Validation-verification of a highly effective, practical human testicular tissue in vitro culture-cryopreservation procedure aimed to optimize pre-freeze and post-thaw motility

Abstract

Purpose: The aim of our paper was to validate a testicular biopsy procedure that simplifies handling, processing, and cryopreservation, while at the same time optimizes sperm motility before freezing and after thawing.

Methods: Two prospective studies were conducted to verify, optimize, and understand the virtues of pre-freeze testicular tissue IVC at different temperatures (21, 30, or 37 °C). Testicular tissue was obtained from clinical specimens designated for whole tissue cryopreservation (i.e., intact mass of tubules) and/or for fresh use in IVF-ICSI cycles. Whole testicular biopsy pieces (1-3 mm(3)) were diluted in glycerol containing freeze solutions, slow cooled to 4 °C and then rapidly frozen in LN2 vapor. Fresh and post-thaw testicular biopsy tissue were evaluated for changes in the quantity (%) and pattern of motility (I-IV: twitching to rapid progression, respectively) over a 1 week duration. The clinical effectiveness of IVC-cryopreserved whole testicular biopsy tissue was also validated analyzing fresh embryo transfers.

Results: More reliable recovery of motile testicular sperm was achieved using whole tissue freeze preservation combined with IVC (24-96 h) post-acquisition at an incubation temperature of 30 °C compared to ambient temperature (21 °C) or 37 °C. Up to 85 % of the pre-freeze motility was conserved post-thaw (+3 h) for easy ICSI selection. Sperm longevity was optimized to fresh tissue levels by implementing testicular biopsy sucrose dilution post-thaw. Favorable clinical outcomes were proven using frozen-thawed testicular biopsy sperm for ICSI.

Conclusions: By employing minimal tissue manipulation, integrating pre-freeze IVC processing at 30 °C and the freezing of whole testicular biopsy tissue, we have reduced the labor and improved the efficacy of processing testicular tissue for freeze-preservation and subsequent ICSI use.

Keywords: In vitro culture (IVC); Motility; Sperm cryopreservation; TESE; Testicular biopsy.

Fig. 1

In panel a, a tricolor-stained histograph (×200 magnification; courtesy of Dr. Charles Sims, M.D.) of thin-sectioned testicular tissue exhibits normal spermatogenesis, from spermatogonia to sperm release in the tubule lumen. Round cell spermatid transition to sperm formation is clearly evident (a). The tissue mass is composed of seminiferous tubules which can be easily dissected into smaller masses (b) for subsequent evaluation or cryopreservation. Using fine needles to shred the tubules of a small testis mass (c) in a droplet of medium under oil, the dispersed cellular contents of the tissue are allowed to settle and equilibrate (d) before evaluation under inverted microscopy (e, hpf = ×400 magnification). This panel depicts normal spermatogenesis, as revealed by a variable size cell population and the presence of >5 sperm/hpf. A styrofoam box containing tube racks (f) is used as a testicular biopsy incubator (when covered), attaining an optimal temperature of 30 °C to culture testicular sperm, while resting on a 37 °C slide warmer surface

Fig. 2

Experimental design schematic reveals the developmental phase of adopting whole testicular tissue cryopreservation and observing improvements in IVC motility characteristics associated with a change of laboratory environments/elevated ambient temperatures. In 2004, a verification study (study 1) was conducted to evaluate the beneficial effect of controlling the IVC temperature at 30 °C with daily repeated measures of motility overtime (1 week) pre-freeze and post-thaw. Subsequently, in 2007, a prospective apriori study of three different temperature treatments (21, 30, 37 °C) with repeated measures of motility was performed to validate the optimum effect of an intermediate temperature on the promotion of progressive sperm motility of longevity. Furthermore, due to the suspected negative effect of residual glycerol in post-thawed testicular tissue sperm longevity in study 1, all freeze-thawed testicular tissue in study 2 was exposed to a 0.5 M sucrose solution prior to dilution in H-HTF medium. In addition, the progressive use of higher glycerol solution concentrations was to insure effective permeation of cryoprotectant into tissue, as well as adopting a practical commercial product (shifting from a 20 % glycerol to 26.7 % glycerol solution in study 1 and 2, respectively). Finally, no randomization was performed as all repeated measures (study 1 and 2) and IVC treatments were equally applied to each patient specimen. The applications of our optimized minimal manipulation treatments (i.e., whole tissue IVC/cryopreservation) over the past 7 years have validated and verified its clinical effectiveness

Fig. 3

The effects of extended IVC (30 °C) on the motility of testicular sperm are graphically shown in stacked columns measuring motility types (I to IV, twitching to rapid progression, respectively; a). In panel b, post-thaw motility patterns of cryopreserved testicular biopsy sperm over time (IVC at 30 °C) are shown in stacked columns measuring the four types of motility. The total motility for each interval, in both panels, is indicated within the lower sub-column. Good post-thaw survival was recovered for cryopreserved whole testicular biopsy specimens in an actively motile state. Upon equilibration, the optimal viability of the post-thaw samples was acquired by +24 h IVC, which significantly declined at +48 and +72 h in study 1 where residual glycerol had a toxic effect on longevity. In each panel, subscript(s) atop each column denote significant differences between intervals

Fig. 4

In study 2, the effect of extended IVC on the motility of testicular sperm was tested at three different incubation temperatures (21, 30, and 37 °C). The four types of motility (graded: I–IV) are graphically shown in stacked columns measuring the total motility at each of four intervals. Differences (p < 0.05) within each column between treatments are reflected by subscript differences adjoining the total motility value. Significant changes in motility patterns between intervals within incubation treatments are indicated by the inclusion of a directional arrow