O-210 Cluster analysis reveals two hidden subgroups among patients with cryptozoospermia
Authors: Di Persio S , Schülke L , Wistuba J , Cremers J.F , Kliesch S , Neuhaus N 
Published: June 2022

Abstract

Study question
Can cryptozoospermic patients be sub classified based on clinical parameters?

Summary answer
Cryptozoospermic patients can be subdivided in two subgroups based on the histological phenotype of their testicular tissues, testicular volume and FSH levels.

What is known already
Cryptozoospermia is a severe form of oligozoospermia in which patients present with a sperm concentration of ≤ 0.1 million per milliliter. Due to such low sperm concentration, cryptozoospermic patients depend on the surgical procedure of testicular sperm extraction (TESE) to retrieve sperm for intracytoplasmic sperm injection (ICSI). The general etiology underlying cryptozoospermia remains hitherto unknown, likely due to the high heterogeneity within this patient cohort regarding histological and endocrine parameters.

Study design, size, duration
We retrospectively selected 132 cryptozoospermic patients (Crypto) who underwent TESE during their fertility treatment. Exclusion parameters were genetic diseases, a history of cryptorchidism or tumors and hormonal treatment. As controls, we selected 160 patients with obstructive azoospermia and normal spermatogenesis. All 292 patients were used for principal component analysis (PCA) followed by hierarchical clustering on principal components (HCPC).

Participants/materials, setting, methods
PCA and hierarchical clustering were performed considering age, testicular volume, hormonal values (FSH, LH, testosterone and free testosterone), ejaculate parameters (Ejaculate pH and volume, sperm concentration and total sperm count) and histological information. Histological analyses were performed on two PAS-stained sections from two independent biopsies per testis and patient. The percentage of seminiferous tubules containing elongated spermatids, round spermatids, spermatocytes or spermatogonia, as well as Sertoli cell only and hyalinized tubules was assessed.

Main results and the role of chance
The PCA and hierarchical clustering subdivided the patient cohort into 3 different clusters. Cluster 1 included all the controls (n = 160) and 2 Crypto patients. Remaining Crypto patients were equally subdivided between cluster 2 (n = 65) and cluster 3 (n = 65). The control patients in cluster 1 were characterized by the highest percentages of tubules with elongated spermatids (&gt;80%), normal testicular volume and hormonal values. Characteristic of Crypto patients in cluster 2 was the arrest of germ cell differentiation at the level of spermatocytes in the majority of seminiferous tubules. In contrast, the majority of seminiferous tubules in Crypto patients in cluster 3 showed a Sertoli cell only phenotype or even tubular shadows. Interestingly, the more severe histological phenotype of the cluster 3 patients was accompanied by higher FSH and LH levels as well as lower testicular volume compared to cluster 2 patients. Despite this, the percentage of tubules with full spermatogenesis was similar in the two Crypto groups and no difference was found in the sperm retrieval rate after TESE in the two patient groups.

Limitations, reasons for caution
Uncovering the existence of subgroups within the cohort of Crypto-patients is the prerequisite for unveiling potentially distinct etiologies. Comprehensive genetic and scRNA-seq analyses of these Crypto-subgroups remain to be performed to validate the findings.

Wider implications of the findings
Overall, this study offers novel approaches towards the understanding of the etiology underlying the reduced sperm formation in Crypto patients and the division into two subgroups will facilitate the strategic search for underlying causes. Moreover, this approach could be applied to any infertility type in order to identify hidden subgroups.

Trial registration number
not applicable