P–023 Innovative ultra-rapid vitrification method for five or fewer testicular spermatozoa from non-obstructive azoospermic men after microsurgical testicular sperm extraction (Micro-TESE)
Authors: Tanaka A , Yanagihara Y , Nagayoshi M , Yamaguchi T , Tanaka I , Itakura A 
Published: August 2021

Abstract

Study question
What technique can be used to successfully cryopreserve five or fewer testicular spermatozoa from non-obstructive azoospermic men?

Summary answer
This method for cryopreserving five or fewer spermatozoa from non-obstructive azoospermic men showed a recovery rate above 90% and a survival rate of about 70%.

What is known already
Clinical outcomes of ICSI when using only five or fewer testicular spermatozoa after cryopreservation have been unsuccessful and are considered to be inferior to those using testicular fresh spermatozoa from Micro-TESE. A possible cause of these poor results has been the lack of a successful freezing technique. In these cases, repeated Micro-TESE and simultaneous oocyte pick up has been the only available treatment.

Study design, size, duration
Evaluation of the efficiency of cryopreservation by modified permeable cryoprotectant-free vitrification method (HTF supplemented with 0.1M sucrose and 10% SPS) for five or fewer testicular spermatozoa from 113 non-obstructive azoospermic men using Micro-TESE was conducted retrospectively at St. Mother Clinic between 2011 and 2018.

Participants/materials, setting, methods
This study included 113 non-obstructive azoospermic men. Each motile spermatozoon was carefully aspirated tail first into the pipette, put into a 2-μl microdroplet media of the vitrification medium near the tip of the Cryotop (Kitazato Corporation, Tokyo, Japan) submerged in liquid nitrogen vapor for 2 min and then immediately plunged in liquid nitrogen. The vitrified spermatozoa were warmed by dipping them into a droplet media. Successfully recovered motile sperm were selected and used for ICSI.

Main results and the role of chance
Number of patients, transfer cycles and collected sperms were 113, 192 and 560. Mean age of patients and their wives were 32.0±3.7y and 28.4±5.8y. Clinical pregnancy rate, miscarriage rate, live birth rate and number of live offspring were 24.0% (46/192), 19.6% (9/46), 19.3% (37/192) and 37 (Male: Female = 17: 20). Sperm recovery rate and survival rate were 90.3% (506/560) and 70.4% (356/506). Fertilization rate and mean number of transferred embryos were 51.6% (99/192) and 1.73 (1–2). Mean gestational weeks and mean body weight at birth were 39.23±5.27w and 2852.31±314.28g. No congenital anomalies were observed in any of the babies.

Limitations, reasons for caution
The maximum number of spermatozoa to which this method can be applied successfully is about 10. When the number of aspirated spermatozoa is over 10, some of them change direction and reach the mineral oil, and once this happens, they cannot be expelled out of the pipette.
Wider implications of the findings: This technique is very useful for the cryopreservation of very small numbers of testicular spermatozoa (fewer than 10) in order to avoid or reduce Micro-TESE interventions.

Trial registration number
N/A