Fertility preservation in boys: recent developments and new insights †.

BACKGROUND: Infertility is an important side effect of treatments used for cancer and other non-malignant conditions in males. This may be due to the loss of spermatogonial stem cells (SSCs) and/or altered functionality of testicular somatic cells (e.g. Sertoli cells, Leydig cells). Whereas sperm cryopreservation is the first-line procedure to preserve fertility in post-pubertal males, this option does not exist for prepubertal boys. For patients unable to produce sperm and at high risk of losing their fertility, testicular tissue freezing is now proposed as an alternative experimental option to safeguard their fertility. OBJECTIVE AND RATIONALE: With this review, we aim to provide an update on clinical practices and experimental methods, as well as to describe patient management inclusion strategies used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss. SEARCH METHODS: Based on the expertise of the participating centres and a literature search of the progress in clinical practices, patient management strategies and experimental methods used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss were identified. In addition, a survey was conducted amongst European and North American centres/networks that have published papers on their testicular tissue banking activity. OUTCOMES: Since the first publication on murine SSC transplantation in 1994, remarkable progress has been made towards clinical application: cryopreservation protocols for testicular tissue have been developed in animal models and are now offered to patients in clinics as a still experimental procedure. Transplantation methods have been adapted for human testis, and the efficiency and safety of the technique are being evaluated in mouse and primate models. However, important practical, medical and ethical issues must be resolved before fertility restoration can be applied in the clinic.Since the previous survey conducted in 2012, the implementation of testicular tissue cryopreservation as a means to preserve the fertility of prepubertal boys has increased. Data have been collected from 24 co-ordinating centres worldwide, which are actively offering testis tissue cryobanking to safeguard the future fertility of boys. More than 1033 young patients (age range 3 months to 18 years) have already undergone testicular tissue retrieval and storage for fertility preservation. LIMITATIONS REASONS FOR CAUTION: The review does not include the data of all reproductive centres worldwide. Other centres might be offering testicular tissue cryopreservation. Therefore, the numbers might be not representative for the entire field in reproductive medicine and biology worldwide. The key ethical issue regarding fertility preservation in prepubertal boys remains the experimental nature of the intervention. WIDER IMPLICATIONS: The revised procedures can be implemented by the multi-disciplinary teams offering and/or developing treatment strategies to preserve the fertility of prepubertal boys who have a high risk of fertility loss. STUDY FUNDING/COMPETING INTERESTS: The work was funded by ESHRE. None of the authors has a conflict of interest.

The initial maturation status of marmoset testicular tissues has an impact on germ cell maintenance and somatic cell response in tissue fragment culture.

Successful in vitro spermatogenesis was reported using immature mouse testicular tissues in a fragment culture approach, raising hopes that this method could also be applied for fertility preservation in humans. Although maintaining immature human testicular tissue fragments in culture is feasible for an extended period, it remains unknown whether germ cell survival and the somatic cell response depend on the differentiation status of tissue. Employing the marmoset monkey (Callithrix jacchus), we aimed to assess whether the maturation status of prepubertal and peri-/pubertal testicular tissues influence the outcome of testis fragment culture. Testicular tissue fragments from 4- and 8-month-old (n = 3, each) marmosets were cultured and evaluated after 0, 7, 14, 28 and 42 days. Immunohistochemistry was performed for identification and quantification of germ cells (melanoma-associated antigen 4) and Sertoli cell maturation status (anti-Müllerian hormone: AMH). During testis fragment culture, spermatogonial numbers were significantly reduced (P < 0.05) in the 4- but not 8-month-old monkeys, at Day 0 versus Day 42 of culture. Moreover, while Sertoli cells from 4-month-old monkeys maintained an immature phenotype (i.e. AMH expression) during culture, AMH expression was regained in two of the 8-month-old monkeys. Interestingly, progression of differentiation to later meiotic stage was solely observed in one 8-month-old marmoset, which was at an intermediate state regarding germ cell content, with gonocytes as well as spermatocytes present, as well as Sertoli cell maturation status. Although species-specific differences might influence the outcome of testis fragment experiments in vitro, our study demonstrated that the developmental status of the testicular tissues needs to be considered as it seems to be decisive for germ cell maintenance, somatic cell response and possibly the differentiation potential.

A diagnostic germ cell score for immature testicular tissue at risk of germ cell loss.

STUDY QUESTION: Can a systematic scoring procedure provide crucial information on the status of highly heterogeneous immature human testicular tissues in the context of cryopreservation for fertility preservation? SUMMARY ANSWER: We developed a systematic histological score as a novel diagnostic tool which differentiates the patient cohort according to the status of germ cell differentiation and number of spermatogonia (normal, diminished and absent), and which could be relevant in the fertility clinic. WHAT IS KNOWN ALREADY: Cryopreservation of testicular tissue of immature boys is currently considered the option for future fertility restoration. However, experimental techniques for the derivation of sperm as well as valid diagnostic scoring of these immature testis tissues are not yet reported. STUDY DESIGN, SIZE, DURATION: Testicular tissues of 39 patients (aged 2-20 years) who attended our clinic for cryopreservation between 2010 and 2015 were analyzed to determine the variability of testicular tissue composition, germ cell numbers and differentiation status. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human testicular tissue samples were divided into three groups. Group NT included patients suffering from diseases which do not directly affect the testes (n = 6; aged 6-14 years), group AT included patients suffering from diseases that directly affect the testes (n = 14; 2-17 years), and group KS (Klinefelter patients, n = 19; 12-20 years). Based on immunohistochemical stainings for MAGEA4, the differentiation status as well as the numbers of gonocytes, spermatogonia and spermatocytes were determined. MAIN RESULTS AND THE ROLE OF CHANCE: Testicular tissue samples from the NT group contained a mean of 100.3 spermatogonia/mm3 (×103). Highly heterogeneous and significantly lower mean numbers of spermatogonia were scored in testes from boys after cytotoxic exposures or with pre-existing disease (AT group: 35.7 spermatogonia/mm3 (×103); KS group: 1.8 spermatogonia/mm3 (×103)). In addition, the germ cell differentiation status was determined and revealed tissues with either spermatogonia and gonocytes, only spermatogonia, spermatogonia and spermatocytes, or all three germ cell types were present. Based on spermatogonial numbers and differentiation status, we developed a germ cell score which we applied to each individual patient sample. LIMITATIONS REASONS FOR CAUTION: Normal human testicular tissue samples are difficult to obtain for ethical reasons and the sample numbers were small. However, six such samples provide a valid baseline for the normal situation. WIDER IMPLICATIONS OF THE FINDINGS: Fertility preservation of immature male tissues is an emerging field and is currently offered in many specialized centers worldwide. Our diagnostic germ cell score delivers an easily applicable tool, facilitating patient counseling and thus ensuring comparability between the centers with regard to future studies. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Funding Initiative: Translational Research, Ministry of Innovation, Science and Research, Federal State of North Rhine Westphalia (z1403ts006). The authors declare that they do not have competing financial interests.

Andrology of male-to-female transsexuals: influence of cross-sex hormone therapy on testicular function.

Patients with gender dysphoria are offered cross-sex hormone therapy and sex reassignment surgery to achieve the transition between the sex assigned at birth and gender identity. According to international guidelines, cross-sex hormone therapy in trans-women should lead to a psychologically and physiologically healthy body with feminized serum hormone levels, resulting in suppression of spermatogenesis. However, in a recently published multi-center study, we discovered a high proportion of patients with male serum hormone levels and qualitatively intact spermatogenesis on the day of sex reassignment surgery. The objective of this study was to review the content of 11 publications that focus on the influence of cross-sex hormone therapy on testicular morphology. These publications were identified based on a PubMed search for the key words transgender/transsexual/gender dysphoria in male-to-female persons, cross-sex hormone therapy, and testicular tissues. Whereas three publications described a marked reduction of the spermatogenic level in all patients examined, eight publications reported inconsistent results. Histological analyses showed highly variable outcomes from qualitatively normal spermatogenesis and undisturbed Leydig/Sertoli cell morphology to full testicular regression with severe cellular damage and hyalinization. Explanations for these heterogeneous findings include insufficient cross-sex hormone therapy regarding dosage or duration. As complete spermatogenesis is associated with virilized serum hormone levels, these patients may face challenges especially after sex reassignment surgery in adjusting to the abruptly established hypogonadal state following removal of the testes. These findings also suggest that contraception should be discussed, and fertility preservation should be offered during/prior to cross-sex hormone therapy. There is a need for more individualized and better-controlled cross-sex hormone therapy and post-treatment regimens. Evidence-based guidelines for attending clinicians need to be established in order to deliver the most appropriate care.

Irradiation affects germ and somatic cells in prepubertal monkey testis xenografts.

Study question: Does irradiation evoke adverse effects in germ and somatic cells in testis xenografts from prepubertal monkeys? Summary answer: In addition to the expected depletion of germ cells, a dose-dependent effect of irradiation was observed at the mRNA and protein level in Sertoli and peritubular myoid cells. What is known already: Testicular irradiation studies in monkeys have focused on the dose-dependent effects on germ cells. Previous studies using intact animals or xenografts reported that germ cells are highly sensitive to irradiation. Their depletion was demonstrated by morphometric and histological analyses. The effect of irradiation on expression of Sertoli and peritubular myoid cell markers, however, has not yet been described. Study design, size, duration: The testes of two prepubertal macaques (Macaca fascicularis) were dissected into testicular fragments. Fragments were randomly exposed in vitro to one of the following three doses of irradiation: 0 Gy, n = 60; 1 Gy, n = 54; 4 Gy, n = 72. Non-irradiated control fragments (0 Gy) were placed into the Faxitron for 6.6 min without irradiation. For 1 Gy and 4 Gy irradiation was applied for 1.7 and 6.6 min, respectively. Grafts were then either immediately analyzed or subcutaneously implanted under the back skin of 39 nude mice and analyzed after 6.5 months. Participants/materials setting methods: Post grafting, 133 testicular xenografts were retrieved. The body weight, serum testosterone level and seminal vesical weight of the host mice as well as the number and weight of retrieved grafts were determined. Larger grafts were used to evaluate both mRNA expression profiles and protein expression patterns. In total, 71 testicular fragments were used for morphometric and histological analysis while 68 fragments were analyzed for gene expression. For PCR arrays, M. fascicularis-specific primer sequences were employed. Irradiation-induced changes in the transcript levels of 34 marker genes were determined for each testicular graft. The effects of irradiation on peritubular myoid cells and Sertoli cells were confirmed by immunohistochemical analysis of chemokine (C-X-C motif) ligand type 11 (CXCL11), alpha smooth muscle actin (SMA) and chemokine (C-X-C motif) ligand type 12 (CXCL12). Main results and the role of chance: The four testes gave rise to 106 xenografts, which were individually analyzed, limiting the role of chance despite using only two monkeys in the study. Prior to grafting, the two donors displayed spermatogonia as the most advanced germ cell type in 95% and 70% of seminiferous tubules, respectively, while remaining tubules contained SCO. No spermatocytes were encountered prior to grafting in either monkey. After 6.5 months, non-irradiated grafts displayed spermatocytes in 15.4% and 1.8% of seminiferous tubules indicating an induction of meiosis. Irradiation resulted in a complete absence of spermatocytes. The percentage of seminiferous tubules containing spermatogonia declined in a dose-dependent manner. In non-irradiated xenografts, ~40% of tubules contained spermatogonia. This proportion was reduced to 3.4% and 4.3% in the 1 Gy treated group and to 1.3% and 0.2% in 4 Gy irradiated grafts. A dose-dependent decline in mRNA levels of selected germ cell marker genes supported the morphologically detected loss of germ cells. Irradiation had no effect on CXCL12 transcript levels. At the protein level, CXCL12-positive Sertoli cells were most abundant in the 1 Gy group compared to the 4 Gy group (P < 0.05), indicating a potential role of CXCL12 during recovery of primate spermatogenesis. The most prominent radiation-evoked changes were for CXCL11, which was localized to smooth muscle cells of blood vessels and seminiferous tubules. Transcript levels declined in a dose-dependent manner in grafts from both monkeys (MM687: P < 0.01 (0 Gy versus 4 Gy), MM627: P < 0.05 (0 Gy versus 4 Gy), P < 0.001 (1 Gy versus 4 Gy)). CXCL11 patterns of protein expression revealed irradiation-dependent changes as well. That peritubular cells are affected by X-irradiation was substantiated by changes at the transcript level between 1 and 4 Gy exposed groups (P < 0.01) and at the protein level of SMA (P < 0.05, 0 Gy versus 4 Gy). Large scale data: n/a. Limitations, reasons for caution: The spermatogonial stem cell system in primates is remarkably different from rodents. Therefore, data from a non-human primate may be more relevant to man. However, species-specific differences amongst primates cannot be fully excluded and the use of only two donors may raise concerns toward the generalization of the findings. There may also be important differences across the prepubertal period (e.g. infancy, early childhood) that are not represented by the ages included in the present study. Wider implications of the findings: This study is the first to indicate relevant testicular somatic cell responses following irradiation of prepubertal primate tissue. In addition to the well-known depletion of germ cells, the changes in Sertoli, and in particular peritubular myoid, cells may have important consequences for spermatogenic recovery. These novel findings should be taken into consideration when irradiation effects are assessed in tumor survivors. Study funding and competing interest(s): Interdisciplinary Center for Clinical Research (IZKF) Münster (Schl2/001/13) and the Excellence Cluster 'Cells in Motion' at the University Münster. There are no conflicts of interest to declare.

Single-cell gene expression analysis reveals diversity among human spermatogonia.

STUDY QUESTION: Is the molecular profile of human spermatogonia homogeneous or heterogeneous when analysed at the single-cell level? SUMMARY ANSWER: Heterogeneous expression profiles may be a key characteristic of human spermatogonia, supporting the existence of a heterogeneous stem cell population. WHAT IS KNOWN ALREADY: Despite the fact that many studies have sought to identify specific markers for human spermatogonia, the molecular fingerprint of these cells remains hitherto unknown. STUDY DESIGN, SIZE, DURATION: Testicular tissues from patients with spermatogonial arrest (arrest, n = 1) and with qualitatively normal spermatogenesis (normal, n = 7) were selected from a pool of 179 consecutively obtained biopsies. Gene expression analyses of cell populations and single-cells (n = 105) were performed. Two OCT4-positive individual cells were selected for global transcriptional capture using shallow RNA-seq. Finally, expression of four candidate markers was assessed by immunohistochemistry. PARTICIPANTS/MATERIALS, SETTING, METHODS: Histological analysis and blood hormone measurements for LH, FSH and testosterone were performed prior to testicular sample selection. Following enzymatic digestion of testicular tissues, differential plating and subsequent micromanipulation of individual cells was employed to enrich and isolate human spermatogonia, respectively. Endpoint analyses were qPCR analysis of cell populations and individual cells, shallow RNA-seq and immunohistochemical analyses. MAIN RESULTS AND THE ROLE OF CHANCE: Unexpectedly, single-cell expression data from the arrest patient (20 cells) showed heterogeneous expression profiles. Also, from patients with normal spermatogenesis, heterogeneous expression patterns of undifferentiated (OCT4, UTF1 and MAGE A4) and differentiated marker genes (BOLL and PRM2) were obtained within each spermatogonia cluster (13 clusters with 85 cells). Shallow RNA-seq analysis of individual human spermatogonia was validated, and a spermatogonia-specific heterogeneous protein expression of selected candidate markers (DDX5, TSPY1, EEF1A1 and NGN3) was demonstrated. LIMITATIONS, REASONS FOR CAUTION: The heterogeneity of human spermatogonia at the RNA and protein levels is a snapshot. To further assess the functional meaning of this heterogeneity and the dynamics of stem cell populations, approaches need to be developed to facilitate the repeated analysis of individual cells. WIDER IMPLICATIONS OF THE FINDINGS: Our data suggest that heterogeneous expression profiles may be a key characteristic of human spermatogonia, supporting the model of a heterogeneous stem cell population. Future studies will assess the dynamics of spermatogonial populations in fertile and infertile patients. LARGE SCALE DATA: RNA-seq data is published in the GEO database: GSE91063. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Max Planck Society and the Deutsche Forschungsgemeinschaft DFG-Research Unit FOR 1041 Germ Cell Potential (grant numbers SCHO 340/7-1, SCHL394/11-2). The authors declare that there is no conflict of interest.