Tharmalingam et al. BMC Medicine(2020) 18:374
https://doi.org/10.1186/s12916-020-01844-y

RESEARCH ARTICLE

Open AccessCisplatin and carboplatin result in similar
gonadotoxicity in immature human testis
with implications for fertility preservation inchildhood cancer
Melissa D. Tharmalingam1,2†, Gabriele Matilionyte1†, William H. B. Wallace3, Jan-Bernd Stukenborg4,
Kirsi Jahnukainen4,5, Elizabeth Oliver4, Anne Goriely6, Sheila Lane7, Jingtao Guo8,9, Bradley Cairns9,
Anne Jorgensen10, Caroline M. Allen11, Federica Lopes1, Richard A. Anderson1, Norah Spears11 and
Rod T. Mitchell1,3*

Abstract
Background: Clinical studies indicate chemotherapy agents used in childhood cancer treatment regimens may
impact future fertility. However, effects of individual agents on prepubertal human testis, necessary to identify later
risk, have not been determined. The study aimed to investigate the impact of cisplatin, commonly used inchildhood cancer, on immature (foetal and prepubertal) human testicular tissues. Comparison was made withcarboplatin, which is used as an alternative to cisplatin in order to reduce toxicity in healthy tissues.
Methods: We developed an organotypic culture system combined with xenografting to determine the effect ofclinically-relevant exposure to platinum-based chemotherapeutics on human testis. Human foetal and prepubertal
testicular tissues were cultured and exposed to cisplatin, carboplatin or vehicle for 24 h, followed by 24–240 h inculture or long-term xenografting. Survival, proliferation and apoptosis of prepubertal germ stem cell populations(gonocytes and spermatogonia), critical for sperm production in adulthood, were quantified.
Results: Cisplatin exposure resulted in a significant reduction in the total number of germ cells (− 44%, p < 0.0001)
in human foetal testis, which involved an initial loss of gonocytes followed by a significant reduction in
spermatogonia. This coincided with a reduction (− 70%, p < 0.05) in germ cell proliferation. Cisplatin exposure
resulted in similar effects on total germ cell number (including spermatogonial stem cells) in prepubertal humantesticular tissues, demonstrating direct relevance to childhood cancer patients. Xenografting of cisplatin-exposed
human foetal testicular tissue demonstrated that germ cell loss (− 42%, p < 0.01) persisted at 12 weeks. Comparison(Continued on next page)* Correspondence: rod.mitchell@ed.ac.uk†
Melissa D. Tharmalingam and Gabriele Matilionyte contributed equally tothis work.
1
MRC Centre for Reproductive Health, The Queen’s Medical ResearchInstitute, The University of Edinburgh, 47 Little France Crescent, Edinburgh
EH16 4TJ, Scotland, UK
3
Edinburgh Royal Hospital for Sick Children, 9 Sciennes Road, Edinburgh EH9
1LF, Scotland, UK
Full list of author information is available at the end of the article© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
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Tharmalingam et al. BMC Medicine(2020) 18:374

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between exposures to human-relevant concentrations of cisplatin and carboplatin revealed a very similar degree ofgerm cell loss at 240 h post-exposure.
Conclusions: This is the first demonstration of direct effects of chemotherapy exposure on germ cell populations inhuman foetal and prepubertal testis, demonstrating platinum-induced loss of all germ cell populations, and similareffects of cisplatin or carboplatin. Furthermore, these experimental approaches can be used to determine theeffects of established and novel cancer therapies on the developing testis that will inform fertility counselling anddevelopment of strategies to preserve fertility in children with cancer.
Keywords: Human, Testis, Cisplatin, Germ cell, Fertility, Prepubertal, Foetal, Xenograft

Background
Survival rates for children with cancer have increased
dramatically over recent decades, and currently, morethan 80% of those affected are expected to survive theirdisease into adulthood [1]. However, the long-term impacts of cancer treatment remain a major concern forthis patient population. The mainstay of cancer treatment includes chemotherapy and radiotherapy, both ofwhich have the potential to damage healthy tissues
resulting in significant long-term morbidity [2]. An association between chemotherapy exposure and effects on
the reproductive system in male and female childhood
cancer survivors is well-recognised and this may resultin infertility in adulthood [3, 4]. Alkylating agents (e.g.
cyclophosphamide and procarbazine) are reported to beamongst the most highly gonadotoxic chemotherapeutic
compounds in both males and females, and cumulative
exposure to these agents can be used to estimate the riskof gonadal damage in patients due to receive such therapies [5]. However, these models do not include nonalkylating agents that may also contribute to gonadotoxicity, nor are they able to determine the direct effects ofchemotherapeutic agents on the gonad.
Major differences exist between the prepubertal andadult testis in humans, especially in terms of germ cell
populations [6]. Spermatogenesis does not start until
after puberty and therefore sperm are not present. The
germ cell populations in the prepubertal testis are predominantly spermatogonia, expressing MAGEA4 protein. This includes a sub-population of spermatogonial
stem cells (SSC), which express UTF1 [7, 8]. In humanfoetal and infantile testis, an additional population of undifferentiated germ cells (gonocytes), which express
AP2γ, are present. Gonocytes differentiate into (pre)spermatogonia over the course of foetal and early postnatal life [6]. Therefore, future fertility is dependent on
differentiation from gonocyte to spermatogonia in infancy and subsequent survival of the SSC population
during childhood.
Platinum-based chemotherapeutic drugs are widelyused in paediatric oncology for the treatment of solid tumours including neuroblastoma, germ cell tumours andosteosarcoma [5, 9, 10]. These agents are considered tobe cell-cycle independent and act to form DNA adducts,
which elicit apoptosis and cell death [11]. Cisplatin is
the most frequently used platinum-based compound,whilst carboplatin may be used as an alternative to cisplatin as it is considered to have a lower risk of ototoxicity and nephrotoxicity [12], although relative
gonadotoxicity remains to be determined.
No previous studies have been conducted to test theeffects of platinum-based chemotherapy on human testicular tissues. However, experimental systems have recently been developed to assess the direct impact ofpharmaceuticals on immature human testicular tissues
including short- (in vitro) and long-term (testicularxenograft) effects of acute exposure [13, 14]. These experimental approaches utilise human foetal testis, whichcontain the major germ cell populations of the childhood testis (gonocytes and spermatogonia), and cantherefore be used to model the acute effects of chemotherapy exposure and potential for recovery of immature
human testicular tissue.
In the present study, we aimed to determine the effects
of exposure to clinically-relevant concentrations ofplatinum-based chemotherapy on germ cells of the immature prepubertal human testis using human foetal
and prepubertal tissues. We also aimed to investigate therelative gonadotoxicity of cisplatin compared to that ofcarboplatin.

Methods
Experimental design

Given that childhood cancer survivors are reported tohave lower fertility rates in adulthood, we aimed tounderstand the effects of exposure to platinum-based
chemotherapy agents (cisplatin and carboplatin) on key
germ cell populations in immature human testis. We
performed controlled laboratory experiments using bothin vitro (short-term) and xenograft (long-term) models(Fig. 1). Experimental protocols were designed prior startof experiments. Immature human testicular tissue pieces
were exposed to clinically relevant chemotherapy doses
based on human plasma serum levels obtained fromTharmalingam et al. BMC Medicine(2020) 18:374

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Fig. 1 Overview of experimental setup. Designed using BioRender.com. Abbreviations: VEH, vehicle; 0.5 CIS, cisplatin (0.5 μg/ml); 1.0 CIS, cisplatin(1.0 μg/ml); CARBO, carboplatin (5 μg/ml)

paediatric cancer patients. Sample size was determinedbased on previous studies using human tissue (at leastn = 5 for each experiment).
Initial experiments using human foetal tissues investigated the effects of cisplatin-exposure on germ cells
using two drug concentrations (0.5 and 1.0 μg/ml) andtwo time-points post-exposure (24 and 96 h). Further experiments were performed to determine the germ celleffects of cisplatin-exposure (0.5 μg/ml only) at intermediate (72 h) and long-term (240 h) time-points. Someof the in vitro cultured tissue pieces were randomly selected at 24 h post-exposure and subsequently xenografted for 12 weeks. Reciprocal grafts of control- or
cisplatin-exposed tissue were grafted into the right andleft dorsal flank of the same mouse, respectively. In
addition, a comparison between exposure to cisplatin(0.5 μg/ml) and therapeutic equivalent concentrations ofcarboplatin (5 μg/ml) was investigated at 72 and 240 h
post-exposure. Due to the limited amount of available

tissue, the effects of cisplatin-exposure on germ cells in
prepubertal human testicular tissue were investigatedusing a single concentration of cisplatin (0.5 μg/ml) at
two time-points post-exposure (24 and 96 h).
All data were analysed once the study was completed
and no outliers were excluded. To compare the effect oftreatment versus vehicle controls, we analysed at least
two tissue sections from two replicates for each treatment. Endpoints included the quantification of key germ
cell populations and assessment of proliferation andapoptosis. Investigators performing the quantification
were blinded to the treatment group.
Study approval

Ethical approval for use of human foetal tissues for research was obtained from the South East Scotland Research Ethics Committee (LREC08/S1101/1), NRES
committee North East – Newcastle and North Tyneside
1 (08/H0906/21+5) and NRES Committee London –Tharmalingam et al. BMC Medicine(2020) 18:374

Fulham (18/10/0822), and written informed consent wasgiven by the women. Ethical approval for collection anduse of human prepubertal testis tissues in research was
obtained from the South East Scotland Research Ethics
Committee (13/SS0145) and Oxford University Hospitals
NHS Foundation Trust (2016/0140). Written informed
consent was obtained from patients’ parents/guardians
and/or the patient themselves (where appropriate).
For studies involving animals, specific approval, including ethical approval, was given by the UK Home Office. All procedures carried out were by the University of
Edinburgh Bioresearch and Veterinary Services and performed in accordance with the Animal (Scientific procedures) Act 1986.
Tissue collection
Human foetal testicular tissues

Testes from second-trimester human foetuses (14–22
weeks gestation; total n = 33) were obtained after medical and surgical termination of pregnancy as described
previously [6]. None of the terminations were due tofoetal abnormalities. Ultrasound scan and subsequent
direct measurement of foot length were used to determine gestational age. Sex was determined by PCR for
the male-specific gene SRY.
For studies involving animals, specific approval, including ethical approval, was given by the UK Home Office and all procedures carried out were in accordancewith the Animal (Scientific procedures) Act 1986.
Prepubertal human testicular tissues

Prepubertal testicular tissues (n = 5 patients; aged 1, 1, 8,
11 and 12 years) were obtained from chemotherapynaïve patients undergoing a testicular biopsy for fertility
preservation at the Royal Hospital for Sick Children in
Edinburgh and John Radcliffe Hospital in Oxford. A
small portion (~ 10%) of the biopsy was donated for research purposes. Testicular tissues from Edinburgh werecollected and immediately transported in Nutristem®
hSEC XF Medium (Biological industries) supplemented
with 1% penicillin/streptomycin (Sigma-Aldrich). Testicular biopsies from Oxford were transported overnightin Hank’s Balanced Salt solution (HBSS) with 10% human serum albumin.
Hanging drop culture system

Human foetal and prepubertal tissue was utilised forin vitro experiments in a hanging drop culture system[15]. Testicular tissue was cut into small pieces (~ 1
mm3) and placed into 30 μl droplets of appropriate
media (for foetal tissue: Alpha MEM (Lonza); 10% foetal
bovine serum, 1% penicillin/streptomycin, 1% nonessential amino acids, 2 mM L-glutamine, 2 mM sodium
pyruvate and 1% insulin transferrin selenium (ITS) (allPage 4 of 16

Sigma-Aldrich); for prepubertal tissue: Alpha MEM(Lonza) and 10% Knockout Serum Replacement (KSR;
Gibco)) and cultured at 37 °C and 32 °C, respectively,
under 5% CO2. Tissue pieces were cultured for 1–3 days
(subject to the exact timing of the tissue arrival) prior to
exposure to cisplatin (Sigma-Aldrich) or vehicle control(ddH2O) for 24 h. Tissue pieces were then transferred to
fresh media without cisplatin and cultured until 24, 72,
96 and 240 h post-cisplatin exposure. In a separate set ofculture experiments, tissues were exposed for 24 h to either cisplatin (0.5 μg/ml), carboplatin (5 μg/ml; Calbiochem) or vehicle control (ddH2O) for 24 h and theexperiment was ended at 72 and 240 h post-exposure. In
some cases, there was sufficient material from a biopsy
sample to allocate to more than one experimental setup.
Human tissue xenografting

Testicular tissue pieces from human foetuses (n = 4) thatwere cultured in vitro were used for xenografting at 24 hpost-cisplatin or vehicle exposure. As previously described [16], tissue fragments were grafted subcutaneously into host adult CD1-nude mice using a 13-gauge
cancer implant needle (Popper and Sons). For eachfoetal sample, a total of 4 pieces (~ 1 mm3) of testicular
tissue was inserted under the dorsal skin of the hostmouse, with random allocation of two vehicle controls
and two 0.5 μg/ml cisplatin-treated pieces along eitherside of the midline. A total of 4 mice were grafted per
foetal sample, and the mice were maintained for 12
weeks post grafting before retrieval of the tissue. Mice
were housed in the same location and exposed to 12 h
light/dark cycles and room temperature, ~ 20–25 °C.
Mice were administered 100 μl of 20 IU human chorionic gonadotrophin (hCG) (Ovitrelle, Merck Serono) beginning a week after xenografting which was continued
three times a week until the end of the experiment. One
mouse was culled before the end of the experiment due
to poor health and was not included in the analysis. For
xenograft retrieval, host mice were culled by inhalationof CO2 and cervical dislocation. Weight of host mouse
body and retrieved grafts were recorded.
Tissue processing

At the end of each experiment, tissue pieces were fixedin Bouin’s fluid (Clin-Tech) for 1 h and transferred to
70% ethanol. Post fixation, samples were paraffinembedded, 5 μm sectioned and assessed by H&E using
standard protocol or used for immunohistochemistry as
described below. Only samples that showed healthy tissue morphology (defined tubules, minimal apoptosis andgerm cell presence) in pre-culture and vehicle controls
were included in the analyses. Two sections from two
replicates for each treatment were stained and analysed.

Tharmalingam et al. BMC Medicine(2020) 18:374

Immunohistochemistry

Double colourimetric immunohistochemistry was performed to detect gonocytes and spermatogonia (detailsof primary antibody in Additional File 1: Table S1).
Sections were dewaxed by incubating in xylene (BDH
Prolabo®, VWR Chemicals) twice for 5 min each. Tissueslides were rehydrated in alcohol series (100% twice, 95%,
80% and 70%) for 20 s and thoroughly rinsed in tap water.
Heat-induced epitope retrieval of all slides was carried out
in a pressure cooker containing 0.01 M citrate buffer. Tissue sections were rinsed in water and blocked with 3% (v/v) hydrogen peroxide in methanol for 15 min on a rocker
at room temperature. To minimise cross-reactivity, tissue
sections were incubated with blocking agent (consisting of
20% appropriate animal serum in Tris-buffered saline(TBS, 0.01 M Tris, 0.85% sodium chloride, pH 7.4) and 5 gof bovine serum albumin (BSA, Sigma-Aldrich, GmbH))
for 30 min at room temperature. Primary antibody diluted
in blocking agent was added to tissue sections for overnight incubation at 4 °C.
To detect the primary antibody, slides were incubatedwith peroxidase (ImmPRESS, Vector Laboratories) for
30 min at room temperature. Each incubation step was
followed by two 5-min washes in TBS. To visualise thefirst antibody, 3,3-diaminobenzidine (DAB, Vector Laboratories) was added and colour development (usually
up to 3 min) was monitored microscopically and stopped
by washing in deionised water. Prior to the second antibody detection, antigen epitopes were retrieved by placing slides in pre-heated 0.01 M citrate buffer andboiling for 2.5 min using a microwave. Once cooled,slides were blocked with appropriate normal serum/
TBS/BSA for 30 min at room temperature, followed byan overnight incubation at 4 °C with subsequent primary
antibody diluted in normal serum/TBS/BSA.
To detect the second antibody, tissue sections were incubated with alkaline phosphatase (ImmPRESS, Vector
Laboratories) for 30 min at room temperature, visualisedby adding FastBlue substrate (Vector Laboratories), andthe reaction was stopped by rinsing in deionised water.
Slides were mounted with PermaFluor aqueous medium(Lab Vision™, Thermo Scientific). Images were taken on
Axio Scan.Z1 slide scanner (Carl Zeiss Microscopy,GmbH) at × 20 magnification.
In addition, each experiment involved positive (all reagents added) and negative controls (primary antibody
replaced with blocking agent) of pre-culture controls
from each foetal sample. Slides for different treatmentsof the same foetal sample were all processed in parallelin a single experiment.
Immunofluorescence

Dewaxing, rehydration, antigen retrieval and blocking ofendogenous peroxidase and appropriate serum blocking

Page 5 of 16

were performed as described above for immunohistochemistry. To detect the primary antibody, slides wereincubated with peroxidase-conjugated secondary antibody for 30 min and visualised using Tyramide signal
amplification kit (PerkinElmer, Inc.) at 1:50 for 10 min(both steps at room temperature). Antigen retrieval was
performed using microwave treatment prior to adding
subsequent primary antibodies. Same detection steps
using different fluorophores were performed for eachprimary antibody. Nuclei were counterstained with
Hoechst (Thermo Fisher Scientific) diluted in TBS at 1:
2000. Tissue sections were mounted with Permafluor(Lab Vision™, Thermo Scientific), and tiled images of thewhole tissue section were captured at × 20 magnification
using LSM 780 Confocal microscope (Carl Zeiss Microscopy, GmbH). Primary and secondary antibody details
used for single and triple immunofluorescent staining
are in Additional File 1: Table S1.
Statistics

Positively stained cells were manually counted and tubular, and tissue areas were measured using Zen 2.3 lite
Blue Edition (Carl Zeiss Microscopy, GmbH) software.
The interstitial area was obtained by subtracting thetubular area from the tissue area. All tubules per tissue
section were included in the analysis. Cell numbers per
tubular or tissue area (mm2) were calculated and plotted.
Percentage in cell number in comparison to control was
also calculated. Data were presented as mean +/− SEM.
Two-way ANOVA statistical analysis was performed inorder to account for inter-individual sample variation
using GraphPad Prism 8 software (La Jolla, CA, USA).
Statistical significance was set to p < 0.05.
Single-cell sequencing analysis

Single-cell RNA sequencing was previously performedon human prepubertal testis ranging from 1 to 11 yearsof age [7]. The resulting dataset was interrogated to define the cell states and populations present in the testisof a 7-year-old, whilst MAGEA4 (spermatogonia) andUTF1 (SSCs) expression was extracted from the dataset
for individuals aged 1, 7 and 11 years. Data were presented as t-SNE and violin plots.

Results
Cisplatin exposure results in differential germ cell loss inhuman foetal testis

Human foetal testicular tissues (n = 11, 14–22 GW)
were exposed in vitro to cisplatin (0.5 and 1.0 μg/ml)
or vehicle for 24 h, and germ cells per tubular area(mm2) were quantified at 24 and 96 h post-exposure(Fig. 2). Germ cell populations consisted of gonocytes(AP2γ+)
and(pre)spermatogonia(MAGEA4+)(Fig. 2a–c). At 24 h post-exposure, there was noTharmalingam et al. BMC Medicine(2020) 18:374

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Fig. 2 Acute effects of cisplatin exposure on germ cell populations in the human foetal testis. AP2γ+ gonocytes (brown) and MAGEA4+(pre)spermatogonia (blue) protein expression in the human foetal testis 24 h (a–c) and 96 h (g–i) following exposure to vehicle or cisplatin (0.5
and 1.0 μg/ml). Scale bars represent 100 μm (or 50 μm for insets). Dotted lines outline seminiferous tubules. Quantification of germ cell counts per
tubular area (mm2) in the human foetal testicular tissues 24 h (d–f) and 96 h (j–l) following exposure to vehicle (VEH) or cisplatin (0.5 and 1.0 μg/ml). Germ cell numbers were unaffected 24 h post-exposure, whilst a significant reduction in number of gonocytes, (pre)spermatogonia and total
germ cells was observed at 96 h following exposure to 0.5 μg/ml and 1.0 μg/ml cisplatin exposure. Data analysed using two-way ANOVA. **p <0.01, ***p < 0.001 and ****p < 0.0001. Values shown are means ± SEM and each data point represents an individual foetus (n = 9–11)

significant difference in either germ cell population in
cisplatin-exposed compared to vehicle-exposed testicular tissues (Fig. 2d–f). However, at 96 h postexposure, germ cell loss was evident for cisplatin exposure at both concentrations (Fig. 2g–i). Quantification showed that both germ cell populations(gonocytes and (pre)spermatogonia) were significantly
reduced in cisplatin-exposed testicular tissues (Fig. 2j,k) and overall total germ cell number (gonocytes +(pre)spermatogonia) was reduced by 44% for both
0.5 μg/ml (301 v 536, p < 0.0001) and 1.0 μg/ml (298 v
536, p < 0.0001) cisplatin exposures (Fig. 2l). Since thedegree of germ cell loss was similar with both concentrations of cisplatin, further experiments were focused on exposure to 0.5 μg/ml cisplatin only.
In order to determine the timing of germ cell loss
for gonocytes and (pre)spermatogonia, we investigatedthe effect of cisplatin exposure at an intermediate

Tharmalingam et al. BMC Medicine(2020) 18:374

time-point (72 h post-exposure, n = 8; Fig. 3a–e).
There was a significant reduction in the number of
gonocytes in cisplatin-exposed testicular tissues (−21%, 380 v 479, p < 0.01; Fig. 3c). However, (pre)spermatogonial number was unchanged in cisplatinexposed testicular tissues compared with vehicle control (270 v 281, p > 0.05; Fig. 3d). Despite the factthat (pre)spermatogonial number was not affected bycisplatin exposure, the loss of gonocytes was sufficient

Page 7 of 16

to cause a significant overall reduction in total germ
cell number (− 15%, 650 v 763, p < 0.01; Fig. 3e).
Progression of cisplatin-induced germ cell loss was investigated in testicular tissues at 240 h post cisplatinexposure (n = 14; Fig. 3f–j). The reduction in gonocyte(− 41%, 194 v 327, p < 0.0001; Fig. 3h), (pre)spermatogonia (− 18%, 324 v 393, p < 0.01; Fig. 3i) and total germ
cell number (− 37%, 433 v 691, p < 0.0001; Fig. 3j) was
maintained over the extended culture period.

Fig. 3 Intermediate and prolonged effects of cisplatin exposure on germ cell populations in the human foetal testis. AP2γ+ gonocytes (brown)
and MAGEA4+ (pre)spermatogonia (blue) protein expression in the human foetal testis 72 h (a, b) and 240 h (f, g) following exposure to vehicle
or cisplatin (0.5 μg/ml). Scale bars represent 100 μm (or 50 μm for insets). Dotted lines outline seminiferous tubules. Quantification of germ cell
subpopulations in the human foetal testis 72 h (c–e) and 240 h (h–j) following exposure to vehicle (VEH) and cisplatin (CIS; 0.5 μg/ml). At 72 h
post-exposure to cisplatin, gonocytes and total germ cells were significantly reduced, whilst (pre)spermatogonia were unaffected. At 240 h postexposure, a decrease in all germ cell populations was observed. Data analysed using two-way ANOVA. *p < 0.05, **p < 0.01 and ****p < 0.0001.
Values shown are means ± SEM and each set of data points represents an individual foetus (n = 8–14)

Tharmalingam et al. BMC Medicine(2020) 18:374

Cisplatin exposure reduces cell proliferation in both germ
cell populations in human foetal testis

To investigate further the reason for germ cell loss in
cisplatin-exposed testicular tissues, we assessed apoptosis
by immunostaining for cleaved caspase 3 (CC3) (Additional File 2: Figure S2). There was no significant difference
in total number of apoptotic (CC3+) cells in the seminiferous cords of cisplatin-exposed testicular tissues, comparedto vehicle controls at either 24 or 96 h post-exposure.

Page 8 of 16

Apoptotic cells in the interstitium were also extremely rare
in both cisplatin-exposed and vehicle control tissues.
Therefore, we assessed proliferation (Ki67+) as an explanation for germ cell population loss in cisplatin-exposed testicular tissues at 24 h post-exposure (Fig. 4a, b). At thistime-point, there were no significant differences in proliferation of either germ cell population following cisplatinexposure (Fig. 4c–e). However, at 96 h post-exposure(Fig. 4f, g), there was a reduction in proliferating germ cells

Fig. 4 Acute effects of cisplatin exposure on germ cell proliferation in the human foetal testis. AP2γ+ gonocytes (red), MAGEA4+ (pre)spermatogonia (blue) and Ki67 (proliferation; green) protein expression in the human foetal testis 24 h (a, b) and 96 h (f, g) following exposure tovehicle (VEH) or cisplatin (CIS; 0.5 μg/ml). Scale bars represent 100 μm (or 20 μm for insets). Quantification of germ cell counts per tubular area(mm2) in the human foetal testis 24 h (c–e) and 96 h (h–j) following exposure to vehicle or cisplatin (0.5 μg/ml). A decrease in proliferating(pre)spermatogonia and total germ cells was observed at 96 h post exposure to cisplatin. *p < 0.05, **p < 0.01. Data analysed using two-way
ANOVA. Values shown are means ± SEM and each set of coloured data points represents an individual foetus (n = 7)

Tharmalingam et al. BMC Medicine(2020) 18:374

in cisplatin-exposed testicular tissues, compared with vehicle controls (Fig. 4h–j). Proliferating (pre)spermatogonia
were significantly reduced (− 81%, 5 v 26, p < 0.01; Fig. 4i)
by cisplatin exposure, and whilst there was a similar trend
towards reduction in proliferating gonocytes (− 71%, 28 v
101, p = 0.06; Fig. 4e), this did not reach statistical significance. Overall, proliferation was decreased in the total germ
cell population (− 70%, p < 0.05; Fig. 4j).
These data demonstrate acute and specific loss of germ
cells from the seminiferous epithelium of human foetal testicular tissues following cisplatin exposure. Furthermore,
there was a differential effect in the timing of germ cell loss,
with gonocyte number being reduced at an earlier timepoint (72 h) compared to (pre)spermatogonial number (96h). This germ cell loss was associated with a reduction in
the number of proliferating germ cells in the testis.

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Exposure to cisplatin results in long-term germ cell loss inhuman foetal testis xenografts

To determine whether there is potential for recovery fromacute cisplatin-induced loss of germ cells, given the ongoingalbeit reduced rate of germ cell proliferation, human foetal(n = 4) testicular tissue pieces that had been exposed to cisplatin in vitro were xenografted subcutaneously into immunocompromised host mice for 12 weeks (Fig. 4). Graft
retrieval rates were similar for mice xenografted withcisplatin-exposed testicular tissues compared with vehicle
controls (90 v 77%, p > 0.05). Seminiferous cord structure
was maintained and both germ cell populations were identified in vehicle (Fig. 5a) and cisplatin-exposed (Fig. 5b) xenografts. However, mean retrieved xenograft weights (mg)were significantly reduced in the cisplatin-exposed groupcompared with vehicle controls (− 50%, 0.50 v 1.02, p <Fig. 5 Long-term effects of cisplatin exposure on human foetal testis xenografts. Immunohistochemistry for AP2γ+ gonocytes (red) and MAGEA4+(pre)-spermatogonia (blue) in human foetal testis tissues exposed to vehicle (VEH; a) or cisplatin (CIS; b) for 24 h prior to xenografting (12 weeks).
Total graft weight (c) was significantly reduced following cisplatin-exposure. Quantification of cell number (d–f) and proliferation (g–i) of germ
cell populations per tubular area (mm2) in xenografts. A significant decrease in all germ cells populations was observed in cisplatin-exposed
tissues. Proliferating (pre)spermatogonia and total germ cells were also significantly reduced in cisplatin-exposed tissues after long-term
xenografting. Data analysed using two-way ANOVA. *p < 0.05, **p < 0.01 and ***p < 0.001. Values shown are means ± SEM and each set ofcoloured data points represents an individual foetus (n = 4)

Tharmalingam et al. BMC Medicine(2020) 18:374

0.001; Fig. 5c). Assessment of xenografts at 12 weeks postgrafting revealed a significant reduction in gonocyte (− 68%,
37 v 115, p < 0.01; Fig. 5d), (pre)spermatogonial (− 35%, 238v 366, p < 0.01; Fig. 5e) and total germ cell (− 42%, 276 v
475, p < 0.01; Fig. 5f) numbers in cisplatin-exposed xenografts compared with vehicle control xenografts. The reduction in graft weight and in total germ cell number in thecisplatin-exposed group could result from either persistentcell loss or a reduction in cell proliferation. Therefore, wequantified proliferation in the germ cell sub-populations(Fig. 5g–i). There was a significant reduction in proliferationof (pre)spermatogonia (− 63%, 11 v 30, p < 0.05; Fig. 5h) andtotal germ cells (− 59%, 27 v 65, p < 0.05; Fig. 5i); however,the similar degree of reduction in gonocytes was not statistically significant (− 53%, 26 v 55, p > 0.05; Fig. 5g).
Taken together, these data indicate that cisplatininduced (pre)spermatogonial loss persists in the immature human testis for several months post-exposure andthat germ cell proliferation is not sufficiently increased
to compensate for this loss.
Cisplatin-exposure leads to loss of spermatogonial stem
cells in the human prepubertal testis

Spermatogonia in the prepubertal human testis havebeen shown to express MAGEA4, with a sub-populationof spermatogonia co-expressing UTF1 believed to represent spermatogonial stem cells (SSCs) [7]. Given that future fertility is dependent on the survival of the SSC
population, we interrogated our recent single-cell transcriptomics data from prepubertal human testicular tissues for MAGEA4 and UTF1 [7]. In tissues obtained
from a 7-year-old boy, germ cells were represented by adistinct cluster separate from somatic cell populations(Fig. 6a). MAGEA4 showed relatively stable expression
in germ cells of boys aged 1, 7 and 11 years, whilst relative expression of UTF1 was reduced with increasing age(Fig. 6b), probably due to progression towards spermatogonial differentiation [7]. To determine whether
cisplatin-exposure in prepuberty results in a similar
germ cell (MAGEA4+) loss to that described for the human foetal testis and if this included the spermatogonial
stem cell (MAGEA4+ /UTF1+) population, we performedin vitro culture of prepubertal testicular tissue obtained
from boys prior to chemotherapy exposure (n = 3–5, 1–
12 years). Tissues were exposed to cisplatin (0.5 μg/ml)
for 24 h, with analysis at 24 (n = 5, aged 1, 1, 8, 11 and12 years) and 96 h (n = 3, aged 1, 1 and 12 years) postexposure (Fig. 6c–h). Cisplatin-exposure did not resultin a significant change in the total number of spermatogonia at 24 h post-exposure (536 v 496, p > 0.05; Fig. 6d).
Overall, SSC number was reduced in prepubertal humantesticular exposed to cisplatin at 24 h post-exposure,compared to vehicle-exposed controls, although this was
not statistically significant (16 v 75, p > 0.05; Fig. 6e). As

Page 10 of 16

there was sufficient testicular tissue available from threeof the patients (aged 1, 1 and 12 years), we also investigated the effects of cisplatin treatment at 96 h postexposure. Quantification showed that there was a significant reduction in total spermatogonial number (− 30%,
310 v 446, p < 0.05; Fig. 6G) and a reduction in SSC
number (− 52%, 6.5 v 13.5, p < 0.05; Fig. 6H), comparedto vehicle-exposed controls. Importantly, there were no
SSCs remaining in the cisplatin-exposed group for 2 outof 3 patients (aged 1 and 12 years) (Fig. 6h).
In summary, these results demonstrate cisplatininduced germ cell loss in prepubertal human testicular
tissues, including a reduction or loss of the SSC population. The degree and timing of spermatogonial cell loss
was similar to that seen in human foetal testicular tissues after cisplatin exposure. This provides further validation for the use of the human foetal testis as a relevant
model system for studying impacts of exposures during
prepuberty, whilst also demonstrating consistent germ
cell effects at different key stages of human testiculardevelopment.
Carboplatin induces similar effects to cisplatin on germ
cell populations in the human foetal testis

Carboplatin is used in clinical practice as an alternative
to cisplatin because of its reported lower risk of side effects. Therefore, we aimed to determine whether carboplatin exposure resulted in a reduction in gonadotoxicity
compared with cisplatin, at equivalent human-relevant
concentrations. Human foetal testicular tissues (n = 6)
were exposed in vitro to cisplatin (0.5 μg/ml), carboplatin (5 μg/ml) or vehicle for 24 h: carboplatin was administered at 10 times the concentration to cisplatin in linewith relative therapeutic doses used in patients. Germcells were quantified at 72 and 240 h post-exposure(Fig. 7). At 72 h post-exposure, there was a significant
reduction in gonocytes in cisplatin-exposed compared tovehicle-exposed tissues (− 28%, 193 v 267, p < 0.01),
whilst carboplatin did not result in a significant change
in gonocyte number (Fig. 7a). (Pre)spermatogonia andtotal germ cell number were unaffected after 72 h forboth platinum-based agents (Fig. 7b, c). However, at
240 h post-exposure, gonocytes were significantly reduced to a similar extent following exposure to cisplatin(− 67%, 76 v 228, p < 0.0001) or carboplatin (− 58%, 96 v
228, p < 0.0001), compared with vehicle-exposed controls(Fig. 7d). (Pre)spermatogonial numbers were similar between cisplatin- and carboplatin-exposed testicular tissues after 240 h and were not significantly different tovehicle controls (Fig. 7e). However, total germ cell number was significantly reduced by a similar amount inboth cisplatin- (− 30%, 492 v 704, p < 0.01) andcarboplatin-exposed tissues (− 28%, 509 v 704, p < 0.05)
compared to vehicle control (Fig. 7f).

Tharmalingam et al. BMC Medicine(2020) 18:374

Page 11 of 16

Fig. 6 Effects of cisplatin exposure on human prepubertal testis. a t-SNE plot of cell populations obtained during single-cell sequencing of
testicular tissue of a 7-year-old male. b Violin plots for normalised expression of (pre)spermatogonium (MAGEA4) and SSC (UTF1) populations intesticular tissues from 1-, 7- and 11-year-old (yo) males. Red dots represent mean expression. Effect of cisplatin (CIS, 0.5 μg/ml) exposure on MAGE
A4+ spermatogonia (blue) and UTF1+ (green) SSCs in the human prepubertal testis 24 h (c–e) and 96 h (f–h) after exposure compared to vehicle(VEH) control. A significant decrease in spermatogonia and SSCs was observed at 96 h post-cisplatin exposure. Data analysed using two-way
ANOVA. *p < 0.05. Values shown are means ± SEM and each set of coloured data points (blue and red—1 year, orange—8 years, green—11 years,
purple—12 years) represents an individual patient (n = 3–5). c, f Arrows—spermatogonium (MAGEA4+), arrowheads—SSC
subpopulation (MAGEA4+/UTF1+)

Taken together, these results demonstrate thatclinically equivalent exposure to carboplatin resultsin similar effects on the germ cell population of theimmature human testicular tissues to those of
cisplatin.

Discussion
The present study, using validated experimental models,demonstrates that exposure to platinum-based chemotherapy agents results in acute loss of germ cells in theimmature human testis and that this germ cell loss doesTharmalingam et al. BMC Medicine(2020) 18:374

Page 12 of 16

Fig. 7 Comparison of cisplatin and carboplatin exposure on germ cell populations of the human foetal testis. Quantification of germ cell
subpopulations in the human foetal testis 72 h (a–c) and 240 h (d–f) following exposure to vehicle (VEH), cisplatin (CIS; 0.5 μg/ml) or carboplatin(CARBO; 5 μg/ml). At 72 h post-exposure, gonocytes were significantly reduced for cisplatin only. At 240 h post exposure, a decrease in gonocytes
and total germ cells was observed following exposure to either cisplatin or carboplatin. Data analysed using two-way ANOVA. *p < 0.05, **p < 0.01
and ****p < 0.0001. Values shown are means ± SEM and each set of data point represents an individual foetus (n = 6)

not recover several months after exposure. The results
also indicate a differential decline in germ cell populations, involving an initial loss of gonocytes followed by areduction of spermatogonia. Importantly, the effects ofcarboplatin were similar to those of cisplatin, based onhuman-relevant drug concentrations.
Cisplatin exposure resulted in a significant reduction
in total germ cells in both foetal and prepubertal humantesticular tissues in culture. This is in keeping with aprevious in vitro study in rodents, which demonstrated a
reduction in germ cell number in prepubertal mouse testicular tissues exposed to cisplatin [17]. In that study,
there was a > 95% reduction in germ cell number 72 h
after cisplatin exposure, whereas the reduction in germ
cells in the present study was ~ 70–80% at 96 h postexposure.
A key finding from the present study was the reduction in the SSC pool in prepubertal human testicular tissues
following
exposuretoplatinum-based
chemotherapy. SSCs represent the key population required for repopulation and restoration of spermatogenesis in the adult testis following gonadotoxic therapy inprimates [18]. Implementation of single-cell transcriptomics has allowed a detailed characterisation of spermatogonial populations present within the adult humantestis [19, 20] and several SSC ‘states’ each with a uniquetranscriptional profile have been described [19]. This approach has also been applied to the human prepubertal
testis, indicating the existence of similar SSC signatures,
including the expression of UTF1 as a key feature of the
SSC pool [7]. The present study demonstrates that exposure to cisplatin in vitro results in a 52% reduction in
SSCs (MAGEA4+ /UTF1+) in the prepubertal humantesticular tissues. Importantly, this overall reduction in
SSCs includes 2 out of 3 patients (aged 1 and 12 years)
in whom no SSCs were identified in the tissue 96 h after
exposure. This suggests that fertility may be permanently
impaired in some individuals receiving therapeutic dosesof cisplatin, consistent with the association of cisplatin
exposure with infertility in male childhood cancer survivors [4]. Further studies involving a larger number of patient samples are required to reduce the impact ofindividual variability and determine whether SSC loss
following platinum-based chemotherapy is dependent on
age or pubertal status. The present findings are of potential importance for counselling patients on future fertility
and for fertility preservation options. Current strategies
to preserve fertility in prepubertal boys involve cryopreservation of testicular material [8, 21]. However, thefuture success of this process to restore fertility later inlife is dependent on the preservation of germ cells withstem cell capability of cryopreserved tissues [22]. It isTharmalingam et al. BMC Medicine(2020) 18:374

conceivable that some of the surviving (MAGEA4+/UTF1−) germ cells in these patients may retain, or acquire, SSC capabilities following gonadotoxic therapy, as
has been demonstrated in mouse models of testicular regeneration and repair following exposure to busulphan,a chemotherapeutic belonging to the highly gonadotoxic
group of alkylating agents [23].
The impacts of exposure to chemotherapy duringchildhood on subsequent reproductive function havebeen investigated in clinical cohorts of childhood cancer
survivors [3–5, 24]. These studies demonstrate that exposure to alkylating agents is associated with infertility inmales [5]. For cisplatin, the data is less consistent. Exposure to platinum-based chemotherapy was not associatedwith a reduction in partner pregnancy rates in one cohortof childhood cancer survivors [25], whilst two other studies involving the Childhood Cancer Survivor Study reported a significant reduction in partner pregnancy rates
in cancer survivors treated with platinum agents compared to their siblings [4, 5]. Clinical studies involving
childhood cancer survivors are limited in their ability todetermine the effects of individual agents as they usually
involve individuals who have received a variety of treatments, including combined therapies which result in seriesof small numbers of patients receiving like-for-like treatments. Therefore, studies involving human-relevant
experimental systems can be useful to determine thespecific effects of an individual agent on testiculargerm cells.
The search for effective cancer therapies with reduced
side-effects has prompted the development of new generations of drugs with lower cytotoxicity. Therefore, we
compared the relative gonadotoxicity of cisplatin withcarboplatin and showed that there were minimal differences in the impact on germ cell numbers between thesetwo platinum-based therapies. Exposure to either agent
resulted in a significant reduction in germ cell number
in human foetal testicular tissues with a similar degreeof germ cell loss in cisplatin- (− 34%) compared tocarboplatin-exposed (− 26%) testicular tissues at 240 h
post-exposure. Although the reduction in (pre)spermatogonial numbers in the cisplatin-exposed group did notreach statistical significance, there was a similar magnitude of reduction to the initial cisplatin-exposure studies
and this is likely to be explained by the smaller numberof foetal testicular samples used in the cisplatincarboplatin studies. Importantly, the present work used
exposures that reflect differences in the doses given topatients in clinical practice: carboplatin is given at ~ 10times the dose of cisplatin and therefore the concentrations used in our in vitro treatments reflected this difference. The finding of no difference in relative
gonadotoxicity between the two agents is consistent witha recent study comparing in vitro exposure inPage 13 of 16

prepubertal murine testis [26]. This study demonstrated
a significant reduction in spermatogonia in both cisplatin- and carboplatin-exposed testis when compared tovehicle-exposed controls with an almost identical proportion of germ cell loss for both drugs [26]. Whilst noadvantage of using carboplatin versus cisplatin is demonstrated in relation to gonadotoxicity, the fact that
there is no worsening of testicular effects coupled with
reported advantages of carboplatin for reducing toxicities in other organs (e.g. ototoxicity) may still support
the preferential use of carboplatin. Comparably, replacement of the alkylating agent procarbazine with an alternative (dacarbazine) from the same class is associatedwith an increased recovery of spermatogenesis without
affecting overall patient survival [27]. Overall, these results demonstrate the importance of establishing therelative toxicity on each tissue of potential replacements
for current therapies, especially when the replacement isin the same class as the existing agent. Because of thelimited availability of human material for research, theanalysis had to be performed across broad age groups,
ranging from ‘second trimester’ and ‘prepuberty’. Hence,a larger study distinguishing early and late foetal andspecific prepubertal periods, e.g. ‘mini-puberty’ in early
postnatal life, may provide further insight into gonadotoxic impacts on the testis at specific stages during
development.
This study demonstrates the utility of these novel experimental approaches for assessing the acute and longterm effects of chemotherapy exposures on the human
prepubertal testicular tissues. In addition, the experimental models may also be used to study the combination of treatments and repeated exposures to mimic
regimens used in clinical practice. This would provide
direct experimental evidence to complement the findings
from clinical studies involving male childhood cancer
survivors [5]. Cancer treatments are rapidly evolving toinclude novel agents including immunotherapy involving
monoclonal antibodies and manipulation of specific cellsignalling pathways. For many of these agents, there isno information on long-term reproductive effects. The
presently used experimental test systems provide ahuman-relevant model that can be used to investigate
the gonadotoxicity of these emerging cancer therapies.
The results of this study in relation to the sensitivity of
the gonocyte population are important and have implications for chemotherapy treatment given to pregnant
women. It is recognised that many chemotherapeutics
can induce foetal loss due to global toxicity to themother; however, when foetal loss is not expected, there
may still be effects on gonadal function and germ cell
survival of the developing foetal testis. During the firsttrimester of pregnancy, the germ cell population in the
foetal gonad consists primarily of gonocytes [16]. DuringTharmalingam et al. BMC Medicine(2020) 18:374

foetal life, there is a gradual transition of the entire germ
cell population from gonocyte to (pre)spermatogonia[16, 28]. In the present study, the acute reduction of
gonocytes resulting from in vitro exposure to platinumbased chemotherapy agents was associated with a subsequent reduction in (pre)spermatogonia in the humanfoetal testis. This indicates that the establishment of the
spermatogonial population in the foetus may be sensitive
to the chemotherapy regimen given to the mother during pregnancy. This is further supported by the findingthat the reduction in (pre)spermatogonial number ismaintained in xenografted testicular tissues several
months after exposure. We have previously shown thathuman foetal testis xenografts exhibit good preservationof testicular structure, seminiferous cord appearance andhistology compared with ungrafted materials and thatdevelopment from gonocytes to spermatogonia continues during the graft period, similar to age-matchedungrafted control testis [16]. Hence, the xenograft model
represents a suitable system to determine the effects of
exposure on germ cells in the human foetal testis.
Few studies have investigated the effects of cisplatin
exposure in pregnancy. Cisplatin administered to pregnant women for cervical cancer during the second trimester crosses the placenta resulting in concentrations
ranging from 23 to 65% (cord blood) and 11–42% (amniotic fluid), compared with those in the maternal circulation [29]. In baboons, administration of carboplatin has
been shown to result in foetal serum levels which were
~ 50% of those in the maternal serum and significant
malformations were noted in 4% of the exposed foetuses[30]. In rodents, cisplatin accumulated in foetal tissue
approximately a week post-administration [31]. Placental
transfer occurs through diffusion, although platinum
agents also commonly utilise active transport mediated
via transporters p-glycoprotein, multi-resistance protein(MRP)-1 and 2 [32], copper influx transporters, ATP7A
and ATP7B [33].
Foetal life is a critical period for programming subsequent testicular development and reproductive function[34]. Exposure to a variety of environmental agents andpharmaceuticals during foetal life has previously beenshown to result in germ cell loss in rodents and humans[35]. Recent studies have demonstrated that exposure tohuman-relevant concentrations of acetaminophen (paracetamol) results in a reduction of gonocytes and total
germ cell number (including (pre)spermatogonia) in human foetal testicular tissues including acute effects ofin vitro exposure and in long-term xenografts [13]. The
gonocyte population was predominantly affected in thepresent study as previously demonstrated for exposure
to phthalates [36]. The mechanism for the acute germ
cell loss following cisplatin exposure is likely to involve
direct damage to the germ cells. We investigated thePage 14 of 16

potential mechanism for cisplatin-induced reduction in
germ cell numbers. As previously reported, there is anincrease in apoptosis (expressed as percentage of tubules
stained with cleaved caspase 3) 8 h prior to the decreasein germ cell number in cisplatin-treated prepubertalmouse testicular tissues [17]. In contrast, the present
study suggests that there is no apparent difference in
apoptotic cell number at any time point studied. However, activation of cleaved caspase 3 pathway could
occur between 24 and 72 h post cisplatin exposure, before the reduction in germ cell number becomes apparent. Interestingly, studies involving acetaminophen- or
phthalate-exposure have shown that testicular effects
may also involve the associated reduction in testosterone
during foetal life [13, 14, 37]. The impact of platinumbased agents on somatic (including Leydig) cell function
in foetal and early postnatal testicular tissues requires
further investigation.
Early postnatal life is also another crucial period forgerm cell development and testicular growth [38]. The
hypothalamo-pituitary-gonadal axis is active and thecompletion of gonocyte differentiation into spermatogonia occurs during this period [38]. As a result, gonocyte loss following platinum exposure during infancy
may impact on the establishment of the normal pool ofSSCs, and fertility in adulthood. This is of particular importance for malignancies (e.g. neuroblastoma) that canarise shortly after birth or during infancy.

Conclusion
In conclusion, we show that exposure to human-relevant
concentrations of platinum-based chemotherapy results
in a reduction in germ cell number, including spermatogonial stem cells, in the immature human testis. We observed a similar germ cell loss for cisplatin andcarboplatin, suggesting that carboplatin may not have reduced gonadotoxicity compared to cisplatin. These experimental approaches using human tissues can bebroadly applied to determining the relative gonadotoxicity of current and novel cancer therapies, which is important for counselling, modification of treatment
regimens and developing strategies to preserve fertility
in children with cancer.
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12916-020-01844-y.
Additional file 1 : Table S1. Antibodies and dilutions used for
immunohistochemistry (IHC) and immunofluorescent (IF) staining.
Additional file 2 : Figure S1. Effect of cisplatin exposure on apoptosis
in human fetal testis. Cleaved Caspase 3 (CC3) protein expression in thehuman fetal testis 24 h following exposure to vehicle (VEH; A) or cisplatin(CIS, 0.5 μg/ml; B). Scale bars represent 100 μm (or 50 μm for insets),
arrowheads indicate positively stained cells: (A) cell in the seminiferous

Tharmalingam et al. BMC Medicine(2020) 18:374

tubule, (B) cell in the interstitium. There was no significant change in thenumber of apoptotic cells within tubules at 24 h (C) and 96 h (D), and no
significant change in the number of apoptotic cells in the interstitium at
24 h (E) and 96 h (F) post-exposure to cisplatin. Data analysed using twoway ANOVA. Values shown are means ± SEM and each set of coloured
data points represents an individual fetus (n = 5).
Acknowledgements
We gratefully acknowledge the work of Anne Saunderson, Rosemary Bayneand the staff of the Bruntsfield Suite of the Royal Infirmary of Edinburgh inprovision of tissue for these studies and the Human Developmental Biology
Resource (www.hdbr.org) for providing some of the foetal material (Joint
MRC/Wellcome Trust; Grant No: 099175/Z/12/Z). We also thank Jill Davies
and the Oxford Cell & Tissue Biobank. We also acknowledge William Mungall
for assistance with the animal work, specifically grafting the tissue andmonitoring the animals post-surgery. We thank Merrill McHoney and Mark
Brougham for assistance with clinical/surgical aspects relating to prepubertal
patients and Richard Sharpe for his helpful comments on the manuscript.

Page 15 of 16100, Singapore 229899, Singapore. 3Edinburgh Royal Hospital for Sick
Children, 9 Sciennes Road, Edinburgh EH9 1LF, Scotland, UK. 4NORDFERTIL
Research Lab Stockholm, Childhood Cancer Research Unit, Department of
Women’s and Children’s Health, Karolinska Institutet and Karolinska University
Hospital, Stockholm, Sweden. 5Division of Haematology-Oncology and Stem
Cell Transplantation, Children’s Hospital, University of Helsinki, Helsinki
University Central Hospital, Helsinki, Finland. 6Radcliffe Department ofMedicine, MRC Weatherall Institute of Molecular Medicine, University of
Oxford, Oxford OX39DS, UK. 7Department of Paediatrics and Child Health,
Oxford University Hospitals NHS Foundation Trust, and Nuffield Department
of Womens and Reproductive Health, University of Oxford, Oxford, UK.
8
Section of Andrology, Division of Urology, Department of Surgery, Universityof Utah School of Medicine, Salt Lake City, UT, USA. 9Howard Hughes
Medical Institute, Department of Oncological Sciences and Huntsman Cancer
Institute, University of Utah School of Medicine, Salt Lake City, UT, USA.
10
Department of Growth and Reproduction, Copenhagen University Hospital(Rigshospitalet), Blegdamsvej 9, 2100 Copenhagen, Denmark. 11Biomedical
Sciences, University of Edinburgh, Edinburgh EH8 9XD, UK.
Received: 22 July 2020 Accepted: 6 November 2020

Authors’ contributions
Conceived and designed the experiments: RTM, MT, GM. Performed theexperiments: MT, GM, FL. Analysed the data: RTM, MT, GM, HW, JBS, KJ, AJ,
FL, NS, AG. Contributed reagents/materials/analysis tools: SL, JBS, EO, KJ, HW,
JG, BC, CA. Wrote the paper: RTM, MT, GM. NS and RTM jointly supervised
PhD student MT. All authors have read and approved the manuscript.
Funding
RTM was supported by a Wellcome Trust Intermediate Clinical Fellowship(Grant No. 098522) and a UK Research and Innovation (UKRI) Future Leaders
Fellowship (MR/S017151/1). JBS was supported by the Swedish Childhood
Cancer Foundation (TJ2020-0026) and the Birgitta and Carl-Axel Rydbeck’s
Research Grant for Pediatric Research (2020-00348). A.G. is supported by Wellcome (Investigator Award 21947) and by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC). Experimental work
was supported by UKRI (MR/S017151/1), Wellcome (Grant No. 098522) andChildren with Cancer UK (15-198). This work was undertaken in the MRC
Centre for Reproductive Health funded by the Grant MR/N022556/1.
Availability of data and materials
The data sets used and/or analysed during the current study are available
from the corresponding author on reasonable request. Single-cell sequencing data is available online at:https://humantestisatlas.shinyapps.io/humantestisatlas1/
Ethics approval and consent to participate
Ethical approval for use of human foetal tissues for research was obtained
from the South East Scotland Research Ethics Committee (LREC08/S1101/1),
NRES committee North East – Newcastle and North Tyneside 1 (08/H0906/
21+5) and NRES Committee London – Fulham (18/10/0822), and written
informed consent was given by the women. Ethical approval for collection
and use of human prepubertal testis tissues in research was obtained fromthe South East Scotland Research Ethics Committee (13/SS0145) and Oxford
University Hospitals NHS Foundation Trust (2016/0140). Written informed
consent was obtained from patients’ parents/guardians and patients, whereappropriate.
For studies involving animals, ethical approval was given by the Animal
Welfare and Ethical Review Board (AWERB) at the University of Edinburgh,
and all procedures carried out were in accordance with UK Home Officeguidelines and the Animal (Scientific procedures) Act 1986.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
MRC Centre for Reproductive Health, The Queen’s Medical ResearchInstitute, The University of Edinburgh, 47 Little France Crescent, Edinburgh
EH16 4TJ, Scotland, UK. 2KK Women’s and Children’s Hospital, Bukit Timah Rd,

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