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Anamaria C Herta, Francesca Lolicato and Johan E J Smitz

The currently available assisted reproduction techniques for fertility preservation (i.e. in vitro maturation (IVM) and in vitro fertilization) are insufficient as stand-alone procedures as only few reproductive cells can be conserved with these techniques. Oocytes in primordial follicles are well suited to survive the cryopreservation procedure and of use as valuable starting material for fertilization, on the condition that these could be grown up to fully matured oocytes. Our understanding of the biological mechanisms directing primordial follicle activation has increased over the last years and this knowledge has paved the way toward clinical applications. New multistep in vitro systems are making use of purified precursor cells and extracellular matrix components and by applying bio-printing technologies, an adequate follicular niche can be built. IVM of human oocytes is clinically applied in patients with polycystic ovary/polycystic ovary syndrome; related knowhow could become useful for fertility preservation and for patients with maturation failure and follicle-stimulating hormone resistance. The expectations from the research on human ovarian tissue and immature oocytes cultures, in combination with the improved vitrification methods, are high as these technologies can offer realistic potential for fertility preservation.

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Hiroyuki Kaneko, Kazuhiro Kikuchi, Junko Noguchi, Manabu Ozawa, Katsuhiko Ohnuma, Naoki Maedomari and Naomi Kashiwazaki

Our objective was to improve the developmental ability of oocytes in porcine primordial follicles xenografted to nude mice, by treating the host mice with gonadotrophins to accelerate follicular growth. Ovarian tissues from 20-day-old piglets, in which most of the follicles were primordial, were transplanted under the kidney capsules of ovariectomized nude mice. Gonadotrophin treatments were commenced around 60 days after vaginal cornification in the mice. Ovarian grafts were obtained 2 or 3 days after treatment with equine chorionic gonadotrophin (eCG-2 and eCG-3 groups), after porcine FSH infusion for 7 or 14 days, or after infusion of porcine FSH for 14 days with a single injection of estradiol antiserum (FSH-7, FSH-14 and FSH-14EA groups, respectively). Gonadotrophin treatments accelerated follicular growth within the xenografts compared with that in control mice given no gonadotrophins, consistent with higher (P < 0.05) circulating inhibin levels in the gonadotrophin-treated mice. In contrast, circulating mouse FSH levels were significantly (P < 0.05) depressed. We recovered large numbers of full-sized oocytes with meiotic competence to the mature stage from the eCG-3, FSH-7, and FSH-14EA, unlike in the control group. Moreover, 56% of matured oocytes with the first polar body (n = 39) were fertilized in vitro in the FSH-14EA group. After in vitro fertilization and subsequent culture for 7 days, one blastocyst was obtained from each of the eCG-3, FSH-7 and, FSH-14EA groups, whereas no blastocysts appeared in the other groups. Exogenous gonadotrophins –not mouse FSH – stimulated the growing follicles that had developed from the primordial follicles in the xenografts: the effects were incomplete but improved to some extent the meiotic and developmental abilities of the oocytes.

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GM Hartshorne

This review offers a practically oriented introduction to follicle culture in vitro, focusing on mouse follicles, but with reference to other species. The main principles of follicle growth are addressed, including the constraints of tissue culture, methods of follicle isolation, and techniques for individual and collective culture of intact follicles. Culture systems that support a spherical or a non-spherical follicular structure in vitro are discussed in terms of follicular and oocyte development, and methods for assessing follicular function in vitro are presented. Oocyte development in most in vitro culture systems is currently suboptimal and the parallel development of oocytes and follicles is discussed, with a view to maintaining the competence of the oocyte. Finally, some potential future applications of follicle growth in vitro are suggested.

Free access

H M Picton, S E Harris, W Muruvi and E L Chambers

The development of technologies to grow oocytes from the most abundant primordial follicles to maturity in vitro holds many attractions for clinical practice, animal production technology and research. The production of fertile oocytes and live offspring has been achieved in mice following the long-term culture of oocytes in primordial follicles from both fresh and cryopreserved ovarian tissue. In contrast, in non-rodent species advances in follicle culture are centred on the growth of isolated preantral follicles. As a functional unit, mammalian preantral follicles are well-suited to culture but primordial and primary follicles do not grow well after isolation from the ovarian stroma. The current challenges for follicle culture are numerous and include: optimisation of culture media and the tailoring of culture environments to match the physiological needs of the cell in vivo; the maintenance of cell–cell communication and signalling during culture; and the evaluation of the epigenetic status, genetic health and fertility of in vitro derived mature oocytes. In large animals and humans, the complete in vitro growth and maturation of oocytes is only likely to be achieved following the development of a multistage strategy that closely mimics the ovary in vivo. In this approach, primordial follicle growth will be initiated in situ by the culture of ovarian cortex. Isolated preantral follicles will then be grown to antral stages before steroidogenic function is induced in the somatic cells. Finally, cytoplasmic and nuclear maturation will be induced in the in vitro derived oocytes with the production of fertile metaphase II gametes.

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V Isachenko, I Lapidus, E Isachenko, A Krivokharchenko, R Kreienberg, M Woriedh, M Bader and J M Weiss

Cryopreservation as a process can be divided into two methods: conventional freezing and vitrification. The high effectiveness of vitrification in comparison with conventional freezing for human oocytes and embryos is shown, whereas data on human ovarian tissue are limited. The aim of this study was to compare the safety and effectiveness of conventional freezing and vitrification of human ovarian tissue. Ovarian tissue fragments from 15 patients were transported to the laboratory within 22–25 h in a special, isolated transport box that can maintain a stable temperature of between 5 and 8 °C for 36 h. Small pieces of ovarian tissue (0.3–1×1–1.5×0.7–1 mm) were randomly distributed into three groups: group 1, fresh pieces immediately after receiving transport box (control); group 2, pieces after vitrification; and group 3, pieces after conventional freezing. After thawing, all the pieces were cultured in vitro. The viability and proliferative capacity of the tissue by in vitro production of hormones, development of follicles, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression after culture were evaluated. A difference between freezing and vitrification was not found in respect to hormonal activity and follicle quality. The supernatants showed 17-β estradiol concentrations of 365, 285, and 300 pg/ml respectively, and progesterone concentrations of 3.82, 1.99, and 1.95 ng/ml respectively. It was detected that 95, 80, and 83% follicles respectively were morphologically normal. The molecular biological analysis, however, demonstrated that the GAPDH gene expression in ovarian tissue after vitrification was dramatically decreased in contrast to conventional freezing. For cryopreservation of human ovarian tissue, conventional freezing is more promising than vitrification, because of higher developmental potential.

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A Nation and L Selwood

A model marsupial culture system has been developed whereby individual primary follicles, obtained from adult ovaries, can be grown in vitro to the antral stage and oocytes retrieved from these follicles can achieve nuclear maturation (metaphase II) in the presence of LH. Primary follicles isolated from adult Sminthopsis macroura ovaries were cultured individually in one of four systems: microdrops under oil, upright, inverted, or roller culture. After 6 days of culture, cumulus–oocyte complexes (COCs) were excised from early antral follicles and incubated for an additional 24 h to assess meiotic competence and the effects of LH and lithium on oocyte maturation. Histology and transmission electron microscopy established normal in vivo standards and verified oocyte and follicular integrity following culture. On day 6 of culture, follicle viability was significantly greater in the inverted system (73%) than in the other three systems (10–46%). The inverted system was the most effective in supporting development with follicles demonstrating progressive growth during culture and showing antral signs by day 4. Meiotic resumption during COC culture was facilitated by LH, but hindered by lithium. The ability to resume meiosis and progress to metaphase II was equivalent in oocytes retrieved following follicle culture and those matured in vivo. This study highlights the importance of oxygen and nutrient availability during marsupial follicle culture, and demonstrates for the first time that primary follicles isolated from adult mammalian ovaries can undergo normal growth and development in vitro, to produce mature, meiotically competent oocytes.

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R. Qvist, L. F. Blackwell, H. Bourne and J. B. Brown

Summary. An in-vitro culture system was developed in which primary mouse follicles from 12–16-day-old mice grew to the preovulatory stage. The important determinants of growth in culture were the inclusion of stroma with the primary follicles, the age of the mouse, the presence of FSH and LH, the use of culture dishes with a hydrophobic membrane and the use of post-menopausal human serum to supply growth factors. During culture the pieces of ovarian tissue containing the primary follicles coalesced to form characteristic spherical clusters. The cultured follicles appeared to be normal as determined by the appearance and organization of the granulosa cells, the appearance of the antrum and the accompanying steroidogenesis, but the ova had not resumed meiosis. The results show that the growth of mouse follicles starting from the primary stage is critically dependent on adequate concentrations of FSH.

Keywords: FSH; mouse; culture; follicle; in vitro

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Marie McLaughlin and Evelyn E Telfer

Quiescent follicles of large mammals initiate growth within cultured pieces of ovarian cortex. Systems capable of sustaining in vitro development from this early stage until oocyte maturation would allow investigation of mechanisms regulating oocyte development in its entirety. The aims of this study were 1) to determine whether bovine follicles initiated to grow in vitro could be isolated from the cortical environment, and could undergo further development and 2) to evaluate the effect of activin and FSH on the development of secondary follicles derived from primordial follicles. Fragments of bovine ovarian cortex were cultured in serum-free medium for 6 days; thereafter, secondary follicles were isolated for further culture. After a maximum total of 21 days in vitro, follicles were either processed for histological assessment or opened to release the oocyte–cumulus complexes for inspection by light microscopy. Compared with control, significant follicle and oocyte growth were observed in activin-exposed follicles, with or without FSH, with some oocyte diameters measuring over 100 microns following a total in vitro period of 15 days. Significant oestradiol secretion was observed in follicles cultured in activin alone after a total of 9 days in vitro compared with other treatment groups; however, this effect was not sustained. In summary, this study demonstrates the promotion of primordial bovine follicle development within a two-step serum-free culture system with oocyte diameters >100 μm achieved over 15 days in vitro. Further development of this system is needed to support complete oocyte growth and thereafter in vitro maturation.

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N. Spears, J. P. de Bruin and R. G. Gosden

An in vitro model of dominant and subordinate ovarian follicles was developed to allow a closer investigation of the phenomenon of follicular dominance. Preantral mouse ovarian follicles were cultured either alone or in pairs. Pairs of follicles were either in direct contact or in shared medium, but without physical contact. The experiments showed that where contact was allowed to develop between follicles one follicle invariably became dominant, while the other would grow and develop little during the culture period. In contrast, there was no effect of co-culture on follicle development in the absence of contact between the follicles. There was, therefore, no evidence of secretion of a diffusible factor by a dominant follicle that could affect the development of neighbouring follicles. After 6 days of co-culture with contact, histological examination of the subordinate follicle showed that it was healthy, in spite of remarkably little growth during culture. In a further experiment, the subordinate follicle was separated from the dominant one after 2 days of co-culture (when a significant difference in size had already developed), and cultured alone. These 'released' follicles exhibited a spurt of growth during the remaining culture period, attaining a size and appearance indistinguishable from those of controls by the end of culture. This confirms that the dominant follicle, while depressing the growth of its neighbour, is not inducing irreversible atresia in the subordinate follicle in this model. The in vitro model will allow a more detailed study of direct influences of dominant–subordinate follicle interactions, and should increase our knowledge of a poorly understood phenomenon.

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Studies of the effect of gonadotrophins on the cumulus and oocyte or on the whole follicle in vitro offer a good tool for understanding the mechanisms of oocyte maturation, the ability of follicles of different sizes to respond to gonadotrophins and the processes of atresia. The birth of normal young from intrafollicular oocytes matured in vitro furnishes proof that in-vitro techniques are valuable as long as follicular integrity is maintained during the whole culture period.