Vitrified human ovaries have fewer primordial follicles and produce less antim ullerian hormone than slow-frozen ovaries

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1 Vitrified human ovaries have fewer primordial follicles and produce less antim ullerian hormone than slow-frozen ovaries Slow-freezing and vitrification methods of human ovarian tissue cryopreservation were compared in terms of primordial follicle count and in vitro antim ullerian hormone (AMH) and estradiol production. Compared with fresh and slow-frozen ovaries, vitrified ovaries contained statistically significantly fewer primordial follicles and produced statistically significantly less AMH in vitro. Estradiol production from slow-frozen and vitrified ovaries was similar but statistically significantly lower than from fresh cultured strips. (Fertil Steril Ò 2011;95: Ó2011 by American Society for Reproductive Medicine.) Key Words: Ovary, cryopreservation, slow freezing, vitrification, AMH, estradiol Pregnancies and live births reported after transplantation of frozen-hawed ovarian grafts in cancer patients have proved that this procedure, albeit still experimental, may help protect the fertility potential of cancer patients who face the risk of premature gonadal failure as a result of cytotoxic chemotherapy and radiation regimens (1 12). Slow freezing is the most commonly employed technique for cryopreservation of human ovarian tissue (13). Whereas vitrification has proven to be a viable alternative to slow freezing in cryopreservation of oocytes and embryos in humans and various animals (14 16), data on its applicability in freezing human ovarian tissue are limited and show inconsistent results. Therefore, we compared slow-freezing and vitrification methods for human ovarian tissue in terms of tissue morphology, Ozgur Oktem, M.D. a Ebru Alper, M.D. a Basak Balaban, M.Sc. a Erhan Palaoglu, M.D. b Kamil Peker, M.D. c Cengiz Karakaya, Ph.D. d Bulent Urman, M.D. a a Women s Health Center, Assisted Reproduction Unit, American Hospital, Istanbul, Turkey b Clinical Laboratories, American Hospital, Istanbul, Turkey c Istanbul Pathology Associates, Istanbul, Turkey d Department of Obstetrics and Gynecology, IVF Laboratory School of Medicine, Gazi University, Ankara, Turkey Received July 29, 2010; revised December 24, 2010; accepted December 28, 2010; published online February 5, O.O. has nothing to disclose. E.A. has nothing to disclose. B.B. has nothing to disclose. E.P. has nothing to disclose. K.P. has nothing to disclose. C.K. has nothing to disclose. B.U. has nothing to disclose. Presented at the 65th annual meeting of American Society for Reproductive Medicine October 17 21, 2009, Atlanta, Georgia. Reprint requests: Ozgur Oktem, M.D., American Hospital Women s Health Center, Assisted Reproduction Unit, Guzelbahce Sok, No. 20, Istanbul, Turkey ( drozguroktem@gmail.com). primordial follicle reserve, and antim ullerian hormone (AMH) and estradiol production in vitro. We enrolled 15 patients undergoing laparoscopic ovarian cyst excisions for benign indications for the study. The study was approved by the institutional review board of the hospital. We obtained cm strips from cortex removed with the cyst capsule, which were further sliced into six equal pieces, each measuring 0.25 cm. Two pieces were allocated to each of three different groups: fresh, slow freezing, and vitrification. One piece from each group was fixed for histologic examination and primordial follicle count. The other piece was cultured for 3 days to measure the in vitro estradiol and AMH production. Slow-frozen or vitrified strips were thawed after 24 hours and then cultured and fixed. The slow-freezing protocol was previously described elsewhere (13). The freezing solution contained 1.5 M dimethyl sulfoxide (DMSO), 0.2 M sucrose, 10% human serum albumin, and buffered phenol-free alpha minimal essential medium (MEM). For vitrification, phenol-free buffered alpha MEM medium containing 15% propanediol, 15% ethylene glycol, 0.2 M sucrose, and 10% human serum albumin was used (freezing protocols are described in detail in the Supplemental Materials, available online). In both techniques, the vials were first placed on a tissue roller during incubation to ensure even penetration of the cryoprotectant for 10 minutes; then they were either loaded into an automated freezer (Planer Kryo 10 series III; Planer PLC, Sunbury-on-Thames, United Kingdom) for slow freezing or were immersed in liquid nitrogen for vitrification. After 24 hours, the vials were thawed at room temperature for 30 seconds andthenplacedina37 C water bath for 2 minutes. The tissues were washed stepwise in media containing progressively lower concentrations of cryoprotectants. Then one piece was immediately fixed for histologic analysis, and the other one was transferred to a culture dish. Cortical samples were cultured for 3 days in 1.5 ml of alpha- MEM culture media supplemented with 100 miu/ml recombinant follicle-stimulating hormone (FSH). Half the culture media was refreshed every other day and stored at 80 C until assayed for AMH and estradiol /$36.00 Fertility and Sterility â Vol. 95, No. 8, June 30, doi: /j.fertnstert Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 For histologic analysis, formalin-fixed paraffin-embedded ovarian cortices were serially sectioned at 7 micron thickness. Primordial follicles were counted in every fifth section (35 microns apart) using an Olympus IX-71 microscope (Olympus Corporation, Tokyo, Japan) under 160 magnification, as previously described elsewhere (17). To avoid duplicate counting in each section, only healthy primordial follicles with visible oocyte nuclei were recorded. Follicles with abnormally shaped oocytes, shrunken ooplasm, and disrupted granulosa cells were considered unhealthy. Follicle density was determined per mm 2 of tissue surface, and a mean value was obtained from all sections. The AMH and estradiol levels in culture fluid were measured as previously described elsewhere (18, 19). Follicle counts and hormone levels were expressed as mean standard deviation (SD). Data were analyzed by analysis of variance (ANOVA) or Kruskal-Wallis tests, where appropriate, and then by multiple comparison post hoc tests. P%.05 was considered statistically significant. The mean age of the patients was years (mean SD). The mean number of primordial follicles (number/mm 2 ) in fresh and slow frozen strips were comparable ( vs , P>.05). However, the vitrified strips contained statistically significantly fewer primordial follicles compared with the fresh and slow-frozen strips ( vs , P<.0001; vs , P<.001, respectively) (Fig. 1A). Histomorphologic analysis revealed that the structures of the primordial follicles were preserved better in the slow-frozen ovaries compared with the vitrified ovaries. As shown in the Figure 1B, the most characteristic morphologic change in the primordial follicles of vitrified ovarian cortices was the shrinkage of ooplasm along with detachment of granulosa cells. Furthermore, these morphologic abnormalities after vitrification were also noted in growing follicles at the preantral and antral stages. After 24 hours of culture, the AMH production from fresh and slow-frozen strips was similar ( vs , respectively, P>.05). However, vitrified ovaries produced statistically significantly less AMH compared with fresh and slow-frozen ovaries ( vs , respectively, P<.05; and vs , respectively, P<.05). Furthermore, at the end of the 3-day culture period, the mean AMH production from vitrified ovaries was statistically significantly lower than from fresh and slowfrozen ovaries ( vs , respectively, P<.05; vs , respectively, P<.05). Fresh and slowfrozen strips produced comparable amounts of AMH ( vs , P>.05, respectively) (see Fig. 1C and D). Estradiol production on the first day of culture was comparable in fresh, slow-frozen, and vitrified ovaries (3, , 1, , and 2, , respectively, P>.05). However, while estrogen production in slow-frozen and vitrified ovaries reached peak levels on culture day 1 then declined on culture day 3, the fresh samples continued to produce the hormone on culture day 3. The mean estradiol production after the 3-day culture period was statistically significantly higher in fresh compared with slowfrozen and vitrified cortical pieces (3, vs. 1, , respectively, P<.05; and 3, vs. 2, , respectively, P<.05). However, estradiol production from slowfrozen and vitrified ovaries was similar (1, vs. 2, , P>.05) (see Fig. 1E and F). We obtained better results with slow freezing than vitrification in human ovarian tissue cryopreservation in terms of primordial follicle counts, tissue integrity, and viability as assessed by in vitro AMH production. The shrinkage of oocytes and disruption of granulosa cells as the most characteristic finding suggest that rapid cooling and/or insufficient penetration of cryoprotectants adversely affects the adaptation of the follicles to instant freezing. To our knowledge, ours is the first study to compare AMH production from frozen-thawed ovarian tissues. In contrast with the lower AMH production from vitrified ovaries, the estradiol production from slow-frozen and vitrified ovaries was comparable. Given that both hormones are produced by granulosa cells of growing follicles, this discrepancy could be explained either by the different sensitivity to freezing of the enzymes that produce estradiol (aromatase) and AMH or by some other unknown mechanism. In fact, regardless of the freezing method, the mean estradiol production was statistically significantly lower compared with fresh tissue samples, suggesting a more profound effect of subzero temperatures on aromatase activity. Studies to date have published inconsistent results mainly due to the heterogeneity in their methods. Keros et al. (20) found that vitrification of human ovarian tissue preserved ovarian stroma better than slow freezing, along with similar degrees of protection of follicles with a combination of propane-1,2-diol (PrOH), ethylene glycol (EG), DMSO, and polyvinylpyrrolidone (PVP) for vitrification and a PrOH-sucrose and EG-sucrose combination for slow freezing. These investigators did not evaluate in vitro hormone synthesis from cryopreserved cortical pieces. Another study failed to find a difference between slow freezing (L-15 Medium [Leibovitz] with L-glutamine þ 1.5 M DMSOþ 0.1 M sucrose þ10% SSS [serum substitute supplement]) and vitrification (Dulbecco s phosphate-buffered solution with SSS and containing 2.62 M DMSO, 2.60 M acetamide, 1.31 M propylene glycol, and M polyethylene glycol) in examining in vitro estradiol and progesterone production and follicle viability. However, they also found that GAPDH gene expression in ovarian tissue after vitrification was dramatically decreased in contrast with conventional freezing (21). Rahimi et al. (22), by use of a human ovarian xenograft model, observed a higher percentage of apoptotic cells in vitrified ovaries after grafting compared with slow-frozen ovaries. To minimize the toxic effect of cryoprotectants at high concentrations, some researchers have used different forms of vitrification, including less concentrated cryoprotectants, direct contact of ovarian tissue with liquid nitrogen (23), or dropping nitrogen onto the tissue (24). Their results suggest that these techniques are superior to conventional vitrification and could facilitate the vitrification process, maximize the cooling rate, or reduce the toxicity of the vitrification solution via a minimal volume of less concentrated cryoprotectants. Data are still limited on the optimal freezing solution and technique for cryopreservation of human ovarian tissue. Our findings do not necessarily mean that vitrification using different cryoprotectants and protocols will not provide better results. Given that the ultimate aim of ovarian tissue freezing is to obtain live births from these grafts after transplantation, perhaps more conclusive results can be obtained by comparing slow freezing versus vitrification in terms of live birth rates and other parameters of IVF outcomes posttransplantation, such as the response of the grafts to ovarian 2662 Oktem et al. Correspondence Vol. 95, No. 8, June 30, 2011

3 FIGURE 1 (A) Primordial follicle density. Graph bars represent the mean number standard deviation of primordial follicles in the groups. Each sample was serially sectioned at 7 micron thickness and primordial follicles were counted in every fifth section (35 microns apart) to obtain a mean value (per mm 2 of tissue surface) from all sections. Each group included 15 samples. Vitrified ovaries contained statistically significantly fewer primordial follicles compared with the fresh and slow-frozen ovaries (P<.05 by ANOVA and multiple comparison post hoc test. a P>.05; b P<.0001; c P<.001). (B) Histologic assessment of fresh and frozen-thawed ovaries. The structure of primordial follicles in slow-frozen ovaries was preserved compared with vitrified slices. In contrast, abnormal morphologic features, characterized by contraction of ooplasm of the oocytes along with granulosa cells, were more frequently observed in the primordial follicles of vitrified ovaries. (A panel, magnification: 320; B panel 160. Scale bars: 100 microns.) (C) Antim ullerian hormone (AMH) production from fresh and frozen-thawed ovaries in culture. Mean AMH production from vitrified ovaries after 3-day culture period was statistically significantly lower than in fresh and slow-frozen strips. (Graph bars: P<.05 by Kruskal-Wallis and multiple comparison posthoc test. a P>.05; b P<.05; c P<.05). (D) AMH production from fresh and frozenthawed ovaries in culture. Daily AMH production as shown in the curves was statistically significantly lower in vitrified samples compared with fresh and slow-frozen slices. (E, F) Estradiol production from fresh and frozen-thawed ovaries in culture. (E) Mean estradiol production (graph bar) and (F) daily estradiol production (curves) from slow-frozen and vitrified ovaries were comparable but were statistically significantly lower than from fresh (P>.05, ANOVA. a P<.05; b P<.05; c P>.05). Oktem. Correspondence. Fertil Steril Fertility and Sterility â 2663

4 stimulation, the follicle growth, and the oocyte yield. Finally, we hope that other researchers using this vitro model may analyze both freezing techniques in a more quantitative manner and use different granulosa and oocyte-derived hormones and factors to gain more information on the impact of different freezing techniques on ovarian function. REFERENCES 1. Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, et al. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 2004;364: Meirow D, Levron J, Eldar-Geva T, Hardan I, Fridman E, Zalel Y, et al. Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy. N Engl J Med 2005;353: Andersen CY, Rosendahl M, Byskov AG, Loft A, Ottosen C, Dueholm M, et al. Two successful pregnancies following autotransplantation of frozen/thawed ovarian tissue. Hum Reprod 2008;23: Silber SJ, DeRosa M, Pineda J, Lenahan K, Grenia D, Gorman K, et al. A series of monozygotic twins discordant for ovarian failure: ovary transplantation (cortical versus microvascular) and cryopreservation. Hum Reprod 2008;23: Demeestere I, Simon P, Buxant F, Robin V, Fernandez SA, Centner J, et al. Ovarian function and spontaneous pregnancy after combined heterotopic and orthotopic cryopreserved ovarian tissue transplantation in a patient previously treated with bone marrow transplantation: case report. Hum Reprod 2006;21: Rosendahl M, Loft A, Byskov AG, Ziebe S, Schmidt KT, Andersen AN, et al. Biochemical pregnancy after fertilization of an oocyte aspirated from a heterotopic autotransplant of cryopreserved ovarian tissue: case report. Hum Reprod 2006;21: Roux C, Amiot C, Agnani G, Aubard Y, Rohrlich PS, Piver P. Live birth after ovarian tissue autograft in a patient with sickle cell disease treated by allogeneic bone marrow transplantation. Fertil Steril 2010;93:2413.e Demeestere I, Simon P, Emiliani S, Delbaere A, Englert Y. Fertility preservation: successful transplantation of cryopreserved ovarian tissue in a young patient previously treated for Hodgkin s disease. Oncologist 2007;12: Demeestere I, Simon P, Moffa F, Delbaere A, Englert Y. Birth of a second healthy girl more than 3 years after cryopreserved ovarian graft. Hum Reprod 2010;25: Sanchez-Serrano M, Crespo J, Mirabet V, Cobo AC, Escriba MJ, Simon C, et al. Twins born after transplantation of ovarian cortical tissue and oocyte vitrification. Fertil Steril 2010;93: 268.e Ernst E, Bergholdt S, Jorgensen JS, Andersen CY. The first woman to give birth to two children following transplantation of frozen/thawed ovarian tissue. Hum Reprod 2010;25: Oktay K. Spontaneous conceptions and live birth after heterotopic ovarian transplantation: is there a germline stem cell connection? Hum Reprod 2006;21: Oktay K, Oktem O. Ovarian cryopreservation and transplantation for fertility preservation for medical indications: report of an ongoing experience. Fertil Steril 2010;93: Son WY, Chung JT, Gidoni Y, Holzer H, Levin D, Chian RC, et al. Comparison of survival rate of cleavage stage embryos produced from in vitro maturation cycles after slow freezing and after vitrification. Fertil Steril 2009;92: Fadini R, Brambillasca F, Renzini MM, Merola M, Comi R, De Ponti E, et al. Human oocyte cryopreservation: comparison between slow and ultrarapid methods. Reprod Biomed Online 2009;19: Hong SW, Sepilian V, Chung HM, Kim TJ. Cryopreserved human blastocysts after vitrification result in excellent implantation and clinical pregnancy rates. Fertil Steril 2009;92: Oktem O, Oktay K. Quantitative assessment of the impact of chemotherapy on ovarian follicle reserve and stromal function. Cancer 2007;110: Freour T, Mirallie S, Bach-Ngohou K, Denis M, Barriere P, Masson D. Measurement of serum anti-m ullerian hormone by Beckman Coulter ELISA and DSL ELISA: comparison and relevance in assisted reproduction technology (ART). Clin Chim Acta 2007;375: Lee JS, Ettinger B, Stanczyk FZ, Vittinghoff E, Hanes V, Cauley JA, et al. Comparison of methods to measure low serum estradiol levels in postmenopausal women. J Clin Endocrinol Metab 2006;91: Keros V, Xella S, Hultenby K, Pettersson K, Sheikhi M, Volpe A, et al. Vitrification versus controlled-rate freezing in cryopreservation of human ovarian tissue. Hum Reprod 2009;24: Isachenko V, Lapidus I, Isachenko E, Krivokharchenko A, Kreienberg R, Woriedh M, et al. Human ovarian tissue vitrification versus conventional freezing: morphological, endocrinological, and molecular biological evaluation. Reproduction 2009;138: Rahimi G, Isachenko V, Kreienberg R, Sauer H, Todorov P, Tawadros S, et al. Re-vascularisation in human ovarian tissue after conventional freezing or vitrification and xenotransplantation. Eur J Obstet Gynecol Reprod Biol 2010;149: Zhou XH, Wu YJ, Shi J, Xia YX, Zheng SS. Cryopreservation of human ovarian tissue: comparison of novel direct cover vitrification and conventional vitrification. Cryobiology 2010;60: Wang Y, Xiao Z, Li L, Fan W, Li SW. Novel needle immersed vitrification: a practical and convenient method with potential advantages in mouse and human ovarian tissue cryopreservation. Hum Reprod 2008;23: Oktem et al. Correspondence Vol. 95, No. 8, June 30, 2011

5 SUPPLEMENTAL MATERIALS OVARIAN TISSUE FREEZING Slow-Freezing and Thawing Processes The slow-freezing solution contained 1.5 M dimethyl sulfoxide (DMSO), 0.2 M sucrose, 10% human serum albumin, and buffered phenol-free alpha minimal essential medium (MEM). The slowfreezing protocol was previously described elsewhere (3). Briefly, two ovarian cortical strips were soaked in cryovials containing 0.5 ml of freezing solution. The vials then were placed on a tissue roller during incubation to ensure even penetration of the cryoprotectant for 10 minutes. After that, they were loaded into an automated freezer (Planer Kryo 10 series III; Planer PLC, Sunbury-on-Thames, United Kingdom) starting at 0 C and cooling at 2 C/minute to 7 C. Manual seeding was performed at 7 C, and the cooling was continued at a rate of 0.3 C/minute to 40 C/minute, then at a faster rate of 10 C/minute to 140 C/ minute. The vials were transferred to a liquid nitrogen Dewar vessel for storage. After 24 hours, the vials were removed from the Dewar vessel, thawed at room temperature for 30 seconds, then placed in a 37 C water bath for 2 minutes. The tissues were washed stepwise in media containing progressively lower concentrations of cryoprotectant with human serum albumin plus sucrose. The duration of each washing step was 5 minutes. The last wash was performed with medium containing human serum albumin only. Then one piece was immediately fixed for histologic analysis, and the other was transferred to the culture dish. Vitrification and Thawing Processes For vitrification, phenol-free buffered alpha MEM containing 15% propanediol, 15% ethylene glycol, 0.2 M sucrose, and 10% human serum albumin was used. The vitrification procedure followed similar steps as the slow freezing method. Briefly, two ovarian cortical pieces were soaked in cryovials containing 0.5 ml of freezing solution. The vials then were placed on a tissue roller during incubation for 10 minutes to ensure even penetration of the cryoprotectant, and subsequently they were immersed in liquid nitrogen. After 24 hours, the vials were removed from the Dewar vessel, thawed at room temperature for 30 seconds, then placed in a 37 C water bath for 2 minutes. The tissues were washed stepwise in media containing progressively lower concentrations of cryoprotectants. The tissues were kept for 5 minutes in each step. The last wash was performed with medium containing human serum albumin only. Then one piece was immediately fixed for histologic analysis, and the other was transferred to culture dish. Fertility and Sterility â 2664.e1

National Physicians Cooperative of the Oncofertility Consortium

National Physicians Cooperative of the Oncofertility Consortium Section 10. Ovarian Tissue Freezing, Thawing, Labeling, and Testing Date Adopted Supersedes Procedure # 11/1/2010 12/15/2009; 11/1/2009;