Human Reproduction Vol.20, No.12 pp. 3554–3559, 2005
doi:10.1093/humrep/dei278
Advance Access publication September 20, 2005.
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, 2Department of
Cardiovascular Surgery, Hadassah University Hospital, P.O.B. 12000, Jerusalem 91120, 3IMT Ltd, 3 Hamazmera St., P.O.B. 2044, Nes
Zyona 70400 and 4Department of Obstetrics and Gynecology, Hadassah University Hospital, P.O.B. 12000, Jerusalem 91120, Israel
5
To whom correspondence should be addressed. E-mail: [email protected]
*These three authors have contributed equally to this work.
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
Cryopreservation of ovaries could help preserve fertility of
women at risk of premature ovarian failure, such as young
cancer patients (Boring, 1991). 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., 1998). Moreover, in order to perform oocyte
retrieval from patients, ovulation is induced, thus, chemotherapy
is postponed putting the patient at risk (Chen, 1986; Porcu
et al., 2000; Fabbri et al., 2001; Revel and Schenker, 2004).
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, 1998; Donnez et al.,
2000). Viable follicles survive after freeze–thawing of human
ovaries (Martinez-Madrid et al., 2004) and ovarian tissue
(Hovatta et al., 1996; Newton et al., 1996; Oktay et al., 1997).
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, 1998; Newton, 1998;
Donnez et al., 2004). In sheep, autotransplantation of frozen–
thawed ovarian cortex (Gosden et al., 1994) and of hemi-ovaries
(Salle et al., 2002) has resulted in pregnancies, deliveries and
prolonged hormone production (Baird et al., 1999; Salle et al.,
2003). 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., 2002). 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., 2004). The first human pregnancy by
cryopreserved ovarian cortex and transplantation was recently
reported (Donnez et al., 2004). 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., 2002).
Most follicles which survive cryopreservation undergo ischaemic
loss during the time required for neovascularization (Nisolle
et al., 2000). Thus, we and others sought to develop a method
of vascular ovarian transplantation which would minimize
ischaemic follicular loss (Wang et al., 2002). The rational
behind this idea is that a vascular transplant would prevent
ischaemic follicular loss and thus the functional lifespan of a
3554 © The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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Transplantation of cryopreserved whole sheep ovary
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., 2004).
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
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., 2004). 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.
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.
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).
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., 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 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
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A.Arav et al.
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). 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 (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., 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 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 18×18 cm, slice
thickness –2.5/0 sp, matrix 256×192, NEX-2.
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.
Results
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).
3556
Figure 3. Embryo development after parthenogenetic activation.
Transplantation of cryopreserved whole sheep ovary
Progesterone (ng/ml)
Progeterone levels of transplanted sheep
2.5
2
1.5
1
0.5
0
94
Sheep no. 1
95
Sheep no. 5
96
96
97
98
111
112
113
Weeks after transplantation
Sheep no. 8
Progesterone (ng/ml)
Progesterone levels of transplanted sheep
2
1.5
1
0.5
0
142 143
144
144 145
146
161 162
163
Weeks after transplantation
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.
Hormonal activity
Progesterone levels measured at 24 and 36 months posttransplantation 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 posttransplantation; 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 posttransplantation; and (ii) 0.8, 1.1, 1.0, 1.1, 1.0 and 1.1 ng/ml
when measured 161–163 weeks post-transplantation.
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).
Discussion
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)
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).
Figure 5. MR images showing an intact frozen thawed transplanted
ovary with small follicles (a) and intact blood vessels (b).
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
3557
A.Arav et al.
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., 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 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., 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 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., 1994) and human (Weissman et al.,
1999) 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.,
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.
3558
This approach could revolutionize the field of cryopreservation
for diverse human applications.
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Submitted on December 19, 2004: revised on May 31, 2005: accepted on
June 3, 2005
3559