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Asian Journal of Andrology logoLink to Asian Journal of Andrology
. 2024 Jul 30;26(6):610–616. doi: 10.4103/aja202437

Testis tissue cryopreservation may be considered in boys with cryptorchidism

Linn Salto Mamsen 1,*,✉, Simone Hildorf 2,*, Elissavet Ntemou 1, Danyang Wang 1, Dina Cortes 3,4, Jens Fedder 5,6, Jørgen Thorup 2,3, Claus Yding Andersen 3,7
PMCID: PMC11614173  PMID: 39075793

Abstract

This study assessed the feasibility of testis tissue cryopreservation (TTC) for fertility preservation in prepubescent boys with cryptorchidism. From January 2014 to December 2022, the University Hospital of Copenhagen (Rigshospitalet, Copenhagen, Denmark) implemented TTC for 56 boys with cryptorchidism to preserve their reproductive potential. Testis tissue samples were collected during orchiopexy (32 cases) or at subsequent follow-up procedures (24 cases), necessitated by an increased risk of infertility as indicated by hormonal assessments and/or findings from initial surgical biopsies. Testis samples were procured for TTC and pathological analysis. The cohort had an average age of 1.3 (range: 0.3–3.8) years at the time of orchiopexy, with 91.1% presenting bilateral cryptorchidism. The study revealed a median germ cell count of 0.39 (range: 0–2.88) per seminiferous tubule, with germ cells detected in 98.0% of the bilateral biopsies and 100% of the unilateral, indicating a substantial potential for fertility in these immature tissues. A dark spermatogonia (Ad) was detected in 37 out of 56 patients evaluated, with a median Ad spermatogonia count of 0.027 (range: 0.002–0.158) per seminiferous tubule. A total of 30.2% of the samples lacked Ad spermatogonia, indicative of potential gonadotrophin insufficiency. The median hormone levels measured were as follows: follicle-stimulating hormone (FSH) at 0.69 (range: 0.16–2.5) U l−1, luteinizing hormone (LH) at 0.21 (range: 0.05–3.86) U l−1, and inhibin B at 126 (range: 17–300) pg ml−1. Despite early orchiopexy, 20%–25% of boys with cryptorchidism remain at risk for future infertility, substantiating the necessity of TTC as a precaution. The study highlights the need for refined predictive techniques to identify boys at higher risk of future infertility.

Keywords: cryopreservation of testis tissue, cryptorchidism, fertility preservation, germ cells, male infertility, prepubescent, spermatogonial stem cells, testis

INTRODUCTION

During puberty, the generation of mature spermatozoa requires a well-functioning testicular environment that includes spermatogonial stem cells (SSCs) and somatic cells such as Sertoli, peritubular myoid, and Leydig cells. Prepubescent boys undergoing gonadotoxic treatments for illnesses like cancer, or for benign conditions such as bilateral cryptorchidism, are at risk of infertility due to the risk of SSC depletion. Given their young age and the resultant inability to produce viable sperm for cryo-storage, testis tissue cryopreservation (TTC) is currently the single, yet experimental, available option to safeguard their fertility in the future.1,2

Originally, TTC was predominantly offered to prepubescent boys diagnosed with cancer before initiating gonadotoxic therapies.2,3 Currently, this fertility preservation method has been extended to include patients with nonmalignant disorders that necessitate similar treatments or bone marrow transplants, including sickle cell disease and thalassemia major.2 Nevertheless, the overall need of TTC is relatively low, attributed to the infrequent occurrence of these medical conditions.

Since the launch of the initial fertility preservation program for prepubertal boys in 2002, many centers worldwide have begun immature tissues for those at risk of infertility.2,4,5 An international survey in 2019 showed that over 1000 prepubescent boys had undergone TTC.5 This represents a four-fold increase since the initial survey in 2012.2 This figure highlights the field’s swift progress, increased interest, and widespread acceptance by both parents and clinicians.6,7,8 Parents generally show support for TTC for their sons, despite the absence of definitive evidence for fertility restoration based on frozen/thawed samples in adult males.8 Notably, animal research, including studies on rodents and primates, has demonstrated that offspring can be successfully produced using frozen/thawed immature testis tissue.9,10 A particularly noteworthy study revealed that the autologous transplantation of frozen/thawed immature testis tissue in rhesus monkeys led to the production of mature sperm. This advancement allowed for the conception of healthy offspring through in vitro fertilization.10

Boys with cryptorchidism might also benefit from TTC as a supplementary treatment in tandem with orchiopexy.8,11,12 Cryptorchidism is significantly more common than the aforementioned diagnoses, with a prevalence of 2%–4% among newborn boys in the Western world.13 Follow-up studies indicate a 13%–46% azoospermia risk in bilateral cryptorchidism patients, even after early surgical intervention.14,15,16,17,18,19,20 In unilateral cryptorchidism patients, the risk of azoospermia is about 4%–13.3%.18,21,22 At orchiopexy, cryptorchid patients with a high risk of infertility may be identified based on testicular histology and hormonal analyses.16,22 Since these young patients possess SSCs, it is possible to obtain immature testis tissue for cryopreservation either during orchiopexy or through a separate procedure.8,12,23 This strategy holds promise for facilitating future fertility restoration.

In University Hospital of Copenhagen (Rigshospitalet, Copenhagen, Denmark), we offer TTC cryopreservation for pediatric patients diagnosed with cryptorchidism who face a potential risk of infertility in their adult years.8,12 The focus of this study is to disseminate our clinical experiences and findings concerning patients with cryptorchidism and its long-term implications.

PATIENTS AND METHODS

Patients

From January 2014 to December 2022, the University Hospital of Copenhagen, Rigshospitalet, implemented TTC for 56 prepubertal boys with cryptorchidism to preserve their reproductive potential. Testis tissue samples were collected during orchiopexy (32 cases) or subsequent follow-up procedures (24 cases). Inclusion criteria were increased risk of infertility as indicated by hormonal assessments and/or findings from initial surgical biopsies. Testis samples were procured for TTC and pathological analysis. Informed consent was obtained from all subjects involved in the study, and for patients below the age of 18 years, consent was obtained from the parents or legal guardians.

Our research adhered to the Declaration of Helsinki. Our center is the only one approved by Danish authorities for testicular tissue cryopreservation per the European Union tissue directive. Ethical approval for biopsy collection for research was obtained from the Scientific Ethics Committees for the Capital Region, Hillerød, Denmark (Approval No. H-22012060 and H-18063061).

Testicular biopsy collection

During anesthesia, bilateral testicular biopsies were retrieved by a pediatric surgeon or urologist from each testis by incision of the tunica albuginea. The biopsied tissue was manually dissected into two fragments. One fragment was immediately fixed in Stieve’s fixative for histological evaluation. One fragment was transported at 34°C in Gibco™ McCoy’s Medium (26600023; Thermo Fisher Scientific, Roskilde, Denmark) within a period of a maximum of 15 min to the laboratory for cryopreservation. If patients had consented to donate a biopsy for research purposes, one additional testis biopsy was obtained and cryopreserved for later research purposes.

Testicular biopsies were either obtained during the initial orchiopexy or in a subsequent independent surgical procedure (Figure 1). Boys with congenital, nonsyndromic bilateral cryptorchidism and testes in a high cryptorchid position were offered cryopreservation during their initial orchiopexy to circumvent an additional surgery. Another cohort of cryptorchid boys was meticulously chosen based on hormonal and histological findings from the primary surgery, indicating an augmented risk of infertility. This cohort comprises highly selective patients. Histological and hormonal data obtained at primary orchiopexy indicated impairment, ranging from moderate to severe. Specifically, the number of germ cells per seminiferous tubule (G/T) was below the lower value of the normal range. For those with bilateral cryptorchidism, a biopsy from each testis was cryopreserved.

Figure 1.

Figure 1

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Patient characteristics. TTC: testis tissue cryopreservation.

Cryopreservation of testis biopsies

In 24 cases, the testis biopsies were weighed before sectioned into small 2 mm3 to 3 mm3 pieces. In bilateral cases, weights of both the right and left biopsies were obtained. A total of 40 biopsy weights were obtained in the 24 cases. Tissue pieces were equilibrated with cryomedium on crushed ice (0°C–4°C) placed on a tilting table for 15 min. The cryomedium comprised 1.5 mol l−1 ethylene glycol, 0.1 mol l−1 sucrose, and 10 mg ml−1 human serum albumin (hSA). Each biopsy was then relocated to individual 1.8 ml cryovials (NUNC A/S, Roskilde, Denmark) filled with 1 ml of fresh cryomedia at 4°C. These vials were subsequently positioned in a pre-equilibrated slow-freezing machine (Planar K10; Planar Products Ltd., Sunbury-On-Thames, UK).

From the moment the tissue was introduced to the cryomedium until the freezing machine’s operation began, the total equilibration time spanned 20 min, inclusive of the transfer to individual tubes. The freezing machine’s program commenced at 1°C, cooled at a rate of –2°C per min to −9°C, paused for a 5-min soak, and then underwent manual seeding for ice crystal formation. Following this, the cooling continued at –0.3°C per min to −40°C, accelerated to –10°C per min to −140°C, after which the samples were directly immersed in liquid nitrogen for extended storage.1

Tissue preparation for histology

Fixated tissues were dehydrated, embedded in paraffin, and cut into 2-µm thick serial sections. Sections were stained with hematoxylin and eosin (H&E) and immunohistochemical staining. The total germ cell number (including fetal gonocytes, prepubertal spermatogonia, and A dark [Ad] spermatogonia) per cross-sectional seminiferous tubule (G/T) and the number of specific type Ad spermatogonia per cross-sectional seminiferous tubule (Ad/T) were counted. Identification of Ad spermatogonia was based on the following criteria: 1) their location at the basement membrane, 2) the presence of a rarefaction zone centrally located within the nucleus, and 3) the homogeneous deeply staining of the nucleus.24,25,26 For each biopsy, at least 100 and 250 cross-sectional seminiferous tubules were counted to obtain G/T and Ad/T measurements, respectively. Normal reference values according to age were presented in mean values, as previously described.27,28 Three patients are missing histological measurements due to storage outside the hospital center.

Hormone levels

Blood samples were obtained at the time of surgery during 8:00 a.m.–11:00 a.m. Following a 10-min centrifugation at 300g (ROTANTA 460 RS; Hettich, Tuttlingen, Germany), serum was isolated and stored at –20°C until analysis. Concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were measured with a time-resolved immunofluorometric assay (AutoDELFIA, Wallac, Turku, Finland), with a lower detection limit of 0.05 IU l−1. Concentrations of inhibin B were measured with an enzyme-linked immunosorbent assay (ELISA; Serotec Ltd., Oxford, UK), with a lower detection limit of 3 pg ml−1.

RESULTS

Characteristics of patients with cryptorchidism having testis tissue cryopreserved

A total of 56 patients with cryptorchidism had testicular tissue cryopreserved between January 2014 and December 2022 (Figure 2a). Patients with cryptorchidism account for 62.4% of the entire Danish TTC cohort, while cancer was the indication in 7.5% of the cases. The indication in the remaining 30.1% was Klinefelter syndrome (15.1%), immunodeficiencies (8.6%), hematological disorders (3.2%), and other nonmalignant conditions (3.2%), as shown in Figure 2a. Patients with cryptorchidism were referred to the Laboratory of Reproductive Biology from the Department of Pediatric Surgery, University Hospital of Copenhagen, Rigshospitalet.

Figure 2.

Figure 2

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Year of TTC and age of patients at TTC. (a) Total number of TTC per year. Black: patients with cryptorchidism; gray: patients with other diagnoses. (b) Age at patients with cryptorchidism having TTC. Black: bilateral cryptorchidism; gray: unilateral cryptorchidism. TTC: testis tissue cryopreservation.

Of the 56 patients with cryptorchidism, 51 (91.1%) were diagnosed with bilateral cryptorchidism. The remaining five had unilateral cryptorchidism and were offered TTC as a second procedure due to reduced histological and/or hormonal parameters at the time of orchiopexy (Table 1). All patients were diagnosed with nonsyndromic cryptorchidism and had no other associated anomalies. The mean age of the patients at orchiopexy was 1.3 (standard deviation [s.d.]: 0.7; range: 0.3–3.8) years. The mean age at TTC was 1.8 (s.d.: 1.1; range: 0.3–5.3) years. Fourteen patients were younger than 1 year of age, 19 were between 1 year and 2 years, 15 were between 2 years and 3 years, 7 were between 3 years and 4 years, and 1 patient was 5.3 years of age (Figure 2b). No postoperative complications arose from the biopsy collections in any case.

Table 1.

Hormonal parameters and characteristics of 56 boys with cryptorchidism who had testicular tissue cryopreserved

Patient number Age at orchiopexy (year) Age at cryopreservation (year) Diagnosis G/T (mean) Ad/T (mean) FSH (IU l−1) LH (IU l−1) Inhibin B (pg ml−1)
1 1.3 2.4 Bilateral cryptorchidism 0.230* 0.015 0.80 0.20 158
2 2.3 3.8 Bilateral cryptorchidism 0.240* 0.005* 0.60 0.10 80
3 0.9 2.1 Bilateral cryptorchidism 0.065* 0* 1.40 0.10 126
4 0.8 3.6 Bilateral cryptorchidism 1.917 0.015 1.10 0.60 155
5 1.5 2.4 Bilateral cryptorchidism 0.071* 0* 0.60 0.10 56*
6 0.8 2.1 Bilateral cryptorchidism 1.320* 0.060 0.50 0.10 104*
7 1.0 2.5 Bilateral cryptorchidism 0.060* 0* 0.60 0.10 165
8 2.2 3.1 Bilateral cryptorchidism 0.061* 0* 1.20 0.40 68*
9 2.2 3.6 Bilateral cryptorchidism 0.055* 0.004* 0.70 0.10 118
10 3.8 5.3 Bilateral cryptorchidism 0.350* 0.004* 0.60 0.10 17*
11 1.4 2.7 Bilateral cryptorchidism 0.368* 0.014 0.90 0.10 102
12 0.6 1.4 Bilateral cryptorchidism 0.065* 0* 0.80 1.10 300
13 0.8 1.6 Bilateral cryptorchidism 0.185* 0* 0.40 0.30 150
14 1.8 At orchiopexy Bilateral cryptorchidism 0.416* 0.054 0.47 0.14 72
15 1.3 At orchiopexy Bilateral cryptorchidism 1.214 0.012 0.35 0.18 139
16 1.7 At orchiopexy Bilateral cryptorchidism 1.372 0.134 0.58 0.20 149
17 0.7 At orchiopexy Bilateral cryptorchidism 1.308* 0.015 0.91 0.46 150*
18 1.0 At orchiopexy Bilateral cryptorchidism 0.502* 0.043 0.75 0.70 186
19 1.1 At orchiopexy Bilateral cryptorchidism 0.501* 0.030 0.68 0.27 85
20 0.7 At orchiopexy Bilateral cryptorchidism 0.759* 0.014 0.52 0.13 189
21 1.0 At orchiopexy Bilateral cryptorchidism 0.744* 0.039 0.46 0.12 96*
22 1.9 At orchiopexy Bilateral cryptorchidism 0.183* 0* 0.69 0.43 66*
23 1.1 At orchiopexy Bilateral cryptorchidism 0.035* 0.004* 1.64 0.28 106
24 0.7 At orchiopexy Bilateral cryptorchidism 0.444* 0.014 0.40 0.07 134*
25 0.8 At orchiopexy Bilateral cryptorchidism 0.272 0.009 1.09 0.20 131
26 1.5 At orchiopexy Bilateral cryptorchidism 0.937* 0.025 0.56 0.65 249
27 1.7 At orchiopexy Bilateral cryptorchidism 0.276* 0* 0.64 0.14 76
28 0.4 At orchiopexy Bilateral cryptorchidism 0.935* 0.012 1.82 3.86 224
29 2.4 At orchiopexy Bilateral cryptorchidism 0.255* 0* 2.50 0.38 44*
30 0.7 At orchiopexy Bilateral cryptorchidism 1.552 0.017 0.57 0.36 222
31 3.5 At orchiopexy Bilateral cryptorchidism 0.565* 0.035 0.56 0.05 57
32 0.8 At orchiopexy Bilateral cryptorchidism 1.699 0.019 1.60 0.37 147
33 0.8 At orchiopexy Bilateral cryptorchidism 1.135* 0.019 0.81 0.08 136
34 1.2 At orchiopexy Bilateral cryptorchidism 0.282* 0.006* 0.64 0.21 158
35 1.1 At orchiopexy Bilateral cryptorchidism 0.490* 0* 0.53 0.06 117
36 1.8 2.7 Bilateral cryptorchidism 0.050* 0* 1.01 0.05 69
37 1.6 2.5 Bilateral cryptorchidism 0.029* 0.002* 0.44 0.41 137
38 0.3 At orchiopexy Bilateral cryptorchidism 1.330* 0.018 1.50 1.61 182
39 0.7 At orchiopexy Bilateral cryptorchidism 0.950* 0.003* 0.45 0.60 207
40 2.5 At orchiopexy Bilateral cryptorchidism NA NA 1.27 0.43 49*
41 1.0 At orchiopexy Bilateral cryptorchidism 0.281* 0.012 0.89 0.05 77
42 1.2 At orchiopexy Bilateral cryptorchidism 0.790* 0.039 0.16 0.28 143
43 1.7 3.4 Bilateral cryptorchidism NA NA 0.91 1.02 115
44 1.4 At orchiopexy Bilateral cryptorchidism 0.960* 0.017 1.49 0.36 105
45 1.5 At orchiopexy Bilateral cryptorchidism 0* 0* 1.20 0.12 60*
46 0.5 At orchiopexy Bilateral cryptorchidism 0.693* 0.004 0.57 0.85 280
47 0.4 At orchiopexy Bilateral cryptorchidism 1.078* 0.005 1.46 1.80 286
48 0.5 At orchiopexy Bilateral cryptorchidism 2.880 0.080 1.27 1.18 264
49 1.3 2.0 Bilateral cryptorchidism 0.806* 0.158 0.61 0.09 170
50 1.3 2.0 Bilateral cryptorchidism 0.488* 0.040 0.69 0.39 173
51 0.7 1.3 Bilateral cryptorchidism 0.297* 0.008 1.55 0.89 135
52 1.4 2.5 Unilateral cryptorchidism 0.066* 0* 0.81 0.11 70*
53 1.2 2.3 Unilateral cryptorchidism NA NA 0.37 0.06 54*
54 1.1 2.7 Unilateral cryptorchidism 0.031* 0* 0.89 0.07 44*
55 0.9 1.5 Unilateral cryptorchidism 0.011* 0* 0.69 0.07 85*
56 1.3 2.2 Unilateral cryptorchidism 0.333* 0* 1.46 0.35 41*
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*Reduced values. Hormonal and germ cell parameters are related to the time of orchiopexy. The total germ cell number (including gonocytes, spermatogonia, and A dark spermatogonia) per cross-sectional seminiferous tubule (G/T) values below the lower value of the normal range was defined according to our hospital references and literature as previously described.29 Ad/T value estimated to be <0.01 according to literature,30 and serum inhibin B levels <2.5 percentile according to our hospital laboratory references from the department of growth and reproduction.61 NA: not available; FSH: follicle-stimulating hormone; LH: luteinizing hormone; Ad/T: A dark spermatogonia per cross-sectional seminiferous tubule; G/T: germ cells per seminiferous tubule

Weight of the cryopreserved testis tissues

In 24 out of the 56 patients, the weight of the cryo-stored testis tissues was obtained. From patients with bilateral cryptorchidism, a biopsy from both the right and left testes was obtained. The mean weight of the cryo-stored biopsies was 12 (s.d.: 7; range: 2–34) mg, as shown in Figure 3a. The mean weight of the cryopreserved testis tissue per patient (i.e., left biopsy plus right biopsy) was 24 (s.d.: 13; range: 7–58) mg.

Figure 3.

Figure 3

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Descriptive data on patients with cryptorchidism who have had testis tissue cryopreservation (TTC). (a) Weight of the testicular biopsies (n = 40) obtained for cryopreservation during orchiopexy in relation to age. (b) The number of germ cells per cross-section of seminiferous tubules (G/T) was examined in a sample of 53 patients with cryptorchidism in relation to age. Biopsy samples were obtained during orchiopexy and each patient represented one value (black dots). Normal age-related G/T (solid line) as previously described.29 (c) The number of A dark spermatogonia per seminiferous tubules (Ad/T) in 53 patients with cryptorchidism in relation to age (black dots), each patient has one value. Reduced number of Ad/T (solid line) is classified as less than 0.01 according to the literature.30

Number of germ cells, Ad spermatogonia, and serum hormonal levels

The number of G/T was measured in 53 of 56 patients. The median number of G/T was 0.416 (interquartile range [IQR]: 0.757; range: 0–2.880; Figure 3b and Table 1). One boy at 1.5 years of age had no germ cells present in the biopsy taken during bilateral orchiopexy. Normal age-related G/T values are included in Figure 3b.29

The number of Ad/T was measured in 53 of 56 patients. Ad spermatogonia were detected in 37 of the patients. In patients with Ad spermatogonia, the median number of Ad/T was 0.027 (IQR: 0.029; range: 0–0.158; Figure 3c and Table 1). Sixteen boys of the 53 (30.2%) had no Ad spermatogonia in the biopsies taken. Normal age-matched Ad/T values are included in Figure 3c.30 Histological data from 41 of the patients have previously been published.8

In the 56 patients, the median level of FSH was 0.69 (IQR: 0.59; range: 0.16–2.50) IU l−1, LH was 0.21 (IQR: 0.33; range: 0.05–3.86) IU l−1, and inhibin B was 126 (IQR: 85; range: 17–300) pg ml−1, as shown in Table 1.

DISCUSSION

This study demonstrates that testis biopsies from boys with cryptorchidism contain germ cells, offering a potential source for future fertility preservation. Importantly, in contrast to biopsies from cancer patients, testis biopsies from cryptorchid patients do not carry the risk of malignant cell contamination. As a result, they are generally considered safe for transplantation. However, it is essential to note that there is an elevated risk of testicular cancer associated with a diagnosis of cryptorchidism.31,32,33,34 This makes transplantation contraindicated if there is a family history of testicular cancer.

The number of germ cells in each biopsy is limited due to the small size, prompting the question of whether these biopsies realistically can be used to restore fertility if the individual becomes infertile in adulthood. To restore fertility from these biopsies, direct SSC transplantation into the rete testis is one viable method that has proven successful in several other species.35,36,37,38 The potential of such a small-sized biopsy was encouraged by our preliminary findings of propagation of SSC-like cells from such small tissue samples.39 Moreover, we did not risk obtaining larger biopsies than our routine, as there is no proven efficacy of fertility restoration in adult life. In our prior studies, SSC-like cells were passaged up to five times, while retaining the expression of several SSC markers demonstrating that these cells from young boys with cryptorchidism can proliferate in vitro.39 The propagation protocol has been refined for clinical application under xeno-free conditions.40 The SSC-like cells were xenotransplanted into the testes of busulfan-sterilized mice. Six weeks after transplantation, these cells formed colonies in the seminiferous tubules, demonstrating that xenotransplantation is feasible, although the proliferation efficiency still needs to be improved before this method can be considered clinically relevant. Moreover, propagating SSCs in vitro might introduce genetic and epigenetic alterations, necessitating thorough testing before clinical application.

To enhance procedure safety, we proposed a method where SSCs from biopsies of cryptorchid boys were directly xenotransplanted to mice without in vitro propagation. This approach demonstrated a colonization efficiency of around 6% posttransplantation,41 suggesting the possibility of fertility restoration through direct transplantation. The technique requires further refinement to improve its efficacy. Moreover, performing transplantation during adolescence will enable SSCs to multiply over the subsequent years, ultimately restoring spermatogenesis before the age of wanted fatherhood. It has previously been shown that a small number of SSCs is sufficient to regenerate spermatogenesis in testicular cancer patients who experienced azoospermia for several years (up to 10 years) following gonadotoxic chemotherapy.42,43 Organotypic studies with human immature testis tissue combined with xenografting can also serve as a helpful model to explore the effect of chemotherapeutic agents.44

Another approach explored the potential of obtaining viable spermatozoa through organotypic culture of cryptorchid testicular biopsies.45 These organotypic cultures supported the testicular structure and partial maturation of the Sertoli cells and peritubular myoid cells during a 60-day culture period.45 Although the number of spermatogonia decreased during this period, some spermatogonia differentiated into boule homolog RNA binding protein (BOLL)-positive spermatocytes.45 Other groups have reported organotypic cultures of prepubertal testis tissue with similar results.46,47,48 Moreover, the organotypic culture of the human fetal testis can support the generation of fertilization-competent spermatids,49 illustrating the potential of very immature testicular tissue.

In this cohort, the mean age of the boys at orchiopexy was 1.3 years (i.e., 15.6 months). The ideal age for corrective surgery for cryptorchidism, as per the American and European Urology Associations, ranges from 6 months to 18 months.50,51 The Nordic countries’ guidelines suggest that orchiopexy should be performed even earlier, between 6 months and 12 months, since the number of germ cells becomes compromised within the first year of age and delays in treatment increase the risk of requiring reproductive assistance later in life.52 Despite early surgery, boys with cryptorchidism may still face fertility challenges. Histological data suggest that 20%–25% of these boys have a diminished count of germ cells, and hormonal treatment might be necessary to preserve SSCs postsurgery.23

The germ cell numbers might improve postsurgery. Yet, during the phase of hindered mini puberty, numbers could further decrease, underscoring cryptorchidism as a valid reason for TTC.44 Evidence suggests that without supplemental hormonal treatment, germ cell deterioration can continue postsuccessful orchiopexy leading to infertility.53 Although Hadziselimovic54 noted that if germ cell count per tubular section is below 0.2, most patients will develop infertility, regardless of whether they have had previous hormonal treatments before orchiopexy or orchiopexy only. The quantification of Ad spermatogonia is a very important marker for male fertility which is believed to represent an SSC. A follow-up study of sperm variables in 31 patients who had undergone early orchiopexy before the age of 2 years showed that if Ad spermatogonia were present in the testicular biopsies in childhood, 94% of the men had a total sperm count of 40 × 106 or greater per ejaculate.55 On the contrary, despite early successful orchiopexy, if Ad spermatogonia were absent in the childhood biopsy, 92% had abnormal spermiograms. These findings have garnered support from other researchers56 and indicate that the presence of Ad spermatogonia serves as a crucial indicator of fertility.

It has been suggested that TTC is not recommended for patients with unilateral cryptorchidism.44 However, it is known that some boys with unilateral cryptorchidism have a bilateral testicular affection due to hypogonadotropic hypogonadism,22,57 assumably around 10%.22 In our study, such boys with unilateral cryptorchidism were eligible for a second-stage testicular biopsy cryopreservation if the germ cell number was low in testicular biopsies and the serum inhibin B was below 2.5 percentile at the time of orchiopexy.8 Based on these criteria, five boys with unilateral cryptorchidism were included in the present study. Boys with unilateral cryptorchidism and hypogonadotropic hypogonadism will have a high risk of infertility and probably not benefit from orchiopexy alone with respect to fertility potential.53,57

The average amount of tissue cryopreserved per patient in our biobank for boys with cryptorchidism was 24 mg, which is considerably lower than the average amount of tissue reported before from the coordinated Fertility Preservation Program of the University of Pittsburgh Medical Center (average amount: 411 mg; s.d.: 837 mg; range: 14–6880 mg).4 The potential of a small-sized biopsy was encouraged by our preliminary findings of the propagation of SSC-like cells from such tissue samples.39 Moreover, we did not risk obtaining larger biopsies than our routine, as there is no proven efficacy of fertility restoration in adult life. It has been demonstrated that testicular growth is not impaired by testicular biopsy intervented in prepubertal cancer patients.58 Similarly, a follow-up study on early-diagnosed Klinefelter patients demonstrated that testicular biopsy procedure has no long-term effect on the gonadal development, testicular volume, and FSH, LH, inhibin B, and testosterone levels.59 The optimal amount of tissue needed to restore fertility remains uncertain. While some advocate for more extensive biopsies, others stress the importance of minimizing the impact on testicular physiology.

We also highlight the effective transport of testicular tissue for longer transportations in cold media (0°C–4°C) advocating for centralized service.1 Cryosurvival of germ and somatic cells, a well-preserved architectural integrity of the tissue, has been feasible with our protocols.1,39 However, given the increased testicular cancer risk in boys with cryptorchidism,31,32,33,34 we must approach transplantation with caution.

The TTC scheme presents several significant strengths within this patient group. Its design, which involves obtaining testis biopsies in conjunction with orchiopexy, is particularly beneficial. This approach spares patients from undergoing additional surgeries.8 Furthermore, the TTC protocol, including tissue thawing, has demonstrated successful methods in ensuring the survival of testis tissue.1,39,40,41,45 In addition, transporting the tissue in cold media has proven not to compromise tissue survival, indicating the potential for a centralized TTC service.1 A significant development has recently been reported: the first successful human autotransplantation of frozen/thawed adult testis tissue. This achievement employed the protocol described here, providing the first human proof of concept that such tissue can survive six months after transplantation.60

However, there are limitations to this method. Most notably, the relatively small amounts of testis tissue stored may be insufficient to restore fertility. Previous research indicates that a few SSCs may be adequate to reinitiate spermatogenesis in testicular cancer patients who have suffered from azoospermia for several years after undergoing gonadotoxic chemotherapy.42,43 This suggests that even small tissue biopsies containing SSCs may have the potential to restore fertility after transplantation. In addition, a limitation lies in the inability to evaluate pregnancy outcomes within the scope of this research. This gap means that while the study can provide insights into certain aspects of testis tissue functionality and characteristics, it falls short of offering a comprehensive understanding of how these findings translate into actual pregnancy rates and outcomes. This leaves a significant aspect of the approach unaddressed, warranting further investigation in future studies.

In conclusion, male fertility restoration is progressing, and clinical tests are on the horizon. Extending this procedure to cryptorchid boys at risk of infertility in adulthood is justifiable. Our work suggests a broader application of TTC, benefiting a larger population.

AUTHOR CONTRIBUTIONS

LSM cryopreserved the TTC, designed the study, analyzed data, and drafted the manuscript. SH designed the study, performed the germ cell calculations, participated in data analysis, and drafted the manuscript. EN and DW were involved in cryopreservation and study design and assisted in drafting the manuscript. DC performed the germ cell calculations and participated in data analysis. JT consulted patients and parents, obtained informed consent, performed orchiopexies, and obtained testis tissues for pathological analysis and cryopreservation. DC, JT, JF, and CYA were involved in the design of the study, funding, project supervision, and revising the manuscript. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.

ACKNOWLEDGMENTS

Marjo Westerdahl from the Department of Gynaecology, Fertility, and Obstetrics, Laboratory of Reproductive Biology, University Hospital of Copenhagen, Rigshospitalet, is acknowledged for excellent technical assistance. Finally, we are grateful to all the patients and parents of underaged children for acceptance of the experimental clinical treatment option of the TTC program and to all involved in the clinical activities in the testis tissue cryopreservation program in Denmark. This study was financially supported by the University Hospital of Copenhagen, Rigshospitalet, the Danish Child Cancer Foundation (2021-7395), Vissing Fonden (519140 AHO/PPT), and the Research Fund between Rigshospitalet and Odense University Hospital (136-A5544).

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