Cryobiology 66 (2013) 233–238

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Cryob iology

journal homepage: www.elsevier .com/locate /ycryo

Viability of human ovarian tissue confirmed 5 years after freezing with spontaneous ice-formation by autografting and chorio-allantoic membrane culture q

Vladimir Isachenko ⇑, Ingrid Orth, Evgenia Isachenko, Peter Mallmann, Doris Peters, Torsten Schmidt,Bernd Morgenstern, Dolores Foth, Bettina Hanstein, Gohar Rahimi University Maternal Hospital, Department of Obstetrics and Gynaecology, Cologne University, Kerpener Str. 34, 50931 Cologne, Germany

a r t i c l e i n f o a b s t r a c t

Article history:Received 29 November 2012 Accepted 11 February 2013 Available online 20 February 2013

Keywords:HumanOvarian tissue FreezingIceTransplantationFolliclesAngiogenesis

0011-2240/$ - see front matter � 2013 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.cryobiol.2013.02.003

q No special funding.⇑ Corresponding author. Fax: +49 221 478 8620.

E-mail address: v.isachenko@yahoo.com (V. Isache

To achieve optimal and uniform outcomes, slow cooling protocols for human ovarian tissues general lyinitiate ice formation at high sub-zero temperatures (�6 to �9 �C). The aim of the study was to investi- gate the function of ovarian tissue that had unintentiona lly self seeded at �20 �C during the freezing step,by examining its development follow ing chicken embryonic chorioallantoic membrane (CAM) grafting and after transplantation back to the patient. Ovarian tissue was frozen in 6% (v/v) dimethyl sulfoxide,6% (v/v) ethylene glycol and 0.15 M sucrose which had self-seeded at �20 �C. Five years after cryopres- ervation, 8 pieces were thawed and transplanted back to the patient. Two small (1 � 2 � 1 mm) pieces ofthis thawed tissue were cultured in a CAM-system for 5 days to assess the tissue viabili ty. The autograft- ed ovarian tissue re-established spontaneous menstrual bleeding within five months and raised serum 17-b Estradiol from 19 to 330 pg/ml. Ultrasound revealed a dominant follicle at the site of the trans- planted tissue in the follicular phase after the menstrual bleed. Analysis of the CAM cultured tissue estab- lished that 88% of the primordial follicles are degenerated and there was limited in growth of blood vessels. In conclusion, in spite of the damage caused by the cryopreservation with spon taneous ice-for- mation the viability could be confirmed by CAM culture and the restora tion of ovarian function after auto-transplantation.

� 2013 Elsevier Inc. All rights reserved.

Introductio n

Cancer is a major public health problem in the world. Cur- rently, in the United States alone one in three women will devel- op cancer in her lifetime [38]. Cancer is also one of the major death causes: in the USA alone a total of 1,638,910 new cancer cases and 577,190 deaths from cancer are projected to occur in2012 [39].

Childhood cancers (from birth to age 14 years) are rare, repre- senting less than 1% of all new cancer diagnoses, but they are the second leading cause of death in children, exceeded only by acci- dents. It is estimated that there are 58,510 survivors of childhood cancer living in the United States, and an additional 12,060 chil- dren will be diagnosed in 2012 [38].

However, due to the increasing of effectiveness of anti-cancer therapy, in USA alone the overall 5-year relative survival rate for childhood cancer has improved markedly over the past three dec-

ll rights reserved.

nko).

ades, from 58.1% for cases diagnose d from 1975 to 1977 to 82.5%for diagnoses during 2001–2007, due to new and improved treat- ments [38].

Cancers in patients of reproductive ages are treated with a com- bination of therapies (surgery, radiation, and chemothera py) cho- sen based on the type and stage of cancer [38]. The problem ofpost-can cer infertility is playing an increasingly significant role be- cause chemothera py can be gonadoto xic and lead to the functional death of ovaries [28,29,43 ,44] .

Cryopres ervation of ovarian tissue before cancer therapy with re-implanta tion after convalescence is one potential key solution of this problem [5,9,43,4 4].

Several cases of restored ovarian function after implantati on ofcryopres erved ovarian cortex in patients with premature ovarian failure after cancer treatment have been published since 1998 and now live birth after thawing and transplantation is reality [1,4,6–8,10,11,28–31,33–37,40].

Investigations show that for optimal cryopres ervation of human ovarian tissue, slow cooling protocols should include a step of ini- tiated ice formation (manual or automate d seeding) and not rely on spontaneou s (‘‘self’’) seeding [12].

234 V. Isachenko et al. / Cryobiology 66 (2013) 233–238

Human ovarian tissue can be cultured on the embryon ic chorio- allantoic membran e (CAM) of hen’s eggs [13,27].

The CAM system is an intermediate stage between in vitro cul- ture and animal experiments , and it could be considered as aninterface between in vitro and in vivo models (including xeno- transplanta tion). Importantly, it does not raise ethical or legal questions, nor does it violate animal protection laws.

The aim of the study was to investigate the health of thawed ovarian tissue that was frozen with spontaneou s ice formatio n byexamining its developmen t following CAM grafting and after trans- plantation back to the patient.

Materials and methods

The protocol of investigatio ns was approved by the Ethics Board of Cologne University.

Except where otherwise stated, all chemicals were obtained from Sigma (Sigma Chemical Co., St. Louis, MO, USA).

Tissue collection and dissection

Patient S. was born in 1983, develope d a Ewing-Sar coma and obtained low dose chemothera py in 1996 (aged 13). In 2007 (aged24) this Patient S. was scheduled to receive a high-dose chemo- therapy because lung-metast ases were diagnosed.

In January 2007, before starting of chemotherapy, this Patient S.underwent laparoscopi c removal and cryopreserv ation of ovarian tissue in the Cologne University Maternal Hospital.

Approximatel y 25% of the ovarian tissue from both ovaries was removed and cryopreserv ed.

Informed written consent for performing investigatio ns was ob- tained before collection of ovaries. According to this protocol 10%of ovarian tissue was used for patient-oriente d research. For trans- portation of ovarian tissue from surgical section, preparation ofovarian strips, freezing and thawing one medium, referred to be- low as ‘basal medium’, was used: Leibovitz L-15 with 5% Serum Substitute Supplement (Irvine Scientific, Santa Ana, CA, USA).

Following the surgical operation (04.01.2007) the fresh ovarian tissue fragments were transported to the laboratory within 20 min with the temperature maintain ed at 32–34 �C. Using tweezers and scalpel no. 22, ovarian fragments were dissected and divided into 20 pieces derived from both ovaries (4–5 � 2–3 � 0.8–1 mm)(length �width � depth) and frozen as described below.

Freezing and thawing

Cooling regime of the protocol for cryopres ervation of ovarian tissue was based on an embryo cryopreservati on protocol [42].

The 20 pieces were each equilibrated in one step in 20 ml offreezing basal medium supplemented with 6% (v/v) dimethyl sulf- oxide, 6% (v/v) ethylene glycol and 0.15 M sucrose for 20 min. atroom temperat ure. After equilibration the pieces were transferred to ten 2 ml cryovials (Nunc, Roskilde, Denmark) (2 pieces in each vial) filled with 1.8 ml of fresh cryopreservati on medium at room temperature and then placed in an IceCube 14S freezer (SyLab,Neupurkers dorf, Austria), with the freezing chamber previously stabilized at �6 �C for 10 min.

The cryopreserv ation programme was as follows: (i) the start- ing temperature was �6 �C; (ii) samples were cooled from �6 �Cto �34 �C at a rate of �0.3 �C/min; (iii) at �34 �C cryo-vials were plunged into liquid nitrogen. The freezing protocol for cryopreser- vation of this ovarian tissue included an autoseeding step at �6 �C.However, the agitation to trigger the ice formation was not per- formed correctly and the cooling ramp commenced without ice formation. One temperature probe monitored the chamber tem-

perature and a second probe was inserted in a dummy cryo-vial without ovarian tissue (Fig. 2).

The procedure of thawing was achieved by holding the cryo- vials for 30 s at room temperature followed by immersion in a100 �C (boiling) water bath for 65 s.

The exposure time of the cryo-vials to the boiling water was visually controlle d by monitoring the amount of ice left in the vial.As soon as the ice at the center of the vial had shrunk to 1–2 mm,the cryo-vial was removed from the boiling water, at which point the temperature of the medium was between +4 and +10 �C. With- in 5–10 s after thawing, the all pieces from the cryo-vial s were ex- pelled into 10 ml thawing solution (‘‘basal medium’’contai ning 0.5 M sucrose) in one single 100 ml specimen container (Sarstedt,Nuembre cht, Germany). The container was placed on a shaker and continuously agitated with 200 osc/min for 15 min at room temperat ure.

The procedure of stepwise dilution of cryoprotect ants was rea- lised by drop-wise addition of 1.6 ml/min basal medium for a fur- ther 30 min at room temperature (see Fig. 1 in Isachenko et al.[14]). The final sucrose concentratio n was 0.083 M, equivalent toalmost isotonic conditions. Finally, the pieces were washed thrice each in basal medium for 10 min, and used for transplanta tion and CAM-cultur e.

CAM-cult ure

Obtainin g of permission from the Ethics Board for use of CAM- Xenotran splantation methodol ogy is not presuppo sed.

Fertilized, newly laid, White Leghorn chicken eggs, were pur- chased from a local hatchery and incubated at 37 �C in air with 60% relative humidity. These were prepared for implantation onday 4 of incubation. Standard microbiology assessment was per- formed to exclude subclinical infections. Preparation of the cho- rio-allant oic membranes was performed essentiall y as previously described [2,13,18]. Each egg was swabbed with warm 70% etha- nol, and a hole then drilled through the pointed pole of the shell.On day 5 the hole was widened into a 1.5–2.0 cm window in the shell. This window was covered with tape until day 10 when for- ceps were used to remove the outer, peridermal , layer of the CAM and to place a 0.5 mm high, 5 mm inner diameter, 6 mm outer diameter, silicone ring on the intact, inner, vascularized CAM basal layer. An ovarian piece was placed within this ring (Fig. 1) and the shell window then covered again. The two eggs were then incu- bated for a further 5 days at 37 �C.

Histology of follicles

For histological investiga tion, one fresh piece collected in 2007,and about half of the CAM cultured tissue was fixed in Bouin’s solu- tion, embedded in paraffin wax, serially sectioned at 4 lm, stained with hematoxylin/e osin, and analyzed under a microscope (�6400,Olympus Co., Tokyo, Japan). Every fifth section was analyzed. The number of viable and degenerated follicles was counted.

Ovarian tissue pieces were sectioned, coded and scored blind.To avoid counting of the same follicles, only the section with a vis- ible oocyte nucleus was counted.

The morphology of the primordi al, primary and secondary folli- cles was evaluated using criteria described by Paynter et al. [32].The primordial follicles had an oocyte surrounded by a single layer of flattened follicular cells while the primary and secondary folli- cles had an oocyte surrounded by one to two layers of spherical granulos a cells. The quality of the follicles was graded on a scale from one to three. A grade 1 follicle was spherical in shape and containe d a spherical oocyte surrounded by evenly distributed granulos a cells. The oocyte had to have homogen ous cytoplasm and slightly granulated nucleus, with condensed chromatin

Fig. 1. Cryopreserved ovarian piece of Patient S. before and after 5 days culture with chorioallantoic membrane (CAM) system. (a) Just after thawing and before placement onthe CAM, (b–d) the same piece after culture on CAM marked by silicone ring as viewed from above (b, c) and below (d). Increased intensiveness of the avian vascularisation inthe place of the seeding of pieces was noted. Bar = 1 mm.

V. Isachenko et al. / Cryobiology 66 (2013) 233–238 235

detectable in the centre of the nucleus. A grade 2 follicle had sim- ilar characteristics, but the oocyte was without condensed chroma- tin within the nucleus and was often irregular in shape; the surrounding granulosa cells could be flat and pulled away from the edge of the follicle. A grade 3 follicle contained a misshapen oo- cyte with or without nuclear vacuolation ; theca and granulos a cells were separated from the edge of the follicle and the partly or fully disrupted granulosa had pyknotic nuclei. Grade 1 and 2 follicles were denoted as normal and those of grade 3 were denoted asdegenerated (for example, see Isachenko et al. [15].

Immunohist ology for angiogen esis

The remaining CAM-cultur ed ovarian pieces were fixed in 4.0%formaldehyd e, embedde d in paraffin wax, serially sectioned at4 lm and stained with antibodies (Dako, Hamburg, Germany) di- rected against von Willebrand factor (1:100).

The intensity of the immunoreactiv ity for von Willebrand factor was semiquantit atively scored [17] as follows: no immunorea ctiv- ity (�), weak immunorea ctivity (+), moderate immunoreactiv ity (++), strong immunoreactiv ity (+++). The extent of vascularisati onwas subjectively estimated based on how much of the total cross-sectio nal area contained cells expressing von Willebrand factor.

Tissue re-transplanta tion

Five years after her ovarian tissue was cryopreserv ed the pa- tient (now aged 30) remained cancer free and returned expressing her wish to have a child. Four of the 10 cryo-vial s with her ovarian tissue was therefore thawed and transplanted in the Cologne Uni- versity Maternal Hospital in 2012. Laparoscopy was used to place these 8 pieces in two peritoneal pockets of the ovarian fossa, one in the right and one in the left pelvic wall.

Statistica l analysis

The histological results were summarized as means and SD.

Results

Freezing with spontaneous ice formation

The temperature probe showed that no ice formation occurred for the first 67 min of cooling from �6 �C at a rate of �0.3 �C/min. At 68 min, at �20.7 �C, ice formation occurred spontaneously in both the dummy and specimen vials and had spread throughout all the cryo-vial s within 2 s (Fig. 2a).

When this spontaneou s ice nucleation took place the tempera- ture rose sharply to �8.4 �C. After this increase in temperat ure, the cooling rate fell to �1 �C/min for 10 min before returning to�0.3 �C/min (Fig. 2a).

The temperat ure profile of freezing that should have taken place with agitated ice formation in a 1.8 ml cryo-tube is shown for com- parison (Fig. 2b).

CAM-cult ure

Both chick embryos survived. After 5 days on the extra-embry- onic chorioallantoic membrane, the ovarian fragments had devel- oped a spherical shape (Fig. 1) and were associate d with several distended blood vessels.

Histology of follicles

Histological examination of the ovarian pieces cultured for 5-days on the CAM revealed ‘‘viable’’ (morphologically normal prean- tral follicles. The estimate d mean preantral (primordial, primary and secondary) follicle density per 1 mm3 was 5.1 ± 0.9 in a single

Fig. 2. Temperature (T) profile during cooling: (lower line) temperature of freezer chamber, (top broken line) temperature of probe, (a) temperature profile of freezing with spontaneous ice formation using a 1.8 ml cryo-tube, (b) temperature profile of freezing with agitated ice formation using a 1.8 ml cryo-tube, (black arrow) beginning of ice formation (note sharp increase in temperature), (white arrow) zone of elevated (–1 to –0.5 C/min) speed of cooling.

236 V. Isachenko et al. / Cryobiology 66 (2013) 233–238

fresh ovarian piece (processed at the time of freezing in 2007) and 4.4 + 2.9 in the two pieces that had been thawed and CAM-cultured for 5 days. A significantly greater proportion of the follicles were classified as normal (grade 1 and 2) in the fresh unfrozen tissue (98.0 + 1.2%) than in the thawed and CAM cultured tissue (11.9 + 4.5%) (P < 0.05).

Immunohist ology for angiogen esis

Weak (+) immunorea ctivity was observed in the frozen-thawed CAM cultured tissues. No comparative values were available from

fresh ovarian tissue as CAM culture was not performed on the fresh tissue in 2007.

Tissue re-transp lantation

Patient S. remained in remission for 5 years and could not con- ceive during this time. The hormone levels looked like postmen o-pausal women with FSH 103.6 mlU/ml, 17- b Estradiol 19 pg/ml and Anti-Mueller -Hormone 0.1 ng/ml.

Five months after re-transplantati on Patient S. informed the clinic about her first spontaneou s menstrual bleeding.

Fig. 3. Ultrasound showing dominant follicle in the area of transplant. Growth offollicle noted: (a) on 05.10.12, (b) on 08.10.12.

V. Isachenko et al. / Cryobiology 66 (2013) 233–238 237

After re-transplan taion the 17- b Estradiol level in the follicular phase was 330 pg/ml and the FSH level 8.0 mIU/ml.

Ultrasound in the spontaneous cycle following her menstrual bleed, confirmed a dominan t follicle in the transplant area (Fig. 3). Timed intercourse was recomme nded.

After ovulation, ultrasoun d detected one normal corpus luteum,but no pregnancy has been established . To the present, a current FSH and 17- b Estradiol level for this patient was not changed. In January 2013 Patient S. was included in standard IVF program of the clinic.

Discussions

In cryopreserv ation many factors contribute to cell and tissue damage, one of these is intracellul ar ice formatio n (Mazur, [26],Mazur et al. [25], Leibo and Mazur, [21]; Leibo, [20]; Leibo and Pool, [22]).

Seeding (initiated ice formation) is a central part of some but not all slow freezing protocols, and has been used in most but not all studies on slow cooling of ovarian tissue. As with embryos a cooling at a rate of �0.3 �C/min from the ice nucleation temper- ature is generally used. Seeding is necessar y so that nucleation and propagation of ice crystals can be controlled, initiating dehydra- tion, increasing the solute concentrations and thereby minimizing the potential damage caused by the physical changes associated with ice formation, with release of latent heat of fusion. At present the temperature of seeding usually varies from �6 to �9 �C.

When permeable cryoprotect ants are used at a concentration of10–12%, as is common for cryopres ervation of ovarian tissue aseeding at a temperat ure of �6 �C should reliably initiate ice for- mation. Without seeding specimens will, as in this case, reach low- er temperature s before self seeding (spontaneous ice formatio n)occurs. Seeding is used for freezing of embryos and other types of cells [3,19,23,45].

Usually freezing of spermatozo a of different species is per- formed without seeding.

However Songassen and Leibo [41] found a positive effect ofseeding at freezing of for mouse spermatozoa. Embryo production by IVF seemed to be improved by using spermatozoa frozen instraws seeded at �4 �C as compared to self seeded control straws [41].

At the same time, some data demonstrates that for slow cooling manual or automated ice formation at a high temperature may not be necessar y. An entire human ovary with its vascular pedicle was frozen [16,24] using the following cryopreserv ation procedure: the ovary was perfused and immersed in 10% dimethyl sulfoxide, and placed in a large volume cryo-vial . The cryovial containing the ovary was then placed in a freezer and frozen at a cooling rate of�1 �C/min, without initiation of ice formation. The authors ob- served high survival rates of follicles, small vessels and stromal cells, as well as a normal histological structure in all ovarian com- ponents after thawing [24,16]. The temperature at which the spec- imens in that publication self seeded is not known, and may have been closer to the conventional seeding temperature than that ob- served in this study (�20 �C), in particular if their cooling chamber had ‘‘cold’’ spots. The fact that we only performed histology after 5 days of CAM culture may have contributed to our poor survival rates, as the CAM cultured tissue may have been deprived of nutri- ents and oxygenati on until revascularization occurred .

Another study compared fresh human ovarian tissue (Group 1)with groups slow cooledeither without or with seeding to initiate ice formation (groups 2 and 3, respectively) [12]. This found no sig- nificant difference between the groups release of Estradiol 17- binto the culture supernat ant, although progesterone concentr a-tions and primordial and primary follicle density were higher inGroup 1 than in Groups 2 or 3. Group 2 (not seeded) had signifi-cantly fewer normal grade follicles than the other two groups. Itwas concluded that for optimal cryopreserv ation of human ovarian tissue, the protocol of conventional freezing should therefore in- clude a step of initiated ice formation [12].

The data of Martinez-Madr id et al. [24] and Jadoul et al. [16]may be explained by the fact that in their study small fragments of ovary were used, whereas in the other studies whole ovaries were cryopreserved that had been equilibrated with cryoprotec- tant via perfusion.

In our opinion, slow cooling of ovarian tissue should be com- bined with seeding i.e. initiation of ice formation usually at a tem- perature of between �6 and �9 �C.

This is in agreement with the investigatio ns of Zhang et al. [46]who investigated the effects of different cooling rates and different ice-seedi ng temperature s on the cryopreserv ation of whole cow ovaries. They found that the optimal protocol for slow freezing ofcow whole ovaries was a cooling rate of -0.2 �C/min and initiated ice formation.

In conclusion, in spite of the damage caused by the cryopreser- vation with spontaneou s ice-formation the surviving follicles could restore ovarian function after transplantation .

Acknowled gment

The author wish to thank Prof. Stanley Leibo from Department of Biological Sciences, University of New Orleans, New Orleans,USA for helpful discussion of these materials.

238 V. Isachenko et al. / Cryobiology 66 (2013) 233–238

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