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 ... -  《-》

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 ... -  《-》

Semen analysis and reproductive hormones in boys with classical Hodgkin lymphoma treated according to the EuroNet-PHL-C2 protocol.

What is the impact of the EuroNet-PHL-C2 treatment for boys with classical Hodgkin lymphoma (cHL) on semen parameters? More than half of the patients (52%, n = 16/31) had oligozoospermia or azoospermia at 2 years from cHL diagnosis; particularly boys treated for advanced-stage cHL had low sperm counts and motility. Chemotherapy and radiotherapy to the inguinal region or testes can impair spermatogenesis and result in reduced fertility. The EuroNet-PHL-C2 trial aims to minimize radiotherapy in standard childhood cHL treatment, by intensifying chemotherapy. The present study aims to assess the (gonadotoxic) impact of this treatment protocol on semen parameters and reproductive hormones in boys aged ≤18 years. This international, prospective, multi-centre cohort study was an add-on study to the randomized phase-3 EuroNet-PHL-C2 trial, where the efficacy of standard cHL treatment with OEPA-COPDAC-28 (OEPA: vincristine, etoposide, prednisone, and doxorubicin; COPDAC-28: cyclophosphamide, vincristine, prednisone, and dacarbazine) was compared to intensified OEPA-DECOPDAC-21 chemotherapy (DECOPDAC-21: COPDAC with additional doxorubicin and etoposide and 25% more cyclophosphamide). Patients were recruited between January 2017 and September 2021. Eligibility criteria included male patients, diagnosed with classical HL before or at the age of 18 years, and treated according to the EuroNet-PHL-C2 protocol in any of the 18 participating sites in the Netherlands, Germany, Belgium, Czech Republic, and Austria. Sperm parameters (sperm concentration, progressive motility, sperm volume, and calculated total motile sperm count) were assessed at diagnosis and 2 years after diagnosis in (post)pubertal boys. Laboratory measurements (serum follicle-stimulating hormone (FSH) and inhibin B) were performed in samples drawn at diagnosis, during treatment (2-3 times), and at 2 years post-diagnosis, and (age-adjusted) analyses were conducted separately for pre-pubertal and (post)pubertal boys. Outcomes were compared between the treatment levels (TL1, TL2, and TL3) and consolidation treatment schemes (COPDAC-28 and DECOPDAC-21). In total, 101 boys were included in the present analysis: 73 were (post)pubertal (median age 15.4 years, (IQR 14.4; 16.6), 10 TL1, 29 TL2, 34 TL3, 62% of TL2/3 patients received COPDAC-28) and 28 boys were pre-pubertal (median age 9.6 years (IQR 6.6; 11.4), 4 TL1, 7 TL2, 17 TL3, 38% of TL2/3 patients received COPDAC-28). The study included six boys who had received pelvic radiotherapy; none were irradiated in the inguinal or testicular area. At diagnosis, 48 (post)pubertal boys delivered semen for cryopreservation; 19 (40%) semen samples were oligospermic and 4 (8%) were azoospermic. Low sperm concentration (<15 mil/ml) appeared to be related to the HL disease itself, with a higher prevalence in boys who presented with B symptoms (76% vs 26%, aOR 2.3 (95% CI 1.0; 3.8), P = 0.001) compared to those without such symptoms. At 2 -years post-diagnosis, 31 boys provided semen samples for analysis, of whom 12 (39%) boys had oligozoospermia and 4 (13%) had azoospermia, while 22 boys (71%) had low total motile sperm counts (TMSC) (<20 mil). Specifically, the eight boys in the TL3 group treated with DECOPDAC-21 consolidation had low sperm counts and low progressive motility after 2 years (i.e. median sperm count 1.4 mil/ml (IQR <0.1; 5.3), n = 7 (88%), low sperm concentration, low median progressive motility 16.5% (IQR 0.0; 51.2), respectively). Age-adjusted serum FSH levels were significantly raised and inhibin B levels (and inhibin B:FSH ratios) were decreased during chemotherapy in (post)pubertal boys, with subsequent normalization in 80% (for FSH) and 60% (for inhibin B) of boys after 2 years. Only 4 out of the 14 (post)pubertal boys (29%) with low sperm concentrations after 2 years had elevated FSH (>7.6 IU/l), while 7 (50%) had low inhibin B levels (<100 ng/l). In pre-pubertal boys, reproductive hormones were low overall and remained relatively stable during chemotherapy. The present analyses included sperm and laboratory measurements up to 2 years post-diagnosis. Long-term reproductive outcomes and potential recovery of spermatogenesis remain unknown, while recovery was reported up to 5- or even 10-year post-chemotherapy in previous studies.Boys who were pre-pubertal at diagnosis were still too young and/or physically not able to deliver semen after 2 years and we could not assess a potential difference in gonadotoxicity according to pubertal state at the time of treatment. Overall, the statistical power of the analyses on sperm concentration and quality after 2 years was limited. Results of the semen analyses conducted among the 31 boys who had provided a semen sample at 2 years post-treatment were generally poor. However, additional long-term and adequately powered data are crucial to assess the potential recovery and clinical impact on fertility. The participating boys will be invited to deliver a semen sample after 5 years. Until these data become available, benefits of intensified chemotherapy in cHL treatment to reduce radiotherapy and lower risk for development of secondary tumours should be carefully weighed against potentially increased risk of other late effects, such as diminished fertility due to the increased chemotherapy burden. Boys with newly diagnosed cHL should be encouraged to deliver sperm for cryopreservation whenever possible. However, patients and clinicians should also realize that the overall state of disease and inflammatory milieu of cHL can negatively affect sperm quality and thereby reduce chance of successful fertility preservation. Furthermore, the measurement of FSH and inhibin B appears to be of low value in predicting low sperm quality at two years from cHL treatment. This study was funded by the Dutch charity foundation KiKa (project 257) that funds research on all forms of childhood cancer. C.M.-K., D.K., W.H.W., D.H., MC, A.U., and A.B. were involved in the development of the EuroNet-PHL-C2 regimen. The other authors declare no potential conflict of interest. N/A.

Drechsel KCE ，Broer SL ，van Breda HMK ，Stoutjesdijk FS ，van Dulmen-den Broeder E ，Beishuizen A ，Wallace WH ，Körholz D ，Mauz-Körholz C ，Hasenclever D ，Cepelova M ，Uyttebroeck A ，Ronceray L ，Twisk JWR ，Kaspers GJL ，Veening MA ... -  《-》

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 ... -  《-》

Evaluating testicular tissue for future autotransplantation: focus on cancer cell contamination and presence of spermatogonia in tissue cryobanked for boys diagnosed with a hematological malignancy.

Kourta D ，Camboni A ，Saussoy P ，Kanbar M ，Poels J ，Wyns C ... -  《-》