Field mapping and measuring the true ERA and power output of our transducers

Field mapping of the output from the therapeutic transducer showed that there was a donut shaped "hotspot" in the 5-cm2 transducer's output (ME7413) at 3 MHz [Figure 2]. The field map was the same regardless of the coupling medium used (DW, degassed DW or 3% (w/v) saline). The beam field of the 5-cm2 transducer changed when it was mapped at 1 MHz: instead of a donut shaped hotspot, there was a discrete peak of energy near the center of the transducer face [Additional file 1 Figure S1].

Figure 2 Beam field map of the Model ME7413 therapeutic ultrasound transducer acquired at 3 MHz. Normalized acoustic pressure is plotted on the Y-axis. The X and Y-axes represent the coordinates used to measure acoustic pressure delivered by the ultrasound transducer. Full size image

Beam plots from both transducers [Additional file 1 Figure S1] were used to determine the area of the beam with energy equal to at least 5% of the peak beam energy when the distance between the hydrophone and transducer was set to 0.5 cm. The ME7413 transducer with a nominal area of 5 cm2 had a true effective radiating area of 4.4 cm2; the ME7410 transducer with a nominal area of 10 cm2 had a true effective radiating area of 9.3 cm2.

The power output of our transducers was determined at intensities indicated by the Mettler Sonicator 740 to be 1 W/cm2 and 2 W/cm2. The 5 cm2 transducer (ME7413) at a nominal intensity setting of 1 W/cm2 had an output of 4.6 W at either 1 or 3 MHz; with a nominal intensity setting of 2 W/cm2 the output varied from 8.9 Watts at 1 MHz to 9.3 Watts at 3 MHz. The 10-cm2 transducer (ME7410) was only measured at 1 MHz and had an output of 10.2 Watts at a nominal intensity setting of 1 W/cm2 and an output of 20.0 Watts at a nominal intensity setting of 2 W/cm2.

True spatially averaged intensities were determined for our transducers

The 5-cm2 transducer (ME7413) had an effective radiating area of 4.4 cm2. At both 1 and 3 MHz frequency the actual intensity for this transducer at an indicated 1 W/cm2 was 1.05 W/cm2. The actual intensity for this transducer at an indicated 2 W/cm2 varied from 2.02 W/cm2 at 1 MHz to 2.11 W/cm2 at 3 MHz. The spatially averaged intensities determined for this transducer were all within 6% of the values indicated by the Mettler Sonicator 740.

The 10-cm2 transducer (ME7410) was only capable of operating at 1 MHz frequency and had an effective radiating area of 9.3 cm2. The actual intensity determined for this transducer at an indicated 1 W/cm2 was 1.1 W/cm2 and at an indicated 2 W/cm2 the actual value was 2.15 W/cm2. The spatially averaged intensities determined for this transducer were within 10% of the values indicated by the Mettler Sonicator 740.

Mitigating thermal bio-effects

In order to create a more even field of ultrasound at both frequencies, we devised a method to rotate the transducer in a horizontal plane coincident with the bottom surface of the ultrasound chamber with the center of rotation offset 8 mm from the center of the transducer face. The movement of the transducer mimics its use as a therapeutic device and results in an averaging of the field output over time.

The distance between the transducer and the scrotum was initially set to 3 cm. In an attempt to increase the energy delivered to the testes, the distance between the scrotum and the transducer was successively decreased. Some rats' testes actually rested on the bottom of the ultrasound chamber, separated from the transducer only by a layer of latex. This may have been responsible for some localized heat damage to the scrotum; these rats would occasionally develop a small circular discoloration on their scrotum.

Constructing a mesh support provided a reproducible offset of 1 cm between the bottom of the treatment chamber and the scrotum; recirculating the coupling medium eliminated any thermal bio-effects localized to the scrotum.

Pilot study 1: published treatment parameters did not alter testis histology

Attempts to cause germ cell loss using a single ten minute dose of ultrasound at 100% duty cycle, 1 MHz and 1 W/cm2 (Pilot Study 1) did not alter testis histology. These were the original parameters that were reported by Fahim to cause the loss of almost all germ cells from the testis [4]. Pilot study 1 used phosphate buffered saline or distilled water as the coupling medium filling the ultrasound chamber. The coupling medium surrounded the scrotum and allowed ultrasound to be efficiently transmitted from the transducer to the scrotum; ultrasound passed through the scrotum and was absorbed by the testes.

Pilot study 2: increased power and degassed coupling medium

An experiment using a single treatment of 1 MHz at 2.2 W/cm2 and 100% duty cycle through degassed water was performed (Pilot Study 2). Treating with 2.2 W/cm2 was more successful than treating with 1 W/cm2. Two weeks after ultrasound treatment, the testis was depleted of developing germ cells and sperm count was reduced to 200 × 103 sperm per cauda epididymis. These sperm were not motile when analyzed in M16 medium.

Fahim reported that his ultrasound conditions caused rats to immediately lose their fertility [4]. When we treated with low frequency and high power (Pilot Study 2), pups were sired during the first and second weeks after treatment. However, there were no motile sperm at the end of this pair of one-week mating trials. Hypothetically, if another mating trial had been performed during the third week after treatment, the rat would have been infertile. This demonstrated that even though motile sperm were not detected at the end of the second mating trial, there were sufficient motile sperm during the initial two-week period after treatment for fertility.

Study 1: two consecutive treatments

In an attempt to bring post-treatment sperm counts closer to zero, the effect of two consecutive treatments separated by two days were tested [Study 1, Table 2]. Two weeks after treatment, total sperm count in the cauda epididymis dropped below 2 × 106 total sperm with essentially no motility when 3 MHz ultrasound was applied at 2.2 W/cm2 through 37°C distilled water at 100% duty cycle [Table 4 Group 4]. Using coupling medium heated to 45°C allowed us to achieve internal testis temperatures comparable to the ultrasound treated testes [Figure 3]. Interestingly, heat alone [Table 4 Group 2] was more effective at reducing epididymal sperm count than the use of 1 MHz ultrasound either when the temperature of the coupling medium was held constant at 37°C [Table 4 Group 3, Tukey's post-test, p < 0.001] or when the temperature of the coupling medium was not regulated [Table 4 Group 6, Tukey's post-test, p < 0.001], however when 1 MHz ultrasound was applied through 3% saline at low power, sperm count was reduced sufficiently so that there was no significant difference from wet heat.

Table 4 Testis temperatures and sperm parameters from Study 1 Full size table

Figure 3 Representative temperature curves during ultrasound or wet heat. A thermal couple was inserted down the long axis of the testis and another was placed in the coupling medium. Coupling medium was re-circulated at 37°C during ultrasound treatments and at 45°C for the wet heat control. The rotation frequency of the transducer correlated with temperature fluctuations at the site of the thermal couple. The wet heat control yielded a testis temperature profile similar to an ultrasound treated testis. Full size image

In contrast, the use of 3 MHz ultrasound resulted in a total epididymal sperm count ~10-fold lower than wet heat alone but with almost 1,000 times fewer motile sperm recovered from the epididymis: 3 MHz treated animals [Table 4 Group 4] had ~ 6 × 103 motile sperm per cauda epididymis while wet heat treated animals [Table 4 Group 2] had ~5 × 106 motile sperm per cauda epididymis (derived from data presented in Table 4; motile sperm = total sperm × % motile).

Study 1: combining heat and ultrasound more effective than heat alone

The normal testis [Figure 4, A-D] had a complex epithelium consisting of many spermatogenic cells in various stages of spermatogenesis. Two weeks after using wet heat to elevate testis temperature there was a significant loss of spermatogenic cells although most seminiferous tubules still retained some spermatogenic cells [Figure 4, E-H].

Figure 4 Representative histology of normal or wet-heat-treated testes and seminiferous tubules. A-D: hematoxylin and eosin stained cross-sections of untreated testis. The tall seminiferous epithelium contains many spermatocytes (sp), round spermatids (rs) and condensing spermatids (cs). Tails (t) of condensing spermatids and newly released testicular sperm are seen in the lumen (Lu) of some tubules. E-H: testis cross-section stained two weeks after wet heat treatment. Almost all tubules have enlarged luminal diameters after treatment with heat alone. The seminiferous epithelium (e) is reduced in height due to the loss of many spermatocytes and spermatids. Some tubules have disorganized epithelium (*). Full size image

In contrast, combining elevated temperature and 3 MHz ultrasound [Table 4 Group 4 or 5] caused testis-wide depletion of germ cells [Figure 5]. The loss of developing spermatocytes and spermatids from the seminiferous epithelium was extensive; almost all tubules examined were effectively depleted by this treatment [Additional file 2 Figure S2]. The loss of spermatogenic output was reflected by sperm counts in these animals below 2 × 106 sperm per cauda epididymis, two weeks after ultrasound treatment [Table 4 Groups 4 and 5].

Figure 5 Testis histology two weeks after 3 MHz ultrasound (Group 4). (A) The loss of spermatogenic cells after this treatment was more complete than after the wet heat treatment. This resulted in a shorter epithelium and a larger diameter lumen. (B) An isolated cluster of tubules in this particular animal showed evidence of thermal damage (td) in addition to the loss of spermatogenic cells. (C) Most tubules had a very short epithelial layer and increased lumen diameter due to the loss of all spermatocytes and spermatids. (D) Tubules that appear to have a larger epithelial layer and smaller diameter lumen were still missing spermatocytes and spermatids. Full size image

Study 2: varying 3 MHz ultrasound treatments

All animals in Study 2 were treated with 3 MHz ultrasound. We varied the temperature of the coupling medium (35 or 37°C), its composition (DW or saline), the number (1 or 2) or duration of treatments (10 or 15 minutes) to determine the effect of these changes in treatment on mean motile sperm count per cauda epididymis [Figure 6]. Except for the group treated through degassed distilled water at 35°C (Group 13), all treatments resulted in a significantly lower mean motile sperm count than the untreated group (Group 8) according to Dunnett's multiple comparison test (p < 0.001).

Figure 6 Average and distribution of motile sperm counts from Study 2. Motile sperm count was determined two weeks after treatment and was plotted as the mean ± SEM (106 per cauda epididymis). The stacked bars represent the proportion of sperm counts that fell into the following ranges of sperm counts (106 per cauda epididymis): < 11, 11 - 20, 21 - 40, 41 - 80, and > 80. Sperm Count Index was calculated as described in the Methods and is reported above each bar. Groups 8 - 12 failed Bartlett's test and were analyzed by the Kruskal-Wallis test with Dunn's post-test, symbols represent groups statistically different from Group 9: *, p < 0.05; **, p < 0.01; ***, p < 0.005. Groups 12 - 14 passed Bartlett's test: symbols represent groups statistically different from Group 12 by Tukey's post-test: §, p < 0.01. Full size image

The most effective treatment in Study 2 (Group 9: treating twice for 15 minutes at 3 MHz and 2.2 W/cm2 intensity through degassed 3% saline held at 37°C) resulted in 3 ± 1 million motile sperm per cauda epididymis and a Sperm Count Index equal to 0. The next three lowest sperm counts were in Groups 10 - 12; all of these treatments resulted in mean motile sperm counts greater than 50 million sperm per cauda epididymis which was significantly higher than observed for Group 9 [Figure 6, Kruskal-Wallis with Dunn's post-test, refer to figure for p-values]. Group 12 had a Sperm Count Index equal to 3.9 and approximately one third of this group's sperm counts fell into the range of 41 - 80 million sperm per cauda epididymis. Group 10 had a Sperm Count Index of 6.0 with a mean sperm count of 67 ± 7 million motile sperm per cauda epididymis. As the higher Sperm Count Index indicated, a much larger proportion (7/8) of this group's sperm counts fell into the range of 41 - 80 million sperm per cauda epididymis.

Study 2: saline was a more effective coupling medium than distilled water at 35°C

When animals were treated once at 37°C for 10 minutes at 2.2 W/cm2 there was not a significant difference in sperm count as a function of coupling medium (degassed distilled water versus degassed 3% saline) so this data was pooled (Group 11). However, when animals were treated twice at 35°C for 15 minutes at 2.2 W/cm2 the composition of the coupling medium did make a significant difference in sperm count (Tukey's post-test, p < 0.01): degassed 3% saline (Group 12) resulted in a sperm count 50% lower than degassed distilled water (Group 13). The use of saline resulted in about half of the sperm counts for Group 12 to be lower than 41 × 106 per cauda epididymis (Sperm Count Index = 3.9) while the use of distilled water (Group 13) resulted in only about 12% of counts below that threshold [Figure 6, Sperm Count Index = 8.1]. In addition, the number of intact sperm was significantly lower (Tukey's post-test, p < 0.05) when treating at 35°C through 3% saline [Figure 7, Group 12] than through degassed distilled water [Figure 7, Group 13].

Figure 7 Percentage of intact sperm recovered in Study 2. Sperm counts tallied both intact sperm and sperm heads not attached to a tail. The number of intact sperm was expressed as a percentage of the total number of sperm recovered. *, Group 9 was statistically lower than Groups 8, 13 and 14 by Tukey's post-test (p < 0.01). §, Group 12 was statistically lower than Group 13 by Tukey's post-test (p < 0.05). Full size image

Most effective treatment

When the four treatments groups (Groups 9 - 12) with the lowest mean sperm counts in Study 2 were compared by one-way ANOVA, Group 9 was found to have a significantly lower mean motile sperm count than the other three groups (Kruskal-Wallis with Dunn's post-test, refer to Figure 6 for p-values). In addition, the percentage of intact sperm in Group 9 [Figure 7] was significantly lower (Tukey's post-test, p < 0.01) than the untreated control [Figure 7, Group 8]. Thus, the treatment that reduced cauda epididymis sperm count two weeks after treatment to the lowest levels was the same in Study 1 (Group 5) and in Study 2 (Group 9): two 15- minute treatments with 3 MHz ultrasound at 2.2 W/cm2 through degassed 3% saline maintained at 37°C.