While fresh cadaveric temporal bones are preferable in middle-ear research, limited availability and strict ethical regulation often require the use of fresh-frozen temporal bones as an alternative. Previous studies have shown that freezing and thawing can alter mechanical properties of soft tissues like ligaments, muscles, and joints, yet the effect of freezing on middle-ear sound transmission has not been fully revealed. This study aims to investigate the effects of freezing by comparing the middle-ear transfer function (METF) between fresh and fresh-frozen temporal bones.

Data of 101 human temporal bones (57 fresh and 44 fresh-frozen) were analyzed. The magnitudes of the METF between 125 and 6000 Hz were averaged frequency-wise and compared between both groups. Further, each frequency-response curve was fitted with a rational polynomial, and all fitted curves were averaged for analysis of shape-preserving information such as the location of natural frequencies.

The average METF magnitude was considerably lower for fresh-frozen temporal bones in frequencies below 3000 Hz with differences of up to 7 dB. Shape-preserving analysis of frequency responses revealed two natural frequencies at 900 and 3487 Hz for fresh and at 988 and 3180 Hz for fresh-frozen temporal bones, with no statistically significant differences between fresh-frozen and fresh temporal bones.

With only a slight reduction in METF magnitude at low frequencies compared to large interindividual variabilities, fresh-frozen temporal bones can be considered as an alternative to fresh temporal bones. However, stiffening of middle-ear structures needs to be taken into account when using fresh-frozen temporal bones.