Biological Therapy for Non-Obstructive Azoospermia Sarah C

EXPERT OPINION ON BIOLOGICAL THERAPY, 2018 VOL. 18, NO. 1, 19–23 https://doi.org/10.1080/14712598.2018.1380622

REVIEW Biological therapy for non-obstructive azoospermia Sarah C. Vija, Edmund Sabanegh Jrb and Ashok Agarwalc aGlickman Urologic and Kidney Institute, Cleveland Clinic Foundation, Cleveland, OH, USA; bDepartment of Urology, Glickman Urologic and Kidney Institute, Cleveland Clinic Foundation, Cleveland, OH, USA; cAndrology Laboratory, Glickman Urologic and Kidney Institute, Cleveland Clinic Foundation, Cleveland, OH, USA

ABSTRACT ARTICLE HISTORY Introduction: Most male patients with non-obstructive azoospermia (NOA) have no therapeutic options Received 26 June 2017 outside of assisted reproductive techniques to conceive a biological child. If mature sperm cannot be Accepted 13 September 2017 obtained from the testes, these patients must rely on options of donor sperm or adoption. Several KEYWORDS techniques are in the experimental stage to provide this patient population alternatives for conceiving. Gene therapy; Areas covered: This review discusses three of the experimental techniques for restoring fertility in men non-obstructive with NOA: spermatogonial stem cell transplantation, the use of adult and embryonic stem cells to azoospermia; develop mature gametes and gene therapy. After this discussion, the authors give their expert opinion spermatogonial stem cell and provide the reader with their perspectives for the future. transplantation; pluripotent Expert opinion: Several limitations, both technical and ethical, exist for spermatogonial stem cell stem cell transplantation, the use of stem cells and gene therapy. Well-defined reproducible protocols are necessary. Furthermore, several technical barriers exist for all protocols. And while success has been achieved in animal models, future research is still required in human models.

1. Introduction Secondary testis failure is caused by impaired pituitary secretion of gonadotropins, which leads to insufficient stimulation of Infertility affects approximately 15% of couples of reproduc- the Sertoli and Leydig cells in the testis. Kallmann syndrome tive age. Male factor infertility plays a role in 50% of these results from a lack of hypothalamic Gonadotropic Releasing cases. Among patients with male factor infertility, approxi- Hormone (GnRH) secretion, which leads to insufficient produc- mately 10–15% have azoospermia [1]. Azoospermia is defined tion of LH and FSH. This subsequently leads to secondary as the absence of spermatozoa in the ejaculate. This condition testicular failure as the Sertoli cells are unable to support sper- is typically classified as obstructive azoospermia (OA) or non- matogenesis and the Leydig cells are insufficiently stimulated to obstructive azoospermia (NOA). After confirmation of NOA provide local testosterone secretion for spermatogenesis. Men with a second semen analysis, the workup of azoospermia who take supplemental testosterone will also develop second- involves a detailed history and physical examination along ary testicular failure because the supplemental circulating tes- with hormonal and genetic testing. Testicular biopsy allows tosterone suppresses FSH and LH production. for a histologic diagnosis although the underlying cause of the Men with NOA have limited options for reproduction. azoospermia may never be determined. OA is characterized by Testicular sperm extraction with Intracytoplasmic Sperm Downloaded by [New York University] at 10:52 20 December 2017 an obstructed flow of spermatozoa at any point along the Injection (ICSI) is possible in these patients, but it is expensive male genital tract. Spermatogenesis in the testis is usually and associated with high morbidity in the female partner. If normal in these patients. The etiology of NOA is more varied. sperm cannot be retrieved by testicular sperm extraction, Normal spermatogenesis requires intact and functioning semi- there are no current options to maintain the reproductive niferous tubules as well as functioning Leydig cells for hormo- potential of these individuals. Therefore, there is significant nal support of spermatogenesis by producing testosterone. clinical demand for alternate therapies for NOA that would NOA can be broadly classified into primary testicular failure allow the production of mature spermatozoa either using and secondary testicular failure. Primary testicular failure refers stem cells or by gene therapy for patients with idiopathic to pathology localized to the testis resulting in elevated causes of infertility. Such a technique might allow for natural Follicle Stimulating Hormone (FSH) and Luteinizing Hormone conception in a population that is otherwise relegated to (LH). Known genetic causes of infertility such as Klinefelter’s assisted reproductive techniques. This review will cover three syndrome and Y chromosome microdeletion will cause pri- experimental approaches for restoring fertility in men with mary testis failure. Local effects from chemotherapy or radia- NOA: spermatogonial stem cell (SSC) transplantation, in vitro tion can also cause primary testis failure and result in NOA. A spermatogenesis using adult or embryonic pluripotent stem significant proportion of primary testicular failure cases are cells, and gene therapy. diagnosed as idiopathic, which limits therapeutic options.

relevant: the transfer of the tissue back into the patient post- Article highlights pubertally with successful regeneration of spermatogenesis. Testicular tissue grafting can be performed in several loca- ● Existing therapeutics for non-obstructive azoospermia (NOA) are limited and depend on the ability to harvest mature sperm from the tions. The ideal location for graft transplantation is the scrotum testis for ICSI because its temperature is lower than that in other sites that ● Spermatogonial stem cell transplantation is a promising option to have been applied in animal models such as the peritoneum or repopulate the germinal epithelium in men with NOA ● The use of embryonic and adult stem cells to develop mature male under the skin on the back [9]. Grafting in ectopic sites also gametes may provide patients with an alternative therapeutic option. requires that the patient use Assisted Reproductive Techniques ● At present, a complete understanding of the stem cell niche is still (ART) for conception. Some authors recommend grafting to lacking ● For cases of NOA caused by a to a mutation at a single gene locus, several sites to optimize yield [10]. Timing of grafting, prepuber- gene therapy may be a definitive treatment for infertility. tal versus postpubertal, has also been debated. High FSH and LH This box summarizes key points contained in the article. concentrations are required for successful proliferation of the graft; therefore, the peri-pubertal period may be an ideal target time, but this has not been explored. Because prepubertal boys do not yet make enough FSH and LH to support spermatogenesis, the ideal timing for grafting may be after patients have reached sexual maturity [10]. 2. SSC transplantation Schlatt et al. performed a grafting procedure in neonatal SSCs are precursors to mature spermatids that reside in the mouse model after castration to maintain high levels of FSH basal compartment of the seminiferous tubules of the testis. and LH [11]. This method, of course, cannot be replicated in SSCs have the capability to self-renew and enter into meiosis humans, but it provides evidence that high levels of gonado- to become a spermatocyte [2]. The spermatocytes then differ- tropins are necessary for successful grafting. entiate into haploid spermatids, which are transformed into SSC injection is the other method of transfer to the recipi- mature spermatozoa. The SSC population is therefore critical ent. The rete testis, efferent ducts, or the seminiferous tubules in the production of sperm. have been identified as injection targets for SSCs in several Sertoli cells surround the SSCs and provide signaling mole- species [12]. Upon injection, the SSCs migrate to the basement cules and factors to support both the self-renewal and differ- membrane of seminiferous tubules and subsequently self- entiation process. This microenvironment is referred to as the renew and differentiate to establish spermatogenesis in the stem cell niche [3]. Recreating the SSC niche in the laboratory recipient. This process should theoretically allow for concep- setting is a critical step in the development of SSC transplan- tion without the requirement of assisted reproductive techni- tation. The concept of transplanting SSCs to enable normal ques, which is a major advantage. Hermann et al. injected spermatogenesis in an otherwise sterile individual has promis- adult rhesus monkey SSCs into the rete testes of recipient ing clinical applications in men with NOA. prepubertal rhesus monkeys and showed that mature sperma- Transplantation of SSCs was first described by Brinster and tozoa were present in the ejaculate when they reached matur- Zimmerman in 1994 when suspensions of testicular cells from ity. These spermatozoa demonstrated fertilizing capabilities fertile mice were transplanted into infertile mice resulting in when used with ICSI [13]. fertility restoration and progeny [4]. Subsequently, it has been At present, SSC transplantation is experimental and has not shown that cryopreservation of SSCs does not impair their been done in human models, but research thus far is promis- ability to produce healthy spermatozoa capable of fertilization ing. Several challenges have plagued the transformation of [5]. Currently, two strategies exist for transplantation of SSCs – this idea into a real clinical therapy for patients: identifying Downloaded by [New York University] at 10:52 20 December 2017 testicular tissue harvest and grafting and SSC isolate injection. the SSC population in the testis, culturing the SSCs, storing of Testicular tissue grafting retains the natural stem cell niche, the SSCs, and reintroducing the cells safely into a recipient. which may be optimal. However, this approach has not yet been Several groups have been able to isolate human SSCs and optimized for cancer patients due to concerns for cancer cell culture and store the cells. However, well-defined protocols contamination. Cryopreservation protocols for testicular tissue that are easily replicated are needed. Additionally, confirming are well established with most centers using a slow freezing the successful introduction of the SSCs into a human recipient protocol with dimethyl sulfoxide [6]. Keros et al. found that a is difficult as one must distinguish between native SSCs and slow freezing protocol with dimethyl sulfoxide led to improved transplanted SSCs as well as native mature spermatozoa and tubule integrity, fewer damaged spermatogonia, and better transplanted spermatozoa [14]. Finally, after successful SSC maintenance of the lamina propria when compared to cryopre- transplantation, it is essential to prove the fertilization poten- servation protocols with glycerol and propanediol [6]. tial of the mature spermatozoa in the recipient. Because there Testosterone production was also better maintained in the is some evidence of SSC loss after transplantation, a sufficient Dimethyl sulfoxide (DMSO) protocol [6]. However, ideal concen- population of SSCs must be harvested. However, the results trations of dimethyl sulfoxide remain to be established. have poor reproducibility and the process is inefficient as Xenograft models have been used to store and support SSCs demonstrated by a study by Wyns et al. showing 14.5% of and testicular tissue in several animal models [7].Wynsetal. the spermatogonial population remained at three weeks after describe using a mouse xenograft model for cryopreserved grafting in a mouse population [15]. immature testicular tissue harvested from prepubertal males SSC transplantation resulting in mature gametes with ferti- [8]. This work still requires a major advancement to be clinically lization potential has not yet been replicated in the human EXPERT OPINION ON BIOLOGICAL THERAPY 21

population, but research thus far has been promising. If it show fertilization potential with ICSI [21]. In order to prompt could be developed successfully for humans, several patient differentiation into germ cells from adult pluripotent stem populations could potentially benefit, including men facing cells, oncogenic factors must be used. Until another protocol gonadotoxic treatment for malignancy who desire biological is developed, adult pluripotent stem cells are not an option as children in the future. Additionally, prepubertal boys with a a therapy for male infertility because of their tumorigenic cancer diagnosis might benefit from this option, particularly risks [17]. given the fact that obtaining mature spermatozoa prior to treatment is not an option. Similarly, adult azoospermic men 4. Gene therapy with Sertoli cell-only syndrome could be treated with transplantation of SSCs to repopulate the germline epithelium. Gene therapy has already been applied in the research setting Patients with Klinefelter’s syndrome demonstrate progressive to many human disease processes. The technique involves fibrosis of the seminiferous tubules over their lifetime and may adding a ‘normal’ gene to a patient’s genome, removing an also be candidates for SSC transplantation if harvested early in ‘abnormal’ gene, or mutating an ‘abnormal’ gene to allow it to life. For patients with genetic defects causing NOA such as function appropriately. This requires a thorough understand- Y-microdeletions, genomic editing would be required to ing of the specific genetic defect underlying the pathologic remove the mutation. If SSC transplantation combined with process. Unfortunately, such knowledge is lacking in the genomic editing was successful, this procedure could also be majority of cases of NOA. applied to fertile patients with genetic diseases at a single Germline genetic defects pose a unique challenge for gene locus to prevent transmission to offspring [16]. therapy in that spermatogenesis originates from stem cells. Therefore, the SSCs would need to be genetically altered in order to pass the altered genetic material to all mature sper- 3. In vitro spermatogenesis using pluripotent stem matozoa and progeny. SSC transplantation with genetic mod- cells ification, as discussed previously, would be required. An alternative method for developing mature gametes in men Although gene therapy for male infertility has been success- with NOA is the use of pluripotent stem cells. This technique ful in mouse models resulting in live progeny, it is not yet could be applied to adult males with idiopathic NOA. In order applicable for clinical practice in infertile patients with NOA to develop germ cells from pluripotent stem cells, several for a number of reasons. As stated earlier, we currently do not discrete steps are required, each with specific stimuli required have enough knowledge to fully understand the single gene for progression to the next stage of development. In addition, defects that cause NOA. For example, altering germline genetics the differentiation process that occurs in vitro does not always is illegal in humans at the present time in most countries, and emulate the process that occurs in vivo. Despite these chal- insertion of genetic material into the germ cell line could lead lenges, pluripotent stem cell lines may enable development of to oncogenesis. Finally, it is evident that gene therapy – which disease models for infertility to allow for development of new is a very expensive therapeutic – would provide therapeutic therapeutics. benefit only for a small subset of patients with NOA. This There are two stem cell sources (in addition to SSCs) that includes some men with NOA caused by known genetic defects may be used for generating germ cells: human embryonic and those with primary ciliary dyskinesia, a genetic disorder stem cells and adult pluripotent stem cells [17]. Human that results in nonmotile spermatozoa. However, men with embryonic stem cell lines have been studied for several Klinefelter’s syndrome and Y-microdeletions would not be can- years. Toyooka et al. generated male germ cells from mouse didates for gene therapy due to the amount of DNA that would embryonic stem cells. However, the fertilization potential of require addition or deletion. Without the knowledge of single Downloaded by [New York University] at 10:52 20 December 2017 the mature sperm was not studied [18]. Subsequent studies gene defects that cause infertility in humans, gene therapy is have shown that haploid spermatids developed from mouse not yet applicable for clinical practice. Substantial ethical and embryonic cells have fertilization potential as demonstrated safety hurdles must be surpassed as well [22]. by live births [19]. Studies with human embryonic stem cells Promising research is being conducted to determine other have yet to yield functional haploid spermatozoa [17].In genetic bases for NOA such as DNA methylation patterns and addition, the use of embryonic stem cells is limited by the sperm mitochondrial genome deletions [23]. As further devel- ethical concerns, further limiting the propagation of this tech- opments occur, more targets may develop for gene therapy. nique toward clinical application. With time, the future may bring answers to these patients as Pluripotent adult stem cells are able to self-renew and to the cause of their fertility [24]. differentiate into all three germ-layer cell types. These stem cells are developed from somatic cells, making them easily 5. Conclusion accessible. Zhu et al. demonstrated that adult mouse pluripotent stem cells can differentiate into SSCs and late-stage male Existing therapies for patients with NOA who wish to conceive a germ cells with the combination of in vitro and in vivo harvest genetically related child are limited and require harvesting [20]. These authors could not demonstrate that these cells mature spermatozoa from the male while the female endures progressed to form mature spermatozoa; therefore, fertiliza- the morbidity of assisted reproductive technology. The use of tion potential was not shown. Hayashi et al. successfully devel- SSCs is promising, although their entrance into the clinical land- oped primordial germ cells from both embryonic stem cells scape awaits further optimization to ensure safety and reprodu- and adult pluripotent stem cells in mice and were able to cibility. The use of SSCs has the broadest applicability to subsets 22 S. C. VIJ ET AL.

of the infertile male population and has been demonstrated Ethical limitations create a significant barrier to performing successfully in animal models. The hurdles that remain include the appropriate studies in humans. In the prepubertal patient identifying ideal protocol for harvest, storage, and transfer in population, storage of testicular tissue, SSCs, or stem cells is humans. Additionally, this option is not available to cancer controversial, given the patient’s inability to provide his own patients, given the risk of oncologic recurrence. Further limiting consent or truly understand the implications of the procedure. the use of stem cell therapies for some patients with NOA is the Moreover, the use of embryonic stem cells is surrounded by potential requirement to manage the underlying genetic defect controversy. Obtaining embryonic stem cells requires destruc- that lead to the disorder in addition to the stem cell transplantation of the embryo. The struggle to balance the need to cure tion. Stem cell therapy using embryonic stem cells or adult disease with the moral obligation to protect future human life pluripotent stem cells is further from the clinical horizon as is inherent to the use of embryonic stem cells. Gene therapy mature spermatozoa with capability to fertilize have not yet for male infertility is similarly wrought with ethical concerns as been developed, likely due to incomplete understanding of the manipulating the germline could have significant implications stem cell niche required for spermatogenesis. as a therapeutic option but also as a gateway to selecting Gene therapy has been very promising to treat certain characteristics to alter the genetics of populations. genetic diseases and does have some applicability to the Standardized protocols for tissue and cell culture and sto- male fertility stage for patients with single gene defects result- rage must be established prior to the use of these therapies in ing in azoospermia. Until further single gene defects are iden- the clinical landscape. Although SSCs have been used with tified and the ethical concerns over gene therapy for the success in several animal models as well as across species, the germline are ameliorated, this option will not be widely applic- results are not easily reproducible and the optimal site, able to the NOA population. method, and timing of injection or grafting are not yet well All novel therapeutics are expensive. Stem cell therapy at understood. The genetic stability of the cells after transplanta- present is used off-label. Insurance does not usually provide tion is critical as any acquired mutations will be passed to any coverage, and the services can be cost prohibitive for progeny. Pretransplantation culture and storage must be per- many patients. If the biologic therapies discussed in this fected to obtain genetic stability. review reach the clinical setting, the procedures will undoubt- Future research is essential for further development of this edly be extremely costly, limiting the patients who can take promising field. Eliminating oncogenic risks must occur before advantage of these options. As supply increases and protocols studies in human subjects can take place. Of the existing novel are optimized, novel treatments tend to become more afford- therapeutics discussed, it is our opinion that SSC transplantation able. However, many existing therapeutics for both male and has the most promise and fewest limitations. It is reasonable to female fertility remain uncovered by insurance plans and offer testicular tissue harvesting to prepubertal patients under- require a significant payment from the patient. Not only will going gonadotoxic treatments, ideally under a clinical research the treatments need to be more affordable, but the viewpoint protocol, with the hope that these technologies will evolve to that fertility treatments need not be covered by insurance in the clinical realm when these patients reach reproductive age. the United States will need to be modified. With regard to assisted reproductive techniques, data suggest that improved Funding insurance coverage results in better outcomes in terms of fewer fresh embryos transferred and fewer multiple gestation This manuscript has not been funded. pregnancies [25,26]. Despite the hurdles that remain, an impressive amount of progress has been made toward novel therapeutics for men Declaration of interest Downloaded by [New York University] at 10:52 20 December 2017 with NOA who desire biological children. The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes 6. Expert opinion employment, consultancies, honoraria, stock ownership or options, expert NOA is amenable to ICSI if mature spermatozoa can be testimony, grants or patents received or pending, or royalties. obtained from the testicle. Unfortunately, not all men with NOA are candidates for ICSI, and the burden for the female References is significant in terms of cost and morbidity. Men with matura- tion arrest and Sertoli cell-only pathology would benefit from Papers of special note have been highlighted as either of interest (•)orof the novel therapeutic options discussed here. The ability to considerable interest (••) to readers. repopulate the germinal epithelium of the seminiferous 1. Cocuzza M, Alvarenga C, Pagani R. The epidemiology and etiology of azoospermia. Clinics. 2013;68(S1):15–26. tubules with SSCs would drastically change the field of male 2. Kanatsu-Shinohara M, Shinohara T. Spermatogonial stem cell infertility. Similarly, propagating adult or embryonic stem cells self-renewal and development. Annu Rev Cell Dev Biol. 2013;29 into mature spermatozoa would provide a therapeutic option (1):163–187. for these patients. Gene therapy is applicable in a smaller 3. Crane GM, Jeffery E, Morrison SJ. Adult haematopoietic stem cell – population of patients but nonetheless would be a major niches. Nat Rev Immunol. 2017;17:573 590. 4. Brinster RL, Zimmermannt JW. Spermatogenesis following male advancement in the field. Despite the major advances made germ-cell transplantation. Dev Biol. 1994;91:11298–11302. in the field of biologic therapy for male infertility, several •• This paper is the first to describe spermatogonial stem cell obstacles exist to bring these to the clinical realm. transplantation in a mouse model. EXPERT OPINION ON BIOLOGICAL THERAPY 23

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