The cup method, a modification of methods used successfully in the rat, the dog, and a smaller primate to suppress spermatogenesis [1, 2], results in reduction of semen quality in adult rhesus monkeys. Unlike in other animal models, treatment did not induce azoospermia, yet the numbers of sperm that were vigorously motile and with normal morphology were exceedingly low. Within exposure method, ultrasound treatment appeared to be most effective for males with smaller testes, suggesting that higher levels of exposure may be required to achieve contraception in individuals and animals with greater testicular mass.
Low intensity therapeutic ultrasound has been demonstrated to exhibit both thermal and non-thermal effects on tissue. It has been well established that acoustic absorption by tissue can result in elevated tissue temperature; the rate at which temperature rises is proportional to the intensity of the ultrasonic beam and inversely proportionate to the density and heat capacity of the tissue . Effects believed to arise from low intensity (0.125–3 W/cm2) ultrasound-induced heating include changes in blood flow, increased flexibility of tendons and scar tissue, and decrease in joint stiffness . Independent of temperature, acoustic radiation produces a time-averaged force that can act on objects in an acoustic field , for review]. Acoustic radiation forces can induce the rotation or spinning of particles as well as produce flow or streaming of fluids. Streaming can alter the local environment of a cell, resulting in altered concentration gradients across cellular membranes . Radiation forces underlie a number of bioeffects including stimulation of cardiac and neural tissue, acceleration of bone healing, enhancement in collagen synthesis, and improvement in both drug uptake by cells (sonoporation), and transdermal delivery of drugs (sonophoresis) .
It is not entirely clear how ultrasound affects sperm production. Increased testicular heat alone has been shown to slow spermatogenesis, induce apoptosis in developing sperm cells, and lead to lower numbers of motile and morphologically normal spermatozoa in an ejaculate , for review]. From rodent and livestock studies, the cells that are most susceptible to damage by acute heat stress are pachytene spermatocytes and spermatids, although B spermatogonia can also be damaged if degree and duration of heat exposure are increased , for review]. In men, elevated scrotal temperatures due to lifestyle, workplace environment, varicocele, or cryptorchidism are inversely correlated with sperm quality [22–24]. From Fahim's work and Tsuruta's studies in the rat, brief exposures of the testes to low intensity ultrasound result in an increase in intratesticular and scrotal temperatures ranging from one up to several degrees Celsius above body temperature [1, 2, 6, 25]. Similarly elevated temperatures can be achieved by warming testicles in a water bath for 15 to 30 minutes, but the effect on sperm production is not nearly as pronounced as when ultrasound is applied [1, 25]. The mechanisms of the extra-temperature effects observed in testicular tissue with ultrasound treatment, however, are not fully understood. Fahim demonstrated that ultrasound exposure results in changes in electrolyte concentrations in fluid from seminiferous tubules and rete testes . Perhaps this finding speaks to ultrasound-induced alterations in the transport of substances across the tubule that may contribute to the stress of spermatogenic cells.
In our study, we demonstrate that the cup method, using warm saline as a coupling medium, is more effective at suppressing sperm production than direct application of the ultrasound probe to the scrotum. Initially, we found this to be a surprising result as we expected the cup method to produce more ultrasound beam scattering and therefore less focused propagation of ultrasound energy through testicular tissue. Moreover, similar conditions of direct application, reproduced by the same model of sonicator, were applied to the testes of dogs and resulted in azoospermia in all animals treated . This discrepancy in response, as well as the lack of a consistent effect with either cup or direct methods, might be explained by differences in testes size between and within species and a general tendency of the testes to resist temperature change due to gonadal vascular and scrotal muscular and glandular mechanisms .
Differences in testes size may account, at least in part, for the differences in efficacy that we see with ultrasound in the rhesus monkey. In both treatment methods, the male with the smaller testes exhibited the greatest reduction in sperm numbers following ultrasound. We speculate that the methods we tested did not sufficiently elevate intratesticular temperatures in males with greater testicular mass. Potentially for this reason, the cup method was superior to the direct because it provided conditions (saline bath maintained at 35-37°C) whereby testicular temperature should stay elevated during the duration of ultrasound exposure. Evidence for this possibility is provided in the rat, where the temperature of the conducting medium was an important factor for observing maximal effects with ultrasound . When saline in the ultrasound treatment cup was maintained at 37°C, the intratesticular temperature in the rat rose rapidly (within 1–2 minutes of ultrasound exposure) above body temperature . By contrast, the direct method in the monkey, by not controlling ambient temperature, likely could not overcome scrotal mechanisms for liberating heat from the testes. It should also be noted that the testes of the monkeys used in our study on average were three times larger (by volume) than testes of the dogs used by Leoci et al. , and adult rhesus monkey testes in general are approximately 15 times larger (by mass) than testes of sexually mature rats [27–29]. It is therefore not surprising that similar ultrasound protocols might not produce highly similar results among species that vary widely with respect to testicle size.
An important finding of the present study is that the effects of ultrasound appear to be fully reversible. The sperm quality of all males, independent of treatment method, recovered somewhat sharply to pretreatment levels between week seven and week nine following ultrasound exposure. This trend was particularly dramatic for male #1, where sperm production was nearly eliminated (total sperm count and normal sperm count were inhibited by over 99%) in the initial weeks post treatment. Fahim reported a similarly robust recovery in sperm production following testicular ultrasound with the smaller cynomolgus monkey (Macaca fascicularis) . Together these studies suggest that primate testes may be particularly resilient to ultrasound radiation, with full recovery of spermatogenic potential. If equally true for men, ultrasound could prove viable as a non-invasive reversible contraceptive provided the duration of prophylaxis can be extended. Additional studies in non-human primates are needed to determine optimal exposure conditions, perhaps including an increase in the ambient temperature, to ensure a prolonged knockdown of spermatogenesis to levels that would be consistent with providing effective contraception.
A concern with any treatment that results in heat stress of the testes is the potential of DNA damage in sperm. Mild, acute, scrotal heat stress leads to DNA strand breaks in spermatogenic cells in mice [30, 31]. Many of these sperm progenitors are eliminated by apoptosis, but as many as 70% of the sperm that developed from thermal-damaged spermatocytes have chromatin abnormalities indicative of DNA defects . In vivo and in vitro fertilization with these sperm results in numerous embryonic abnormities and sharply reduced rates of blastocyst formation . These studies mirror a growing concern in the field of reproductive medicine that certain long-term health adversities seen in children are potentially associated with conception achieved with sperm possessing damaged DNA . Before ultrasound treatment should be considered as a method of reversible contraception in humans, future studies should determine whether DNA damage persists in spermatozoa following recovery of sperm numbers and normal morphology in the months following testicular ultrasound exposure.