P-347 In search of the most efficient device to freeze low numbers of spermatozoa: a comparative evaluation of various vitrification device
Authors: Krunic M , Tarantino M , Toumpa O , Magni B , Schoemans C , Mateizel I , Wouters K , Tournaye H , De Munck N 
Published: July 2024

Abstract

Study question
Can different vitrification devices be used for cryopreservation of few spermatozoa for patients with varying sperm concentrations and which device is the most efficient?

Summary answer
The study demonstrates the efficacy of Cryotop, CBS, and Vitrifit devices in cryopreservation of low numbers of spermatozoa, with Cryotop Open showing superior performance.

What is known already
Sperm cryopreservation is crucial for managing male-factor infertility, yet conventional methods, reporting significant reduction in motility with only 30 to 40% viability post-thawing, fall short of optimal cryopreservation of small numbers of spermatozoa. Currently, ongoing efforts to enhance cryopreservation techniques seek to minimize cryodamage and optimize outcomes, particularly for spermatozoa from patients with conditions like oligozoospermia, cryptozoospermia, or azoospermia, where each spermatozoa is of substantial importance. Several devices have shown promising results regarding cryopreservation of small numbers of spermatozoa. However, to date, no comparison has been performed between multiple embryo-vitrification devices in terms of recovery and viability post thawing.

Study design, size, duration
Between January 2023 and May 2023, this study included 50 fresh ejaculated routine samples: 25 with spermatozoa concentrations &gt;1 million/ml and 25 samples with concentrations &lt;1 million/ml. For each sample, 10 motile spermatozoa were loaded on four different embryo-vitrification devices performed in triplicate: Cryotop Open (Kitazato), Cryotop Closed (Kitazato), CBS (Cryo BioSystem) and Vitrifit (Cooper Surgical). Recovery, motility, immotile vitality rates, user-friendliness as well as the duration of the freezing/thawing procedures were examined.

Participants/materials, setting, methods
Ten spermatozoa, selected in Hepes-HSA, were loaded into a 2 µl mixture of SpermFreeze and HEPES-HSA. Held 3 cm above LN2 for 2 minutes, straws were plunged into LN2. Thawing involved immersing straws in 2 µl Hepes-HSA under oil. Hypo-Osmotic-Swelling test was used for the vitality assessment of the immotile spermatozoa. Thawing protocols showed minor variations based on the carrier type. The study was approved by the local Ethics Committee.

Main results and the role of chance
This study conducted a detailed analysis of four vitrification devices for cryopreservation of low spermatozoa numbers. For concentrations &gt;1 million/ml, Vitrifit, Cryotop Open, Cryotop Closed, and CBS displayed comparable recovery rates (mean % ± SD) (85±0.09, 89±0.06, 84±0.10, 86±0.07); motility rates (76±0.12, 79±0.08, 78±0.13, 80±0.08); immotile vitality rates (16±0.08, 13±0.07, 13±0.09, 12±0.07). In this group, Vitrifit and Cryotop Open exhibited similar freezing and recovery times (4’10’’/7’42’’ and 4’11’’/7’40’’ respectively). Cryotop Closed and CBS had slightly longer freezing times (4’33’’ and 5’31’’ respectively), with Cryotop Closed showing the fastest recovery (7’11’’). Similarly, for sperm concentrations &lt;1 million/ml, devices demonstrated similar recovery (87±0.09, 91±0.06, 88±0.07, 83±0.12), motility (55±0.10, 61±0.09, 56±0.07, 50±0.09), and immotile vitality (26±0.07, 26±0.06, 27±0.07, 27±0.06). For concentrations &lt;1 million/ml, all devices showed similar freezing and recovery times. Statistical analyses, including ANOVA and Levene’s test, revealed no significant inter-device differences, substantiated by minimal Cohen’s d effect sizes. Regarding the practical aspect, due to the ergonomic design and plastic composition that allows better microscopic visibility, Cryotop (both closed and open) demonstrated superior handling performance to CBS. Considering both statistical validation and manipulation, Cryotop Open distinguishes itself as the most efficient and user-friendly device for cryopreservation of low spermatozoa numbers.

Limitations, reasons for caution
The study’s main limitation is the limited sample size. Further research with larger cohorts and exploration of fertilization potential is necessary to validate these findings. Additionally, our study did not explore potential variations in cryopreservation outcomes related to additional sperm sample characteristics, which could impact the results.

Wider implications of the findings
Successful cryopreservation of low sperm numbers provides hope for cryptozoospermia and (non-)obstructive azoospermia patients. It allows the use of their own sperm, potentially avoiding donation. With superior frozen sperm survival, recovery rates, and a shorter search time, this method enhances fertility preservation accessibility, guiding patients in future IVF treatments effectively.

Trial registration number
EC-2022-272