermo G, Joris H, Devroey P, Van Steirteghem A C.
Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte.
Lancet.
1992;
340
17-18
20
Perez G I, Tilly J L.
Cumulus cells are required for the increased apoptotic potential in oocytes of aged
mice.
Hum Reprod.
1997;
12
2781-2783
21
Porter R N, Smith W, Craft I L, Abdulwahid N A, Jacobs H S.
Induction of ovulation for in vitro fertilization using buserelin and gonadotropins.
Lancet.
1984;
2
1284-1285
22
Raman R S, Chan P J, Corselli J U, Patton W C, Jacobson J D, Chan S R, King A.
Comet assay of cumulus cell DNA status and the relationship to oocyte fertilization
via intracytoplasmic sperm injection.
Hum Reprod.
2001;
16
831-835
23
Rattanachaiyanont M, Leader A, Leveille M C.
Lack of correlation between oocyte-corona-cumulus complex morphology and nuclear maturity
of oocytes collected in stimulated cycles for intracytoplasmic sperm injection.
Fertil Steril.
1999;
71
937-940
24
Rutherford A J, Subak-Sharpe R J, Dawson K J.
Improvement of in vitro fertilization after treatment with buserelin, an agonist of
luteinising hormone releasing hormone.
Br Med J.
1988;
296
1765-1768
25
Scott L F, Sundaram S G, Smith S.
The relevance and use of mouse embryo bioassays for quality control in an assisted
reproductive technology program.
Fertil Steril.
1993;
60
559-568
26
Singh N P, McCoy M T, Tice E L, Schneider E L.
A simple technique for quantification of low levels of DNA damage in individual cells.
Exp Cell Res.
1988;
175
184-191
27
Testart J, Belaisch-Allart J C, Forman R, Gazengel A, Strubb N, Hazput A, Strubb N,
Frydman R.
Influence of different stimulation treatment on oocyte characteristics and in vitro
fertilization ability.
Human Reprod.
1989;
4
22-29
28
Vinatier D, Dufour P H, Subtil D.
Apoptosis: A programmed cell death involved in ovarian and uterine physiology.
Eur J Obstet Gynecol Reprod Biol.
1996;
67
85-102
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