Title: Ovarian follicle culture: advances and challenges for human and nonhuman primates
Abstract: The removal and cryostorage of ovarian cortical biopsies is now offered as a fertility preservation option for young women. The only available option to restore fertility using this tissue is by transplantation, which may not be possible for all patients. The full potential of this tissue to restore fertility could be achieved by the development of in vitro systems that support oocyte development from the most immature stages to maturation. The techniques of in vitro growth (IVG) combined with in vitro maturation (IVM) are being developed with human tissue, but comparing different systems has been difficult because of the scarcity of tissue so nonhuman primates are being used as model systems. There are many challenges to developing a complete culture system that would support human oocyte development, and this review outlines the approaches being taken by several groups using tissue from women and nonhuman primate models to support each of the stages of oocyte development. The removal and cryostorage of ovarian cortical biopsies is now offered as a fertility preservation option for young women. The only available option to restore fertility using this tissue is by transplantation, which may not be possible for all patients. The full potential of this tissue to restore fertility could be achieved by the development of in vitro systems that support oocyte development from the most immature stages to maturation. The techniques of in vitro growth (IVG) combined with in vitro maturation (IVM) are being developed with human tissue, but comparing different systems has been difficult because of the scarcity of tissue so nonhuman primates are being used as model systems. There are many challenges to developing a complete culture system that would support human oocyte development, and this review outlines the approaches being taken by several groups using tissue from women and nonhuman primate models to support each of the stages of oocyte development. Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/telfere-follicle-culture-human-oocytes-primates/ Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/telfere-follicle-culture-human-oocytes-primates/ The development of culture systems with the aim of growing oocytes from the earliest stage of follicle through to maturity for in vitro fertilization (IVF) could have a lasting impact on clinically assisted reproduction, particularly for the growing number of women who are surviving cancer only to face infertility as a result of the gametotoxic effects of cancer therapies (1Jeruss J.S. Woodruff T.K. Preservation of fertility in patients with cancer.N Engl J Med. 2009; 360: 902-911Crossref PubMed Scopus (479) Google Scholar). Recent success with ovarian tissue transplantation (2Donnez J. Silber S. Andersen C.Y. Demeestere I. Piver P. Meirow D. et al.Children born after autotransplantation of cryopreserved ovarian tissue. A review of 13 live births.Ann Med. 2011; 43: 437-450Crossref PubMed Scopus (290) Google Scholar, 3Andersen C.Y. Kristensen S.G. Greve T. Schmidt K.T. Cryopreservation of ovarian tissue for fertility preservation in young female oncological patients.Future Oncol. 2012; 8: 595-608Crossref PubMed Scopus (57) Google Scholar, 4Silber S.J. Barbey N. Scientific molecular basis for treatment of reproductive failure in the human: an insight into the future.Biochim Biophys Acta. 2012; 1822: 1981-1996Crossref PubMed Scopus (13) Google Scholar) has expanded interest in and efforts to cryopreserve and store ovarian tissue for future fertility. Ovarian tissue cryopreservation is the only option available for young patients without partners or who cannot undergo ovarian stimulation for oocyte/embryo cryopreservation and for patients for whom the risk for reintroduction of malignant cells precludes transplantation. Thus, the need for follicle culture systems that can efficiently use all classes of ovarian follicles, derived from clinically cryopreserved ovarian tissue, as sources of gametes would maximize reproductive potential for future fertility. However, complete in vitro growth (IVG) from immature primordial stages with subsequent IVF of oocytes followed by embryo transfer and production of live offspring has, so far, been achieved only in mice (5Eppig J.J. O'Brien M.J. Development in vitro of mouse oocytes from primordial follicles.Biol Reprod. 1996; 54: 197-207Crossref PubMed Scopus (785) Google Scholar, 6O'Brien M.J. Pendola J.K. Eppig J.J. A revised protocol for in vitro development of mouse oocytes from primordial follicles dramatically improves their developmental competence.Biol Reprod. 2003; 68: 1682-1686Crossref PubMed Scopus (433) Google Scholar). Several groups have focused on culturing later stages of follicle development from rodents and have produced developmentally competent oocytes and viable offspring (7Cortvrindt R. Smitz J. Van Steirteghem A.C. A morphological and functional study of the effect of slow freezing followed by complete in-vitro maturation of primary mouse ovarian follicles.Hum Reprod. 1996; 11: 2648-2655Crossref PubMed Scopus (68) Google Scholar, 8Eppig J.J. Schroeder A.C. Capacity of mouse oocytes from preantral follicles to undergo embryogenesis and development to live young after growth, maturation, and fertilization in vitro.Biol Reprod. 1989; 41: 268-276Crossref PubMed Scopus (489) Google Scholar, 9Roy S.K. Greenwald G.S. Hormonal requirements for the growth and differentiation of hamster preantral follicles in long-term culture.J Reprod Fertil. 1989; 87: 103-114Crossref PubMed Scopus (121) Google Scholar, 10Spears N. Boland N.I. Murray A.A. Gosden R.G. Mouse oocytes derived from in vitro grown primary ovarian follicles are fertile.Hum Reprod. 1994; 9: 527-532PubMed Google Scholar, 11Xu M. Kreeger P.K. Shea L.D. Woodruff T.K. Tissue-engineered follicles produce live, fertile offspring.Tissue Eng. 2006; 12: 2739-2746Crossref PubMed Scopus (285) Google Scholar). The success of these techniques has encouraged the demanding challenge of adapting them for humans and other primates. Although these techniques have been used to study the regulation of follicle development, the ultimate aim of follicle culture is to increase the availability of developmentally competent oocytes from what would be available from conventional methods, and there is still much to do before follicle culture can be used as a strategy for obtaining competent oocytes. In recent years a great deal of progress has been made in developing culture techniques for humans and nonhuman primates, and in this review we describe the technologies and discuss the prospects as well as the problems of applying them clinically. Female reproductive function requires cyclic development and maturation of ovarian follicles on a background of continuous activation from the pool of primordial follicles. Primordial follicles are formed before birth and represent a population of germ cells from which recruitment for growth occurs throughout the woman's reproductive life. Follicular growth and development involves a series of complex and precisely regulated events: 1) initiation of primordial follicle growth and development to the preantral follicle stage; 2) formation of antral follicles where expansion to the preovulatory or graafian follicle is associated with granulosa cell proliferation and antral fluid accumulation within the basement membrane; and 3) rupture of the graafian follicle, releasing a cumulus-oocyte complex at ovulation in response to the midcycle LH surge (Fig. 1A). As the oocyte grows within the follicle it is held in meiotic arrest at the dictyate stage of prophase I, but during development within the follicle it must acquire the ability to resume meiosis (meiotic competence) and the ability to support fertilization and embryonic development (developmental competence). Thus the oocyte depends on the local environment within the follicle for subsequent function as a gamete, and the formation and maintenance of connections facilitating bidirectional communication between the oocyte and granulosa cells are key to oocyte development in all species. The development of culture conditions for immature germ cells (both eggs and sperm) is one of the greatest technical challenges of reproductive technology. An understanding of the physiologic requirements of the oocyte, granulosa, theca, and perhaps even the stromal cells is needed. These requirements are complex and change during growth, so a major consideration is the starting point of the culture system, i.e., which stage of follicle to start with. The majority of follicles within the ovary in all young mammalian females are at the primordial stage of development, and those follicles are continually being used during reproductive life (12Gougeon A. Chainy G.B. Morphometric studies of small follicles in ovaries of women at different ages.J Reprod Fertil. 1987; 81: 433-442Crossref PubMed Scopus (218) Google Scholar). We do not know if this pool represents a homogeneous population but it is thought that at this stage follicles have not yet been exposed to selection processes that lead to follicle degeneration (13Picton H.M. Harris S.E. Muruvi W. Chambers E.L. The in vitro growth and maturation of follicles.Reproduction. 2008; 136: 703-715Crossref PubMed Scopus (209) Google Scholar, 14Thomas F.H. Walters K.A. Telfer E.E. How to make a good oocyte: An update on in vitro models to study follicle regulation.Hum Reprod Update. 2003; 9: 1-15Crossref PubMed Scopus (99) Google Scholar). Although rodents are excellent models for pioneering technologies, intermediate species are needed to test feasibility for human applications. Follicles of some domestic animals (cows, sheep, and goats) can resemble those of humans regarding growth rates and size, but the protracted length of folliculogenesis in vivo, estimated in women to be 90 days from the entrance of a preantral follicle into the growing pool to preovulatory follicles (15Gougeon A. Regulation of ovarian follicular development in primates: facts and hypotheses.Endocr Rev. 1996; 17: 121-155Crossref PubMed Scopus (1134) Google Scholar), and the long length of the follicular phase of the spontaneous menstrual cycle in nonhuman primates (∼2 weeks compared with a few days in domestic mammals) more closely reflect that of women. Therefore, nonhuman primates are emerging as an important translational model to advance technologic developments in follicle culture. Several approaches have been taken to develop human follicles in vitro with the use of fresh (16Telfer E.E. McLaughlin M. Ding C. Thong K.J. A two step serum free culture system supports development of human oocytes from primordial follicles in the presence of activin.Hum Reprod. 2008; 23: 1151-1158Crossref PubMed Scopus (354) Google Scholar, 17Hovatta O. Silye R. Abir R. Krausz T. Winston R.M. Extracellular matrix improves survival of both stored and fresh human primordial and primary ovarian follicles in long-term culture.Hum Reprod. 1997; 12: 1032-1036Crossref PubMed Scopus (242) Google Scholar) and thawed cryopreserved (17Hovatta O. Silye R. Abir R. Krausz T. Winston R.M. Extracellular matrix improves survival of both stored and fresh human primordial and primary ovarian follicles in long-term culture.Hum Reprod. 1997; 12: 1032-1036Crossref PubMed Scopus (242) Google Scholar, 18Picton H.M. Gosden R.G. In vitro growth of human primordial follicles from frozen-banked ovarian tissue.Mol Cell Endocrinol. 2000; 166: 27-35Crossref PubMed Scopus (61) Google Scholar) human cortical tissue. It is now clear that if we are to achieve complete development of human oocytes, a dynamic multistep culture system is required to support each of the transitional stages (16Telfer E.E. McLaughlin M. Ding C. Thong K.J. A two step serum free culture system supports development of human oocytes from primordial follicles in the presence of activin.Hum Reprod. 2008; 23: 1151-1158Crossref PubMed Scopus (354) Google Scholar, 19Telfer E.E. McLaughlin M. In vitro growth (IVG) systems for human oocytes: from primordial to maturation.in: Kim S.S. Donnez J. Principles and practice in fertility preservation. Cambridge University Press, Cambridge, UK2011: 397-408Crossref Scopus (2) Google Scholar, 20Telfer E.E. McLaughlin M. Human follicle activation and development in vitro.Semin Reprod Med. 2011; 29: 15-23Crossref PubMed Scopus (76) Google Scholar, 21Smitz J. Dolmans M.-M. Donnez J. Fortune J.E. Hovatta O. Jewgenow K. et al.Current achievements and future research directions in ovarian tissue culture, in vitro follicle development and transplantation: implications for fertility preservation.Hum Reprod Update. 2010; 16: 395-414Crossref PubMed Scopus (234) Google Scholar). The first step is to support the initiation of primordial follicle development and early growth, the second stage is to optimize the growth of follicles from preantral to antral stages, with the completion of oocyte growth being achieved during the third stage. The focus should be primarily on oocyte development, which may not require the development of large follicular structures but rather the maintenance of differentiated somatic cells in contact with the developing oocyte. A multistep system for IVG follicles has been proposed (22McLaughlin M. Telfer E.E. Oocyte development in bovine primordial follicles is promoted by activin and FSH within a two-step serum-free culture system.Reproduction. 2010; 139: 971-978Crossref PubMed Scopus (98) Google Scholar, 23Telfer E.E. McLaughlin M. Strategies to support human oocyte development in vitro.Int J Dev Biol. 2012; 56: 901-907Crossref PubMed Scopus (36) Google Scholar) to produce competent oocytes from human ovarian cortical tissue. The multistep approach needs to support the changing requirements of the developing oocyte and its surrounding somatic (granulosa) cells with the main focus being on maintaining oocyte–somatic cell interactions. Several groups have worked on each of the steps required to support human oocyte development in vitro: 1) activation of primordial follicles through culturing ovarian cortex (16Telfer E.E. McLaughlin M. Ding C. Thong K.J. A two step serum free culture system supports development of human oocytes from primordial follicles in the presence of activin.Hum Reprod. 2008; 23: 1151-1158Crossref PubMed Scopus (354) Google Scholar, 17Hovatta O. Silye R. Abir R. Krausz T. Winston R.M. Extracellular matrix improves survival of both stored and fresh human primordial and primary ovarian follicles in long-term culture.Hum Reprod. 1997; 12: 1032-1036Crossref PubMed Scopus (242) Google Scholar); 2) isolation and culture of growing preantral follicles to achieve oocyte growth and development (16Telfer E.E. McLaughlin M. Ding C. Thong K.J. A two step serum free culture system supports development of human oocytes from primordial follicles in the presence of activin.Hum Reprod. 2008; 23: 1151-1158Crossref PubMed Scopus (354) Google Scholar, 24Roy S.K. Treacy B.J. Isolation and long-term culture of human preantral follicles.Fertil Steril. 1993; 59: 783-790Crossref PubMed Google Scholar, 25Abir R. Franks S. Mobberley M.A. Moore P.A. Margara R.A. Winston R.M. Mechanical isolation and in vitro growth of preantral and small antral human follicles.Fertil Steril. 1997; 68: 682-688Abstract Full Text PDF PubMed Scopus (141) Google Scholar, 26Hovatta O. Wright C. Krausz T. Hardy K. Winston R.M. Human primordial, primary and secondary ovarian follicles in long-term culture: effect of partial isolation.Hum Reprod. 1999; 14: 2519-2524Crossref PubMed Scopus (189) Google Scholar, 27Xu J. Bernuci M.P. Lawson M.S. Yeoman R.R. Fisher T.E. Zelinski M.B. Stouffer R.L. Survival, growth, and maturation of secondary follicles from prepubertal, young and older adult, rhesus monkeys during encapsulated three-dimensional (3D) culture: effects of gonadotropins and insulin.Reproduction. 2010; 140: 685-697Crossref PubMed Scopus (105) Google Scholar, 28Xu J. Lawson M.S. Yeoman R.R. Zelinski M.B. Stouffer R.L. 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Hirshefeld-Cytron J. et al.In vitro oocyte maturation and preantral follicle culture from the luteal phase baboon ovary produce mature oocytes.Biol Reprod. 2011; 84: 680-697Google Scholar); and 3) aspiration and maturation of oocyte-cumulus complexes (32Alak B.M. Coskun S. Friedman C.I. Kennard E.A. Kim M.H. Seifer D.B. Activin A stimulates meiotic maturation of human oocytes and modulates granulosa cell steroidogenesis in vitro.Fertil Steril. 1998; 70: 1126-1130Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 33Cavilla J.L. Kennedy C.R. Byskov A.G. Hartshorne G.M. Human immature oocytes grow during culture for IVM.Hum Reprod. 2008; 23: 37-45Crossref PubMed Scopus (48) Google Scholar). The aim of ongoing research in this field has been to combine each of these steps to achieve complete development of human oocytes (16Telfer E.E. McLaughlin M. Ding C. Thong K.J. A two step serum free culture system supports development of human oocytes from primordial follicles in the presence of activin.Hum Reprod. 2008; 23: 1151-1158Crossref PubMed Scopus (354) Google Scholar). Progress in achieving this goal and the use of nonhuman primate models will be reviewed. The majority of follicles within ovarian cortical tissue are quiescent primordial, so the first consideration of an IVG system should be to optimize initiation of primordial follicles in vitro and support early follicle development. The factors regulating follicle initiation and early growth are still not well defined, but the process requires a combination of inhibitory, stimulatory, and maintenance factors (34Nelson S.M. Telfer E.E. Anderson R.A. The ageing ovary and uterus: new biological insights.Hum Reprod Update. 2013; 19: 67-83Crossref PubMed Scopus (159) Google Scholar). Studies using knockout mouse models have demonstrated the importance of the phosphatidylinositol-3′-kinase (PI3K)–Akt signaling pathway within the oocyte in regulating follicle activation (35Reddy P. Liu L. Adhikari D. Jagarlamudi K. Rajareddy S. Shen Y. et al.Oocyte-specific deletion of Pten causes premature activation of the primordial follicle pool.Science. 2008; 319: 611-613Crossref PubMed Scopus (565) Google Scholar). The phosphatase and tensin homologue deleted on chromosome ten (PTEN) acts as a negative regulator of this pathway and suppresses initiation of follicle development (35Reddy P. Liu L. Adhikari D. Jagarlamudi K. Rajareddy S. Shen Y. et al.Oocyte-specific deletion of Pten causes premature activation of the primordial follicle pool.Science. 2008; 319: 611-613Crossref PubMed Scopus (565) Google Scholar). The transcription factor FOXO3a is a downstream effector of this pathway and acts to inhibit follicle recruitment (36Castrillon D.H. Miao L. Kollipara R. Horner J.W. DePinho R.A. Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a.Science. 2003; 301: 215-218Crossref PubMed Scopus (705) Google Scholar). However, primordial follicles of fetal and juvenile macaque ovaries lack FOXO3a expression, suggesting that alternative transcription factors may mediate follicle activation in primates (37Ting A.Y. Zelinski M.B. Distribution of FOXO transcription factors in ovaries of fetal and juvenile rhesus macaques.Biol Reprod. 2012; 191Google Scholar). Other components of this pathway depend on the mammalian target of rapamycin complex 1 (mTORC1), a serine/threonine kinase that regulates cell growth and proliferation in response to growth factors and nutrients and regulates primordial follicle activation (38Adhikari D. Liu K. mTOR signaling in the control of activation of primordial follicles.Cell Cycle. 2010; 9: 1673-1674Crossref PubMed Scopus (60) Google Scholar). How these pathways regulate human follicle development is unclear, but culture models facilitate the study of these processes. Using pharmacologic inhibitors of PTEN in vitro, increased activation of presumably human primordial follicles has been demonstrated after xenotransplantation of ovarian cortical strips into immunodeficient mice (39Li J. Kawamura K. Cheng Y. Liu S. Klein C. Liu S. et al.Activation of dormant ovarian follicles to generate mature eggs.Proc Natl Acad Sci U S A. 2010; 107: 10280-10284Crossref PubMed Scopus (264) Google Scholar). A recent study using a human culture model has also demonstrated that treatment with rapamycin (an inhibitor of mTORC1) results in decreased activation of primordial follicles as well as oocyte loss in growing follicles (40McLaughlin M. Patrizio P. Kayisli U. Luk J. Thomson T.C. Anderson R.A. et al.mTOR kinase inhibition results in oocyte loss characterized by empty follicles in human ovarian cortical strips cultured in vitro.Fertil Steril. 2011; 96: 1154-1159Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Figure 1B illustrates components of the PI3K pathway and their influence on follicle activation. One limitation of determining primordial follicle activation with the use of cultured human ovarian cortex includes difficulty in assessing the follicular cohort within the starting material. In human (41Schmidt K.L. Byskov A.G. Andersen A.N. Andersen C.Y. Density and distribution of primordial follicles in single pieces of cortex from 21 patients and in individual pieces of cortex from three entire human ovaries.Hum Reprod. 2003; 18: 1158-1164Crossref PubMed Scopus (176) Google Scholar) and nonhuman primate (42Hornick J.E. Duncan F.E. Shea L.D. Woodruff T.K. Isolated primate primordial follicles require a rigid physical environment to survive and grow in vitro.Hum Reprod. 2012; 27: 1801-1810Crossref PubMed Scopus (109) Google Scholar) ovaries, the distribution of follicles within the ovarian cortex is heterogeneous. It is important to note that follicular density can vary more than two orders of magnitude in pieces of human cortical tissues from within the same ovary (41Schmidt K.L. Byskov A.G. Andersen A.N. Andersen C.Y. Density and distribution of primordial follicles in single pieces of cortex from 21 patients and in individual pieces of cortex from three entire human ovaries.Hum Reprod. 2003; 18: 1158-1164Crossref PubMed Scopus (176) Google Scholar). Each cortical piece has variation in number and stage distribution of follicles, so it is difficult to compare numbers of follicles in a piece of control starting tissue with those in pieces subjected to different treatments in vitro (43Rice S. Ojha K. Mason H. Human ovarian biopsies as a viable source of pre-antral follicles.Hum Reprod. 2008; 23: 600-605Crossref PubMed Scopus (29) Google Scholar, 44Ting A.Y. Yeoman R.R. Lawson M.S. Zelinski M.B. In vitro development of secondary follicles from cryopreserved rhesus macaque ovarian tissue after slow-rate freeze or vitrification.Hum Reprod. 2011; 26: 2461-2472Crossref PubMed Scopus (91) Google Scholar). Recently, a procedure using the vital dye neutral red was developed for visualizing preantral follicles within ovine cortical pieces and successfully applied to determining follicular density in human cortical tissue (45Chambers E.L. Gosden R.G. Yap C. Picton H.M. In situ identification of follicles in ovarian cortex as a tool for quantifying follicle density, viability and developmental potential in strategies to preserve female fertility.Hum Reprod. 2010; 25: 2559-2568Crossref PubMed Scopus (43) Google Scholar, 46Kristensen S.G. Rasmussen A. Byskov A.G. AndersenCY. Isolation of pre-antral follicles from human ovarian medulla tissue.Hum Reprod. 2011; 26: 157-166Crossref PubMed Scopus (68) Google Scholar). Whether primordial follicles can develop normally within the human or nonhuman primate ovarian cortex during in vitro culture after exposure to neutral red is not known, but if so, this could prove to be a useful tool for identifying pieces of cortex containing viable primordial follicles for experiments on follicle activation, as well as for screening pieces of tissue before ovarian transplantation. Human primordial follicles can be activated to grow and develop within mechanically loosened cortical pieces. Multilaminar preantral (secondary) stage follicles can be detected in this system after 6 days of culture (16Telfer E.E. McLaughlin M. Ding C. Thong K.J. A two step serum free culture system supports development of human oocytes from primordial follicles in the presence of activin.Hum Reprod. 2008; 23: 1151-1158Crossref PubMed Scopus (354) Google Scholar). This system differs from those described in other studies (17Hovatta O. Silye R. Abir R. Krausz T. Winston R.M. Extracellular matrix improves survival of both stored and fresh human primordial and primary ovarian follicles in long-term culture.Hum Reprod. 1997; 12: 1032-1036Crossref PubMed Scopus (242) Google Scholar, 26Hovatta O. Wright C. Krausz T. Hardy K. Winston R.M. Human primordial, primary and secondary ovarian follicles in long-term culture: effect of partial isolation.Hum Reprod. 1999; 14: 2519-2524Crossref PubMed Scopus (189) Google Scholar) because the culture medium is serum free and no supporting matrix is present. The vital step in this process lies in the preparation of tissue which involves the removal of most of the underlying stromal tissue and any growing follicles (Fig. 2). When these small fragments of human ovarian cortex are cultured, there is a significant shift of follicles from the quiescent to the growing pool over short culture periods of 6–10 days (16Telfer E.E. McLaughlin M. Ding C. Thong K.J. A two step serum free culture system supports development of human oocytes from primordial follicles in the presence of activin.Hum Reprod. 2008; 23: 1151-1158Crossref PubMed Scopus (354) Google Scholar), an observation repeated in cows, where extensive primordial activation has been reported within 2 days in vitro (22McLaughlin M. Telfer E.E. Oocyte development in bovine primordial follicles is promoted by activin and FSH within a two-step serum-free culture system.Reproduction. 2010; 139: 971-978Crossref PubMed Scopus (98) Google Scholar, 47Wandji S.A. Srsen V. Voss A.K. Eppig J.J. Fortune J.E. Initiation in vitro of growth of bovine primordial follicles.Biol Reprod. 1996; 55: 942-948Crossref PubMed Scopus (260) Google Scholar, 48Wandji S.A. Srsen V. Nathanielsz P.W. Eppig J.J. Fortune J.E. Initiation of growth of baboon primordial follicles in vitro.Hum Reprod. 1997; 12: 1993-2001Crossref PubMed Scopus (190) Google Scholar), indicating that activation results from a release from intraovarian factors that act to inhibit the initiation of follicle growth (49McLaughlin E.A. McIver S.C. Awakening the oocyte: controlling primordial follicle development.Reproduction. 2009; 37: 1-11Crossref Scopus (143) Google Scholar) (Fig. 2). It is clear that tissue shape and stromal density are important factors that contribute to the regulation of follicle growth initiation in vitro, because solid cubes or strips (1 mm thick) of human cortical tissue show less growth initiation (17Hovatta O. Silye R. Abir R. Krausz T. Winston R.M. Extracellular matrix improves survival of both stored and fresh human primordial and primary ovarian follicles in long-term culture.Hum Reprod. 1997; 12: 1032-1036Crossref PubMed Scopus (242) Google Scholar) and a high proportion of atretic follicles (50Sanfilippo S. Canis M. Romero S. Sion B. Déchelotte P. Pouly J.L. et al.Quality and functionality of human ovarian tissue after cryopreservation using an original slow freezing procedure.J Assist Reprod Genet. 2013; 30: 25-34Crossref PubMed Scopus (30) Google Scholar) compared with cortex cultured as flattened "sheets" where much of the underlying stroma is removed (16Telfer E.E. McLaughlin M. Ding C. Thong K.J. A two step serum free culture system supports development of human oocytes from primordial follicles in the presence of activin.Hum Reprod. 2008; 23: 1151-1158Crossref PubMed Scopus (354) Google Scholar). Likewise, relatively thin pieces of macaque cortical tissue (0.5 × 1 × 1 mm) exhibit atretic oocytes and follicles within contracted nonviable tissue by 7 days of culture (Ting, personal communication). The physical environment of the follicles within the cortical tissue affects their response to stimulatory and inhibitory factors and thus influences their ability to grow (49McLaughlin E.A. McIver S.C. Awakening the oocyte: controlling primordial follicle development.Reproduction. 2009; 37: 1-11Crossref Scopus (143) Google Scholar). Primordial follicles isolated enzymatically from nonhuman primate (42Hornick J.E. Duncan F.E. Shea L.D. Woodruff T.K. Isolated primate primordial follicles require a rigid physical environment to survive and grow in vitro.Hum Reprod. 2012; 27: 1801-1810Crossref PubMed Scopus (109) Google Scholar) and human (50Sanfilippo S. Canis M. Romero S. Sion B. Déchelotte P. Pouly J.L. et al.Quality and functionality of human ovarian tissue after cryopreservation using an original slow freezing procedure.J Assist Reprod Genet. 2013; 30: 25-34Crossref PubMed Scopus (30) Google Scholar, 51Amorim C.