Introduction
Given the advances in the treatment of cancers, more people are surviving their battles with cancer such that around 0.4% of all young women are now cancer survivors2. The main treatment modalities in cancer are surgery, chemotherapy and radiotherapy. Chemotherapeutic agents can be gonadotoxic, though this is also dependent on the patient's age, the cumulative dose of the agent and previous cancer treatment3. Radiotherapy involving the pelvis or adjacent areas will almost invariably lead to abrupt and permanent ovarian failure4, and treatments not involving these areas still incur a risk of such dysfunction, from around 50% for breast cancer to around 15% for acute myeloid leukemia, for example5. Thus, a substantial number of young female cancer survivors have lost their fertility owing to their treatments, and, therefore, there is an increased demand for preservation of fertility among such patients as their numbers continue to grow. Fortunately, the clinical area of assisted reproduction has evolved such that this demand may perhaps be met by a combination of current techniques and those being perfected in the laboratory.
The ovary contains a pool of about 400,000 resting follicles at birth, and about 90% of these are of the earliest developmental stage (primordial follicles), when each oocyte is surrounded by a single layer of flattened granulosa cells6. After initiation of growth during fertility, the primordial follicle becomes a primary follicle (in which the granulosa cells become cuboidal) and then eventually transforms into secondary follicles by acquiring steroidogenically active theca cells that secrete the hormones needed for proper reproduction. At a later growth stage, a fluid-filled antrum forms inside the granulosa cell layer as the follicle forms a tertiary stage in preparation for preovulation. Whereas the duration of transition from a primordial follicle into a preovulatory follicle in vivo is relatively long (more than 200 days in humans6 and around 60 days in rodents7), during fertility, less than 0.1% of primordial follicles grow all the way into mature antral follicles that can ovulate, with the rest degenerating and being absorbed by the body at any stage of their development. Thus, the supply of mature follicles is limited.
Today, the only clinically established fertility preservation method is embryo cryopreservation after in vitro fertilization (IVF). However, this method is only possible in adult women and requires a male partner or a sperm donor. Cryopreservation of unfertilized oocytes or ovarian tissue should still be considered an experimental procedure with a low success rate in humans. Indeed, the live-birth rate after oocyte cryopreservation is around 2% per oocyte thawed8, and only five live births have been reported after ovarian tissue cryopreservation and subsequent autotransplantation9, 10, 11, 12. The largest number of female gametes is preserved by cryopreservation of the ovarian cortex, as occurs during ovarian tissue cryopreservation, or by cryopreservation of a complete ovary. However, whole-ovary cryopreservation is to date also an experimental procedure only tested in animal models and with a low success rate, as well13, 14.
Two major problems with ovarian tissue cryopreservation followed by autotransplantation are that only a small portion of the primordial follicles survive the ischemia that results from the nonvascular autotransplantation15 and that there is a risk of reintroducing metastatic cancer cells, which may be present within the freeze-thawed ovarian cortex16, if the tissue is derived from a cancer patient in the hope of restoring fertility after treatment. The report by Xu et al.1 describes a method to achieve maturity of immature mouse follicles in vitro, fertilizing them and producing normal offspring after embryo transfer into pseudopregnant mice. This methodology, if applicable in humans, would have the potential to cryopreserve an almost unlimited source of female gametes with no risk of cancer cell transmission after restoration of fertility.
In their study, Xu et al.1 mechanically and enzymatically isolated secondary follicles, belonging to the cohort of early growing follicles, from ovaries of prepubertal female mice. The follicles were placed individually into droplets of alginate, a copolymer extracted from seaweed. The alginate droplets were then transformed into hydrogel beads by chemical crosslinking. By this method, the follicles were cultured in a three-dimensional environment, which allowed follicle growth in all directions. Encapsulated follicles were cultured for eight days, after which the alginate beads were enzymatically removed by lyase treatment. During culture, the follicle size more than doubled, the oocyte size increased similarly to that of in vivo controls and a fluid-filled antrum was formed. Secretion of steroids from the cultured follicles mimicked the normal pattern. The
liberated antral follicles were then cultured in media containing human chorionic gonadotropin to induce oocyte maturation. Around 70% of oocytes completed meiosis, which was 20% lower than the in vivo maturation rate. The fertilization rate at IVF was 68% and 82% in oocytes from in vitro cultured follicles and in vivo controls, respectively. Live-birth rates from the limited number of fertilized oocytes that were transferred into the uteri of pseudopregnant mice were 20% for zygotes derived from in vitro grown follicles and 25% from in vivo controls. Both male and female offspring showed normal fertility after sexual maturation.
The report by Xu et al.1 is a considerable advancement in the field of in vitro maturation of female gametes, although it is not the first report of live births from in vitro grown mouse follicles. Previous studies have used conventional two-dimensional culture conditions17, 18, 19, though, notably, culture of isolated follicles has previously only been achieved with follicles of greater maturity than those used in the study by Xu et al.1 However, the method of Xu et al.1 cannot be directly applied to humans. One obstacle would be that the vast majority of follicles that survive ovarian tissue cryopreservation and thawing are the primordial follicles20. They are at a considerably earlier developmental stage than the secondary follicles used in the study by Xu et al.1 and do not contain any theca cells, which are formed from the surrounding interstitial stroma at a later developmental stage. Moreover, the culture period of eight days with development in vitro from a secondary to an antral follicle is only around one-third of the normal developmental time in vivo in the mouse7. This accelerated follicular development in vitro in an artificial milieu could result in epigenetic changes within the oocyte that may lead to late-onset diseases—an issue of concern with new techniques in assisted reproduction. Indeed, two signs of warning in this respect are that the first mouse produced by complete in vitro maturation encountered late-onset health problems including obesity and neurological abnormalities21, and that aberrant fetal growth and malformation patterns were seen after in vitro culture of sheep zygotes22. It is quite possible that a human oocyte grown in vitro will be even more sensitive, as the culture period would be much longer23. If the in vitro conditions could be further optimized, for example, by adding the sequential presence of various key growth factors24, there may be a possible way for efficient fertility preservation in humans on the basis of the technique developed by Xu et al.1
A suggested methodology (Fig. 1) would be to separately cryopreserve small pieces of human ovarian cortex, with each piece probably containing hundreds of primordial follicles. When fertility restoration is desired, the cortical pieces could be thawed one at a time and then cultured for a period of two to three weeks to allow for development of primordial follicles into secondary follicles23. Secondary follicles could then be isolated and cultured in a three-dimensional environment as described by Xu et al.1. At the isolation step of the secondary follicles from the ovarian cortex, any possible metastatic cancer cells in the ovarian stroma would be excluded, as the stroma would be separated away from the follicles. If any cancer cells were present within the isolated secondary follicles, it could easily be noted by the uninhibited proliferation of cancer cells, and those particular alginate beads could be excluded. A large number of mature oocytes could then be fertilized by IVF and the embryos stored for future use.
Figure 1: A plausible effective method for fertility preservation in humans on the basis of the methodology put forward by Xu et al. and previous observations.
Cryopreserved tissue slices of human ovarian cortex would be thawed individually and cultured in vitro for three weeks to allow for growth of follicles from the primordial to the secondary stage. These follicles could then be isolated and further cultured individually in vitro in a three-dimensional environment within alginate beads. When the follicles have reached the antral stage, they would be retrieved from the beads and exposed to human chorionic gonadotropin (hCG) to induce oocyte maturation. In vitro fertilization of mature oocytes would give a large number of embryos, with one or two of them transferred directly to the uterine cavity of the woman and the majority cryopreserved for future use.
Full size image (110 KB)Thus, the study of Xu et al.1 may open up new ways for efficient and safe fertility preservation in females that acquire cancer during childhood or normal reproductive ages. The technique should preferably be refined in several animal models, including a nonhuman primate species, before subsequent application in humans.
