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

Abstract

STUDY QUESTION

What is the contamination rate by cancer cells and spermatogonia numbers in immature testicular tissue (ITT) harvested before the start of gonadotoxic therapy in boys with a hematological malignancy?

SUMMARY ANSWER

Among our cohort of boys diagnosed with acute lymphoblastic leukemia (ALL) and lymphomas, 39% (n = 11/28) had cancer cells identified in their tissues at the time of diagnosis and all patients appeared to have reduced spermatogonia numbers compared to healthy reference cohorts.

WHAT IS KNOWN ALREADY

Young boys affected by a hematological cancer are at risk of contamination of their testes by cancer cells but histological examination is unable to detect the presence of only a few cancer cells, which would preclude autotransplantation of cryobanked ITT for fertility restoration, and more sensitive detection techniques are thus required. Reduced numbers of spermatogonia in ITT in hematological cancer patients have been suggested based on results in a limited number of patients.

STUDY DESIGN, SIZE, DURATION

This retrospective cohort study included 54 pre- and peri-pubertal boys who were diagnosed with a hematological malignancy and who underwent a testicular biopsy for fertility preservation at the time of diagnosis before any gonadotoxic therapy between 2005 and 2021.

PARTICIPANTS/MATERIALS, SETTING, METHODS

Among the 54 patients eligible in our database, formalin-fixed paraffin-embedded (FFPE) testicular tissue was available for 28 boys diagnosed either with ALL (n = 14) or lymphoma (n = 14) and was used to evaluate malignant cell contamination. Hematoxylin and eosin (H&E) staining was performed for each patient to search for cancer cells in the tissue. Markers specific to each patient’s disease were identified at the time of diagnosis on the biopsy of the primary tumor or bone marrow aspiration and an immunohistochemistry (IHC) was performed on the FFPE ITT for each patient to evidence his disease markers. PCR analyses on the FFPE tissue were also conducted when a specific gene rearrangement was available.

MAIN RESULTS AND THE ROLE OF CHANCE

The mean age at diagnosis and ITT biopsy of the 28 boys was 7.5 years (age range: 19 months–16 years old). Examination of ITT of the 28 boys on H&E stained sections did not detect malignant cells. Using IHC, we found contamination by cancerous cells using markers specific to the patient’s disease in 10 of 28 boys, with a higher rate in patients diagnosed with ALL (57%, n = 8/14) compared with lymphoma (14%, n = 2/14) (P-value < 0.05). PCR showed contamination in three of 15 patients who had specific rearrangements identified on their bone marrow at the time of diagnosis; one of these patients had negative results from the IHC. Compared to age-related reference values of the number of spermatogonia per ST (seminiferous tubule) (Spg/ST) throughout prepuberty of healthy patients from a simulated control cohort, mean spermatogonial numbers appeared to be decreased in all age groups (0–4 years: 1.49 ± 0.54, 4–7 years: 1.08 ± 0.43, 7–11 years: 1.56 ± 0.65, 11–14 years: 3.37, 14–16 years: 5.44 ± 3.14). However, using a cohort independent method based on the Z-score, a decrease in spermatogonia numbers was not confirmed.

LIMITATIONS, REASONS FOR CAUTION

The results obtained from the biopsy fragments that were evaluated for contamination by cancer cells may not be representative of the entire cryostored ITT and tumor foci may still be present outside of the biopsy range.

WIDER IMPLICATIONS OF THE FINDINGS

ITT from boys diagnosed with a hematological malignancy could bear the risk for cancer cell reseeding in case of autotransplantation of the tissue. Such a high level of cancer cell contamination opens the debate of harvesting the tissue after one or two rounds of chemotherapy. However, as the safety of germ cells can be compromised by gonadotoxic treatments, this strategy warrants for the development of adapted fertility restoration protocols. Finally, the impact of the hematological cancer on spermatogonia numbers should be further explored.

STUDY FUNDING/COMPETING INTEREST(S)

The project was funded by a grant from the FNRS-Télévie (grant n°. 7.4533.20) and Fondation Contre le Cancer/Foundation Against Cancer (2020-121) for the research project on fertility restoration with testicular tissue from hemato-oncological boys. The authors declare that they have no conflict of interest.

TRIAL REGISTRATION NUMBER

N/A.

Introduction

Owing to effective chemo- and radiotherapy treatments, survivorship of children diagnosed with leukemia and lymphoma is high, with a remission rate estimated at more than 80% (Botta et al., 2022). Having survived their disease, these patients are at risk of impaired fertility due to the gonadotoxic effects of their treatments (Wyns et al., 2015).

For prepubertal patients, in whom there is no onset of spermatogenesis yet, the only available option to preserve fertility is the cryopreservation of their immature testicular tissue (ITT) containing spermatogonial stem cells (SSCs), a subpopulation of spermatogonia. Different methods to restore the patients’ fertility have been proposed: IVM of the testicular tissue or cells, and transplantation of ITT fragments or isolated SSCs allowing in vivo maturation (Wyns et al., 2020).

In the case of patients diagnosed with a hematological malignancy, there is a well-known risk of cancer cells being present in the patient’s ITT owing to the hematogenous spreading nature of the disease, especially in leukemic patients where the testis has been shown to be a preferential site for leukemic relapse (Pollanen et al., 1990; Ritzén, 1990) and where there is a high risk of reseeding cancer cells back to the patient in the case of autotransplantation.

In the 1980s and 1990s, several teams studied the testicular tissue of prepubertal patients diagnosed with a hematological cancer using histology and immunohistochemistry (IHC) to search for contamination by cancerous cells. Contamination rates ranging from 21% to 76% were identified (Reid and Marsden, 1980; Chessells et al., 1986). Since then, more sensitive detection techniques have been developed. PCR to detect minimal residual disease (MRD) has already been widely utilized in ovarian tissue and has allowed the detection of malignant cells that were not identified through histology and IHC (Dolmans et al., 2010; Rosendahl et al., 2010; Greve et al., 2012; Asadi-Azarbaijani et al., 2016). This method has not yet been applied to detect the malignant contamination in ITT of prepubertal boys.

In this study, using histology, IHC, and PCR on formalin-fixed paraffin-embedded (FFPE) tissue samples that were harvested at the time of ITT cryobanking for fertility preservation, we evaluated testis tissue fragments of our cohort of young boys diagnosed with leukemia or lymphoma for the presence of cancer cells. As most studies have evaluated spermatogonia numbers in cases where the patients have already been treated by chemotherapy for their cancerous disease (Medrano et al., 2021; Rives-Feraille et al., 2022), we quantified and assessed the impact of cancer on spermatogonia counts in the largest cohort of pre- and peripubertal patients who had not received any gonadotoxic treatment at the time of tissue harvesting.

Materials and methods

Patients and tissue samples

Eligible patients were selected from the records of the Andrology Department of the Cliniques Universitaires Saint-Luc, between 2005 and 2021. Patients diagnosed either with acute lymphoblastic leukemia (ALL) or lymphoma, and who underwent a testicular biopsy with ITT cryopreservation for fertility preservation before any gonadotoxic therapy, were included.

The medical files of all patients were systematically reviewed to collect data on Tanner stages, and age at the time of biopsy as well as the presence of markers specific to the patient’s disease identified at the time of diagnosis through immunophenotyping and PCR on the bone marrow, blood, or initial tumor.

All patients had given their informed consent to cryopreserve a fragment of testicular tissue as part of a fertility preservation procedure approved by the ethics committee since 2005. At the time of sampling, part of the tissue was fixed in 4% paraformaldehyde, alcohol-dehydrated in xylene, and paraffin embedded for routine anatomopathological analysis of the tissue content. Residual FFPE material was used for this study with the approval of the Ethics committee of the Cliniques Universitaires Saint-Luc (2021/26JAN/032). Part of the tissue was obtained from the biobank in the form of paraffin blocs or, for some patients, as a limited number of consecutive 5 µm thin slices of tissue fragments.

Histology

A single 5 µm slice was stained with hematoxylin and eosin (H&E) per patient to detect the presence of cancer cells as described in the most recent ‘WHO classification of tumours of haematopoietic and lymphoid tissues’ (2017) as well as to assess the presence of spermatogonia.

The slides were scanned at 400× using a Leica SCN400 slide scanner (Leica Microsystems, Wetzlar, Germany) and images were analyzed with QuPath software (Bankhead et al., 2017).

Immunohistochemistry

To evaluate the presence of cancer cells, IHC was performed on consecutive 5 µm slices, using an automated IHC assay on the BenchMark ULTRA system (Ventana Medical Systems, Inc., USA) with markers specific to each patient’s disease. The markers of cancer cells for each patient were selected among those found following immunophenotyping performed on the patient’s bone marrow aspirations or his initial tumor at the time of diagnosis of his hematological malignancy. One marker per slide was analyzed for each patient due to the scarcity of the FFPE tissue. Positively marked cells were identified on the whole slide and counted. Since some of the IHC markers used can also be expressed in normal lymphoid cells, the result was confirmed by examining the morphology of the stained cells by an experienced hemato-pathologist. The results were expressed as the number of positive cells over the total number of cells on the slide.

To evidence spermatogonia, MAGEA4 staining was performed as previously described (de Michele et al., 2018). The total number of positively marked spermatogonia per round/oval shaped seminiferous tubules (STs) with a maximum diameter of 1.5 were counted (Ntemou et al., 2019). The results were expressed as the total number of germ cells per round/oval STs on the whole fragment on the slide. Briefly, sections were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked by incubating the slides for 30 min with 0.3% H₂O₂. After washing in deionized water for 5 min, the slides were placed in citrate buffer for 75 min at 98°C. Subsequently, they were washed in 0.05 M Tris-buffered saline (TBS) and 0.05% Triton X-100 (Sigma-Aldrich). To block nonspecific binding sites, they were incubated with 10% normal goat serum and 1% bovine serum albumin (Invitrogen) for 30 min. The primary antibody MAGEA4 (gifted by Giulio Spagnoli, dilution 1/500–1/250) was added to the sections and incubated overnight at 4°C. The following day, the slides were washed in 0.05 M TBS and 0.05% Triton X-100. The slides were then incubated with the secondary anti-mouse antibody (Envision+ System-Labeled Polymer-HRP, DAKO K4001) for 60 min at RT, followed by washing in 0.05 M TBS and 0.05% Triton X-100 three times for 2 min each. The sections were then incubated for 10 min with the chromogen diaminobenzidine (DAKO K3468). The nuclei were counterstained with hematoxylin. Finally, the sections were dehydrated and mounted.

All antibodies used as well as their dilutions are listed in Table 1.

All slides were scanned at 400× using a Leica SCN400 slide scanner (Leica Microsystems, Wetzlar, Germany) and analyzed using QuPath software (Bankhead et al., 2017).

PCR and RT-PCR

Specific fusion transcripts and primers sequences were selected based on the analysis of the bone marrow, blood, or primary tumor at the time of diagnosis and are listed in Table 2.

In these initial samples, T-cell rearrangements (TCR) and clonal immunoglobulin (IGH) were identified through qualitative PCR with a sensitivity of 10⁻² that was determined and validated on samples from a large cohort of patients before routine implementation of the assay in clinical care. Fusion transcripts were identified through quantitative RT-PCR with a sensitivity of 10⁻⁴ to 10⁻⁵, normalized with ABL-1 reference gene.

For TCR and IGH, 10 sections of 10 µm slices were used to extract DNA using the GeneJET FFPE DNA Purification kit (ThermoFisher) according to manufacturer’s instructions (Table 3). PCR to detect IGH and TCR followed the BIOMED-2 guidelines (sensitivity 10⁻²) (van Dongen et al., 2003).

For detection of TEL/AML1 and E2A/PBX1, 10 sections of 10 µm slices were used to extract RNA, using the QIAGEN RNeasy FFPE kit (QIAGEN) following manufacturer’s instructions. Following the Europe Against Cancer Program (Gabert et al., 2003), a homemade mix of primers were used to detect both TEL/AML1 and E2A/PBX1. Real-time quantitative PCR (RT-PCR) was performed with 5 µl cDNA in a final volume of 15 µl per PCR. The results were normalized with ABL-1 reference gene. Results are expressed in quantitative cycle (Cq) value (36.0) (Table 4).

All RT-PCR experiments were performed in duplicates.

Statistical analysis

All analyses were performed using the GraphPad Prism 9 software. Continuous variables are presented as means or medians and SDs. A Chi-squared test was performed to compare contamination rates between patients according to their disease.

P-value <0.05 was considered statistically significant.

Results

Patients’ characteristics

There were 54 eligible patients identified in our database. Figure 1 shows the patients’ numbers and the analyses performed according to their disease. Slides or blocs of FFPE tissue were retrieved for 28 patients for whom H&E and IHC analyses of cancer cell contamination were performed. The mean age at biopsy of the overall cohort was 7.5 years ± 4.4 ranging from 19 months to 16 years old with a mean age of 7.5 ± 4.4 years in the ALL group and 7.9 ± 4.1 years in the lymphoma group.

The characteristics of the study population are summarized in Table 5. Of the 28 patients, 24 were Tanner Stage 1 and four patients were Tanner Stage II or III but were either not able to give a semen sample or the sample was of too poor quality (i.e. azoospermia, cryptozoospermia) and thus they also underwent ITT cryopreservation to optimize the fertility preservation procedure.

Cancer cell contamination of ITT

Histology

There were 26 H&E-stained slides which did not show the presence of malignant cells (Figure 2). Two slides were excluded from analysis due to absence of tissue on the slide after staining.

Immunohistochemistry

The combination of disease- and patient-specific markers that were selected are summarized in Table 2.

The tissue of 10 out of 28 patients showed a positive staining for at least one marker (ALL, n = 8; Lymphoma, n = 2) (Table 2), corresponding to an overall contamination rate of 35.7%.

Significantly higher rates were observed for patients with ALL as shown in Figure 3.

PCR/RT-PCR

PCR for the presence of either TCR or Ig rearrangements was performed on samples of twelve patients. Another three patients had genetic rearrangements in their fusion transcripts, either TEL/AML1 or E2A/PBX1 and were eligible for RT-PCR. The remaining thirteen patients of the cohort either did not have a genetic rearrangement identified at diagnosis (n = 8) or there was no available FFPE testicular tissue (n = 5) to perform the analysis, either because the remaining slides did not contain testicular tissue or the available blocs at the biobank were emptied by the previous analyses (Fig. 1).

While the amount of extracted material quantified using the Nanodrop spectrophotometer showed the presence of RNA in the three samples (Table 4) used for RNA extraction, RT-PCR to quantify the fusion transcripts (TEL/AML1 and E2A/PBX1) was inconclusive as the housekeeping gene was not detected (sensitivity superior to 10⁻¹).

Among the remaining patients’ samples (n = 12) analyzed for TCR and Ig rearrangements, three patients showed the presence of rearrangements in their testicular tissue (Table 2).

Detection and quantification of spermatogonia

Histology was able to identify the presence of spermatogonia in all patients (Table 5).

MAGEA4 staining was performed on the slides with available testicular tissue that were retrieved in all 28 patients. In four patients, no MAGEA4 positive spermatogonia were identified on the slide. In the remaining 24 patients, MAGEA4 positive spermatogonia were identified. The number of spermatogonia per tubule are presented in Table 6. The numbers of round/oval shaped STs analyzed for counting ranged from 18 to 274 STs.

Figure 4 represents these numbers according to the patient’s age.

Supplementary Table S1 represents the Spg/ST Z-scores that were calculated for each patient sample based on the means and standard deviations (SD) of their respective age groups.

Discussion

Many teams have investigated the presence of cancer cells in prepubertal testicular tissue as it was long considered a preferential site for leukemia and lymphoma relapse (Givler, 1969; Kamiyama and Funata, 1976; Reid and Marsden, 1980; Ortega et al., 1984; Suc et al., 1989). Prior to the introduction of effective imaging and molecular biology techniques to evaluate the efficacy of care and to monitor the patient after treatment completion, many teams have used testis tissue sampling to assess the presence or not of residual cancer cells as an indicator for the risk of relapse (for review, see Kourta et al., 2023). In these studies, the tissue fragments were assessed with histology and IHC and the contamination rates ranged from 15% to 45%. These figures are informative of the spreading potential of cancer cells and their ability to persist in the testis following treatment, but they do not allow the clinician to estimate the cancer cell contamination of cryopreserved ITT for fertility preservation purposes as these fragments are most often harvested prior to the start of therapy to avoid its gonadotoxic effects.

In this study, we aimed to assess the contamination rate by cancer cells among ITT samples harvested for fertility preservation prior to the start of any chemo- or radiotherapy from our cohort of pre- and peripubertal boys diagnosed with a hematological cancer. The main goal was to determine whether the cryopreserved ITT bore the risk of disease relapse and to exclude a tissue sample that is not eligible for autotransplantion to the patient after cure.

As some preliminary data suggest that the disease itself could be responsible for a reduced number of spermatogonia contained in the tissue (Stukenborg et al., 2018; Masliukaite et al., 2023) and as the presence of spermatogonia is a prerequisite for the reproductive potential after autotransplantation, a secondary outcome, was to determine whether leukemia or lymphoma had an impact on the spermatogonia counts of these patients.

Cancer cell contamination was observed in 39% of our patients (n = 11/28). Only one other team has previously assessed the presence of malignant cells, before the initiation of cancer therapy, in a cohort of 24 patients diagnosed solely with leukemia, and they found a contamination rate of 21% detected through histology alone (Kim et al., 1981). In our population, histology was unable to identify the presence of cancer cells, whereas IHC detected 10 positive patients. Both the specificity and sensitivity of these techniques in the testis can only be extrapolated from what is already known in the bone marrow. For a histological examination alone, sensitivity and specificity are respectively 86% and 77% (Suchita Pant, 2020). When integrating IHC in the analysis, it enhances the accuracy of the diagnosis making it clearly superior to histology alone. However, for clinical contamination analysis in fertility preservation patients, IHC and histology lack the required specificity and sensitivity, and the use of PCR, which is widely known as the most sensitive technique (as it can detect MRD in as little as 1 MRD cell in 10 000 cells in the bone marrow; van Dongen et al., 2003) is needed. Even though it has never been performed on cryopreserved ITT, PCR to detect MRD on frozen-thawed ovarian tissue of pre- and post-pubertal patients has already been performed by several teams based on the patients’ specific TCR/Ig or genetic rearrangements identified on the bone marrow at diagnosis (Dolmans et al., 2010; Rosendahl et al., 2010; Greve et al., 2012; Asadi-Azarbaijani et al., 2016). We therefore applied such techniques to detect specific cancer cell markers in ITT. However, as evaluation on frozen-thawed samples is limited in young boys by the small size of the testis and thus the amount of tissue sample, we applied the detection methods to FFPE testicular sections. On one hand, the RT-PCR performed on the extracted RNA did not allow us to draw any conclusion about the malignant contamination status of the ITT as the extracted material, though detected with the spectrophotometer, was insufficient and could not be amplified and further analyzed.

On the other hand, the PCR performed on the extracted DNA was able to identify the same TCR or Ig rearrangements as those present in the bone marrow at diagnosis in three patients. Among those three patients, we observed a cancer cell contamination of the tissue in one patient that was not detected by histology or IHC. This finding is in line with previous findings in ovarian tissue showing a discrepancy between histology, IHC, and PCR results in two studies (Rosendahl et al., 2010; Greve et al., 2012) and between histology and PCR in another (Dolmans et al., 2010), warranting the use of more sensitive detection methods.

The risks of cancer cell contamination of the tissue in hematological cancers have led many teams to harvest the ITT after a few rounds of chemotherapy (Braye et al., 2019; Valli-Pulaski et al., 2019; Goossens et al., 2020) as it has been shown to decrease the amount of contamination in ovarian tissue by cancer cells in both prepubertal and adult females without altering the fertility potential (Poirot et al., 2019; Dolmans et al., 2021).

Furthermore, although high, we believe our contamination rates could have been underestimated. We hypothesize this was due to the small size of the ITT FFPE samples that did not allow us to reach the optimal surface of tissue when performing the nucleic acid extractions. We suggest that clinicians take this into account when performing the testicular biopsy at diagnosis whenever possible, knowing the very small testis size of the youngest prepubertal patients. Moreover, the fixation with formalin, causing nucleic acid crosslinking, affects the quality of the extracted nucleic acids thus altering the sensitivity in the downstream applications (Williams et al., 1999; McDonough et al., 2019). While clinicians should take full advantage of the FFPE material in this context, they must remain aware of the quality of such tissue samples. Indeed, when comparing the yields of extracted RNA and DNA as well as sequencing between frozen and FFPE tissue (colon, bladder, and prostate carcinoma tissue, as well as normal colon and liver tissue) from the same patients, Hedegaard et al. (2014) have found similar yields at different time points, ranging from 2 months to 14 years, but have noted differences in the sequencing quality of the FFPE nucleic acids when there was a prolonged storage time, up to 14 years (Hedegaard et al., 2014), which is in line with results in another study where the tissue was also analyzed after 14 years of storage (Boeckx et al., 2011). In our study, the longer median storage time of the FFPE tissue from which RNA (14 ± 1 years) and DNA (13 ± 1.5 years) respectively had been extracted could explain the poor results obtained when performing the RT-PCR on RNA and further support the hypothesis of an undervaluation of the contamination rate in the overall cohort. More factors are key to obtaining reliable as well as reproducible results and should be closely monitored when dealing with FFPE tissue, such as shortening the time between removal and fixation of the fragment (i.e. perioperative ischemia time) as well as the archival storage conditions; some studies have advised storing the FFPE blocks at 4°C (von Ahlfen et al., 2007; Boeckx et al., 2011), as opposed to room temperature in our case.

Besides the risk for cancer cell contamination to be evaluated before considering any autotransplantation, the reproductive potential of the tissue, notably based on the presence and number of spermatogonia, is also important for a successful fertility restoration strategy. By performing IHC to identify and quantify MAGEA4+ cells, we were able to prove the presence of spermatogonia on 24 of 28 available slides. Our results show an increase in the number of MAGEA4+ spermatogonia by age which is consistent with findings in the literature in cohort studies using either MAGEA4 (Heckmann et al., 2018) or UTF1 and DDX4 staining (Valli-Pulaski et al., 2019). Furthermore, a larger meta-analysis, where results from a cohort of 372 boys, mainly control groups from multiple studies, without a known disease affecting spermatogenesis (i.e. cryptorchidism, varicocele, cancer) were combined, the authors determined age-related reference values of the number of spermatogonia per ST throughout prepuberty. In our cohort, we were able to observe a clear increase in 10-year-old boys, followed by a further increase in older age groups, which concurs with spermatogonia number evolutions described in the Masliukaite study (Masliukaite et al., 2016).

Our secondary aim was to determine whether the cancerous disease itself has a negative impact on the presence and numbers of Spg/ST, as suggested by other teams in smaller series of patients who had not been exposed to gonadotoxic therapies (Stukenborg et al., 2018; Masliukaite et al., 2023). In 2018, Stukenborg et al. (2018) had already observed fewer spermatogonia on biopsies of only two prepubertal leukemia patients (AML, juvenile myelomonocytic leukemia (JMML)) stained with DDX4 and identified on biopsies containing ≥25 tubular cross sections. Very recently, Masliukaite et al. (2023) reported, in a cohort of 18 prepubertal patients with a hematological malignancy (ALL, AML, lymphoma), a decreased number of spermatogonia in 44% of boys across all age groups except for the 11–14 year old boys where the authors observed a higher number of Spg/ST values, compared to the simulated dataset of healthy controls (Masliukaite et al., 2016); they used MAGEA4 staining on testicular biopsies collected before treatment and containing ≥30 round/oval shaped STs (P-value > 0.05). When comparing our patients’ samples with the same reference age groups, we found that even though we included a few patients (n = 7) in whom MAGEA4+ cells were counted on less than 25–30 round/oval shaped STs, spermatogonia numbers were decreased in all age groups including the 11–14 years boys (Table 6), concurring with the hypothesis of a deleterious impact of the disease on the testicular tissue of prepubertal boys, irrespective of their age at biopsy. However, to standardize age-dependent differences of spermatogonia numbers, we computed Z-scores of Spg/STs of the patients’ samples in these age groups with reference age means and SDs from the simulated controls cohort (Supplementary Table S1). Based on the Funke et al. study (Funke et al., 2021), mean Spg/ST Z-scores higher than −3SD are found in non-treated cancer patients and indicate a normal spermatogonia count, while patients who received a treatment with an alkylating agent showed reduced counts. In our cohort, none of the mean Z-scores were lower than—3SD, and thus considered within the normal range which is in line with the figures of Funke et al. (2021) for non-treated cancer patients (Funke et al., 2021). This raises the question of whether there is an impact or not of the disease on spermatogonia numbers in these age categories. Furthermore, we performed a subcategory analysis between contaminated and non-contaminated tissue that did not show a difference in Z-score numbers (Supplementary Table S1) between the two subgroups. We could hypothesize that the cohorts in which an adverse effect of the disease was found (Stukenborg et al., 2018; Masliukaite et al., 2023) were comprised of patients with higher contamination rates.

Data for the peripubertal group (14–16 years) are even less clear, as they are beyond the age range of the Masliukaite et al. (2016) study (Masliukaite et al., 2016) so neither a comparison of Spg/ST figures nor the calculation a Z-score were possible. We however did observe lower Spg/ST values than those of the oldest age group of blood cancer patients (11–14 years) (Masliukaite et al., 2023) which could suggest an adverse effect of the disease in this age range, but since the comparison of the results to healthy controls could not be made, these results remain uncertain.

Conclusion

To our knowledge, this is the first study assessing the contamination rate by malignant cells prior to the start of a gonadotoxic therapy in ITT from young boys diagnosed with either leukemia or lymphoma using three different detection methods, i.e. histology, IHC, and PCR. We obtained a contamination rate by cancer cells of 39% in our study population. Such high levels of cancer cell contamination open the debate regarding harvesting the tissue after one or two rounds of chemotherapy. We also observed the superiority of more sensitive detection techniques. Nonetheless, these results should be considered with caution, as the evaluation of part of the tissue may not be representative of the contamination risk of the entire cryopreserved ITT. While negative results will not ensure the safety of the tissue for clinical application, clinicians should consider introducing these methods into routine clinical practice as they allow the exclusion of some patients from fertility restoration through autotransplantation of cryopreserved ITT.

Supplementary data

Supplementary data are available at Human Reproduction online.

Data availability

The data underlying this article will be shared upon reasonable request to the corresponding author.

Acknowledgements

The authors would like to thank Prof. Selda Aydin (Pathology Department, Cliniques Universitaires Saint Luc) for her help in retrieving the FFPE tissues from the biobank.

Authors’ roles

D.K. designed the study, collected the data, performed the experiments, analyzed the results, and wrote the article. A.C. and P.S. participated in data collection and contributed to the interpretation of the results. M.K. and J.P. contributed to the interpretation of the results and scientific discussion. C.W. was responsible for the project concept, supervision of the study, as well as critical review and approval of the final version of the article.

Funding

D.K. is funded by a grant from the FNRS-Télévie (grant no. 7.4533.20) and the Fondation Contre le Cancer/Foundation Against Cancer (2020-121) for the research project on fertility restoration with testicular tissue from hemato-oncological boys.

Conflict of interest

The authors declare no conflict of interest.

References