Bench to Bedside
As chemotherapy and radiation treatments for cancer or other conditions can cause infertility, patients are encouraged to take steps to preserve their fertility before initiating gonadotoxic therapies. Sperm cryopreservation is standard for adult males and allows them to father genetically related children using intrauterine insemination (IUI), in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), which require millions, thousands or only one sperm, respectively. The cumulative live-birth rates using these standard assisted reproductive technologies is approximately 50%1,2. Unfortunately, these options are not available to prepubertal boys who do not make sperm, which poses an important health concern, as most of these young patients will survive their cancer and will have their reproductive years still in front of them. For this group and for adult survivors who did not preserve a sperm sample, several techniques exist in the research pipeline that may offer new opportunities to recover fertility (Fig. 1).
Although prepubertal boys are not producing sperm, they do have spermatogonial stem cells (SSCs) in their testes3,4 that are poised to initiate spermatogenesis at puberty. Several clinics around the world are cryopreserving testicular tissue or cells from prepubertal boys hoping that SSCs in that tissue may be used to generate spermatogenesis, for example by transplanting into the endogenous niches of the seminiferous tubules or autologous grafting of intact seminiferous tubules (Fig. 1). Successes in several animal models5 suggest that these techniques can be translated to the human fertility clinic. Yet, it is important to weigh the risk of the testicular biopsy procedure against the uncertain benefit of experimental therapies. Also, for individuals with blood-borne cancers or cancers that metastasize to the testes, there is a potential risk of reintroducing malignant cells when transplanting testicular tissues or heterogeneous testicular cell suspensions back into them. These concerns about clinical application have fueled the search for in vitro alternatives for generating transplantable germline stem cells (primordial germ cells or SSCs) or fertilization-competent sperm.
SSC transplantation as a means to preserve fertility (Fig. 1) is challenging owing to the small amount of tissue, and therefore SSCs, that can be obtained from testicular biopsy of prepubertal boys. Nonetheless, its feasibility is enhanced by observations in rodents that SSCs can be greatly expanded in culture, transplanted into the seminiferous tubules of infertile recipients, undergo spermatogenesis and give rise to live offspring6,7,8. These results suggest that stem cells may be generated from a small testicular biopsy. Two laboratories recently reported progress culturing human SSCs from the testes of prepubertal patients with cancer4,9. Although these human tissue studies are essential and promising, they will require a robust bioassay to assess the spermatogenic potential of cultured cells. Preclinical studies in nonhuman primate models of male infertility10 may help to fill this key technology gap.
There are conflicting reports about whether the risk of reintroducing occult malignant cells into survivors can be eliminated by presorting potentially therapeutic SSCs from heterogeneous testis cell suspensions, for instance by FACS before SSC transplantation5. But sorting is not an option for the autologous tissue transplantation paradigm (Fig. 1), where intact testicular tissues would be transplanted back into the patient. The risk of reintroducing cancer into an individual could be circumvented by the development of methods to produce sperm in vitro.
This year, Sato et al.11 demonstrated for the first time that fertilization-competent haploid germ cells from mice can be generated in organ culture. Testis fragments from 2.5- to 3.5-day-old mice that were initially devoid of haploid germ cells were placed on an agarose gel that was half-soaked in medium containing knockout serum replacement—a modification of a gas-liquid interface method devised to keep differentiated organs alive in vitro12. Haploid round spermatids or sperm were retrieved after 23 or 42 days in culture, respectively, and used to successfully fertilize mouse oocytes in vitro by round spermatid injection (ROSI) and ICSI. The resulting embryos generated live offspring with normal development and fertility11. Preliminary data indicated that establishing organ culture from frozen and thawed testis tissue might also be possible, but the fertilization potential of the resulting sperm was not tested11. Thus, a prepubertal individual with cancer could cryopreserve testicular tissue before gonadotoxic treatment that can be thawed and placed in organ culture when needed (Fig. 1). A single sperm can then be extracted and used for ICSI, eliminating the risk of reintroducing cancer cells.
In the last decade, the generation of germ cells from human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) has emerged as a possible way to treat azoospermic men who did not preserve semen samples before gonadotoxic treatment13,14,15. iPSC technology is particularly germane, because it could theoretically enable an infertile man to produce genetically related transplantable SSCs or sperm from iPSCs derived from his own somatic cells, including skin or blood cells. Putative primordial germ cells—which are SSC precursors—can now be created by differentiating human iPSCs in the presence of fetal gonadal stromal cells14 or the bone morphogenic proteins BMP4, BMP7 and BMP8b (ref. 15). The latter study further showed meiotic progression and generation of putative gametes by overexpression of deleted-in-azoopermia (DAZ) family proteins in iPSC-derived primordial germ cells15. These meiotic cells were unfortunately not clinical grade, as lentiviral vectors were used to introduce the DAZ proteins; however, this work provides important proof of principle and an opportunity to study human meiosis, in vitro.
In the human system, it is not possible to test experimentally whether the iPSC-derived primordial germ cells are transplantable or whether they require further differentiation into SSCs to acquire regenerative (spermatogenic) potential. Valuable insights were provided in a recent study in mice showing that mouse ESCs and iPSCs can be induced to an epiblast-like cell state that efficiently gives rise to cells similar to primordial germ cells when cultured with BMP4 (ref. 16). The resulting in vitro–derived mouse germ cells were transplanted into the testes of W/Wv mice, which lack endogenous spermatogenesis. Colonies of donor spermatogenesis that yielded functional sperm, which produced viable offspring after ICSI, were observed in recipient mice ten weeks after transplantation. These exciting results may open a new avenue for treating male infertility, but caution is warranted because some of the offspring obtained using this approach died prematurely with tumors around the neck region16. Additional studies are needed to characterize the genetic and epigenetic mechanisms of germ cell specification and pathogenesis and also to determine whether this work can be translated to higher primates where pluripotent stem cells and germ lineage development may differ from mice. If safety concerns are appropriately addressed in animal models, iPSC-derived primordial germ cells, SSCs or haploid germ cells may provide fertile opportunities for patients who did not preserve testicular tissue before undergoing gonadotoxic treatment (Fig. 1).
The germ cell research field is experiencing a renaissance of new discoveries as well as maturing technologies that are nearing clinical translation. This progress substantially improves the capacity to study germ lineage development, spermatogenesis and mechanisms of male fertility and infertility. Thus, it is reasonable to expect in the next decade or two that the options for preserving and restoring male fertility will expand from the current available approach of sperm freezing followed by IVF or ICSI to also include organ culture, tissue grafting, stem cell transplantation and in vitro derivation of gametes (Fig. 1). As with any rapidly developing field that has potential to affect the clinic, it will be essential to establish strict criteria to monitor progress and avoid sensationalism while giving careful attention to issues of safety and feasibility.
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Nature Reviews Urology (2015)