In vitro maturation of primordial follicles after cryopreservation of human ovarian tissue: Problems remain

June 4, 2017 | Autor: Luigi Selvaggi | Categoria: Cryopreservation, Humans, Female, Ovarian Follicle, Neoplasms, Ovary, Oocytes, Ovary, Oocytes
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Med Pediatr Oncol 2002;38:153±157

OVERVIEW In Vitro Maturation of Primordial Follicles After Cryopreservation of Human Ovarian Tissue: Problems Remain Raffaella Depalo, MD,* Giuseppe Loverro,

MD,

and Luigi Selvaggi, MD

Key words: in vitro maturation; primordial follicle; human ovarian cortical tissue; cryopreservation

INTRODUCTION

Cryopreservation of fragments of ovarian cortex offers an opportunity to preserve gametes of women at risk of premature menopause. It is particularly indicated in women of pre- or post-puberal age affected by cancer that have to be subjected to chemo and/or radiotherapy. Fragments of ovarian cortex can easily be obtained by laparoscopic biopsy, and a small fragment contains a large number of quiescent primordial follicles. Moreover, these primordial follicles are freeze-resistant because they are made up of small cells, are less differentiated, and lack the zona pellucida and cortical granules. As they are metabolically quiescent, they have longer time to recover from any damage caused by freeze-thaw procedures during the long period of follicular maturation, so that they are less exposed to the risk of cytogenetic error due to structural damage. In the future, frozen-thawed ovarian tissue may be used for autotransplantation, or to induce oocyte growth and maturation by means of procedures for in vitro maturation (IVM) of primordial and primary follicles [1]. Although transplantation of frozen-thawed ovarian tissue has been successfully performed and has resulted in pregnancy in various animal species [2±5], one of the main obstacles to this procedure in patients affected by neoplastic disease is the risk of freezing cancerous cells that would then be transferred into the patient, now cancer-free, during autotransplant procedures [6]. IVM of immature oocytes from primordial and primary follicles is a viable alternative to autotransplantation of fragments of ovarian cortex. This technique has still to be developed for clinical application but might offer a chance of conception to young women affected by premature menopause following antineoplastic treatment, or by early ovarian failure. IVM of primordial and primary follicles could optimize assisted reproduction techniques, as it would minimize the costs of treatment, increase the number of oocytes recovered for fertilization, and avoid the risks of ovarian hyperstimulation syndrome. Other ß 2002 Wiley-Liss, Inc. DOI 10.1002/mpo.10053

clinical applications could include transferring the nucleus of oocytes contained in primordial and primary follicles into enucleated mature oocytes, and constituting a sort of oocyte bank for oodonation. Finally, IVM of primordial follicles into antral follicles that can release an MII-stage fertilizable oocyte would promote a better knowledge of the physiological events that take place during the process of oocyte development [7]. Pregnancy and live offspring have been obtained from fresh and frozen-thawed primary follicles [8,9], from IVM of mouse primordial follicles [10], and from fragments of bovine fetal ovarian cortex [11]. In 1965, Edwards succeeded in producing maturation of oocytes from antral follicles of humans, mice, sheep, pigs, and monkeys [12]. In 1991, Cha reported a successful pregnancy after IVM of human oocytes from antral follicles [13]. Fertilization induced by intracytoplasmatic injection of IVM oocytes obtained from small antral follicles during unstimulated cycles was reported by Trounson in 1994 [14]. Despite the ample volume of research, clinical application of IVM of human oocytes is con®ned to immature oocytes obtained from antral follicles that did not respond to gonadotropic stimulation by adequate nuclear maturation. Unfortunately, the excellent results obtained in animal species with IVM of oocytes from primordial follicles have still to be emulated in humans. OPEN PROBLEMS

One of the ®rst problems to be faced in IVM is the decision whether to subject single primordial follicles or the whole fragment of ovarian cortex to in vitro culture. ÐÐÐÐÐÐ

Institute of Obstetrics and Gynaecology II, University of Bari, Bari, Italy *Correspondence to: Raffaella Depalo, MD, Institute of Obstetrics and Gynaecology II, University of Bari, Piazza G. Cesare, 11-70124 Bari, Italy. E-mail: [email protected] Received 12 March 2001; Accepted 9 October 2001

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From the technical point of view, it is dif®cult to isolate the follicles from the dense ovarian stroma. In 1993, Roy and Treacy [15] succeeded in obtaining enzymatic isolation of preantral follicles by means of digestion with collagenase, while Cortvrindt et al [16] in 1996 and Abir et al in 1997 [17], isolated primordial follicles by disaggregation under a stereomicroscope, and Oktay et al., in 1996 [18] and Hovatta et al. [19] in 1999, employed a combination of both methods. Manual dissection is more laborious but avoids the chemical damage to cells and follicles caused by enzymatic digestion. The latter results in a loss of the stromal component surrounding the oocyte, of the layer of thecal cells, and the basal membrane that surrounds the granulosa cells (GC). The loss of these cellular interactions explains why follicles obtained with this method can only be maintained in culture for a very few days. Moreover, in vitro, follicles lacking the stromal cells and thecal layer rapidly attach to the culture dishes, GC quickly spread, and contact with the substrate hinders intact preservation of the unity of the follicle. Attachment to the surface of the culture wells can result in premature interruption of the cumulus±oocyte communications and thus impair the potential maturation process of the oocyte [20]. Even the normal spherical shape is lost during in vitro culture, or alternatively, the normal spherical shape can be maintained after embedding in collagen gel or agar, but fewer oocytes become fertile with this system [20]. It is known that the development of mammalian follicles from the primordial follicle stage to the preovulatory stage is a complex process that requires coordination between the oocyte and the surrounding thecal cells and granulosa. The oocyte and the somatic cells regulate the reciprocal functions by means of gap junctions and autocrine, paracrine, and endocrine signals. In view of these complex cellular interactions, in vitro culture systems must replicate the in vivo growth conditions and preserve the normal dynamic interactions between the oocyte and the follicle and stromal cells [20]. For these reasons, culture of whole fragments of the fragment of ovarian cortex is the in vitro culture model that best preserves the structural integrity of the follicle. A second problem is the question of the culture time needed for the oocyte to grow and develop into a mature oocyte. In different animal species, IVM is protracted for variable periods of time: in mice, a period of 3 weeks, corresponding more or less to the complete growth period in vivo, induces development of the primordial follicle up to the antral stage. An equivalent culture system of human follicles would take much longer, up to about 6 months, to allow growth and development of an oocyte to stage MII [21]. Moreover, the ®nal size of a mature human antral follicle is much greater than that of the other animal species studied.

Yet another problem is the role played by the use of normal (20%) or reduced (5%) oxygen tensions in the culture plant for primordial follicles. Data in the literature are highly controversial and the most appropriate oxygen tension for producing growth and maturation of follicular cells and hence the oocyte is still not known. Anoxia results in reduced production of steroids whereas high oxygenation reduces the rate of embryo development [22]. A controlled study by Smitz and Cortvrindt (1998), comparing 5 and 10% oxygen tension, demonstrated that lower oxygen tensions reduce follicle survival and impair the meiotic ability of the oocyte. These alterations are due to reduced availability of oxygen in the innermost layer of the GC [23]. In vitro culture conditions must allow oxygen to penetrate the various compartments of the follicle freely: high oxygen tension may be essential to regulate the penetration of the innermost cell layers. CULTURE SETUP

The speci®c IVM conditions need to be optimized, particularly when the immature oocytes have been subjected to stress during freeze-thaw procedures. Freezing induces a series of biophysical and osmotic stresses that can impair the normal process of fertilization and development of the oocyte. These result in a subtle structural change rather than in gross morphological destruction. Maturation of human ovarian follicles is a complex process that probably requires a very dynamic, variable system in the different stages of follicular growth and demands an optimal hormonal environment and the presence of various growth factors, some of which are known while others have still to be identi®ed. Gonadotropins may not be determinant in the preantral phases of folliculogenesis in vivo, but they acquire a preeminent role in vitro, as they activate cell proliferation and differentiation, increase the acquisition of gap junctions, and reduce atresia phenomena [24]. The presence of FSH and LH in the culture medium seems to be essential for expansion of the cumulus cells around the oocyte. LH has been shown to act on the somatic cells of the follicle by ``uncoupling'' the communication between the cumulus cells and the oocyte, thus allowing it to resume meiosis [25]. Abir et al. observed that formation of the antrum in human preantral follicles can only occur in the presence of adequate concentrations of FSH, while growth in size can be attained only when LH is added to the medium [17]. The inclusion of serum affords a complex blend of hormones, nutrients, growth and adhesion factors, including collagen and ®bronectin, that are needed to support cell proliferation, but the serum exerts an inhibitory effect

In Vitro Maturation Human Primordial Follicles

on follicular-type functions of the GC. Serum contains many components, such as amino acids, proteins, carbohydrates, growth factors, and many others that have not yet been identi®ed [26]. A serum-free culture system reduces the number of unde®ned elements, and is important to help understand the mechanisms that regulate growth and maturation of the oocyte. However, prolonged exposure to serum-free culture medium produces undesired effects on the oocyte, like premature release of cortical granules that leads to hardening of the zona pellucida, and reduction of the fertilizing potential of the oocyte. In a recent work on the ovarian cortex of bovines and primates, Wandij hypothesizes that serum factors in the culture medium inhibit the growth of the pre-GC [27]. When fragments of ovarian tissue are incubated in a serum-free medium, primordial follicles pass on to the primary stage. Once this has occurred, the physical and biochemical conditions are adapted to sustain further growth of the follicles. FOLLICULOGENESIS AND OOCYTE MATURATION

In vivo growth of a primordial follicle up to the antral stage1 is a very long, complex process during which the follicular volume increases approximately 300-fold [28]. What mechanism may trigger the growth of one follicle, while hundreds of others remain quiescent, is still a mystery. It is possible that local factors deriving from the interstitial tissue or factors secreted by other follicles as they grow, or else systemic factors, may play a determinant part [29]. The follicular maturation process is subdivided into two parts: initial and late folliculogenesis. Initial folliculogenesis is independent of the gonadotropins. The growth of the follicle and oocyte is regulated by paracrine and autocrine factors and lasts at least 180 days; the transition from a primordial to a primary follicle is not a process of growth but rather of slow maturation. At this stage, the oocyte has a diameter of 35 mm and is arrested in the diplotene meiotic process [30±31]. The follicle then becomes sensitive to gonadotropic stimulation. In response to incretions of FSH and LH, proliferation of the GC is observed, together with the appearance of thecal cells and an increase in the diameter of the follicle. During this phase of folliculogenesis, the oocyte forges ever closer relationships with the surrounding environment. The relationships between oocyte and GC arise through a communication network mediated by 1

The appearance of an antral cavity starts with the development of small ¯uid-®lled cavities of 40 mm in diameter that aggregate to form the antrum. From this point, the GC surrounding the oocytes constitute the cumulus oophorus [28].

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specialized junctions of gap junction type, so that the follicle and oocyte respond in a predestined manner to endocrine stimuli and those produced locally. The interaction between the oocyte and the follicular cells is twodirectional: many studies have shown that the oocyte probably plays a role in modulating steroidogenesis, differentiation of the GC, expansion of the cumulus, and the three-dimensional shape of the follicle itself. Thus, the importance of communication between the somatic and the germ compartments before the resumption of meiosis is clear. In the ®rst stages of follicular growth, there is no increase in the size of the oocyte or reactivation of meiosis. Oocyte activity in this stage is expressed by the synthesis of large aliquots of ribosomes and mRNA, the latter being used to form a reserve of essential proteins needed for the ®nal stage of oocyte maturation [32]. Steroid and androgen receptors have been demonstrated in the earliest phases of folliculogenesis and in primary follicles, while estrogen receptors have been observed on the ovarian cortex [33]. The follicle gradually becomes multilayered and is then de®ned as secondary. Beyond this period, the oocyte secretes glycoproteins that condense around it forming an acellular, translucid layer known as the zona pellucida. Contact between the oocyte and GC is maintained by cytoplasmatic processes that penetrate the zone to form gap junctions. In the pre-ovulatory phase, after a process that lasts 90 days in vivo, the oocyte with competent meiotic activity has reached the diameter of 120 mm. In vitro, with the technology now available, it is possible to culture human primordial follicles. In vitro, when the cortical tissue with its primordial follicles is transferred to any kind of culture system, spontaneous and massive follicular growth starts in the ®rst days of culture. This phenomenon makes it seem likely that in vitro follicular transformation is triggered by the loss of an inhibitory in¯uence of the ovarian network. After a few days of culture, mass transformation of the follicles from the primordial stage to the primary stage is observed. This transformation of primordial follicles into primary follicles signals the passage from a form of quiescence to growth, and is characterized by a series of morphological changes that include a transformation in the shape of the GC from ¯attened to cuboid, proliferation of the GC and alterations in the oocyte [34]. The oocyte is now about 35 mm in diameter. If the follicles have undergone stress from freeze-thaw techniques, or if the culture conditions are not ideal, necrosis and atresia of the oocyte and surrounding stromal cells are observed at this stage. Provided the follicular structures are intact and the culture conditions suitable, the culture system can be maintained for 4 weeks and some follicles can attain the secondary stage. In this next stage, the follicle acquires a layer of thecal cells and the

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oocyte increases in size from 25 to about 90 mm and acquires the zona pellucida. WORK REMAINING

It is vitally important to ®nd a culture system that can rapidly induce not only proliferation of the granulosa and stroma cells but also an increase in oocyte volume. CONCLUSIONS

Cryopreservation of ovarian cortical tissue and IVM of primordial and primary follicles offer new lines of study in the research and basic science ®elds. However, the scarcity of human oocytes has up to now delayed the progress of biological and developmental studies and at present the most advanced knowledge is based on the results of research conducted in other species that are often phylogenetically far from man. Studies on in vitro growth and maturation of primordial human follicles provide better insights into the metabolism of these cells, along with the markers of cell vitality and the causes of fertilization failure. This research therefore should open new frontiers in the treatment of female infertility. ACKNOWLEDGMENTS

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