Follicle development requires complex and coordinated interactions between both the oocyte and its associated somatic cells. In ovarian dysfunction, follicle development may be abnormal due to defective somatic cell function; for example, premature ovarian insufficiency or malignancies. Replacing defective somatic cells, using the reaggregated ovary (RO) technique, may ‘rescue’ follicle development. ROs containing mature follicles have been generated when transplanted to a host mouse to develop. We have developed a RO culture technique and the aims were to determine how follicle development differed between transplanted and cultured ROs, and the influence of ovarian age (P2 vs P6). Mouse ROs were cultured for 14 days; P2 and P6 ovaries cultured as Controls. Follicle development was compared to ROs transplanted for 14 days and ovaries from P16 and P20 mice. ROs generated from either P2 or P6 exhibited similar follicle development in culture whereas in vivo follicle development was more advanced in P6 ROs. Follicles were more developed in cultured ROs than transplanted ROs. However, follicles in cultured ROs and ovaries had smaller oocytes with fewer theca and granulosa cells than in vivo counterparts. Our results demonstrate the fluidity of follicle development despite ovary dissociation and that environment is more important to basal lamina formation and theca cell development. Furthermore, follicle development within cultured ROs appears to be independent of oocyte nest breakdown and primordial follicle formation in source ovaries. Our results highlight the need for understanding follicle development in vitro, particularly in the development of the RO technique as a potential fertility treatment.
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- Abstract: cryopreservation x
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- Abstract: in vitro follicle culture x
Belinda K M Lo, Sairah Sheikh and Suzannah A Williams
C L Lu, J Yan, X Zhi, X Xia, T R Wang, L Y Yan, Y Yu, T Ding, J M Gao, R Li and J Qiao
Fertility preservation is an important type of frontier scientific research in the field of reproductive health. The culture of ovarian cortices to i) initiate primordial follicle growth and ii) procure developing follicles for later oocyte maturation is a promising fertility preservation strategy, especially for older women or cancer patients. At present, this goal remains largely unsubstantiated in primates because of the difficulty in attaining relatively large follicles via ovarian cortex culture. To overcome this hurdle, we cultured macaque monkey ovarian cortices with FSH, kit ligand (KL), basic fibroblast growth factor (bFGF), and/or epidermal growth factor (EGF). The various factors and factor combinations promoted primordial follicle development to different extents. Notably, both bFF (bFGF, 100 ng/ml and FSH, 50 ng/ml) and KF (KL, 100 ng/ml and FSH, 50 ng/ml) contributed to the activation of primordial follicles at day 12 (D12) of culture, whereas at D18, the proportions of developing follicles were significantly higher in the bFF and KF groups relative to the other treatment groups, particularly in the bFF group. Estradiol and progesterone production were also highest in the bFF group, and primary follicle diameters were the largest. Up until D24, the bFF group still exhibited the highest proportion of developing follicles. In conclusion, the bFGF–FSH combination promotes nonhuman primate primordial follicle development in vitro, with the optimal experimental window within 18 days. These results provide evidence for the future success of human ovarian cortex culture and the eventual acquisition of mature human follicles or oocytes for fertility restoration.
Benjamin Fisch and Ronit Abir
Anti-cancer therapy, particularly chemotherapy, damages ovarian follicles and promotes ovarian failure. The only pharmacological means for protecting the ovaries from chemotherapy-induced injury is gonadotrophin-releasing hormone agonist, but its efficiency remains controversial; ovarian transposition is used to shield the ovary from radiation when indicated. Until the late 1990s, the only option for fertility preservation and restoration in women with cancer was embryo cryopreservation. The development of other assisted reproductive technologies such as mature oocyte cryopreservation and in vitro maturation of oocytes has contributed to fertility preservation. Treatment regimens to obtain mature oocytes/embryos have been modified to overcome various limitations of conventional ovarian stimulation protocols. In the last decades, several centres have begun cryopreserving ovarian samples containing primordial follicles from young patients before anti-cancer therapy. The first live birth following implantation of cryopreserved-thawed ovarian tissue was reported in 2004; since then, the number has risen to more than 130. Nowadays, ovarian tissue cryopreservation can be combined with in vitro maturation and vitrification of oocytes. The use of cryopreserved oocytes eliminates the risk posed by ovarian implantation of reseeding the cancer. Novel methods for enhancing follicular survival after implantation are presently being studied. In addition, researchers are currently investigating agents for ovarian protection. It is expected that the risk of reimplantation of malignant cells with ovarian grafts will be overcome with the putative development of an artificial ovary and an efficient follicle class- and species-dependent in vitro system for culturing primordial follicles.
Xiaoqian Wang, Sally Catt, Mulyoto Pangestu and Peter Temple-Smith
Cryopreservation of ovarian tissue is an important option for preserving the fertility of cancer patients undergoing chemotherapy and radiotherapy. In this study, we examined the viability and function of oocytes derived in vitro from pre-antral follicles as an alternative method for restoring fertility. Pre-antral follicles (specified as secondary follicle with a diameter around 100–130 μm) were mechanically isolated from vitrified-warmed and fresh adult mouse ovarian tissues and cultured for 12 days followed by an ovulation induction protocol at the end of this period to initiate oocyte maturation. Oocytes were then released from these follicles, fertilized in vitro, and cultured to the blastocyst stage and vitrified. After storage in liquid nitrogen for 2 weeks, groups of vitrified blastocysts were warmed and transferred into pseudo-pregnant recipient females. Although most of the isolated mouse pre-antral follicles from fresh (79.4%) and vitrified (75.0%) ovarian tissues survived the 12-day in vitro culture period, significantly fewer mature oocytes developed from vitrified-warmed pre-antral follicles than from the fresh controls (62.2 vs 86.4%, P<0.05). No difference was observed in embryo cleavage rates between these two groups, but the proportion of embryos that developed into blastocysts in the vitrification group was only half that of the controls (24.2 vs 47.2%, P<0.05). Nevertheless, live births of healthy normal pups were achieved after transfer of vitrified blastocysts derived from both experimental groups. This study shows that successful production of healthy offspring using an in vitro follicle culture system is feasible, and suggests that this procedure could be used in cancer patients who wish to preserve their fertility using ovarian tissue cryopreservation.
K. Jewgenow, L. M. Penfold, H. H. D. Meyer and D. E. Wildt
About 1500 preantral follicles can be recovered from a single cat ovary by mechanical dissection. This is a potentially rich source of genetic material if ova could be preserved and grown in vitro, especially from rare or endangered species that die abruptly or are ovariectomized for medical reasons. The aims of this study were to examine cryoprotectant toxicity and then the potential of successfully cryopreserving preantral cat follicles. In the initial toxicity trial, isolated cat follicles (40–90 μm) were exposed to dimethylsulfoxide, glycerol, 1,2-propandiol or ethylene glycol at 0°C for 15 min. Follicle viability was assessed by supravital staining using a combination of Trypan blue and Hoechst 33258 at 0 h, and after 18 h and 1 week of culture. Percentages of follicles with intact oocytes and granulosa cells were similar (P >0.05) among control (no cryoprotectant), dimethylsulfoxide, 1,2-propandiol and ethylene glycol treatments at all time points, but were reduced (P <0.05) after glycerol exposure. On the basis of this finding, dimethylsulfoxide and 1,2-propandiol were used to cryopreserve intact follicles, and post-thaw viability was assessed by supravital staining and 5-bromo-2′-deoxyuridine uptake into oocytes and granulosa cells during culture. Of control (noncryopreserved) follicles, 31.4% ± 2.9%, 18.8% ± 1.9% and 16.2% ± 1.6% were intact after 0 h, 18 h and 1 week of culture, respectively. Uptake of 5-bromo-2′-deoxyuridine occurred in approximately 20% of follicles at all time points. On the basis of the presence of both a healthy oocyte and granulosa cells, cryopreservation in dimethylsulfoxide or 1,2-propandiol allowed approximately 19% of follicles to survive. Approximately 10% demonstrated clear evidence of cell activity that was sustainable for 1 week. In conclusion, the cat ovary contains a population of preantral follicles that are not adversely affected by short-term exposure to most conventional cryoprotectants. Furthermore, there is a subpopulation of these follicles capable of surviving cryopreservation, remaining structurally intact and physiologically active after thawing.
Evelyn E Telfer
Ovarian cryopreservation rapidly developed from basic science to clinical application and can now be used to preserve the fertility of girls and young women at high risk of sterility. Primordial follicles can be cryopreserved in ovarian cortex for long-term storage and subsequently autografted back at an orthotopic or heterotopic site to restore fertility. However, autografting carries a risk of re-introducing cancer cells in patients with blood-born leukaemias or cancers with a high risk of ovarian metastasis. For these women fertility restoration could only be safely achieved in the laboratory by the complete in vitro growth (IVG) and maturation (IVM) of cryopreserved primordial follicles to fertile metaphase II (MII) oocytes. Culture systems to support the development of human oocytes have provided greater insight into the process of human oocyte development as well as having potential applications within the field of fertility preservation. The technology required to culture human follicles is extremely challenging, but significant advances have been made using animal models and translation to human. This review will detail the progress that has been made in developing human in vitro growth systems and consider the steps required to progress this technology towards clinical application.
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.
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.
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.
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.