Die vaskularisierte Chorioallantoismembran (CAM): ein Kultursystem für kryokonserviertes menschliches Ovarialgewebe als Alternative zur Xenotransplantation

Zusammenfassung

Fragestellung: Bisher waren 2 Möglichkeiten zur Beurteilung der Effektivität von Kryokonservierungsprotokollen für Ovarialgewebe nach dem Auftauen bekannt: die Xenotransplantation und die In-vitro-Kultur in einem großen Mediumvolumen unter permanenter mechanischer Bewegung. Die belgische Arbeitsgruppe von J. Donnez hat gezeigt, dass ein Chorioallantoismembran-(CAM-)Kultursystem die Kultivierung von menschlichem Ovarialgewebe vor der Transplantation, in den ersten (ischämischen) Phasen vor der Neovaskularisation ermöglicht. Das Ziel dieser Studie war daher der Vergleich der Effektivität der In-vitro-Kultur von menschlichem Ovarialgewebe nach dem Auftauen in einem großen Mediumvolumen unter permanenter mechanischer Agitation mit dem CAM-Kultursystem. Material und Methodik: Ovarialgewebefragmente von 5 Patientinnen wurden innerhalb von 20 Minuten bei 32–34 °C in das Labor transportiert, die Fragmente in kleinere Stücke aufgeteilt (1–2 × 0,7–1 mm), eingefroren, aufgetaut und zufällig in die 2 folgenden Gruppen aufgeteilt: Gruppe 1: In-vitro-Gewebekultur für 7 Tage in einem großen Mediumvolumen unter mechanischer Agitation, Gruppe 2: Gewebekultur im CAM-System für 5 Tage. Die Vitalität des Gewebes wurde durch die Entwicklung der Follikel und die Proliferation von follikulären Zellen immunohistologisch (Zytokeratin und Ki-67) nach der Kultivierung bewertet. Ergebnisse: In den Gruppen 1 und 2 waren 85 bzw. 87 % der Follikel morphologisch normal. Die immunhistologische Analyse zeigte jedoch, dass die proliferativen Eigenschaften der Follikelzellen nach der Kultur im CAM-System deutlich höher waren. Schlussfolgerung: Somit ist anzunehmen, dass für die Kultur von kryokonserviertem humanem Ovarialgewebe das CAM-System besser geeignet ist als die In-vitro-Kultur in einem großen Mediumvolumen unter permanenter mechanischer Bewegung.

Abstract

Purpose: Previously there were only two effective ways to determine the quality of cryopreservation procedures for ovarian tissue after thawing: xenotransplantation and in vitro culture in a big volume of medium with permanent mechanical agitation. The Belgian group of J. Donnez has shown that the chorioallantoic membrane (CAM) culture system offers a new approach to study human ovarian tissue transplantation in its first ischemic stages, yielding information on the timing of tissue changes before neovascularization is established. The aim of this study was to compare the effectiveness after thawing of human ovarian tissue cultured in vitro in a big volume of medium with agitation with a CAM culture system. Material and Methods: Ovarian tissue fragments from 5 patients were transported within 20 min at 32–34 °C to the laboratory. The fragments were divided into smaller pieces (1–2 × 0.7–1 mm), frozen, thawed and randomly divided into the following two groups: Group 1: tissue cultured in vitro for 7 days in a big volume of medium with mechanical agitation; Group 2: tissue cultured in a CAM system for 5 days. The viability of the tissue from the respective method of cultivation was evaluated by immunohistochemistry (cytokeratin and Ki-67) and assessed according to the development of follicles and follicular cell proliferation. Results: 85 and 87 % of the follicles were morphologically normal in group 1 and group 2, respectively. Immunohistochemical analysis showed that the proliferative characteristics of follicular cells after culture in the CAM system were significantly increased. Conclusion: Both the CAM system and in vitro culturing in a big volume of medium with permanent mechanical agitation are suitable for culturing human ovarian tissue. However, the CAM system provides more information.

Schlüsselwörter

Ovarialgewebe - Kryokonservierung - CAM - Kultur - Follikel - Zytokeratin - Ki-67

Key words

tissue - cryopreservation - CAM - culture - follicles - cytokeratin - Ki-67

Literatur

• 1 Krebs in Deutschland 2005/2006. Häufigkeiten und Trends. Eine gemeinsame Veröffentlichung des Robert Koch-Instituts und der Gesellschaft der epidemiologischen Krebsregister in Deutschland e.V. 2010 120P
• 2 Altekruse S F, Kosary C L, Krapcho M Hrsg et al. Surveillance, Epidemiology, and End Results Cancer Statistics Review, 1975–2007. Bethesda, MD: National Cancer Institute; 2010
• 3 Dittrich R, Maltaris T, Hoffmann I et al. Fertility preservation in cancer patients.  Minerva Ginecol. 2010;  62 63-80
• 4 von Wolff M, Montag M, Dittrich R et al. Fertilitätsprotektion bei Frauen. Empfehlungen des Netzwerks FertiPROTEKT.  Geburtsh Frauenheilk. 2010;  70 85-100
• 5 Isachenko V, Isachenko E, Kreienberg R et al. Eine Kryobank für humanes Ovarialgewebe: Konzept und Perspektiven.  Frauenarzt. 2008;  49 518-521
• 6 Donnez J, Bassil S. Indications for cryopreservation of ovarian tissue.  Hum Reprod Update. 1998;  4 248-259
• 7 Oktay K, Newton H, Aubard Y et al. Cryopreservation of immature human oocytes and ovarian tissue: an emerging technology?.  Fertil Steril. 1998;  69 1-7
• 8 Oktay K, Karlikaya G. Ovarian function after transplantation of frozen, banked autologous ovarian tissue.  New Engl J Med. 2000;  342 1919
• 9 Meirow D, Nugent D. The effects of radiotherapy and chemotherapy on female reproduction.  Hum Reprod Update. 2001;  7 534-543
• 10 Meirow D, Baum M, Yaron R et al. Ovarian tissue cryopreservation in hematologic malignancy: ten years' experience.  Leuk Lymphoma. 2007;  48 1569-1576
• 11 Gosden R G. Prospects for oocyte banking and in vitro maturation.  J Natl Cancer Inst Monogr. 2005;  34 60-63
• 12 Schmidt K L, Andersen C Y, Loft A et al. Follow-up of ovarian function post-chemotherapy following ovarian cryopreservation and transplantation.  Hum Reprod. 2005;  20 3539-3546
• 13 Donnez J, Martinez-Madrid B, Jadoul P et al. Ovarian tissue cryopreservation and transplantation: a review.  Hum Reprod Update. 2006;  12 519-535
• 14 Donnez J, Dolmans M M, Demylle D et al. Restoration of ovarian function after orthotopic (intraovarian and periovarian) transplantation of cryopreserved ovarian tissue in a woman treated by bone marrow transplantation for sickle cell anaemia: case report.  Hum Reprod. 2006;  21 183-188
• 15 Silber S J, Gosden R. Ovarian transplantation in a series of monozygotic twins discordant for ovarian failure.  New Engl J Med. 2007;  356 1382-1384
• 16 Donnez J, Squifflet J, Pirard C et al. Live birth after allografting of ovarian cortex between genetically non-identical sisters.  Hum Reprod. 2011;  26 1384-1388
• 17 Revel A, Laufer N, Ben Meir A et al. Micro-organ ovarian transplantation enables pregnancy: a case report.  Hum Reprod. 2011;  26 1097-1103
• 18 Donnez J, Dolmans M M, Demylle D et al. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue.  Lancet. 2004;  364 1405-1410
• 19 Meirow D, Levron J, Eldar-Geva T et al. Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy.  New Engl J Med. 2005;  353 318-321
• 20 Demeestere I, Simon P, Emiliani S et al. Fertility preservation: successful transplantation of cryopreserved ovarian tissue in a young patient previously treated for Hodgkin's disease.  Oncologist. 2007;  12 1437-1442
• 21 Andersen C Y, Rosendahl M, Byskov A G et al. Two successful pregnancies following autotransplantation of frozen/thawed ovarian tissue.  Hum Reprod. 2008;  23 2266-2272
• 22 Silber S J, DeRosa M, Pineda J et al. A series of monozygotic twins discordant for ovarian failure: ovary transplantation (cortical versus microvascular) and cryopreservation.  Hum Reprod. 2008;  23 1531-1537
• 23 Sánchez-Serrano M, Crespo J, Mirabet V et al. Twins born after transplantation of ovarian cortical tissue and oocyte vitrification.  Fertil Steril. 2010;  93 268.e11-268.e13
• 24 Ernst E, Bergholdt S, Jørgensen J S et al. The first woman to give birth to two children following transplantation of frozen/thawed ovarian tissue.  Hum Reprod. 2010;  25 1280-1281
• 25 Rosendahl M, Schmidt K T, Ernst E et al. Cryopreservation of ovarian tissue for a decade in Denmark: a view of the technique.  Reprod Biomed Online. 2011;  22 162-171
• 26 Donnez J, Squifflet J, Jadoul P et al. Pregnancy and live birth after autotransplantation of frozen-thawed ovarian tissue in a patient with metastatic disease undergoing chemotherapy and hematopoietic stem cell transplantation.  Fertil Steril. 2011;  95 1787.e1-1787.e4
• 27 Schmidt K T, Rosendahl M, Ernst E et al. Autotransplantation of cryopreserved ovarian tissue in 12 women with chemotherapy-induced premature ovarian failure: the Danish experience.  Fertil Steril. 2011;  95 695-701
• 28 Oktay K, Turkcuoglu I, Rodriguez-Wallberg K A. Four spontaneous pregnancies and three live births following subcutaneous transplantation of frozen banked ovarian tissue: what is the explanation?.  Fertil Steril. 2011;  95 804.e7-804.e10
• 29 Roux C, Amiot C, Agnani G et al. Live birth after ovarian tissue autograft in a patient with sickle cell disease treated by allogeneic bone marrow transplantation.  Fertil Steril. 2010;  93 2413.e15-19
• 30 Imthurn B, Cox S L, Jenkin G et al. Gonadotrophin administration can benefit ovarian tissue grafted to the body wall: implications for human ovarian grafting.  Mol Cell Endocrinol. 2000;  163 141-146
• 31 Radford J. Restoration of fertility after treatment for cancer.  Horm Res. 2003;  59 (Suppl.) 21-23
• 32 Oktay K, Newton H, Mullan J et al. Development of human primordial follicles to antral stages in SCID/hpg mice stimulated with follicle stimulating hormone.  Hum Reprod. 1998;  13 1133-1138
• 33 Newton H, Aubard Y, Rutherford A et al. Low temperature storage and grafting of human ovarian tissue.  Hum Reprod. 1996;  11 1487-1491
• 34 Gook D, Edgar D, Borg J et al. Oocyte maturation, follicle rupture and luteinization in human cryopreserved ovarian tissue following xenografting.  Hum Reprod. 2003;  18 1772-1781
• 35 Rahimi G, Isachenko E, Isachenko V et al. Comparison of necrosis in human ovarian tissue after conventional slow freezing or vitrification and transplantation in ovariectomized SCID mice.  Reprod Biomed Online. 2004;  9 187-193
• 36 Maltaris T, Kaya H, Hoffmann I et al. Comparison of xenografting in SCID mice and LIVE/DEAD assay as a predictor of the developmental potential of cryopreserved ovarian tissue.  In Vivo. 2006;  20 11-16
• 37 Rahimi G, Isachenko V, Todorov P et al. Apoptosis in human ovarian tissue after conventional freezing or vitrification and xenotransplantation.  CryoLetters. 2009;  30 300-309
• 38 Rahimi G, Isachenko V, Kreienberg R et al. Re-vascularisation in human ovarian tissue after conventional freezing or vitrification and xenotransplantation.  Eur J Obstet Gyn R B. 2010;  149 63-67
• 39 Lotz L, Montag M, van der Ven H et al. Xenotransplantation of cryopreserved ovarian tissue from patients with ovarian tumors into SCID mice – no evidence of malignant cell contamination.  Fertil Steril. 2011;  95 2612-2614.e1
• 40 Isachenko V, Montag M, Isachenko E et al. Effective method for in-vitro culture of cryopreserved human ovarian tissue.  Reprod Biomed Online. 2006;  13 228-234
• 41 Isachenko V, Isachenko E, Reinsberg J et al. Cryopreservation of human ovarian tissue: comparison of rapid and conventional freezing.  Cryobiology. 2007;  55 261-268
• 42 Isachenko V, Isachenko E, Reinsberg J et al. Cryopreservation of human ovarian tissue: effect of spontaneous and initiated ice formation.  Reprod Biomed Online. 2008;  16 336-345
• 43 Isachenko V, Isachenko E, Reinsberg J et al. Simplified technique of human ovarian tissue freezing: quick cooling from − 36 degree C.  CryoLetters. 2008;  29 261-268
• 44 Isachenko E, Isachenko V, Nawroth F et al. Human ovarian tissue preservation: is vitrification acceptable method for assisted reproduction?.  CryoLetters. 2008;  29 301-314
• 45 Isachenko V, Lapidus I, Isachenko E et al. Vitrification and conventional freezing of human ovarian tissue: morphological, endocrinological and molecular biological evaluation.  Reproduction. 2009;  138 319-327
• 46 Isachenko E, Isachenko V, Nawroth F et al. Effect of long-term exposure at suprazero temperatures on activity and viability of human ovarian cortex.  Fertil Steril. 2009;  91 1556-1559
• 47 Isachenko V, Isachenko E, Weiss J M et al. Cryobanking of human ovarian tissue for anti-cancer treatment: comparison of vitrification and conventional freezing.  CryoLetters. 2009;  30 449-454
• 48 Isachenko V, Isachenko E, Nawroth F et al. Genexpression und Morphologie der Follikel nach konventionellem Einfrieren und Vitrifikation von humanem Ovarialgewebe.  Geburtsh Frauenheilk. 2010;  70 561-567
• 49 Isachenko E, Isachenko V, Weiss J M et al. Cryopreservation of human Oocytes and Embryos either by direct Plunging into liquid Nitrogen or by using an aseptic Approach. In: Chian R-C, Quinn P, Hrsg. Fertility Cryopreservation. New York, USA: Cambridge University Press; 2010: 157-168
• 50 Isachenko V, Gagsteiger F, Isachenko E et al. Concept of human Ovarian Tissue Cryobanking. In: Chian R-C, Quinn P, Hrsg. Fertility Cryopreservation. New York, USA: Cambridge University Press; 2010: 213-217
• 51 Martinez-Madrid B, Donnez J, Van Eyck A S et al. Chick embryo chorioallantoic membrane (CAM) model: a useful tool to study short-term transplantation of cryopreserved human ovarian tissue.  Fertil Steril. 2009;  91 285-292
• 52 Auerbach R, Kubai L, Knighton D et al. A simple procedure for the long-term cultivation of chick embryos.  Devel Biol. 1974;  41 391-394
• 53 Kunzi-Rapp K, Genze F, Küfer R et al. Chorioallantoic membrane assay: vascularized 3-dimensional cell culture system for human prostate cancer cells as an animal substitute model.  J Urol. 2001;  166 1502-1507
• 54 Cushman R A, Wahl C M, Fortune J E. Bovine ovarian cortical pieces grafted to chick embryonic membranes: a model for studies on the activation of primordial follicles.  Hum Reprod. 2002;  17 48-54
• 55 Gigli I, Cushman R A, Wahl C M et al. Evidence of a role for antimullerian hormone in the suppression of follicle activation in mouse ovaries and bovine ovarian cortex grafted beneath the chick chorioallantoic membrane.  Mol Reprod Dev. 2005;  71 480-488
• 56 Berube M, Deschambeault A, Boucher M et al. MMP-2 expression in uveal melanoma: differential activation status dictated by the cellular environment.  Mol Vis. 2005;  11 1101-1111
• 57 Nap A W, Dunselman G A, de Goeij A F et al. Inhibiting MMP activity prevents the development of endometriosis in the chicken chorioallantoic membrane model.  Hum Reprod. 2004;  19 2180-2187
• 58 Maas J W, Groothuis P G, Dunselman G A et al. Endometrial angiogenesis throughout the human menstrual cycle.  Hum Reprod. 2001;  16 1557-1561
• 59 Kunzi-Rapp K, Rück A, Kaufmann R. Characterization of the chick chorioallantoic membrane model as a short-term in vivo system for human skin.  Arch Dermatol Res. 1999;  291 290-295
• 60 Katoh M, Nakada K, Miyazaki J. Liver regeneration on chicken chorioallantoic membrane.  Cells Tissues Organs. 2001;  169 125-133
• 61 Nakada K, Yao Y, Mashima J et al. Skeletal muscle regeneration induced by chorio-allantoic grafting.  J Muscle Res Cell Motil. 1998;  19 169-177
• 62 Leng T, Miller J M, Bilbao K V et al. The chick chorioallantoic membrane as a model tissue for surgical retinal research and simulation.  Retina. 2004;  24 427-434
• 63 Valdes T I, Kreutzer D, Moussy F. The chick chorioallantoic membrane as a novel in vivo model for the testing of biomaterials.  J Biomed Mater Res. 2002;  62 273-282
• 64 Borges J, Tegtmeier F T, Torio-Padron N et al. Angiogenesis investigations in tissue engineering. The cylinder model on the chorioallantoic membrane.  Chirurg. 2004;  75 284-290
• 65 Ribatti D, Vacca A, Roncali L et al. The chick embryo chorioallantoic membrane as a model for in vivo research on antiangiogenesis.  Curr Pharm Biotechnol. 2000;  1 73-82