Individuals may regard reproduction as optional but sufficient number of them must be productive to perpetuate the species. The reproductive system is surprisingly vulnerable and depends, among other things, on a limited endowment of oocytes, controlled proliferation of spermatogonial stem cells and the genetic integrity of both. The developmental competence of oocytes and spermatogonial stem cells is maintained by evolved mechanisms for cellular detoxification and genomic stability, and excess or damaged cells are eliminated by apoptosis. Gonadal failure as a result of germ cell depletion can occur at any age, and from the effects of chemical cytotoxicity, disease and infection as well as genetic predisposition. Among extrinsic factors, alkylating agents and ionizing radiation are important causes of iatrogenic gonadal failure in young women and men. In animal models, there is evidence that hormonal manipulation, deletion of genes involved in apoptotic pathways and dietary manipulation can protect against natural and induced germ cell loss, but evidence in humans is absent or unclear. Assisted reproductive technologies (ARTs) provide an ensemble of strategies for preserving fertility in patients and commercially valuable or endangered species. Semen cryopreservation was the first technology for preserving male fertility, but this cannot serve prepubertal boys, for whom banking of testicular biopsies may provide a future option. In sterilized rodents, cryopreserved spermatogonial stem cells can recolonize seminiferous tubules and reinitiate spermatogenesis, and subcutaneous implantation of intact tubules can generate spermatozoa for fertilization in vitro by intracytoplasmic sperm injection. Transplantation of frozen-banked ovarian tissue is well-established for restoring cyclicity and fertility and is currently undergoing clinical evaluation for cancer patients. When restoration of natural fertility is unnecessary or reimplantation is unsafe, it is desirable to culture the germ cells from thawed tissue in vitro until they reach the stage at which they can be fertilized. Low temperature banking of immature germ cells is potentially very versatile, but storage of embryos and, to a lesser extent, mature oocytes is already practised in a number of species, including humans, and is likely to remain a mainstay for fertility preservation.
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- Abstract: Fertility preservation x
- Abstract: cryopreservation x
- Abstract: chemotherapy x
- Abstract: ovary transplantation x
- Abstract: cancer treatment x
- Abstract: ovarian transposition x
- Abstract: testicular transplantation x
- Abstract: xenograft x
- Abstract: oncofertility x
- Abstract: sperm banking x
R Gosden and M Nagano
R A Anderson
Human fertility is dependent on maturation of germ cells through meiosis and their association with supporting cells, which in the female are also the source of sex steroids. These processes are sensitive to both chemotherapy and radiotherapy thus can be damaged by anti-cancer treatments. The uterus is also sensitive to radiotherapy. Our understanding of and the ability to manipulate fertility has increased together with survival rates from many cancers, particularly those affecting children, younger men, and women. The growth of interest in fertility preservation for cancer patients is a natural union of these two fields. Sperm banking has been available for many years, and is a recognized and evidence-based option for men that should be available to all. Options for women and pre-pubertal boys and girls are, however, more experimental, other than for women of committing oocytes to fertilization and cryopreservation as embryos. This Focus Issue of Reproduction aims to address the current status of some of the clinical and laboratory aspects of this burgeoning subspecialty to highlight not only areas of progress but also areas of uncertainty where future developments are required to allow the provision of accurate information, and safe and effective treatments.
Teresa K Woodruff
In 2007, I was asked by the University of Calgary to participate in a symposium called ‘Pushing the Boundaries – Advances that Will Change the World in 20 Years’. My topic was oncofertility, a word I had just coined to describe the intersection of two disciplines – oncology and fertility – and I was thrilled to share my passion for this new field and help young women with cancer protect their future reproductive health. Fertility preservation in the cancer setting lacked a concerted effort to bridge the disciplines in an organized manner. In early 2015, I was delighted to deliver a presentation for the Society for Reproduction and Fertility titled ‘Sex in Three Cities’, where I gave an update on the oncofertility movement, a remarkable cross-disciplinary, global collaboration created to address the fertility preservation needs of young cancer patients. During my tour of the UK, I was impressed by the interest among the society and its members to engage colleagues outside the discipline as well as the public in a dialogue about cutting-edge reproductive science. In this invited review, I will describe the work of the Oncofertility Consortium to provide fertility preservation options in the cancer setting and accelerate the acceptance of this critical topic on a global scale. I hope that one day this word and field it created will change the world for women who had been left out of the equation for far too long.
Jill A Jenkins, Bruce E Eilts, Amy M Guitreau, Chester R Figiel, Rassa O Draugelis-Dale and Terrence R Tiersch
Flow cytometry (FCM) and computer-assisted sperm motion analysis (CASA) methods were developed and validated for use with endangered razorback suckers Xyrauchen texanus collected (n=64) during the 2006 spawning season. Sperm motility could be activated within osmolality ranges noted during milt collections (here 167–343 mOsm/kg). We hypothesized that sperm quality of milt collected into isoosmotic (302 mOsm/kg) or hyperosmotic (500 mOsm/kg) Hanks' balanced salt solution would not differ. Pre-freeze viabilities were similar between osmolalities (79%±6 (s.e.m.) and 76%±7); however, post-thaw values were greater in hyperosmotic buffer (27%±3 and 12%±2; P=0.0065), as was mitochondrial membrane potential (33%±4 and 13%±2; P=0.0048). Visual estimates of pre-freeze motility correlated with total (r=0.7589; range 23–82%) and progressive motility (r=0.7449) by CASA and were associated with greater viability (r=0.5985; P<0.0001). Count (FCM) was negatively correlated with post-thaw viability (r=−0.83; P=0.0116) and mitochondrial function (r=−0.91; P=0.0016). By FCM-based assessments of DNA integrity, whereby increased fluorochrome binding indicated more fragmentation, higher levels were negatively correlated with count (r=−0.77; P<0.0001) and pre-freeze viabilities (r=−0.66; P=0.0004). Fragmentation was higher in isotonic buffer (P=0.0234). To increase reproductive capacity of natural populations, the strategy and protocols developed can serve as a template for use with other imperiled fish species, biomonitoring, and genome banking.
Jose R Rodriguez-Sosa, Robert A Foster and Ann Hahnel
Xenografting of testicular tissue is an attractive new strategy for studying postnatal development of spermatogenesis and to preserve male genetics in large mammals. Typically, small cubes of immature testis (1 mm3) are grafted under the dorsal skin of immune-deficient mice. We attempted to increase the total number of seminiferous tubules in each xenograft with spermatogenesis by grafting flat strips of testis (∼9×5×1 mm) from ram lambs in immune-deficient mice. The percentage of grafts that survived and percentage of seminiferous tubules that developed spermatogenesis were the same as those reported after xenografting small cubes of lamb testis. Partially purified sheep spermatogonia were labeled with the fluorescent dye carboxy fluorescein diacetate succinyl diester and transplanted into the seminiferous tubules of one of the donor testis just before engraftment. The temporary label in the donor cells was detected for 4 weeks after xenografting, suggesting that co-engraftment of spermatogonia with testicular tissue may be a way to rapidly determine the effect of a specific gene on spermatogenesis. Finally, Sertoli cell lesions in xenografts of lamb testes were quantified, and their number and severity were found to increase, especially after grafts had been in place for 4 weeks. Although this coincided with the development of spermatogenesis, the extent of germ cell differentiation negatively correlated with severity of the lesions.
Jose R Rodriguez-Sosa, Guilherme M J Costa, Rahul Rathi, Luiz R França and Ina Dobrinski
Testis tissue xenografting is a powerful approach for the study of testis development and spermatogenesis, and for fertility preservation in immature individuals. In bovine testis xenografts, maturation and spermatogenesis are inefficient when compared to other species. To evaluate if exogenous modulation of the endocrine milieu in recipient mice will affect spermatogenic efficiency in xenografts from newborn calves, recipient mice were treated with the GnRH antagonist acyline (5 mg/kg s.c. every 2 weeks) to reduce testosterone production in xenografts, or with 6-N-propyl-2-thiouracil (PTU, 0.1% in drinking water for 4 weeks), to induce transient hypothyroidism in recipient mice respectively. Both treatments altered developmental parameters of testis xenografts and reduced germ cell differentiation. While the effects of acyline treatment can be attributed to inhibition of GnRH and gonadotropin action, lower Sertoli cell numbers and decreased seminiferous tubule length observed after PTU treatment were opposite to effects reported previously in rats. Regardless of treatment, Sertoli cells underwent only partial maturation in xenografts as Müllerian inhibiting substance and androgen receptor expression were lower than in donor and adult tissue controls respectively. In conclusion, although treatments did not result in improvement of maturation of bovine testis xenografts, the current study demonstrates that exogenous modulation of the endocrine milieu to affect xenograft development in recipient mice provides an accessible model to study endocrine control of spermatogenesis in large donor species.
Michiko Nakai, Hiroyuki Kaneko, Tamas Somfai, Naoki Maedomari, Manabu Ozawa, Junko Noguchi, Junya Ito, Naomi Kashiwazaki and Kazuhiro Kikuchi
Xenografting of testicular tissue into immunodeficient mice is known to be a valuable tool for facilitating the development of immature germ cells present in mammalian gonads. Spermatogenesis in xenografts and/or in vitro embryonic development to the blastocyst stage after ICSI of xenogeneic sperm has already been reported in large animals, including pigs; however, development of the embryos to term has not yet been confirmed. Therefore, in pigs, we evaluated the in vivo developmental ability of oocytes injected after ICSI of xenogeneic sperm. Testicular tissues prepared from neonatal piglets, which contain seminiferous cords consisting of only gonocytes/spermatogonia, were transplanted under the back skin of castrated nude mice. Between 133 and 280 days after xenografting, morphologically normal sperm were recovered, and a single spermatozoon was then injected into an in vitro matured porcine oocyte. After ICSI, the oocytes were electrostimulated and transferred into estrus-synchronized recipients. Two out of 23 recipient gilts gave birth to six piglets. Here, we describe for the first time that oocytes fertilized with a sperm from ectopic xenografts have the ability to develop to viable offspring in large mammals.
V von Schönfeldt, R Chandolia, L Kiesel, E Nieschlag, S Schlatt and B Sonntag
Improvements in cancer survival rates have renewed interest in the cryopreservation of ovarian tissue for fertility preservation. We used the marmoset as a non-human primate model to assess the effect of different cryoprotectives on follicular viability of prepubertal compared to adult ovarian tissue following xenografting. Cryopreservation was performed with dimethylsulfoxide (DMSO), 1,2-propanediol (PrOH), or ethylene glycol (EG) using a slow freezing protocol. Subsequently, nude mice received eight grafts per animal from the DMSO and the PrOH groups for a 4-week grafting period. Fresh, cryopreserved–thawed, and xenografted tissues were serially sectioned and evaluated for the number and morphology of follicles. In adult tissue, the percentage of morphologically normal primordial follicles significantly decreased from 41.2±4.5% (fresh) to 13.6±1.8 (DMSO), 9.5±1.7 (PrOH), or 6.8±1.0 (EG) following cryopreservation. After xenografting, the percentage of morphologically normal primordial (26.2±2.5%) and primary follicles (28.1±5.4%) in the DMSO group was significantly higher than that in the PrOH group (12.2±3 and 5.4±2.1% respectively). Proliferating cell nuclear antigen (PCNA) staining suggests the resumption of proliferative activity in all cellular compartments. In prepubertal tissues, primordial but not primary follicles display a similar sensitivity to cryopreservation, and no significant differences between DMSO and PrOH following xenografting were observed. In conclusion, DMSO shows a superior protective effect on follicular morphology compared with PrOH and EG in cryopreserved tissues. Xenografting has confirmed better efficacy of DMSO versus PrOH in adult but not in prepubertal tissues, probably owing to a greater capacity of younger animals to compensate for cryoinjury.
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.
Karen R Kilcoyne and Rod T Mitchell
Transplantation of testicular tissues and cells has been proposed as a future clinical option for patients who have had testicular tissue cryopreserved prior to receiving gonadotoxic therapies. Whilst this approach remains experimental, success using animal models and successful transplantation of ovarian tissue resulting in live births in female patients provides optimism for the development of clinical applications involving transplantation of testicular tissue in males. Careful consideration must be given to patient groups that may benefit from this approach in the future. Current research is focused on optimising patient selection, methods for tissue cryopreservation and development of transplantation techniques that might restore sperm production or future fertility in males. Crucially, attention must be focused on ensuring safety of transplantation, including eliminating the potential for infection or re-introducing malignancy. Furthermore the genetic/epigenetic integrity of any gametes generated must be ensured to allow generation of normal offspring. This review will provide an overview of the current status of transplantation of testicular tissue and cells for fertility preservation in males.