Microsurgical testicular sperm extraction (micro‐TESE) has been shown to successfully retrieve sperm in azoospermic men in 54% of cases 13 . In the setting of a surgical sperm retrieval (SSR) in a non‐malignant testis, the underlying pathological changes in the testis correlate to the successful chance of SSR. In patients with hypospermatogenesis, maturational arrest, and Sertoli‐cell‐only syndrome, retrieval rates of 85%, 49%, and 37% have been reported, respectively, using micro‐TESE 13 . Micro‐TESE may also be used to extract sperm from the ipsilateral testis at the time of an orchidectomy, a technique known as an onco‐micro‐TESE. At present, there is no clear way to predict which testicular units affected by cancer may yield sperm at the time of SSR. The purpose of the present study was to investigate potential markers of spermatogenesis by assessing the relationship of tumour characteristics (type, grade), tumour size, presence of testicular microlithiasis (TML), and tumour markers, with the presence of spermatogenesis in the affected testis of patients with a TGCT.

Testicular germ cell tumours (TGCTs) are the most common malignancy in young men in their peak fertile years 1 . The inherent subfertility risk 2 and treatment induced (often permanent) infertility 3 , 4 , quantified as an average 30% decrease in fertility 5 , 6 in patients with testicular cancer, warrants significant interest in predicting and managing these patients’ infertility. At first presentation, >50% of patients with testicular cancer are oligozoospermic 7 , whilst up to 24% may be azoospermic 8 . After combined therapy with surgery and chemotherapy, 10‐year post‐treatment natural paternity rates have been reported at 50% 9 . Pre‐treatment, but optimally before surgery 10 , semen cryopreservation is strongly recommended for all patients with testicular cancer 11 , 12 . However, this is invariably unsuccessful for the azoospermic patient and this sub‐group of patients present a unique challenge.

In 2013, LDH assays were changed, which shifted the numerical upper and lower normal boundaries, thus negating the statistical analysis of the association of LDH with spermatogenesis. The relationship between FSH and LH levels, and testosterone, with the presence of spermatogenesis could not be conducted due to insufficient data.

To evaluate how patient and/or tumour characteristics were predictive of spermatogenesis a univariate logistic regression was conducted for each hypothesised predictor of spermatogenesis, including: age, tumour stage, type, tumour size (PTTO), presence of microcalcification, and raised tumour markers. Multivariable logistic regression with backward stepwise elimination (α = 0.20) was then used to identify the best predictors of spermatogenesis. Statistical significance was defined as P < 0.05.

Tumour markers (αFP, βhCG and LDH) before orchidectomy were identified from patient records and correspondence documents. Only αFP and βhCG data were used in the analysis. Raised αFP was defined as >10 ng/L, and βhCG as ≥1 ng/L. Testicular function markers, including FSH, LH, testosterone, and preoperative semen analysis, were also sought from the patient records.

Macroscopic planar measurements were taken from the pathological specimen. Tumour diameter was recorded and testes were measured in three planes. From these dimensions, the PTTO was calculated as a ratio after calculating the volumes of the testis and tumour.

The presence of spermatogenesis was determined as being either ‘focal’ or ‘widespread throughout the testis’ and its distance from the tumour was described as ‘within tumour’, ‘next to the tumour’, or ‘away from tumour’. ‘Next to tumour’ was defined as the presence of spermatogenesis abutting the tumour. ‘Away from the tumour’ was defined as the presence of spermatogenesis occurring at any distance from the tumour where there was normal testicular parenchyma separating the tumour and the presence of spermatogenesis.

Spermatogenesis was defined as the presence of fully formed, mature spermatozoa, which would be suitable for in vitro fertilisation/intracytoplasmic sperm injection. Spermatids alone were not considered as indicative of spermatogenesis. A Johnson score was not used in this series, as this scoring system was not developed for whole orchidectomy specimens. The Johnson score is calculated by grading the most advanced level of sperm maturation on a scale of 1–10 in at least 100 different seminiferous tubules from a testis biopsy, and then calculating the mean score in that small sample by dividing the total score by the number of tubules in the biopsy 14 .

All specimens had been initially reported by local pathologists. Slide review of all cases with evaluation of spermatogenesis, and confirmation of identification of tumour type, staging, and size in relation to the testis, were then conducted by one expert pathologist (C.H.).

Our primary outcome measures included the presence of spermatogenesis, identified as ‘present’ or ‘absent’. Secondary outcome measures included the nature of the spermatogenesis: whether it was focal or widespread, and its relation to the tumour, whether it was within the tumour, adjacent to the tumour, or distant from the tumour. Other outcome measures included: the size of the tumour, measured as the percentage tumour testis occupation (PTTO), the subtype of tumour, the presence of microcalcification, and the levels of tumour markers (lactate dehydrogenase [LDH], βhCG, α‐fetoprotein [αFP]) before orchidectomy. All secondary outcome measures were assessed in their ability to predict spermatogenesis.

Males aged >16 years at the time of orchidectomy with TGCTs and spermatogenesis data were included. Patients were excluded if they had a testicular neoplasm other than a GCT (three patients), or if they had no available data on the presence of spermatogenesis (three patients), leaving a final cohort of 103 patients.

In all, 97/103 (94%) patients had pre‐orchidectomy βhCG levels measured and 48/97 (49%) had an elevated βhCG value. Spermatogenesis was not present in 17/48 (35%) of the patients with a raised βhCG compared to 14/49 (29%) with a normal value. In all, 94/103 (91%) patients had pre‐orchidectomy αFP levels measured and 23/94 (24%) had an elevated αFP, of which seven of the 23 (30%) did not have spermatogenesis. Neither elevated βhCG nor αFP was statistically related to presence of spermatogenesis on univariate or multivariate analysis.

Age was not found to be a predictor of spermatogenesis on univariate nor multivariate analysis. Although a decline in the rate of spermatogenesis occurred after the age of 50 years, the number of men in this age group in this series was too small to make any statistically meaningful conclusion (Fig. 4 ).

Microcalcification was identified on pathological review in 43/103 specimens. In those with microcalcification, nine (21%) did not have spermatogenesis. Microcalcification was not significantly related to spermatogenesis on univariate or multivariate analysis. However, spermatogenesis was 7.25‐times more likely to occur in areas away from microcalcification than where microcalcification was present; spermatogenesis only occurred within the area of microcalcification in four patients (9%).

An age‐adjusted logistic regression model found that for every 1% increase in PTTO, the chance of the presence of spermatogenesis decreased by 4% (odds ratio [OR] 0.96, 95% CI 0.95–0.98, P < 0.001). When categorising PTTO based on a 50% threshold, it was found that if the patient had a PTTO >50% they were 82% (OR 0.19, 95% CI 0.07–0.48, P < 0.001) less likely to have spermatogenesis than those with a PTTO <50%. Figure 2 shows the presence of spermatogenesis in tumours of varying percentages of testis volume. In order to use age‐adjusted PTTO as a means to predict an individual's likelihood of spermatogenesis, the logistic regression model was plotted (Fig. 3 ) to show the negative relationship between PTTO and probability of spermatogenesis.

It was possible to measure the PTTO in 99 of the 103 patients. The average PTTO was 34%: 25 patients had <10%, 39 had 10–49%, 33 had 50–75%, and two had >75%. The average PTTO in those with spermatogenesis was 28%, and in those without spermatogenesis it was 48%. Figure 2 shows the frequency of spermatogenesis with increasing PTTO. A discrepancy exists between the presence of spermatogenesis in patients whose PTTO was <50% and in those whose PTTO was >50%.

Spermatogenesis was present in seminomas, non‐seminomas, and mixed tumours in 70% (44/63), 58% (11/19), and 81% (17/21) of the patients, respectively. Spermatogenesis was widespread in the majority of patients with all tumour subtypes; widespread spermatogenesis was present in 59% of seminomas (26/44), 73% of non‐seminomatous GCTs (eight of 11), and 65% (11/17) of mixed tumours. Regarding tumour stage, 75% (46/61) of T1 tumours had spermatogenesis, as did 85% (11/13) of T2 tumours, and 52% (15/29) of T3 tumours.

Overall, 70% (72/103) of the patients had spermatogenesis present. Of these, spermatogenesis was widespread in 63% (45/72) and focal in 38% (27/72). Spermatogenesis occurred away from the tumour in 50% (36/72) of the patients and in 44% (32/72) it occurred both near to and away from the tumour. Only 1% (1/72) of spermatogenesis found occurred within the tumour alone and 4% (3/72) occurred adjacent to the tumour alone (Fig. 1 ).

A total of 103 patients were included in the study, and all patients underwent radical orchidectomy from February 2011 to December 2015. The mean (sd; range) age of the cohort was 35 (11; 17–77) years; 62 patients were aged ≤35 years and 41 were aged >35 years. Table 1 summarises the patients’ characteristics.

Discussion

The present results demonstrate that increasing size of tumour relative to testis size (or PTTO) is associated with reduced likelihood of spermatogenesis. Our present findings can be summarised into a rule of 50s: men with PTTO of >50% have a <50% chance of spermatogenesis in their affected testis. Neither tumour type, TMN stage, presence of microcalcification, nor raised tumour markers, were found to predict spermatogenesis on either univariate or multivariate analysis.

Recently, several other studies have also investigated spermatogenesis predictors. Our present overall rate of spermatogenesis (70%) is comparable to previous findings of 68% 18, 62% 19 and 79% 20. These studies also found a statistically significant negative relationship between testicular tumour size and presence of spermatogenesis. Choy et al. 19, using their logistic model, estimated the probability of spermatogenesis in a tumour of 1 cm was 86% and for a tumour of 5 cm it was 57%. One of the strengths of our present study is that we took into account the testis size when comparing tumour size, which is more clinically relevant than tumour diameter alone for two reasons: (i) we know that spermatogenesis is more likely to occur away from the tumour, therefore smaller testis may be more impacted by the same tumour size than someone with a larger testis; and (ii) tumours are not symmetrical, so an estimate of testis occupation is more representative of tumour bulk compared to a single measurement. One other study took testis size into consideration when measuring tumour size: Suzuki et al. 21 found that patients with a non‐cancerous testicular tissue width of <7.5 mm had a significantly lower retrieval rate (41%) compared to those whose width was >7.5 mm (93%). However, the measurement we used (PTTO) is more likely to be reproducible on ultrasonography, as tumour‐free width will vary depending on the side of the tumour.

The relationship between microcalcification and spermatogenesis is intriguing. Whilst there was no clear association between the presence of microcalcification and spermatogenesis on univariate or multivariate analysis, spermatozoa where 7.25‐times more likely to be found away from areas of microcalcification, and were only seen in areas of microcalcification in 9% of patients. This certainly warrants further investigation, as presence of widespread microcalcification or TML on preoperative ultrasonography may have a negative predictive impact on the chance of subsequent successful sperm retrieval.

The relationship between age and presence of spermatogenesis is less consistent in the literature and remains an area of controversy. Our present results support Suzuki et al. 21 who did not find age to be associated with the presence of spermatogenesis in a similar cohort size of 104 specimens. However, in a cohort of 145 patients, Shoshany et al. 18 found on univariate analysis that older age was a negative predictor of spermatogenesis (P = 0.05). Our present results showed that men aged ≥50 years were much less likely to have spermatogenesis (45%) compared to men aged <50 years (75%), although these data were only based on 11 patients, and it is too small to draw any statistically significant conclusions; a larger cohort is required to strengthen this finding. Indeed, in the background population, increasing age has been shown to be strongly associated with decreased semen quality, including decreases in sperm motility, normal morphology, and increased genetic malformations, and any changes with age may relate to this 22.

With regards to other markers, Delouya et al. 20 and Shoshany et al. 18 found that raised tumour markers and more advanced tumour stage were negatively associated with presence of spermatogenesis, but neither of these results have been reproduced in our present study or other studies 19, 21.

There was a significant paucity of testicular functional markers such as FSH and LH, testosterone, and semen analysis measurements taken before orchidectomy, so fertility markers could not be assessed as predictors of spermatogenesis as it was intended. This lack of data demonstrates an endemic lack of focus on determining patient testis function and fertility status preoperatively. In this respect, this study's retrospective design therefore is a significant limitation. On extensive review of patients notes it was clear there was little information available about patient's desires for parenthood, or known risk factors for infertility. Since 2016, our team has changed our approach to proactively assess patients’ fertility status preoperatively, with assessment of the preoperative FSH, LH, and analysis of semen parameters before any planned orchidectomy to be able to prospectively assess this relationship. In other surgical disciplines, baseline organ function is a mandatory assessment before the removal of a paired organ, such as a kidney; it seems anomalous therefore that testis are removed without measuring testicular function first.

Assessing the desire of patients with testicular cancer for future paternity, measuring their fertility markers (FSH, LH and testosterone), determining patient age and PTTO at diagnosis, and conducting semen analysis are important steps in fertility planning and preservation. Identification of the severely oligozoospermic or azoospermic patient before orchidectomy can allow referral for onco‐TESE, and PTTO measurements can help determine sperm retrieval success.

Based on the present study's findings, the following fertility planning in men with testicular cancer is recommended (Fig. 5).

The findings of the present study are limited by its retrospective design and sample size. This limits the amount of semen analysis and testis function data available, as well as other relevant clinical features, which may have an impact on overall fertility (i.e. cryptorchidism). Future directions would be to expand the dataset in a prospective manner with baseline testicular functional data to correlate to the histopathological findings and preoperative ultrasonography assessment. A closer assessment of the location of the tumour and any TML on preoperative imaging with correlation to the final histopathological pattern of spermatogenesis would clarify the potential impact and predictive role of TML, and rete testis involvement, in determining the chances of finding sperm in testicular cancer.