1 Institute of Animal Science, Agricultural Research Organization (ARO), the Volcani Center, P.O.B. 6, Bet Dagan 50250, 2 Department of Cardiovascular Surgery, Hadassah University Hospital, P.O.B. 12000, Jerusalem 91120, 3 IMT Ltd, 3 Hamazmera St., P.O.B. 2044, Nes Zyona 70400 and 4 Department of Obstetrics and Gynecology, Hadassah University Hospital, P.O.B. 12000, Jerusalem 91120, Israel * These three authors have contributed equally to this work.
5 To whom correspondence should be addressed. E-mail: nathan{at}cryo-imt.com
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Abstract |
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Key words: autotransplantation/cryopreservation/ovary/ovulation/sheep
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Introduction |
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The aim of the present study was to determine whether reanastomosis of cryopreserved whole ovaries to a blood supply could restore full ovarian function in a large species. A sheep model was selected for this research due to similarities to human ovaries such as dense fibrous stroma and relatively high primordial follicle density in the ovarian cortex.
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Materials and methods |
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In vitro studies
The aim of the in vitro studies was to determine the perfusion time of the freezing solution and the post-thaw recovery of the ovarian follicles and of the blood vessels.
Ovarian perfusion, cryopreservation and thawing
In vitro studies to determine perfusion time and follicular survival were performed on freshly collected sheep ovaries from the slaughter house. University of Wisconsin solution (UW) (Madison, WI, USA), supplemented with 10% (v/v) dimethylsulphoxide (DMSO) (Sigma, St Louis, USA), was selected for vascular perfusion andovaries were perfused for 1, 3 and 10 min duration with 10 ml of UW supplemented with 10% DMSO (v/v). We found that after 3 min DMSO reaches saturation in the ovarian cortex (unpublished data). Therefore, our perfusion time for the other in vitro and in vivo studies was 3 min.
Ovaries were inserted into a 16 mm diameter glass test tube (Manara, Israel) containing 10 ml freezing solution. Slow freezing was performed as follows: slow cooling to 6°C when seeding was performed. Directional freezing was then performed to the final temperature of 30°C at 0.01 mm/s, resulting in a cooling rate of 0.3°C/min, after which the tubes were plunged into liquid nitrogen. Thawing was performed 2 weeks to 2 months after cryopreservation, by plunging the test tube into a 68°C water bath for 20 s and then into a 37°C water bath for 2 min.
Follicular survival
Follicular survival evaluations were calculated by live/dead ratio following fluorescein diacetate (FDA) and 4,6-diamidino-2-phenylindoldihydrochloride) (DAPI) stains on whole frozenthawed ovaries. After thawing, slices of ovarian cortex were incubated in 1 ml HEPESTalp supplemented with 5 µl of FDA and DAPI stain solution (5 mg/1 ml DMSO) (Sigma, USA) for 5 min at room temperature. Scoring of live/dead follicular ratio was performed using a fluorescent microscope (Zeiss, Germany). Comparison of follicular survival after freezethawing whole ovaries with that of fresh follicles was done on seven fresh and seven frozenthawed sheep ovaries. At least 100 follicles were counted from each ovary (Figure 1). Statistical analysis was performed by t-test using the general linear model procedure of JMP (SAS Institute, 1994).
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Immunohistochemistry and histological evaluations
Ovarian tissue samples that were freshly collected from the slaughter house were frozen as described above, at a cooling rate of 0.3°C/min, and thawed. Ovaries were then fixed in 4% paraformaldehyde in PBS at 4°C. Serial 5 µm sections were prepared after the samples had been dehydrated in graded ethanol solutions, cleared in chloroform and embedded in Paraplast (Sigma, USA).
For immunohistochemistry, factor VIII-related antigen was detected using Polyclonal anti-human von Willebrand factor (vWf, factor VIII) (Zymed Laboratories, Israel) diluted in 1% normal goat serum in phosphate-buffered saline at 1:700 dilution and LSAB2 detection kit (Dako Corp., USA) according to the manufacturers instructions.
In vivo studies
The aim of the in vivo studies was to confirm the findings of the in vitro experiments and to find out if this model of freezing a whole ovary with its blood vessels is feasible in a large animal model.
Ovarian resection, perfusion, cryopreservation and thawing
Nine month old Assaf sheep were used for in vivo experiments (n = 8). This research was approved by the animal ethics committee. Under general anaesthesia, longitudinal low median laparotomy was performed. Dissection and isolation of the right ovarian vascular pedicle enabled disconnection of the ovary and pedicle at a point near the origin of the ovarian artery. The ovarian artery was perfused under a microscope with 10 ml of cold (4°C) UW supplemented with 10% DMSO for 3 min and then inserted into a freezing tube containing 10 ml of the same cryoprotectant. Slow freezing and thawing were performed as described above. Careful temperature measurements were taken to avoid heating the ovaries to >20°C during thawing.
Transplantation of intact ovary
Within 314 days of resection, sheep were prepared for ovarian autotransplantation to the contralateral ovarian vascular pedicle of the same sheep as previously described (Revel et al., 2004). In short, under laparotomy the contralateral ovary was resected and the ovarian artery and vein isolated and prepared for grafting. Cryoprotectants were rinsed out from the thawed ovary under the microscope (Zeiss) via the ovarian artery using 10 ml HEPESTalp medium supplemented with 0.5 mol/l sucrose and 10 IU/ml heparin (Sigma). Ovarian vascular transplantation was performed by reanastomosing the ovarian artery and vein with 10/0 interrupted sutures (Ethilon; Johnson & Johnson, USA). A surgical microscope (OP-Mi6; Zeiss) was used for magnification during end-to-end vascular anastomosis. Blood flow was verified by observing pulsation in the ovarian artery and venous return causing normal distention of the ovarian vein. In order to reduce adhesions, a gel containing hyaluronic acid (Intergel; Johnson & Johnson) was applied to the grafted ovary.
Ovarian function post-transplantation
Oocyte aspiration and parthenogenetic activation. Four weeks after autotransplantation, we administered 600 IU pregnant mares serum gonadotrophin (PMSG) (Intervet SA 49100 Boxmeer, The Netherlands), and performed explorative laparotomy the next day. Follicular aspiration from the grafted ovary was carried out using a syringe and a 20G needle. The aspirated follicular content was transported at 37°C in HEPESTalp (Sigma, USA) to the animal fertility laboratory and inspected for oocytes under the microscope. Aspirated oocytes were matured in vitro for 24 h using a method described by Zeron et al. (2001). Parthenogenetic activation was induced by Ionomycin and 6DMAP and oocytes were put in SOF medium in a 5% O2, 5% CO2 at 38.5°C incubator for another 48 h (Roth et al., 2001
). Oocyte aspiration was repeated 4 months after autotransplantation.
Hormonal measurements. In order to assess ovarian activity, we sampled bi-weekly progesterone levels for 3 weeks. Blood sampling was obtained 2 (94113 weeks) and 3 years (142163 weeks) after transplantation. Venous sheep blood was collected into lithium heparin-coated test tubes (Greiner Labortechnic, Austria), centrifuged and plasma was stored at 20°C until analysis. Progesterone was measured using a Coat-A-Count kit (Diagnostic Products Corp., USA) as previously reported (Revel et al., 2004). In brief, 200 µl of plasma was put in a test tube coated with progesterone antibodies. To the test tubes we added 1 ml of progesterone labelled with 125I, which was incubated overnight at room temperature and readings were obtained in a gamma counter (Kontron Gamma Counting System, Switzerland). The sensitivity of the kit is 0.1 ng/ml, and progesterone of >1 ng/ml is considered as evidence for a functional corpus luteum (Amir and Gacitua, 1985
).
Magnetic resonance imaging (MRI). This was performed 24 months after transplantation on two sheep; one with a frozenthawed transplanted ovary and the second on an untreated sheep as control. All MR images were performed with a 1.5-T system (Sigma LX; General Electric, USA), using a GP5 coil. Multiplanar, T2-weighted fast spin-echo (FSE) imaging was performed (axial, coronal and sagital planes). TE 98; TR 3020, EC 1/1, 15.6 kHz, field of view 18x18 cm, slice thickness 2.5/0 sp, matrix 256x192, NEX-2.
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Results |
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Histology
HE stains also performed on frozenthawed ovaries revealed normal morphology of the frozenthawed ovaries.
Immunohistochemistry
Immunohistochemistry of factor VIII showed that in ovaries that were frozen at a cooling rate of 0.3°C/min and thawed, endothelial cells produced factor VIII (Figure 2).
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In vivo studies
Oocyte aspiration
Laparotomy, performed 1 month following successful autotransplantation (n = 5), revealed severe adhesions in one sheep, mild adhesions in three sheep and no adhesions in one sheep. Follicular aspiration was possible, following adhesiolysis, in the sheep with mild adhesions (n = 4). This procedure was not possible in the sheep with severe adhesions (n = 1).
Two oocytes were retrieved from two sheep, one from each. Repeated oocyte aspiration 4 months after autotransplantation was successful in one sheep and four oocytes were retrieved (now or ever).
Parthenogenic activation resulted in normal development of all the six retrieved oocytes. Normal oocyte division and development (Figure 3) suggests that the retrieved oocytes were healthy.
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Hormonal activity
Progesterone levels measured at 24 and 36 months post-transplantation demonstrates that two sheep maintained their cyclicity during this time-period (Figure 4). Plasma progesterone levels measured in sheep number 1 were: (i) 0.3, 0.1, 1.2, 1.7, 2.1 and 1.4 ng/ml when measured 9496 weeks post-transplantation; and (ii) 1.9, 1.6, 0.1, 0.3, 0.7 and 1.5 ng/ml when measured 142146 weeks post-transplantation. Plasma progesterone levels of sheep number 8 were: (i) 0.9, 1.2, 1.2, 1.1, 1.1 and 1.3 ng/ml when measured 111113 weeks post-transplantation; and (ii) 0.8, 1.1, 1.0, 1.1, 1.0 and 1.1 ng/ml when measured 161163 weeks post-transplantation.
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MRI results
MRI revealed an intact transplanted ovary with small follicles. Diameter of the transplanted ovary was 15-16 mm as compared to 19-20 mm in the control sheep ovary. This variation between the ovaries is still within the normal size of ovaries in sheep that were never pregnant. In addition, ovarian blood vessels were found intact (Figure 5).
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Discussion |
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We conclude from these in vitro studies that freezing and thawing using the MTG freezing apparatus maintains ovarian architecture and blood vessel integrity. Following in vitro studies we performed ovarian transplantation studies.
The second stage of this project was vascular autotransplantation using frozenthawed whole ovaries. Intact sheep ovaries were perfused and frozen with the vascular stump intact. Following thawing of the ovary, cryoprotectants were flushed out by perfusing cold medium. All thawed ovaries remained intact, without any visible cracks. In this model we performed the autotransplantation by end-to-end anastomosis into either the original site or to the pedicle of the contralateral ovary, hoping to achieve natural pregnancy. Due to the depth of these sites in the sheep, this is far more challenging technically than transplantation to superficial blood vessels in the abdominal wall or the neck. Five of eight ovaries were successfully transplanted, as was confirmed by immediate resumption of blood flow. Failures could be technical in three cases (damage to blood vessels) or secondary to endothelial damage by the freezingthawing process (Zook et al., 1998). It could also be due to prolonged ischaemic time until successful completion of the anastomoses. However, in our more recent experience (unpublished data) we abandoned transplantation into the original site (because of the adhesions precluding natural conception) and have transplanted the frozenthawed ovaries to the neck vessels using end-to-side anastomosis with long-term patency and viability approaching 100%. MRI performed in one case showed a morphologically normal ovary with intact blood vessels. This would suggest that the blood supply that was restored through transplantation maintained ovarian morphology and vascular supply for up to 2 years (Figure 5).
We have been informed that there is ischaemic damage when processing ovarian cortex slices, which is done at room temperature, for the purpose of cryopreservation (Prof. Ronel, personal communication). Our method involves the immediate perfusion of the harvested ovary with cold UW solution, thereby minimizing the ischaemic damage caused before cryopreservation.
Fertility restoration was confirmed by follicular development. Since the number of oocytes retrieved waslow (one to four oocytes) and the success of IVF in sheep depends on the ram sperm quality, we decided to perform parthenogenic activation of the oocytes. Thus, the development of the embryo solely depends on the oocyte quality. Follicular growth enabled follicular aspiration and oocyte retrieval 1 month and again 4 months after transplantation and has resulted in normal development of parthenogenic embryos (Figure 3). However, adhesions that interfere with the aspiration process might prevent natural conception.
In our previous study we showed that three sheep were cyclic for a period of 715 months after transplantation (Revel et al., 2004). In the present study we demonstrate that 24 and 36 months after transplantation two of the three sheep are hormonally active; one is cyclic and the other has a persistent corpus luteum (Figure 4). Serum progesterone levels of >1 ng/ml that were maintained for
7 days indicate that there is an active corpus luteum (Amir and Gacitua, 1985
). The persistent corpus luteum might reflect impairment of the prostaglandin feedback due to adhesions caused by the surgery.
We conclude that transplantation of a frozenthawed ovary with its blood supply has allowed long-term fertility restoration. In the present study, ovarian activity was detected as early as 2 months after transplantation (as observed by oocyte aspiration and development) compared to what has been shown in previous studies, where a period of 3-4 months was necessary to enable follicular growth by transplantation of sheep (Gosden et al., 1994) and human (Weissman et al., 1999
) frozenthawed slices of ovarian cortex. This may be due to survival of some of the larger follicles such as small antral follicles which have allowed the immediate continuation of follicular growth.
Restoration of fertility by transplantation of intact ovary and reproductive tract in rats has been demonstrated (Wang et al., 2002). We now report long-term intact organ cryopreservation, with restored function following thawing and transplantation, in a large animal for
36 months post-transplantation.
This approach could revolutionize the field of cryopreservation for diverse human applications.
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References |
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Baird DT, Webb R, Campbell BK, Harkness LM and Gosden RG (1999) Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at 196 C. Endocrinology 140,462471.
Boring CC, Squires TS and Tong T (1991) Cancer statistics. CA Cancer J Clin 41,1936.
Chen C (1986) Pregnancy after human oocyte cryopreservation. Lancet 1,884886.[CrossRef][Medline]
Donnez J and Bassil S (1998) Indications for cryopreservation of ovarian tissue. Hum Reprod Update 4,248259.
Donnez J, Godin PA, Qu J and Nisolle M (2000) Gonadal cryopreservation in the young patient with gynecological malignancy. Curr Opin Obstet Gynecol 12,19.[CrossRef][ISI][Medline]
Donnez J, Dolmans M M, Demylle D, Jadoul P, Pirard C, Squifflet J, Martinez-Madrid B and Van Langendonckt A (2004) Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet.
Fabbri R, Porcu E, Marsella T, Rocchetta G, Venturoli S and Flamigni C (2001) Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod 16,411416.
Gosden RG, Baird DT, Wade JC and Webb R (1994) Restoration of fertility to oophorectomized sheep by ovarian autografts stored at 196 degrees C. Hum Reprod 9,597603.[Abstract]
Hovatta O, Silye R, Krausz T, Abir R, Margara R, Trew G, Lass A and Winston RM (1996) Cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediol-sucrose as cryoprotectants. Hum Reprod 11,12681272.[Abstract]
Liu J, Van der Elst J, Van den Broecke R and Dhont M (2002) Early massive follicle loss and apoptosis in heterotopically grafted newborn mouse ovaries. Hum Reprod 17,605611.
Martinez-Madrid B, Dolmans MM, Van Langendonckt A, Defrere S and Donnez J (2004) Freezethawing intact human ovary with its vascular pedicle with a passive cooling devise. Fertil Steril 82,13901394.[CrossRef][ISI][Medline]
Newton H, Aubard Y, Rutherford A, Sharma V and Gosden R (1996) Low temperature storage and grafting of human ovarian tissue. Hum Reprod 11,14871491.
Newton H (1998) The cryopreservation of ovarian tissue as a strategy for preserving the fertility of cancer patients. Hum Reprod Update 4,237247.
Nisolle M, Casanas-Roux F, Qu J, Motta P and Donnez J (2000) Histologic and ultrastructural evaluation of fresh and frozenthawed human ovarian xenografts in nude mice. Fertil Steril 74,122129.[CrossRef][ISI][Medline]
Oktay K, Nugent D, Newton H, Salha O, Chatterjee P and Gosden RG (1997) Isolation and characterization of primordial follicles from fresh and cryopreserved human ovarian tissue. Fertil Steril 67,481486.[CrossRef][ISI][Medline]
Oktay K, Buyuk E, Veeck L, Zaninovic N, Xu K, Takeuchi T, Opsahl M and Rosenwaks Z (2004) Embryo development after heterotopic transplantation of cryopreserved ovarian tissue. Lancet 363(9412),837840.
Porcu E, Fabbri R, Damiano G, Giunchi S, Fratto R, Ciotti PM, Venturoli S and Flamigni C (2000) Clinical experience and applications of oocytes cryopreservation. Mol Cell Endocrinol 169,3337.[CrossRef][ISI][Medline]
Revel A and Schenker J (2004) Ovarian tissue banking for cancer patients: is ovarian cortex cryopreservation presently justified? Hum Reprod 19,1419.
Revel A, Elami A, Bor A, Yavin S, Natan Y and Arav A (2004) Whole sheep ovary cryopreservation and transplantation. Fertil Steril, in press.
Roth Z, Arav A, Bor A, Zeron Y, Braw-Tal R and Wolfenson D (2001) Improvement of quality of oocytes collected in the autumn by enhanced removal of impaired follicles from previously heat-stressed cows. Reproduction 122,737744.
Salle B, Demirci B, Franck M, Rudigoz RC, Guerin JF and Lornage J (2002) Normal pregnancies and live births after autograft of frozenthawed hemi-ovaries into ewes. Fertil Steril 77,403408.[CrossRef][ISI][Medline]
Salle B, Demirci B, Franck M, Berthollet C and Lornage J (2003) Long-term follow-up of cryopreserved hemi-ovary autografts in ewes: pregnancies, births, and histologic assessment. Fertil Steril 80,172177.[ISI][Medline]
Tucker MJ, Morton PC, Wright G et al (1998) Clinical application of human egg cryopreservation. Hum Reprod 13,31563159.[Abstract]
Wang T, Banker MC, Clydon M, Hicks GL and Layne JR (1992) Freezing preservation of mammalian cardiac explant V. Cryoprotection by ethanol. Cryobiology 29,470477.[CrossRef][ISI][Medline]
Wang X, Chen H, Yin H, Kim SS, Lin Tan S and Gosden RG (2002) Fertility after intact ovary transplantation. Nature 415,385.
Weissman A, Gotlieb L, Colgan T, Jurisicova A, Greenblatt EM and Casper RF (1999) Preliminary experience with subcutaneous human ovarian cortex transplantation in the NOD-SCID mouse. Biol Reprod 60,14621467.
Zeron Y, Ocherteny A, Kedar O, Borochov A, Sklan D and Arav A (2001) Seasonal changes in bovine fertility: relation to developmental competence of oocytes, membrane properties and fatty acid composition of follicles. Reproduction 121,447454.
Zook N, Hussmann J, Brown R, Russell R, Kucan J, Roth A and Suchy H (1998) Microcirculatory studies of frostbite injury. Ann Plast Surg 40,246253; discussion 5455.
Submitted on December 19, 2004; resubmitted on May 31, 2005; accepted on June 3, 2005.
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