Impact of low- or moderate-risk gonadotoxic chemotherapy prior to testicular tissue freezing on spermatogonia quantity in human (pre)pubertal testicular tissue.

What is the impact of low- or moderate-risk gonadotoxic chemotherapy received prior to testicular tissue freezing (TTF), and of the cancer itself, on spermatogonia quantity in testicular tissue from (pre)pubertal boys? Vincristine, when associated with alkylating agents, has an additional adverse effect on spermatogonia quantity, while carboplatin has no individual contribution to spermatogonia quantity, in testicular tissue of (pre)pubertal boys, when compared to patients who have received non-alkylating chemotherapy. The improved survival rates after cancer treatment necessitate the inclusion of fertility preservation procedures as part of the comprehensive care for patients, taking into consideration their age. Sperm cryopreservation is an established procedure in post-pubertal males while the TTF proposed for (pre)pubertal boys remains experimental. Several studies exploring testicular tissue of (pre)pubertal boys after TTF have examined the tubular fertility index (TFI, percentage of seminiferous tubule cross-sections containing spermatogonia) and the number of spermatogonia per seminiferous tubule cross-section (S/T). All studies have demonstrated that TFI and S/T always decrease after the introduction of chemotherapeutic agents, especially those which carry high gonadotoxic risks such as alkylating agents. Testicular tissue samples from 79 (pre)pubertal boys diagnosed with cancer (from 6 months to 16 years of age) were cryopreserved between May 2009 and June 2014. Their medical diagnoses and previous chemotherapy exposures were recorded. We examined histological sections of (pre)pubertal testicular tissue to elucidate whether the chemotherapy or the primary diagnosis affects mainly TFI and S/T. (Pre)pubertal boys with cancer diagnosis who had been offered TTF prior to conditioning treatment for hematopoietic stem cell transplantation were included in the study. All the patients had previously received chemotherapy with low- or moderate-risk for future fertility. We have selected patients for whom the information on the chemotherapy received was complete. The quantity of spermatogonia and quality of testicular tissue were assessed by both morphological and immunohistochemical analyses. A significant reduction in the number of spermatogonia was observed in boys treated with alkylating agents. The mean S/T values in boys exposed to alkylating agents were significantly lower compared to boys exposed to non-alkylating agents (P = 0.018). In contrast, no difference was observed for patients treated with carboplatin as the sole administered alkylating agent compared to the group of patients exposed to non-alkylating agents. We observed an increase of S/T with age in the group of patients who did not receive any alkylating agent and a decrease of S/T with age when patients received alkylating agents included in the cyclophosphamide equivalent dose (CED) formula (r = 0.6166, P = 0.0434; r = -0.3759, P = 0.0036, respectively). The TFI and S/T decreased further in the group of patients who received vincristine in combination with alkylating agents (decrease of 22.4%, P = 0.0049 and P < 0.0001, respectively), but in this group the CED was also increased significantly (P < 0.0001). Multivariate analysis, after CED adjustment, showed the persistence of a decrease in TFI correlated with vincristine administration (P = 0.02). This is a descriptive study of testicular tissues obtained from (pre)pubertal boys who were at risk of infertility. The study population is quite heterogeneous, with a small number of patients in each sub-group. Our results are based on comparisons between patients receiving alkylating agents compared to patients receiving non-alkylating agents rather than chemotherapy-naive patients. The French national guidelines for fertility preservation in cancer patients recommend TTF before highly gonadotoxic treatment. Therefore, all the patients had received low- or moderate-risk gonadotoxic chemotherapy before TTF. Access to testicular tissue samples from chemotherapy-naive patients with comparable histological types of cancer was not possible. The functionality of spermatogonia and somatic cells could not be tested by transplantation or in vitro maturation due to limited sample sizes. This study summarizes the spermatogonial quantity of (pre)pubertal boys prior to TTF. We confirmed a negative correlation between the cumulative exposure to alkylating agents and spermatogonial quantity. In addition, the synergistic use of vincristine in combination with alkylating agents showed a cumulative deleterious effect on the TFI. For patients for whom fertility preservation is indicated, TTF should be proposed for chemotherapy with a predicted CED above 4000 mg/m2. However, the data obtained from vincristine and carboplatin use should be confirmed in a subsequent study including more patients. This study had financial support from a French national research grant PHRC No. 2008/071/HP obtained by the French Institute of Cancer and the French Healthcare Organization. The sponsors played no role in the study. The authors declare no conflicts of interest. N/A.

Feraille A ，Liard A ，Rives N ，Bubenheim M ，Barbotin AL ，Giscard d'Estaing S ，Mirallié S ，Ancelle A ，Roux C ，Brugnon F ，Daudin M ，Schneider P ，Dumont L ，Rondanino C ... -  《-》

Spermatogonial quantity in human prepubertal testicular tissue collected for fertility preservation prior to potentially sterilizing therapy.

Stukenborg JB ，Alves-Lopes JP ，Kurek M ，Albalushi H ，Reda A ，Keros V ，Töhönen V ，Bjarnason R ，Romerius P ，Sundin M ，Norén Nyström U ，Langenskiöld C ，Vogt H ，Henningsohn L ，Mitchell RT ，Söder O ，Petersen C ，Jahnukainen K ... -  《-》

Long-term follow-up of boys who have undergone a testicular biopsy for fertility preservation.

What is the long-term reproductive health outcome of patients who have undergone testicular sampling for fertility preservation (FP) before and during the pubertal transition period? In long-term follow-up after testicular sampling for FP, hormonal data showed that 33% of patients had primary seminiferous tubule insufficiency (high FSH) while semen analyses showed 52% of patients having a severe reduction in total sperm counts or complete absence of ejaculated sperm. During childhood and adolescence, both treatments for cancer and benign haematological diseases that require a bone marrow transplantation, can be detrimental to spermatogenesis by depleting the spermatogonial stem cell population. A testicular biopsy prior to chemotherapy or radiotherapy, even though still an experimental procedure, is now recommended for FP by European and USA oncofertility societies if performed within an institutional research setting. While short-term follow-up studies showed little to no post-operative complications and a normal testicular development after 1 year, data regarding the long-term follow-up of boys who have undergone this procedure are still lacking. This is a longitudinal retrospective cohort study that reports on the long-term follow-up of pre- and peri-pubertal boys who have undergone a testicular biopsy for FP between May 2005 and May 2020. All the patients included in this study were referred to our programme by haematologists-oncologists who are part of a regional multi-centric collaborative care pathway. Of the 151 boys referred to our FP programme, 139 parents/legal guardians accepted that their child undergo a testicular biopsy. Patient characteristics (i.e. age at biopsy, urogenital history, pubertal status at diagnosis), indications (disease type and dosage of gonadotoxic treatments), operative and post-operative data (biopsy volume, surgical complications), anatomopathological analyses (presence/absence of spermatogonia, Johnsen score) and reproductive data (semen analyses, FSH, LH, testosterone levels) were collected from the institutions' FP database and medical records or from the 'Brussels Health Network'. Cumulative alkylating agent treatment was quantified using the cyclophosphamide equivalent dose (CED). Patients who were 14 years or older at the time of the follow-up and in whom the testicular tissue was shown to contain spermatogonia were included in the reproductive outcome analysis. Comparison of the sperm count findings (absence/presence of spermatozoa) and FSH levels (high (≥10 IU/l)/normal) between patients who were either pre- (Tanner 1) or peri-pubertal (Tanner >1) at the time of the biopsy was done using the Mann-Whitney U or Fisher's tests. A multiple logistic regression was used to study the relationship between the hormone reproductive outcome (high versus normal FSH), as a proxy marker for fertility, and both the pubertal status (Tanner 1 versus Tanner >1) and Johnsen score at the time of the biopsy, while adjusting for CED. A testicular biopsy was performed in 139 patients either before (129/139) or after (10/139) the start of a gonadotoxic treatment. Post-operative complications occurred in 2.1% (3/139). At the time of the procedure, 88% (122/139) of patients were pre-pubertal and 12% (17/139) were peri-pubertal. The presence of spermatogonia was documented in 92% (128/139) of cases. Follow-up data were available for 114 patients after excluding 23 deceased and two patients lost to follow-up. A paediatric endocrinologist's follow-up including clinical examination and data on reproductive hormones was available for 57 patients (age ≥14) and 19 (33%) of these were found to have high FSH levels (20 ± 8.8 IU/l). There were 37 patients who had returned to the reproductive specialist's consultation for post-treatment fertility counselling and results on semen analysis were available in 27 of these cases; 14/27 (52%) had severely impaired semen parameters including 8 who were azoospermic. Among patients who received an alkylating agent-based treatment (n = 42), a peri-pubertal status (Tanner >1) at the time of diagnosis/biopsy was found to be associated with a higher risk of having primary testicular failure (defined by an FSH ≥ 10 IU/l) after treatment completion with an OR of 6.4 (95% CI 1.22-33.9; P = 0.03). Of all the patients, 2.6% had already fulfilled their wish to build a family or were actively seeking parenthood. Although this is the largest cohort with follow-up data providing proxy markers of the reproductive potential of boys in whom a testicular biopsy for FP was performed before puberty or during the pubertal transition period, the amount of data provided is limited, and originating from a single programme. Further data collection to confirm the observations in other settings is therefore awaited. Testicular sampling for FP should be offered to boys at risk of losing their fertility (and is recommended for those at high risk) as part of ethically approved research programmes. Long-term follow-up data on increasing numbers of boys who have undergone an FP procedure will help improve patient care in the future as patient-specific factors (e.g. urogenital history, age at gonadotoxic therapy) appear to influence their reproductive potential after gonadotoxic therapies. FNRS-Télévie, the Salus Sanguinis Foundation and the Belgian Foundation against Cancer supported the studies required to launch the FP programme. The authors declare that they have no conflict of interest. N/A.

Kanbar M ，de Michele F ，Giudice MG ，Desmet L ，Poels J ，Wyns C ... -  《-》

Childhood cancer and hematological disorders negatively affect spermatogonial quantity at diagnosis: a retrospective study of a male fertility preservation cohort.

What is the impact of cancer or hematological disorders on germ cells in pediatric male patients? Spermatogonial quantity is reduced in testes of prepubertal boys diagnosed with cancer or severe hematological disorder compared to healthy controls and this reduction is disease and age dependent: patients with central nervous system cancer (CNS tumors) and hematological disorders, as well as boys <7 years are the most affected. Fertility preservation in pediatric male patients is considered based on the gonadotoxicity of selected treatments. Although treatment effects on germ cells have been extensively investigated, limited data are available on the effect of the disease on the prepubertal male gonad. Of the few studies investigating the effects of cancer or hematologic disorders on testicular function and germ cell quantity in prepuberty, the results are inconsistent. However, recent studies suggested impairments before the initiation of known gonadotoxic therapy. Understanding which diseases and at what age affect the germ cell pool in pediatric patients before treatment is critical to optimize strategies and counseling for fertility preservation. This multicenter retrospective cohort study included 101 boys aged <14 years with extra-cerebral cancer (solid tumors), CNS tumors, leukemia/lymphoma (blood cancer), or non-malignant hematological disorders, who were admitted for a fertility preservation programme between 2002 and 2018. In addition to clinical data, we analyzed measurements of testicular volume and performed histological staining on testicular biopsies obtained before treatment, at cryopreservation, to evaluate number of spermatogonia per tubular cross-section, tubular fertility index, and the most advanced germ cell type prior to chemo-/radiotherapy. The controls were data simulations with summary statistics from original studies reporting healthy prepubertal boys' testes characteristics. Prepubertal patients with childhood cancer or hematological disorders were more likely to have significantly reduced spermatogonial quantity compared to healthy controls (48.5% versus 31.0% prevalence, respectively). The prevalence of patients with reduced spermatogonial quantity was highest in the CNS tumor (56.7%) and the hematological disorder (55.6%) groups, including patients with hydroxyurea pre-treated sickle cell disease (58.3%) and patients not exposed to hydroxyurea (50%). Disease also adversely impacted spermatogonial distribution and differentiation. Irrespective of disease, we observed the highest spermatogonial quantity reduction in patients <7 years of age. For ethical reasons, we could not collect spermatogonial quantity data in healthy prepubertal boys as controls and thus deployed statistical simulation on data from literature. Also, our results should be interpreted considering low patient numbers per (sub)group. Cancers, especially CNS tumors, and severe hematological disorders can affect spermatogonial quantity in prepubertal boys before treatment. Consequently, these patients may have a higher risk of depleted spermatogonia following therapies, resulting in persistent infertility. Therefore, patient counseling prior to disease treatment and timing of fertility preservation should not only be based on treatment regimes, but also on diagnoses and age. This study was supported by Marie Curie Initial Training Network (ITN) (EU-FP7-PEOPLE-2013-ITN) funded by European Commision grant no. 603568; ZonMW Translational Adult stem cell research (TAS) grant no. 116003002. No competing interests. N/A.

Masliukaite I ，Ntemou E ，Feijen EAM ，van de Wetering M ，Meissner A ，Soufan AT ，Repping S ，Kremer LMC ，Jahnukainen K ，Goossens E ，van Pelt AMM ... -  《-》

Prior exposure to alkylating agents negatively impacts testicular organoid formation in cells obtained from childhood cancer patients.

Can human pre- and peri-pubertal testicular cells obtained from childhood cancer patients, previously treated with chemotherapy, form testicular organoids (TOs)? Organoid formation from testicular tissue collected from childhood cancer patients positively correlates with SRY-Box transcription factor 9 (SOX9) expression in Sertoli cells, which in turn negatively correlates with previous exposure to alkylating chemotherapy. Pre- and peri-pubertal boys exposed to highly gonadotoxic therapies can only safeguard their fertility potential through testicular tissue cryopreservation. Today, there is no established clinical tool to restore fertility using these testicular samples. Organoids hold promise in providing fundamental early insights in creating such platforms. However, the generation of TOs that closely resemble the innate testis, to enable a thorough monitoring of the necessary steps for germ cell differentiation and somatic functionalities, remains a challenge. We used a Matrigel-based three-layer gradient culture system to generate human TOs and to reveal whether chemotherapy exposure affects TO formation capacity and the functionality of pre- and peri-pubertal testicular somatic cells. Testicular cells of 11 boys (aged 7.7 ± 4.1 (mean ± SD) years) were assessed for TO formation in relation to previous chemotherapy exposure and SOX9 expression in histological sections of paraffin-embedded testicular tissue samples collected on the day of biopsy and compared with testicular tissue samples obtained from 28 consecutive patients (aged 6.9 ± 3.8 (mean ± SD) years). All 39 patients were part of the fertility preservation project NORDFERTIL; an additional 10 samples (from boys aged 5.5 ± 3.5 (mean ± SD) years, without an underlying pathology) in an internal biobank collection were used as controls. We obtained 49 testicular tissue samples from boys aged 0.8-13.4 years. Fresh samples (n = 11) were dissociated into single-cell suspensions and applied to a three-layer gradient culture system for organoid formation. Histological sections of another 28 samples obtained as part of the fertility preservation project NORDFERTIL, and 10 samples from a sample collection of a pathology biobank were used to evaluate the effects of prior exposure to alkylating agents on testicular samples. Testicular organoid formation was defined based on morphological features, such as compartmentalized structures showing cord formation, and protein expression of testicular cell-specific markers for germ and somatic cells was evaluated via immunohistochemical staining. Hormone secretion was analysed by specific enzyme-linked immunosorbent assays for testosterone and anti-Müllerian hormone (AMH) production. Our results revealed that 4 out of 11 prepubertal testicular samples formed TOs that showed compartmentalized cord-like structures surrounded by interstitial-like areas and increasing levels of both testosterone as well as AMH over a 7-day culture period. We observed that SOX9 expression was correlated positively with TO formation. Moreover, exposure to alkylating agents before biopsy was inversely correlated with SOX9 expression (P = 0.006). N/A. Due to the limited amount of material available, only 11 out of the 39 pre- and peri-pubertal testicular tissue samples could be used for the organoid formation experiments. The testicular tissue samples obtained from a sample collection of the internal biobank of Department of Pathology, Karolinska University Hospital were considered normal and included in the study if no testicular pathology was reported. However, detailed information regarding previous medical treatments and/or testicular volumes of the patients included in this biobank was not available. Our observations suggest that SOX9 expression may serve as a putative indicator of TO formation, indicating a critical role of Sertoli cells in promoting organoid formation, seminiferous tubule integrity, and testicular function in pre- and peri-pubertal testicular tissue. This study was supported by grants from the Swedish Childhood Cancer Foundation (PR2019-0123; PR2022-0115; TJ2020-0023) (J.-B.S.), Finnish Cancer Society (K.J.), Finnish Foundation for Paediatric Research (K.J.), Swedish Research Council (2018-03094; 2021-02107) (J.-B.S.), and Birgitta and Carl-Axel Rydbeck's Research Grant for Paediatric Research (2020-00348; 2020-00335; 2021-00073; 2022-00317) (J.-B.S. and K.J.). Y.C. and Y.Y. received a scholarship from the Chinese Scholarship Council. J.P.A-L. was supported by a Starting Grant in Medicine and Health (2022-01467) from the Swedish Research Council. R.T.M. was supported by a UKRI Future Leaders Fellowship (MR/S017151/1). The MRC Centre for Reproductive Health was supported by an MRC Centre Grant (MR/N022556/1). The authors declare no competing interests.

Cui Y ，Harteveld F ，Ba Omar HAM ，Yang Y ，Bjarnason R ，Romerius P ，Sundin M ，Norén Nyström U ，Langenskiöld C ，Vogt H ，Henningsohn L ，Frisk P ，Vepsäläinen K ，Petersen C ，Mitchell RT ，Guo J ，Alves-Lopes JP ，Jahnukainen K ，Stukenborg JB ... -  《-》