Oocyte recovery, embryo development and ovarian function after cryopreservation and transplantation of whole sheep ovary

A. Arav1,*, A. Revel4,*, Y. Nathan3,5,*, A. Bor1, H. Gacitua1, S. Yavin1, Z. Gavish1, M. Uri3 and A. Elami2

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Successful cryopreservation of a whole ovary may provide a solution for women with premature ovarian failure. The aim of this study was to evaluate the function of cryopreserved whole sheep ovaries both in vitro and in vivo. METHODS: Transplantation of frozen–thawed intact ovaries was performed on eight sheep by artery and vein anastomosis to the contralateral ovarian artery and vein. The remaining ovary was removed. Oocyte aspiration was performed 1 and 4 months post-transplantation. Serum progesterone levels were measured after 24 and 36 months. Magnetic resonance imaging (MRI) was carried out 12 months after transplantation. RESULTS: Progesterone activity was detected in three sheep from 24 to 36 months post-transplantation. Oocyte retrieval was successful in two sheep and parthenogenic activation has resulted in embryonic development up to the 8-cell stage. MRI revealed an intact ovary with small follicles and intact blood vessels. CONCLUSIONS: Whole ovaries, and the follicles and blood vessels they contain, are able to survive cryopreservation. In addition, MRI has shown that blood vessels were intact and that normal blood flow had resumed to the transplant. We conclude that immediate and long-term hormonal restoration and normal ovulation is possible after cryopreservation and transplantation of whole ovaries in sheep.

Key words: autotransplantation/cryopreservation/ovary/ovulation/sheep


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cryopreservation of ovaries could help preserve fertility of women at risk of premature ovarian failure, such as young cancer patients (Boring, 1991Go). One of the options for preserving fertility offered to patients is cryopreservation of oocytes. The pregnancy rate using frozen–thawed human oocytes is <2% (Tucker et al., 1998Go). Moreover, in order to perform oocyte retrieval from patients, ovulation is induced, thus, chemotherapy is postponed putting the patient at risk (Chen, 1986Go; Porcu et al., 2000Go; Fabbri et al., 2001Go; Revel and Schenker, 2004Go). Cryopreservation of ovarian cortex tissue which is rich in primordial and primary follicles has been suggested as an alternative to ovulation induction and oocyte cryopreservation for preserving fertility (Donnez and Bassil, 1998Go; Donnez et al., 2000Go). Viable follicles survive after freeze–thawing of human ovaries (Martinez-Madrid et al., 2004Go) and ovarian tissue (Hovatta et al., 1996Go; Newton et al., 1996Go; Oktay et al., 1997Go). This has aroused interest in this procedure as a potential means of preserving the fecundity of patients at risk of premature ovarian failure (Donnez and Bassil, 1998Go; Newton, 1998Go; Donnez et al., 2004Go). In sheep, autotransplantation of frozen–thawed ovarian cortex (Gosden et al., 1994Go) and of hemi-ovaries (Salle et al., 2002Go) has resulted in pregnancies, deliveries and prolonged hormone production (Baird et al., 1999Go; Salle et al., 2003Go). Nevertheless, in all these cases there was a reduction in the total follicular number due to ischaemia, therefore ovarian function was transient (Liu et al., 2002Go). Only eight of 80 human oocytes aspirated from a cryopreserved transplanted ovary were suitable for IVF and only one oocyte fertilized normally (Oktay et al., 2004Go). The first human pregnancy by cryopreserved ovarian cortex and transplantation was recently reported (Donnez et al., 2004Go). It appears that the main obstacles to successful restoration of fertility from frozen–thawed ovarian cortex are adhesions and the massive ischaemic damage to follicles until neovascularization develops (Liu et al., 2002Go). Most follicles which survive cryopreservation undergo ischaemic loss during the time required for neovascularization (Nisolle et al., 2000Go). Thus, we and others sought to develop a method of vascular ovarian transplantation which would minimize ischaemic follicular loss (Wang et al., 2002Go). The rational behind this idea is that a vascular transplant would prevent ischaemic follicular loss and thus the functional lifespan of a vascular ovarian transplant would be considerably extended. Previously, we have demonstrated that hormonal activity was restored in sheep that were autotransplanted with frozen–thawed whole ovaries (Revel et al., 2004Go).

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cryopreservation apparatus
For this research a freezing apparatus based on the technology of directional freezing was used, named multi-thermal gradient (MTG), which enabled freezing at a very slow and accurate cooling rate and the control of ice crystal morphology (A.Arav, US Patent 5,873,254) (Revel et al., 2004Go). The cooling rate was set to 0.3°C/min by varying the speed (0.01 mm/s) at which the tube passes through the temperature gradient (0.514°C/mm).

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 frozen–thawed ovaries. After thawing, slices of ovarian cortex were incubated in 1 ml HEPES–Talp 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 freeze–thawing whole ovaries with that of fresh follicles was done on seven fresh and seven frozen–thawed 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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Figure 1. Photos of live/dead fluorescent stains using FDA/DAPI of a frozen thawed sheep ovary. FDA staining is green and we can see many primordial follicles. Black arrow is pointing on a small antral follicle which has survived. DAPI staining is shown in blue; the black circles that were not stained are where the primordial follicles are.

 

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 manufacturer’s 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 3–14 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., 2004Go). 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 HEPES–Talp 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 mare’s 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 HEPES–Talp (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)Go. 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., 2001Go). 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 (94–113 weeks) and 3 years (142–163 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., 2004Go). 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, 1985Go).

Magnetic resonance imaging (MRI). This was performed 24 months after transplantation on two sheep; one with a frozen–thawed 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In vitro studies
Follicular and vascular survival
Live/dead fluorescent stains showed no significant difference in follicular survival between fresh ovaries (99.7 ± 0.7%) and frozen–thawed ovaries (97.7 ± 3.1%) (Figure 1).

Histology
HE stains also performed on frozen–thawed ovaries revealed normal morphology of the frozen–thawed 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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Figure 2. Histological section of a frozen thawed ovary. Pictures A and C are of sections that were stained using anti-factor VIII antibodies and pictures B and D were stained using H&E.

 

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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Figure 3. Embryo development after parthenogenetic activation.

 

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 94–96 weeks post-transplantation; and (ii) 1.9, 1.6, 0.1, 0.3, 0.7 and 1.5 ng/ml when measured 142–146 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 111–113 weeks post-transplantation; and (ii) 0.8, 1.1, 1.0, 1.1, 1.0 and 1.1 ng/ml when measured 161–163 weeks post-transplantation.



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Figure 4. Progesterone levels measured in transplanted sheep approximately 2 and 3 years post transplantation. Other sheep had negligible progesterone levels and therefore are not presented.

 

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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Figure 5. MR images showing an intact frozen thawed transplanted ovary with small follicles (a) and intact blood vessels (b).

 


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human organ transplantations such as heart, kidney and liver are performed using only fresh grafts. Human whole ovary transplantation or freezing are not clinically performed. To test the feasibility of such a procedure we started with in vitro studies. Fluorescent and haematoxylin and eosin (H&E) stains performed on frozen–thawed ovaries revealed normal ovarian morphology including primordial follicle survival of 97.7%. These results are similar to the results achieved by Martinez-Madrid et al. (2004)Go on human frozen–thawed ovary. Blood vessels are shown to have normal morphology and production of factor VIII by the endothelial cells as demonstrated by H&e stains (Figure 2) and factor VIII immunohistochemistry (Figure 2).

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 frozen–thawed 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 freezing–thawing process (Zook et al., 1998Go). 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 frozen–thawed 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 7–15 months after transplantation (Revel et al., 2004Go). 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, 1985Go). The persistent corpus luteum might reflect impairment of the prostaglandin feedback due to adhesions caused by the surgery.

We conclude that transplantation of a frozen–thawed 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., 1994Go) and human (Weissman et al., 1999Go) frozen–thawed 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., 2002Go). 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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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on December 19, 2004; resubmitted on May 31, 2005; accepted on June 3, 2005.





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