Introduction. Key Message AOGS MAIN RESEARCH ARTICLE HELLE N. WILKEN-JENSEN, STINE G. KRISTENSEN, JANNI V. JEPPESEN & CLAUS YDING ANDERSEN.

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1 A C TA Obstetricia et Gynecologica AOGS MAIN RESEARCH ARTICLE Developmental competence of oocytes isolated from surplus medulla tissue in connection with cryopreservation of ovarian tissue for fertility preservation HELLE N. WILKEN-JENSEN, STINE G. KRISTENSEN, JANNI V. JEPPESEN & CLAUS YDING ANDERSEN Laboratory of Reproductive Biology, Juliane Marie Center for Women, Children and Reproduction, University Hospital of Copenhagen, University of Copenhagen, Copenhagen, Denmark Key words Immaturity, oocytes, in-vitro maturation, fertility preservation, embryoscope, epidermal growth factor Correspondence Claus Yding Andersen, Laboratory of Reproductive Biology, Section 5712, University Hospital of Copenhagen, Blegdamsvej 9, Rigshospitalet, DK-2100 Copenhagen, Denmark. yding@rh.dk Conflict of interest The authors have stated explicitly that there are no conflicts of interest in connection with this article. The authors alone are responsible for the content and writing of the article. Please cite this article as: Wilken-Jensen HN, Kristensen SG, Jeppesen JV, Yding Andersen C. Developmental competence of oocytes isolated from surplus medulla tissue in connection with cryopreservation of ovarian tissue for fertility preservation. Acta Obstet Gynecol Scand 2014; 93: Received: 1 June 2013 Accepted: 6 September 2013 DOI: /aogs Abstract Objective. Evaluating the developmental competence of immature oocytes collected from surplus medulla tissue in connection with ovarian tissue cryopreservation for fertility preservation. Design. Cohort comparative study. Setting. University laboratory in Denmark from Population. 69 girls and women (0 38 years of age) who each had one ovary cryopreserved for fertility preservation. Methods. Ovaries were obtained directly from the local hospital or from collaborating hospitals (two to five hours transport on ice). Immature oocytes were aspirated from large antral follicles visible on the ovaries, and collected from the saline solution, containing surplus medulla tissue, following dissection of the ovarian cortical tissue for cryopreservation. The immature oocytes were cultured for 48 h in an Embryoscope TM Time-lapse System or in culture dishes overlaid with liquid paraffin using commercial and in-house supplemented culture media. Main outcome measures. Maturation rate for immature oocytes reaching metaphase II. Results. With a maturation rate of 3.1%, only 21 of 682 immature oocytes reached metaphase II. Immature oocytes from ovaries that had been transported on ice for two to five hours performed significantly poorer than those recovered immediately after surgery. Addition of epidermal growth factor and follicle fluid from human small antral follicles to the culture medium did not augment the maturation rate. Immature oocytes cultured in the Embryoscope performed significantly better than those in conventional culture dishes. Conclusions. In vitro maturation of immature oocytes should only be attempted clinically from visible antral follicles and where the ovary is not subjected to a cooling period prior to recovery of immature oocytes. Abbreviations: EGF, epidermal growth factor; FSH, follicle stimulating hormone; IVM, in vitro maturation; LH, luteinizing hormone; MII, metaphase II. Introduction New methods for preserving the fertility in female cancer patients at risk of becoming sterile due to gonadotoxic treatment are currently undergoing rapid development. This progress is heralded by the increasing number of longterm cancer survivors that are concerned about their future fertility and the possibility of having a biological child (1). Most cancer treatments are gonadotoxic but only aggressive protocols may, in the worst case, eliminate the ovarian Key Message Immature oocytes collected in connection with ovarian tissue cryopreservation for fertility preservation could potentially be used clinically; however, current results show a very poor in vitro maturation rate of the immature oocytes, especially when the ovary is subjected to cooling prior to recovery. 32 ª 2013 Nordic Federation of Societies of Obstetrics and Gynecology, Acta Obstetricia et Gynecologica Scandinavica 93 (2014) 32 37

2 pool of follicles and render the patient infertile. However, many patients are likely to increase their chances of conceiving by having fertility preservation performed. Currently, three methods of fertility preservation are in use; these include oocyte and embryo cryopreservation, but ovarian tissue cryopreservation is also becoming an established technique (2,3). The latter method has proved attractive because, contrary to oocyte and embryo freezing, no hormone pre-treatment is required, which often causes a delay that is incompatible with a need for urgent cancer therapy. Furthermore, ovarian tissue cryopreservation is not only for the adult fertile women, but also for pre-pubertal girls (4,5). Current practice in connection with cryopreservation of ovarian tissue in many centers is to excise one entire ovary and leave one ovary in situ. Only the cortical tissue is frozen, whereas the medulla tissue with all the growing follicles is normally discharged. However, the medulla tissue contains the majority of antral follicles and there will invariably be immature oocytes present in the discharged medulla tissue. Although these ovaries are unprimed and have received no stimulation with exogenous hormones, they do harbor immature oocytes, which could, if recovered, be matured and preserved and thereby represent an extra number of oocytes that potentially could be used for fertility purposes. The aim of present study was to evaluate the number of immature oocytes available in the surplus medulla tissue and to test the developmental competence for maturing to the metaphase II (MII) stage using commercial and in-house supplemented culture media. Material and methods From September 2011 to October 2012, 79 patients had their ovarian tissue cryopreserved at Rigshospitalet, Copenhagen, Denmark; a total of 69 girls and women (0 38 years of age) were included in the present study. The indications for entering the fertility preservation program were breast cancer (n = 29), Hodgkin lymphoma (n = 11), non-hodgkin lymphoma (n = 2), lymphoma (n = 4), cervix cancer (n = 4), sarcoma (n = 8), other malignancies (n = 9), and non-malignant indications (n = 12). Eleven of these women had received chemotherapy prior to excision of ovarian tissue. In eight of the 69 women, no oocytes were found. The menstrual history and fertility of the women were not known. Oocyte collection and in vitro maturation The excised ovaries were transported to the laboratory either from the local hospital (5 10 min transport) or from collaborating hospitals (two to five hours transport on ice) (4,6,7). The ovaries were suspended in culture medium, which from the local hospital had a temperature of 37 C, whereas from collaborating hospitals the temperature was close to 0 C. Before isolation of the cortical tissue, all visible antral follicles were aspirated using 23-gauge syringe needles. The aspirated follicular fluid was transferred to a Petri dish and examined for the presence of oocytes under a stereomicroscope. During the process of isolating the ovarian cortex, the medulla was removed. This required careful and meticulously cutting with sharp scalpels to prepare the cortical tissue to the required thickness. The cryopreservation of the ovarian cortex has been described elsewhere (7). During this process the medulla usually became minced into many small pieces of tissue. The discarded material and the saline solution in which the preparation of the cortical tissue took place were subsequently examined for the presence of oocytes under a stereomicroscope. All oocytes found were retrieved and transferred to LAG-medium (no: , Origio A/S, Maløv, Denmark) at 37 C and 5.6% CO 2, which served as a holding medium during the process of oocyte collection. The oocytes were classified into one of three groups: (1) those found in fluid from aspirated antral follicles, (2) cumulus-enclosed oocytes found in the saline solution of discharged ovarian tissue and (3) naked oocytes found in the saline solution of discharged ovarian tissue. Each individual oocyte was subsequently transferred to modified pre-equilibrated in-vitro maturation (IVM)- medium (Origio A/S) containing one of the different hormone supplements as outlined in Table 1. To secure a fully equilibrated medium, the dishes was prepared the previous day and left overnight in a humidified incubator at 37 C and 5.6% CO 2, with reduced oxygen tension, 7% O 2. Each individual oocyte was cultured in drops (30 ll) of IVM-medium plus supplements in cell culture dishes (no ; Nunc, Roskilde, Denmark) overlaid with liquid paraffin (Origio A/S) in an incubator. Oocytes were examined under a stereomicroscope after 24 h for possible extrusion of the first polar body. After 48 h the oocytes were denuded and checked again. Images of each individual oocyte were acquired at zero hour and, if there was any extrusion of polar body, also at 24 or 48 h, using a AxioCamERc5s digital camera attached to an AxioVert 135 microscope. AXIOVISION (Rel ) was used to obtain and analyze the images. Baseline oocyte diameter was calculated by determining the mean of the longest and shortest diameters. Another incubation method was also used involving an Embryoscope TM Time-lapse System (Unisense Fertilitech A/S, Aarhus, Denmark), where each oocyte was kept in individual wells in the Embryoslide TM Incubation Tray (Unisense). The oocytes were kept at 37 C and 5.6% CO 2 ª 2013 Nordic Federation of Societies of Obstetrics and Gynecology, Acta Obstetricia et Gynecologica Scandinavica 93 (2014)

3 Table 1. Maturation rate for immature oocytes cultured in different in vitro maturation (IVM) and in-house supplemented culture media. Supplements Patients, n Mean age SD (range) Oocytes retrieved, n Mean oocytes retrieved/ patients SD (range) IVM rate n % 1 IVM-media IU/L FSH IU/L LH (1 32) IVM-media IU/L FSH IU/L LH + 5 a (4 18) ng/ml EGF 3 IVM-media IU/L FSH IU/L LH + 5 a (2 20) % FF 4 IVM-media IU/L FSH IU/L LH (1 31) ng/ml EGF 5 IVM-media IU/L FSH IU/L LH (1 18) % FF 6 IVM-media IU/L FSH IU/L LH (2 16) All patients ( ) (1 32) a Two patients had half of their oocytes cultured in Medium 2 and the other half in Medium 3, and are therefore represented in both groups. EGF, epidermal growth factor; FSH, follicle stimulating hormone; IVM, in vitro maturation; LH, luteinizing hormone. in a humidified atmosphere for 48 h as above and checked for the presence of polar body extrusion by evaluating the time lapse sequence recorded and by denudating the oocyte after 48 hours incubation. This project was approved by the ethical committee of the municipalities of Copenhagen and Frederiksberg (J. no. H ). Statistical analysis Statistical analysis was performed using SAS9.3 software (SAS Institute, Cary, NC, USA). The analysis was performed on the linear correlation between age and number of collected oocytes (Pearson, p < 0.05). A chi-square contingency table (2 9 2) was used to examine whether the incubation method had an effect on oocyte maturation. Logistic regression was used to examine whether the oocyte collection site or the transportation time of the ovaries before oocyte collection had an effect on oocyte maturation. A p-value of <0.05 was considered significant. Results A total of 682 immature oocytes from 61 patients were recovered from the follicular fluid aspirates and from the saline solution containing the surplus medulla tissue. The average number of oocytes collected per patient was (mean SD) (Table 1). The number of oocytes was significantly positively correlated with the age of the patients (R = 0.31, n = 61; p < 0.05) (Figure 1). A positive correlation was also found between the ovarian volume and the recovered number of oocytes (R = 0.44, n = 61; p < 0.05) (data not shown). Number of oocytes found per pa ent Age (years) Figure 1. Relationship between the age of the patient and the number of oocytes retrieved. Linear regression line y = x , R 2 = (p < 0.05). Triangles designate patients who have had previous chemo- or radiotherapy. Circles show the nine patients in whom mature metaphase II oocytes were obtained after culture. In vitro maturation rates of immature oocytes All immature oocytes were placed into in vitro cultures. The overall maturation rate of oocytes reaching MII and extruding a first polar body (i.e. the IVM rate) amounted to 3.1% (Table 1). Following classification, the highest percentage of maturation was found in the immature oocytes aspirated directly from the antral follicles compared with immature oocytes collected from the saline solution containing the surplus medulla tissue (Table 2). A significant difference in maturation rate was found between cumulus-oocyte-complexes and nude oocytes collected from the saline solution (Table 2) (logistic regression; p < 0.05). Immature oocytes that matured in 34 ª 2013 Nordic Federation of Societies of Obstetrics and Gynecology, Acta Obstetricia et Gynecologica Scandinavica 93 (2014) 32 37

4 Table 2. Maturation rates for the different classes of immature oocytes. Statistical analysis was performed for oocytes cultured in Medium 1 (IVM-media IU/L FSH IU/L LH).There was a significant difference in maturation rate between cumulus-oocyte-complex and nude oocytes collected from the saline solution (logistic regression analysis; p < 0.05). Oocytes collected from Oocytes, n Patients, n Mean age (years) SD Metaphase II oocytes, n In vitro maturation (%) Antral follicles Cumulus-oocyte-complex a,b Nude oocytes Saline solution Cumulus-oocyte-complex a Nude oocytes b Values with different superscripts are significantly different (p < 0.05). FSH, follicle stimulating hormone; IVM, in vitro maturation; LH, luteinizing hormone. vitro and reached the MII stage were all enclosed by cumulus cells, with the exception of two. The diameter of 254 of 631 oocytes was measured. The diameter of the oocytes varied greatly, from approximately lm. The mean diameter (SD) was lm. The oocytes that matured were lm in diameter, which represented 87% of the immature oocytes collected (data not shown). Maturation rate in relation to different culture media supplements used In the first trial, luteinizing hormone (LH) and follicle stimulating hormone (FSH) (100 IU/L) were added to the IVM-media and 31 patients were included. The mean age of the patients (SD) was years. A total of 385 oocytes were included in the study (Table 1). The IVM-rate was 5.2%. The other trials outlined in Table 1, with different supplements to the IVM-media, did not have a positive outcome, except in the media supplemented with 40% follicular fluid, where one of 69 oocytes matured to a MII oocyte (Table 1). Comparison of two different incubation systems Two different incubation methods were used with the maturation media supplemented with 100 IU/L LH and FSH. A significant difference was found between immature oocytes cultured with the Embryoscope and immature oocytes cultured in conventional culture in dishes, referred to as droplets (Table 3). Maturation rate in relation to time of transport prior to oocyte collection Ovaries were surgically removed at three different hospitals in Denmark and transported for zero to five hours to the Laboratory of Reproductive Biology, Copenhagen, Denmark, where the cryopreservations were performed. To determine whether the transportation time prior to oocyte collection had an effect on the developmental competence of the immature oocytes, the maturation rates from oocytes collected from the different hospitals were compared. A significantly higher maturation rate was found in immature oocytes collected from ovaries recovered at the local hospital with a very short transport time, compared with the other two hospitals, with two to five hours transportation time prior to oocyte collection (Table 4). Discussion This study demonstrates a poor overall developmental competence for all immature oocytes collected from surplus medulla tissue during cryopreservation of ovarian tissue for fertility preservation. These immature oocytes derived from antral follicles represented follicles with Table 3. Comparison of the maturation rates of oocytes cultured with the Embryoscope or conventional culture in dishes (referred to as droplets). Statistical analysis was performed for oocytes cultured in Medium 1 (IVM-media IU/L FSH IU/L LH). A significant difference was found between the two incubation methods (Fisher s exact test; p < ). Incubation method Total number of patients Number of oocytes Number of matured oocytes Number of patients with matured oocytes IVM rate,% Embryoscope a Droplets b Values with different superscripts are significantly different (p < 0.05). IVM, in vitro maturation; LH, luteinizing hormone; FSH, follicle stimulating hormone. ª 2013 Nordic Federation of Societies of Obstetrics and Gynecology, Acta Obstetricia et Gynecologica Scandinavica 93 (2014)

5 Table 4. Maturation rate in relation to time of transport prior to oocyte collection. Statistical analysis was performed for oocytes cultured in Medium 1 (IVM-media IU/L FSH IU/L LH). Hospital 1, with a short transport time, showed a significantly higher maturation rate compared with Hospitals 2 and 3 (logistic regression analysis; p < 0.05). Hospital Transport time, hours Patients, n Oocytes, n MII oocytes, n IVM, % b a a Values with different superscripts are significantly different (p < 0.05). FSH, follicle stimulating hormone; IVM, in vitro maturation; LH, luteinizing hormone; MII, metaphase II. diameters from around 2 9 mm, and although atresia is common in many of these antral stage follicles (8), their ability to resume meiosis and reach the MII stage was low, with an overall maturation rate of only 3.1%. Furthermore, this study showed that if the ovary was transported on ice four to five hours prior to oocyte collecting, representing the majority of the ovaries included, the immature oocytes showed a significantly lower maturation rate, demonstrating that the developmental competence of the collected oocytes is likely to deteriorate during cooling and transport. Immature oocytes collected immediately after excision of the ovary had the highest maturation rate but still, only one in nine oocytes had the capacity to mature to the MII phase. These results show that immature oocytes collected from antral follicles immediately after excision may represent a source worth collecting to provide mature oocytes for either cryopreservation or fertilization. However, the maturation rate is likely to be low and the subsequent results in terms of children born remains unknown. In the current study we showed that surplus medulla tissue which is normally discharged in connection with preparation of the ovarian cortex for fertility preservation represents a substantial source of immature oocytes and that oocytes were found in almost 90% of all patients irrespective of age. The number of oocytes was correlated positively to the age of the patients even when reaching their mid-30s, which is surprising, since circulating levels of anti-m ullerian hormone (AMH) at that age have started to drop, reflecting a diminished number of antral follicles (9). It is also of interest to notice that it is not uncommon to collect in excess of 30 oocytes from one ovary from women aged years, and that the likelihood of achieving mature oocytes increases with the number of oocytes retrieved. In addition, five immature oocytes were collected from a three-year-old girl. To our knowledge this is the youngest age reported for ovarian oocyte retrieval. However, none of the oocytes was capable of maturing. Several different media supplements were tested to augment the maturation rate. It has recently been shown that the mid-cycle surge of gonadotropins elicits a surge of epidermal growth factor (EGF)-like peptides such as amphiregulin and epiregulin from the mural granulosa cells which have a direct effect on the cumulus cells, causing oocyte resumption of meiosis (10). This signal obviously is lacking in the in vitro culture systems tested in the present study, as there are no mural granulosa cells being exposed to a surge of gonadotropins. To compensate for this activity, EGF was added to the culture medium in concentrations similar to those found in follicle fluid in connection with the mid-cycle surge (11). EGF did not augment the maturation rate, which indicates that immature cumulus cells are incapable of responding to this signal. We also tested whether increased concentrations of gonadotropins from IU/L could augment oocyte maturation, but no effect was observed. The same results were obtained when the immature oocytes were cultured with different concentrations of follicular fluid from small antral follicles in order to mimic the natural environment. Collectively, these results demonstrate that the follicle is a highly specialized microenvironment and that our understanding of how oocytes develop in human small antral follicles is currently far too limited to mimic these conditions in vitro and produce mature oocytes with a high frequency. Two different incubation systems were used in the present study. We found that immature oocytes cultured in the Embryoscope performed significantly better than those in conventional culture dishes. The Embryoscope has been developed for embryos to minimize the disturbance to the culture environment by integrating the incubation and inspection of embryos into one time-lapse imaging system. This capacity was beneficial in the current study, as the more stable culture environment in the Embryoscope resulted in a significantly better IVM outcome for the immature oocytes than for conventional culture. Many studies have shown a much higher rate of IVM than found here (12 16). There are several reasons for the observed low rate of maturation in the present study: The diameter of the follicles from which the oocytes where obtained was lower than in most IVM programs, where the diameter of the aspirated follicles normally ranges from 10 to 15 mm. The results of IVM programs are significantly improved when priming with FSH takes place and a bolus trigger of human chorionic gonadotropin is used 36 h prior to aspiration. This is in contrast to the present study where ovaries were collected at variable times during the menstrual cycle. 36 ª 2013 Nordic Federation of Societies of Obstetrics and Gynecology, Acta Obstetricia et Gynecologica Scandinavica 93 (2014) 32 37

6 Most of the oocytes collected in the present study were derived from ovaries that had been transported prior to cryopreservation and cooled to slightly above 0 C, which profoundly attenuated their developmental competence. To improve the maturation rate we now plan to focus on a specific group of patients in order to obtain better quality oocytes patients from whom the ovary is available immediately after excision and where it will be possible to prime with FSH administration for three to four days (awaiting for surgery to be performed) and possibly to administer human chorionic gonadotropin 36 h prior to surgery. We also found that the majority of oocytes from these immature follicles have a diameter of lm. The oocytes undergoing maturation were also located in this group, but the number of oocytes outside this range is too small to predict their ability to mature. In conclusion, the present study demonstrates that substantial numbers of human immature oocytes can be obtained from one ovary in connection with cortical tissue freezing and fertility preservation. However, developmental competence in terms of resuming meiosis is poor for these oocytes and is negatively affected by transport of the ovary prior to freezing. This method may be attempted clinically only in women whose ovaries are available immediately after excision, focusing primarily on cumulus-enclosed oocytes from visible antral follicles. Funding Financial support from the Danish Cancer Society (DP05112/R2-A41-09-S2), the Danish Medical Research Council ( ; ), the Novo Nordisk Foundation, Svend Andersen Foundation and the University Hospital of Copenhagen is gratefully acknowledged. References 1. Partridge AH, Gelber S, Peppercorn J, Sampson E, Knudsen K, Laufer M, et al. Web-based survey of fertility issues in young women with breast cancer. J Clin Oncol. 2004;22: Andersen CY, Kristensen SG, Greve T, Schmidt KT. Cryopreservation of ovarian tissue for fertility preservation in young female oncological patients. Future Oncol. 2012;8: Donnez J, Dolmans M. Preservation of fertility in females with haematological malignancy. Br J Haematol. 2011;154: Rosendahl M, Schmidt KT, Ernst E, Rasmussen PE, Loft A, Byskov AG, et al. Cryopreservation of ovarian tissue for a decade in Denmark: a view of the technique. Reprod Biomed Online. 2011;22: Schmidt KT, Larsen EC, Andersen CY, Andersen AN. Risk of ovarian failure and fertility preserving methods in girls and adolescents with a malignant disease. Br J Obstet Gynaecol. 2010;117: Schmidt KL, Ernst E, Byskov AG, Nyboe Andersen A, Yding Andersen C. Survival of primordial follicles following prolonged transportation of ovarian tissue prior to cryopreservation. Hum Reprod. 2003;18: Tryde Schmidt KL, Yding Andersen C, Starup J, Loft A, Byskov AG, Nyboe Andersen A. Orthotopic autotransplantation of cryopreserved ovarian tissue to a woman cured of cancer follicular growth, steroid production and oocyte retrieval. Reprod Biomed Online. 2004;8: Gougeon A. Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr Rev. 1996;17: Wallace WH, Kelsey TW. Human ovarian reserve from conception to the menopause. PLoS ONE. 2010;27(5):e Conti M, Hsieh M, Park JY, Su YQ. Role of the epidermal growth factor network in ovarian follicles. Mol Endocrinol. 2006;20: Humaidan P, Westergaard LG, Mikkelsen AL, Fukuda M, Yding Andersen C. Levels of the epidermal growth factor-like peptide amphiregulin in follicular fluid reflect the mode of triggering ovulation: a comparison between gonadotrophin-releasing hormone agonist and urinary human chorionic gonadotrophin. Fertil Steril. 2011;95: Azem F, Hasson J, Cohen T, Shwartz T, Mey-Raz N, Almog B, et al. Retrieval of immature oocytes after chemotherapy for Hodgkin s disease and prolonged ovarian down-regulation with gonadotropin-releasing hormone agonist. Fertil Steril. 2009;92(828):e Fasano G, Moffa F, Dechene J, Englert Y, Demeestere I. Vitrification of in vitro matured oocytes collected from antral follicles at the time of ovarian tissue cryopreservation. Reprod Biol Endocrinol. 2011;9: Huang JY, Buckett WM, Gilbert L, Tan SL, Chian RC. Retrieval of immature oocytes followed by in vitro maturation and vitrification: a case report on a new strategy of fertility preservation in women with borderline ovarian malignancy. Gynecol Oncol. 2007;105: Isachenko E, Rahimi G, Isachenko V, Nawroth F. In-vitro maturation of germinal-vesicle oocytes and cryopreservation in metaphase I/II: a possible additional option to preserve fertility during ovarian tissue cryopreservation. Reprod Biomed Online. 2004;8: Revel A, Koler M, Simon A, Lewin A, Laufer N, Safran A. Oocyte collection during cryopreservation of the ovarian cortex. Fertil Steril. 2003;79: ª 2013 Nordic Federation of Societies of Obstetrics and Gynecology, Acta Obstetricia et Gynecologica Scandinavica 93 (2014)