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Original Basic Science—General

Safeguarding Fertility With Whole Ovary Cryopreservation and Microvascular Transplantation

Higher Follicular Survival With Vitrification Than With Slow Freezing in a Ewe Model

Torre, Antoine MD, PhD; Vertu-Ciolino, Delphine MD; Mazoyer, Claire MS; Selva, Jacqueline MD, PhD; Lornage, Jacqueline MD, PhD; Salle, Bruno MD, PhD

Author Information
doi: 10.1097/TP.0000000000001296

The past 30 years have witnessed a steady increase in the number of children and young adults with malignancies, together with impressive therapeutic advances that have considerably improved survival.1 A common side effect of cancer treatments in the growing population of young cancer survivors is permanent gonadal damage responsible for infertility.2 Fertility preservation has thus become a major treatment objective.

For nonpubertal girls and for adults who are ineligible for ovarian stimulation, ovarian tissue cryopreservation, although still considered experimental, is the only option.3 Either ovarian-cortex fragments or the whole ovary with its vascular pedicle can be cryopreserved. Two cryopreservation methods exist, slow freezing, and vitrification. In slow freezing, ice crystal formation is minimized by lowering the temperature slowly and using a small dose of cryoprotectant. Vitrification, a more recent method, seeks to avoid ice crystal formation by cooling the tissue rapidly and using high doses of cryoprotectants.4

Nonvascular grafting of both slow-frozen3,5 and vitrified6 ovarian cortex has permitted healthy births. However, ischemia of the fragments before the development of effective neovascularisation causes the loss of 60% of the follicles7,8 and shortens the graft lifespan.

Cryopreservation of the whole ovary with its vascular pedicle, followed by microvascular transplantation, has been suggested to minimize ischemia and improve fertility.9 With cryopreservation, both the function and the vascular supply of the ovary must be maintained. Animal studies are needed to determine how these objectives can be reached. Ewes have ovaries similar in size and fibrous consistency to human ovaries10 and are therefore used by most of the research groups working on ovary cryopreservation.

Although the implantation of autologous slow-frozen and thawed whole ewe ovaries has led to the delivery of healthy lambs,11,12 the success rate is low. Slow freezing adversely affects the physiology and permeability of the ovarian pedicle.13 Vitrification might be more effective.14 After autologous transplantation, vitrified and warmed rabbit kidneys durably regained their function and ensured survival.15 In vitro, vitrification preserves follicular morphology and viability.16-18 However, the autologous microvascular transplantation of vitrified whole ewe ovaries has failed to produce successful pregnancies.19 One possible explanation for this failure is that the vitrifying solution used was a previous generation vitrification solution (VS4), a first-generation product designed to achieve vitrification at 1000 atmospheres.20,21 Calorimetric studies established that vitrification was incomplete with VS4.16,19 The last-generation vitrifying solution VM3 can induce vitrification at atmospheric pressure and provides calorimetric conditions more likely to allow whole-organ cryopreservation.22

We hypothesized that whole-ovary vitrification using VM3 would be effective in preserving fertility. We assessed this hypothesis in the ewe model by comparing follicle survival and fertility outcomes after autologous microvascular transplantation of whole ovaries cryopreserved using either VM3 vitrification or slow freezing.


This study was approved by the ethics committee of the Veterinary School of Lyon (ENVL 0804). Animal experiments were carried out at the Institute Claude Bourgelat, Veterinary School of Lyon, France, in compliance with French government accreditation (A691270505), and with French and European legislation concerning animal research.


Twelve Ile-de-France pubertal ewes aged 6 months were obtained from a local breeder and housed as a group.

Animals were allocated at random in a 1:1 ratio to slow freezing or vitrification as the method of whole-ovary cryopreservation. The whole process is summarized in Figure 1. For technical reasons, ovaries scheduled for slow freezing had to be retrieved before those scheduled for vitrification. At liquid nitrogen temperature, any biological process is stopped, and the storage duration has no impact. We thus decided to graft vitrified ovaries first, accepting a planned lower duration of liquid nitrogen storage in this group, to ensure that ewes of both groups were grafted at the same age.

Short description for the experimental design for whole ovary cryopreservation and microvascular auto-transplantation. VM3 is a vitrificant solution of the last generation.

Surgical Collection of the Whole Left Ovary With Its Vascular Pedicle

Each ewe underwent a subumbilical midline laparotomy under general anaesthesia. Electrical scalpels were not used. The left ovarian pedicle was dissected by following the lumbo-ovarian vessels up to their junction with the aorta and inferior vena cava. Each secondary vessel was ligated as near as possible to the vascular pedicle, using nylon suture (Prolene 6-0, Ethicon, Issy Les Moulineaux, France), then divided. Care was taken to protect the vascular supply to the tube and ovary. The lumbo-ovarian artery and vein were then ligated and divided as near as possible to the aorta and inferior vena cava, respectively, and the ovary with its pedicle was harvested (Figure 2A).

Whole-ovary cryopreservation and micro-vascular transplantation in a ewe. A, Harvesting of the ovary with its pedicle. B, Exposure of the harvested specimen to the last-generation vitrification solution VM3 or to the slow-freezing cryoprotector dimethyl sulfoxide (2 M), by both immersion and perfusion. The specimen was then sealed in an ethylene-vinyl acetate cryobag. Note that during VM3 exposure, toxicity was minimised by lowering the temperature gradually. C, Vitrification (right) or slow freezing (left) of the whole ovary, followed by storing in liquid nitrogen. D, After warming/thawing, microvascular transplantation of the cryopreserved ovary to the contralateral ovarian pedicle after removal of the remaining ovary. E, After removal of the arterial clamp, venous filling was checked, and the venous clamp was then removed. F, Orthotopic transplantation of the ovary.

The ovarian artery was cannulated with a 24G catheter (BD Insyte, BD, Le Pont de Claix, France), as described elsewhere.19 The final preparation was composed only of the ovary and of its artery (with a catheter) and vein.

Cryopreservation Procedures

The ovaries were transported to the cryopreservation laboratory by car in X-Vivo survival medium (Lonza, Verviers, Belgium) at 10°C. Transport time was 30 minutes. Impregnation of the ovaries by the cryoprotective agents was obtained by both immersion and arterial perfusion at 0.35 mL/minute via multichannel peristaltic pumps (Watson Marlow 505 OF, Dreux, France) with silicone tubing.

Slow Freezing and Thawing of Whole Ovaries (n = 6)

The ovaries were perfused for 10 minutes at room temperature with BM1 (Eurobio, Courtaboeuf, France) containing 2 M dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Saint-Louis, MO) and 10% foetal calf serum (Eurobio).23 Each ovary was then sealed into an ethylene-vinyl acetate (EVA) cryobag (Macropharma, Mouvaux, France) containing 10 mL of BM1 with 2 M of DMSO (Figure 2B). Slow freezing was then carried out using a MiniCool device (Air Liquide Santé, Puteaux, France), allowing temperature control as follows24: from room temperature to −35°C at −2°C/minute with automatic seeding at −7°C then from −35°C to −150°C at −25°C/minute. This process lasted 33 minutes. The frozen ovary was stored in liquid nitrogen at −196°C (Figure 2C).

For thawing, the ovary in the EVA cryobag was placed in liquid nitrogen vapors for 1 minute to avoid thermal shock then warmed in a water bath at 45°C until thawing was complete. The ovary was then removed from the cryobag, and the cryoprotectors were rinsed out by both immersion and perfusion at room temperature, in three 5-minute steps, using 1.5 M DMSO and 300 mM mannitol (CDM Lavoisier, Paris, France) diluted in BM1 for the first step, 0.5 M DMSO and 100 mM mannitol diluted in BM1 for the second step, and BM1 (Eurobio) for the third step. Thawing lasted 17 minutes.

Vitrification and Warming of Whole Ovaries (n = 6)

Vitrification was achieved by immersion in and perfusion with the last-generation solution VM3 (21th Century Medicine, Fontana, CA). VM3 contains 8.4 M of cryoprotective agents consisting of DMSO (2.86 M), ethylene glycol (2.71 M), formamide (2.86 M), polyvinylpyrrolidone, and the antifreeze molecules Supercool X-1000 and Supercool Z-1000.14 VM3 should be used in gradually increasing concentrations, during a slow decrease in temperature, to minimize toxicity. We achieved the gradual concentration increase by diluting pure VM3 in its recommended diluent LM5 (21st Century Medicine). Figure 3 describes the setup and the VM3 concentration (C(t)) in the immersion and perfusion solution computed based on time (t), initial VM3 concentration (C(0)), and concentration (Cd) and flow (fd) of the diluent. Dilution was performed at constant volume (V), under high-flow mixing (fm). Air bubbles were carefully purged from each tube. Tubes outside the controlled-temperature chamber were thermally insulated. The MiniCool parameters needed to achieve the temperature decrease kinetics described above were determined in preliminary tests. The ovary with its immersion and perfusion system was placed in the MiniCool device. Over a period of 18 minutes, the temperature decreased from 10°C to −3°C and the VM3 concentration increased from 2 M to 5 M (Cd = 8.4 M, C(0) = 2 M, V = 10 mL, fd = 0.35 mL/min). After 10 minutes equilibration at −3°C with 5 M VM3 for immersion and perfusion, the ovary was immersed in and perfused with pure VM3 (8.4 M) at −3°C for 20 minutes.22 So the total VM3 impregnation time was 48 minutes. The whole ovary was placed in an EVA cryobag with 10 mL of pure VM3 (Figure 2B). The cryobag was sealed, taking care to eliminate all air bubbles, immersed and stored in liquid nitrogen (Figure 2C).

Vitrification procedure: setup used to gradually change the VM3 concentration (C(t)) while exposing or rinsing the ovary (A), and calculation of the VM3 concentration (B). The ovarian immersion-perfusion device was placed in the chamber of a programmable freezer. (C(t)) was dependent on time, initial VM3 concentration (C(0)), and Cd and fd. Note that dilution was conducted at constant volume (V), under mixing at high flow (fm).

Warming of the ovary involved 2 steps. The vitrified ovary in the EVA cryobag was allowed to warm slowly by natural convection in liquid nitrogen vapours, to −133°C, suggested to be the temperature of glass transition of vitrified ovaries. Ovarian temperature was evaluated using a test ovary containing a thermocouple and exposed to VM3. The ovary was then quickly warmed to 0°C by conduction and forced convection in a 45°C water bath, removed from the EVA cryobag and placed in its immersion and perfusion system in the MiniCool. Finally, the ovary was immersed in and perfused with 4.2 M VM3 and 0.3 M mannitol at −3°C for 10 minutes. Over the next 30 minutes, the VM3 and mannitol concentrations were decreased gradually to 1 M and 0 M, respectively, whereas the temperature was increased from −3°C to 20°C, by starting the dilution and evacuation flow. Cd was 0 M for VM3 and 0.1 M for mannitol; C(0) was 4.2 M for VM3 and 0.3 M for mannitol. Dilution volume (V) was 10 mL, and fd was 0.7 mL/min. The cryoprotector removal process lasted 40 minutes. The ovary was finally perfused with BM1 for 10 minutes before transplantation.

Microvascular Transplantation of the Cryopreserved Whole Ovary

Each ewe underwent a second subumbilical midline laparotomy under general anaesthesia. The autologous left ovary was transplanted on the right side 3 to 6 weeks after the ovary harvesting and cryopreservation procedure. Before transplantation, thawing/warming of the ovary was conducted in a synchronized manner to minimize the duration of warm ischemia. The transplantation site was prepared microsurgically by dissecting artery and vein as described for the harvesting procedure. An intravenous bolus of 5000 IU of heparin11,19,25 was given just before clamping the right lumbo-ovarian artery and vein and then repeated hourly. End-to-end transplantation of the left ovarian vessels onto the right ovarian pedicle was performed under a binocular magnification glass (4.0-340 mm; Heine, Herrsching, Germany) (Figure 2D). The donor and receiver blood vessels were approximated using a double vascular clamp (Teleflex, Faget, France) then anastomosed using separate nylon sutures (Ethilon 9-0, Ethicon). A microcannula (Moria, Antony, France) was used to instill the pedicle with 40 IU/mL of heparinized saline (Héparine Choay, Sanofi, Paris, France). After completion of the vascular anastomoses, the arterial clamp was removed, whereas the venous clamp was left in place. Ovarian color and venous filling were monitored (Figure 2E) to assess permeability of the arterial anastomosis while eliminating bias due to retrograde filling from the vein. Finally, the venous clamp was removed, and the ovary was fixed in orthotopic position (Figure 2F), using polyglactin suture (Vicryl 2-0, Ethicon).

The removed right ovary was fixed in formaldehyde for histological analysis.

Ischemia duration was defined as the time from blood vessel clamping for the harvesting procedure and the release of blood vessel clamping during the transplantation procedure, the storage time in liquid nitrogen being subtracted from the clamping-to-unclamping time.

Follow-Up of the Animals

The assessors were not blinded to the randomization group.

Postoperative Care

After each surgical procedure, 1 mg/kg of low molecular weight heparin was injected daily for 10 days (enoxaparin sodium; Sanofi-Aventis, Paris, France). Animals received antibiotic injections for 5 days (170 mg/10 kg of procain benzylpenicillinate and 250 mg/10 kg of dihydrostreptomycine, ie, 1.5 ml/10 kg per day of Penigectyl, Virbac, Carros, France). Nonsteroidal anti-inflammatory drug was injected for 5 days (1.4 mg/kg per day of carpofen; Rimadyl, Zoetis, Paris, France). The ewes were closely monitored concerning their scar, pain, appetite, and rectal temperature for 5 days.

Sexual Behavior

The ewes were kept together in a pasture, with a fertile ram. The ram wore a harness bearing an identifying chalk. Ewes that mated with the ram were marked on the rump.

Progesterone Assays

Blood was drawn twice a week, starting 2 months after the ovary transplant procedure, and used to perform progesterone assays (Vidas Progesterone; Biomérieux, Marcy l'Etoile, France), as described earlier.19 Serum progesterone concentrations greater than 1 ng/mL were taken to indicate ovulation.26 Durable progesterone elevation was presumed to indicate gestation.

Ewe Offspring

Lambs delivered by the study ewes underwent a careful physical examination. They were left in the flock. Their reproductive function was assessed.


Euthanasia and Gross Examination

A postmortem examination was performed as promptly as possible after accidental deaths. Surviving ewes were killed about 3 years after transplantation. A laparotomy was performed to retrieve the transplanted ovary with its blood vessels, which was fixed for histological assessment. Any anatomical modifications of the reproductive organs were recorded.

Histological Examination

The ovaries and their vessels were fixed for 1 day in Bouin solution and prepared in paraffin wax. Follicular count was performed as previously described19 and compared between slow-frozen and vitrified ovaries.


Data were analyzed using Stata version 11.0 (Stata Corp., College Station, TX). Means and 95% confidence intervals (95% CIs) were used as measures of central tendency and variability, respectively. Qualitative and quantitative variables were compared with Fisher exact test or Wilcoxon paired test, respectively. Values of P less than 0.05 were considered significant.


Immediate Outcomes of the Surgical Procedures

Harvesting, perfusion, and cryopreservation of the left ovary, as well as removal of the right ovary, were successful in all 12 ewes. Details are reported in Table 1. For technical reasons, these procedures were performed first in the ewes allocated to the slow-freezing group, which were therefore significantly younger at the time of the harvesting procedure compared with the ewes allocated to vitrification. To correct for this difference, transplantation was performed first in the vitrification group, which therefore had a significantly shorter time from harvesting to transplantation compared with the slow-freezing group.

Characteristics of the study animals (6 ewes in each group)

Despite the small ovarian vessel diameter of about 1 mm, microanastomosis of the cryopreserved left ovarian vessels to the right ovarian pedicle was successful in all 12 animals. After transplantation, blood flow was restored in all 6 ovaries in the slow-freezing group and in 4 of the 6 ovaries in the vitrification group. The 2 primary failures were probably ascribable to a haemorrhagic suffusion (kink) within the pedicle, at a distance from the vascular sutures, at clamp removal. No visible fracture of the ovarian pedicle occurred. The ovarian arteries filled with blood downstream of the anastomoses before the blood flow was interrupted. Venous filling did not occur. Table 2 reports data on outcomes.

Outcomes after transplantation of autologous whole ovaries cryopreserved by slow freezing or VM3 vitrification

Immediate Evaluation of the Cryopreservation Protocols

As previously observed with VS4,27 each ovary vitrified with VM3 had whitish patches at the cortical surface, suggesting local ice formation. No whitish patches were visible at the vascular pedicle surface. As also reported with VS4,27 warming phase crystallisation occurred invariably in the VM3-vitrified ovaries. No ovarian pedicle fractures were recorded.

Follow-Up Data

All 12 ewes survived the surgical procedures, but 1 died in each group, both from infection unrelated to the study. These deaths occurred 4 weeks after transplantation in the slow-freezing group (before any hormonal assessment) and 7 months posttransplantation in the vitrification group (before the ovarian stimulation). Thus, 11 ewes (5 in the slow-freezing group and 6 in the vitrification group) were available for the analysis of outcomes.

Sexual Behavior

Apart from the ewe which died prematurely, all ewes were marked by the ram's chalk, including those having received a vitrified ovary with immediate failure of blood flow reestablishment. So it is sure that each surviving ewe mated.

Serum Progesterone Concentrations

Hormone production resumed in all ewes of both groups (Figure 4).

Serum progesterone concentrations over time in ewes after whole-ovary cryopreservation and micro-vascular transplantation. Black arrows with “M” letter indicate when the ewe mated. A gestation occurred in ewe 80099.

In the slow-freezing group, a typical progesterone plateau (5 ng/mL) occurred in 3 ewes (80068, 80109, and 80136). Two-week-long progesterone elevations to 3 to 15 ng/mL occurred in 2 ewes (80327, 80099).

In the vitrification group, a typical progesterone plateau occurred in 3 ewes (80155, 80369, and 80203). Two-week-long progesterone elevations occurred in 3 ewes (80176, 80353, and 80010). Two of them (80010 and 80353) were the animals with primary failure of ovarian blood flow restoration.


In the slow-freezing group, 1 ewe (80099) delivered a twin gestation, 1 year 9 months and 12 days after transplantation. Both lambs, a male and a female, were healthy. The female grew and reproduced normally, whereas the male died of maternal neglect. No gestations were obtained in the vitrification group.

Data From the Transplanted Ovaries

The transplants from each of the 2 ewes that died accidentally were taken and fixed late (about 36 hours) after death due to difficulties in finding a surgeon. The transplantation site was not assessable in either ewe because of necrosis, and in 1 ewe, the transplant was not identified by histological examination. Therefore, the analysis included 10 transplantation sites (5 in each group) and 11 transplants (5 slow frozen and 6 vitrified).

Transplantation Sites

Of the 5 transplantation sites examined in the slow-freezing group, 2 exhibited adhesions and 1 hydrometrium. The ovaries appeared normal and contained follicles in all 5 animals, whereas only 3 had normal-appearing pedicles.

Of the 5 transplantation sites in the vitrification group, 3 had adhesions. In 2 animals, the ovaries had a normal appearance with follicles. The pedicles looked normal in 4 animals.

Histological Assessment

In native ovaries, 9605 (95% CI, 6450-12760) follicles of all kinds, including 6747 (95% CI, 4310-9184) primordial follicles were counted. Nine follicles of all kinds including 1 primordial follicle were counted in all 5 slow frozen ovaries. Eighty-five follicles of all kinds including 1 primordial follicle were counted in all 5 vitrified ovaries. The follicle counts were thus very low in both groups (Table S1, SDC, The distribution of follicle stages was similar in the 2 groups, except for a higher proportion of secondary follicles in the vitrification group. When we considered all viable follicles (excluding atretic follicles), we found that follicular survival was very low in both groups but was significantly higher after vitrification than after slow freezing (Table 2).


In this study of autologous transplantation of cryopreserved whole ovaries in ewes, 1 gestation occurred after cryopreservation by slow freezing versus none after vitrification. However, follicle survival was significantly better with vitrification, although very low with both methods. After transplantation, neither progesterone production nor sexual behavior differed between the 2 groups.

Our study has several limitations. We used a novel vitrification protocol involving continuous changes in temperatures and cryoprotector concentrations over time. The temperatures were measured directly but the cryoprotector concentrations were estimated by computation. Moreover, ovarian cryogenic parameters were assessed after VS4 exposure,16,19 but not after VM3 exposure. Consequently, we cannot be certain that our protocol resulted in complete ovarian vitrification. In addition, slow frozen ovaries were retrieved from animals that were 50 days younger, which could have optimized follicular survival in this group. However, this difference is negligible compared with the 12-year life expectancy of sheep and cannot call into question our conclusion that vitrification provides higher follicular survival.

Fertility preservation with whole ovary falls in the category of research, and no attempt has ever been made in woman. We and many others chose the ewe as a large animal model for studying fertility restoration with cryopreserved ovarian tissue. Indeed, ewe and women share similarities in body size, ovarian anatomy, and reproduction characteristics.10 However, the extrapolation of the present results to woman should be considered with caution.

There have been several reports of microvascular transplantation of autologous cryopreserved ewe ovaries,11,12,19,25,28-33 but very few of them were performed in orthotopic position,11,12,19,31-33 or provided information on subsequent fertility.11,12,19 Orthotopic ovarian transplantation as performed in our study requires considerable skill and experience.19 In some studies, technical difficulties required a change in the experimental design from orthotopic to heterotopic transplantation for rescue, at the time of surgery.25 Spontaneous fertility was recorded in 2 of these earlier studies.11,12 In 1 of them, 1 ovary was slow frozen from each of 8 ewes. Cryprotector perfusion was achieved under constant pressure (<40 mm Hg), contrasting with the constant flow used in most other reports. Antithrombotic therapy was not used. Progesterone production was documented in 3 ewes after 12 to 14 months, and 1 of these ewes delivered 527 days after transplantation. At study completion 19 months after transplantation, follicular survival was low (<7.6%), and 2 ewes had thrombosis of the pedicle with atrophy of the ovary.12 Our results with slow freezing were similar to those obtained in this earlier study. In the other study, both ovaries from each of 14 ewes were cryopreserved by slow freezing before autologous transplantation. Oestrogen and progesterone were given before transplantation to promote vasodilation, and an antiplatelet agent was added to the anticoagulant treatment. Ovarian hormonal function was restored in all 14 ewes, 9 (64%) got pregnant, and 4 (29%) delivered 7 normal lambs. At study completion 22 months after transplantation, follicular survival was 60%.11 These better follicular survival and fertility outcomes compared with ours may be ascribable in part to the more aggressive thrombosis prevention strategy and in part to the larger amount of transplanted ovarian tissue, that is, both ovaries in each animal instead of a single ovary in our study. However, bilateral oophorectomy is not currently used for fertility preservation. Fertility outcomes in ewes have been compared after autologous microvascular transplantation of fresh versus vitrified whole ovaries with VS4, using an analogous protocol to ours. Of the 5 ewes that received fresh ovaries, 2 had gestations, with a follicular survival of 0.5% and 24.6%. Neither gestation nor follicular survival occurred in the vitrification group.19 Thus, autologous microvascular ovarian transplantation per se had detrimental effects on the ovaries. Follicular survival was considerably higher in our vitrification group, suggesting better performance of VM3 compared with VS4. The 3 abovementioned studies11,12,19 suggest the following: both the cryopreservation procedure and the transplantation procedure contribute to the low fertility seen after whole-ovary cryopreservation; gestations can be obtained even in ewes with very small numbers of follicles; and fertility depends on the amount of ovarian tissue transplanted and on the thrombosis prophylaxis strategy used at transplantation.

Advances in transplantation protocols have led to substantial improvements in fertility outcomes.11 Few attempts have been made to improve ovarian harvesting and perfusion. Nevertheless, we previously showed heterogeneity in the in vitro perfusion of the ovarian parenchyma.34 The nonperfused zones are not sufficiently exposed to the cryoprotectors and undergo either destruction in slow-frozen ovaries or ice nucleation followed by warming-phase crystallization in vitrified ovaries.27 Therefore, the harvesting and perfusion steps are crucial, and their optimization would be expected to improve fertility after whole-ovary cryopreservation. In 1 study, the ewes received anticoagulant injections at the time of the harvesting procedure.25 However, after microvascular transplantation, the ovaries lost over 90% of their follicles. Combined antiplatelet and anticoagulant therapy at the time of harvesting deserves evaluation. Furthermore, the risk of bovine ovary perfusion failure has been shown to increase when the perfusion is administered at a high flow rate and more than 3 hours after harvesting.35 In ewes, the presence of nonperfused ovarian zones was more common when the harvesting operator was inexperienced.34 Therefore, ovarian harvesting should be performed by an experienced operator, perhaps with antiplatelet and anticoagulant treatment, and the ovary should be perfused immediately after harvesting.


Progress must be made at every stage (harvesting, cryopreservation, and transplantation) before whole-ovary cryopreservation can be considered as an option for safeguarding fertility in women. Whole-ovary vitrification was associated with better follicular survival and may therefore hold promise for improving fertility after autologous transplantation of cryopreserved ovaries.


English editing was supported by the nonprofit Association Maternité Médecine et biologie de la Reproduction de l’Hôpital de Poissy.

The authors thank Myriam Momier for helping to set up the vitrification protocol, as well as Odile Lepinasse and all the personnel at the pathology department of the Edouard Heriot hospital (Lyon) for their skilled technical assistance.


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