P-080 Enzymatic tissue processing after testicular biopsy in non-obstructive azoospermia enhances sperm retrieval and cumulative live birth rates
Authors: Vloeberghs V , Racca A , De Munck N , Mateizel I , Tournaye H 
Published: June 2023

Abstract

Study question
What is the added value of enzymatic processing of testicular biopsies on testicular sperm retrieval rates in patients with non-obstructive azoospermia (NOA)?

Summary answer
In addition to mechanical mincing, enzymatic digestion increased sperm retrieval rates in testicular biopsies of NOA patients with 48.9%.

What is known already
Many studies focus on the surgical approach to optimize recovery of testicular sperm in NOA, and in spite of that, there is still controversy whether the type of surgery makes any difference as long as multiple biopsies are taken. Few studies, however, focus on the role of the IVF laboratory and the benefit of additional lab procedures, like enzymatic digestion, in order to optimize sperm retrieval rates and CLB rate per TESE.

Study design, size, duration
A retrospective single-center cohort study including all patients who underwent their first TESE by open multiple-biopsy method from January 2004 till July 2022. Only patients with a normal karyotype, absence of Y-q deletions and a strict diagnosis of NOA based on histology were included. Primary outcome was sperm retrieval after mincing or enzymes for intracytoplasmic sperm injection (ICSI). Secondary outcome was CLB after ICSI with fresh TESE, and subsequent ICSI cycles with frozen TESE.

Participants/materials, setting, methods
Multiple biopsies were obtained from the testis, unilateral or bilateral, on the day of oocyte retrieval. Upon mechanical mincing, dishes were searched for 30 min; when no or insufficient numbers of spermatozoa were observed, enzymatic treatment was performed using collagenase type IV.
Multivariable regression analysis was performed to predict CLB by adjusting for the following confounding factors: male age, male FSH level, cryptorchidism, enzymatic digestion, number of oocytes and female age.

Main results and the role of chance
Hundred-eighteen patients were included of which 61.0% had successful sperm retrieval. Spermatozoa were retrieved after mechanical mincing (23.7%; 28/118) or after additional enzymatic digestion of the remaining 90 patients (48.9%; 44/90).
Mean male characteristics were not different between patients with sperm retrieval after mincing or enzymes: age (34.5 vs 34.5 y), testis volume (10.2 vs 10.6 ml), FSH (17.8 vs 16.9 IU/l), cryptorchidism (21.4 vs 34.1%) and histological diagnosis (Sertoli Cell Only 53.6 vs 47.7%, maturation arrest 21.4 vs 38.6%, sclerosis/atrophy 25.0 vs 13.6%), respectively.
Of the 72 patients with sperm available for ICSI, 23/72 (31.9%) obtained a LB after the injection with fresh testicular sperm (fresh and frozen embryo transfers). Forty-nine patients remained without LB, of which 34 had supernumerary testicular sperm frozen. Of these, 9/47 (47.4%) had a LB after ICSI with frozen testicular sperm, giving rise to a total CLB per TESE of 32/118 (27.1%) or 32/72 (44.4%) CLB per TESE with sperm.
Of the female characteristics (couples with sperm available), only female age (30.3 vs 32.7 y – p = 0,042) was significantly lower in the group with a live birth.
Multivariable logistic regression analysis showed that enzymatic digestion was associated with significant decrease of CLB per TESE.

Limitations, reasons for caution
Limitations of the study are related to the retrospective design. The selection of only NOA patients with specific characteristics (normal karyotype and absence Y-q deletion) and having their first TESE ever strengthens our findings. Whether enzymatic digestion after a failed TESE without digestion may improve retrieval rate remains undecided.

Wider implications of the findings
Enzymatic processing increases the sperm retrieval rate from testicular biopsies of NOA patients compared to mechanical mincing, demonstrating the importance of an appropriate laboratory protocol. NOA patients should be counselled that if sperm has been found after enzymatic digestion, their chances to father a genetically own child will be lower.

Trial registration number
not applicable