Scrotal Heat Stress Research Articles

Potential confounding factors in measurement of specific cell-free seminal mRNAs and microRNAs derived from human reproductive organs.

Cell-free seminal RNA (cfs-RNA) is mixed transcripts derived from male reproductive organs, and is potential biomarker for the research and diagnosis of male reproductive-related diseases. However, some clinical factors, including age, asymptomatic Ureaplasma urealyticum (UU) infection, scrotal heat stress, abstinence period, and the storage condition of semen samples, may interfere with sperm parameters and the measurement of seminal biomarkers. Accordingly, this study was designed to evaluate the effect of above clinical factors on the measurement of cfs-RNA, aiming to lay a foundation for its research use and potential clinical application. Semen samples were collected according to the selected clinical factors. Cell-free seminal plasma was obtained by centrifugation and total RNA was extracted with TRIzol LS. Selective male reproductive organ-specific cfs-mRNAs and cfs-miRNAs were quantified by quantitative real-time PCR. The concentration and total amount of cfs-mRNAs and cfs-miRNAs in one ejaculate were calculated and compared. ACTB, DDX4 (testis-specific), WFDC9 (epididymis-specific), and miR-514a-3p (testis-specific) significantly increased after scrotal heat stress. SEMG1 (seminal vesicle-specific) showed declining tendency with the prolonged abstinence period. Age, asymptomatic UU infection, and the storage condition showed no significant impact on the measurement of cfs-RNA. These results indicate that scrotal heat stress significantly interfere with the selected cfs-RNA derived from the testis and epididymis, and abstinence period may affect the yield of cfs-mRNA from seminal vesicle, while other clinical factors has no significant impact on the measurement. Thus, heat exposure and abstinence period should be considered for the cfs-RNA measurement in its research or clinical application.

Selected sperm traits are simultaneously altered after scrotal heat stress and play specific roles in in vitro fertilization and embryonic development

Improvements in the estimation of male fertility indicators require advances in laboratory tests for sperm assessment. The aims of the present work were (1) to apply a multivariate analysis to examine sperm set of alterations and interactions and (2) to evaluate the importance of sperm parameters on the outcome of standard IVF and embryonic development. Bulls (n = 3) were subjected to scrotal insulation, and ejaculates were collected before (preinsulation = Day 0) and through 56 days (Days 7, 14, 21, 28, 35, 42, 49, and 56) of the experimental period. Sperm head morphometry and chromatin variables were assessed by a computational image analysis, and IVF was performed. Scrotal heat stress induced alterations in all evaluated sperm head features, as well as cleavage and blastocyst rates. A principal component analysis revealed three main components (factors) that represented almost 89% of the cumulative variance. In addition, an association of factor scores with cleavage (factor 1) and blastocyst (factor 3) rates was observed. In conclusion, several sperm traits were simultaneously altered as a result of a thermal insult. These sperm traits likely play specific roles in IVF and embryonic development.

Recovery of normal testicular temperature after scrotal heat stress in rams assessed by infrared thermography and its effects on seminal characteristics and testosterone blood serum concentration

Reestablishment of testicular normal temperature after testicular heat stress is unknown and its effect varies widely. The aim of this study was to investigate the impact of scrotal insulation (IN) on testicular temperature and its relation to semen quality and testosterone blood serum concentration. For this, 33 rams were used; 17 submitted to IN for 72 hours (using bags involving the testes) and 16 not submitted to IN (control group). The experiment was performed between August and December 2013 in Pirassununga, Brazil (21°56″13″ South/47°28′24″ West). Seminal characteristics, testosterone blood serum concentration, rectal temperature (RT), respiratory frequency, scrotal superficies mean temperature (SSMT), and eye area mean temperature (EAMT) were analyzed 7 days before IN and 21, 35, 49, 63, and 90 days afterward. Scrotal superficies mean temperature and EAMT were measured by thermography camera FLIR T620. Testosterone was evaluated by radioimmunoassay. Analysis of variance was used to determine the main effects of treatment, time, and treatment-by-time interaction using PROC MIXED of SAS software adding command REPEAT. Pearson correlation test was used to verify correlation between SSMT, EAMT, RT, and respiratory frequency. Significant difference was considered when P ≤ 0.05. At the end of IN, SSMT was higher (P < 0.05) in insulated group (32.26 ± 0.19oC) than in control group (30.58 ± 0.18oC), and the difference between rectal and testicular (deduced from SSMT) temperatures was 1.12 °C; in the other times of the evaluation this difference was between 2.91 and 4.25 °C in IN group. Scrotal superficies mean temperature was reestablished 24 hours after IN. Rectal temperature and EAMT presented correlation (r = 0.59; P < 0.0001). There was time-by-treatment interaction for total sperm (P = 0.0038) and progressive motility (P = 0.01), abnormal spermatozoa (P < 0.0001), membranes integrity (P < 0.0001), induced thiobarbituric acid reactive substances (TBARSs; P = 0.05), and DNA integrity (P = 0.0004). These semen characteristics were negatively affected 21 days after IN, and excluding induced TBARSs and abnormalities, recovered 35 days afterward; induced TBARSs just were affected after 49 days of IN; sperm abnormalities just recovered after 63 days. Testosterone blood serum concentration was lesser in insulated rams (P = 0.03). Thus, the difference of 1.12 °C between RT and testicular temperature impacts semen quality and testosterone blood serum concentration. Moreover, this study shows that rams can recover testes temperature efficiently toward IN and that infrared thermography is an efficient tool to identify differences on SSMT.

A novel antioxidant formulation designed to treat male infertility associated with oxidative stress: promising preclinical evidence from animal models.

Does a novel antioxidant formulation designed to restore redox balance within the male reproductive tract, reduce sperm DNA damage and increase pregnancy rates in mouse models of sperm oxidative stress? Oral administration of a novel antioxidant formulation significantly reduced sperm DNA damage in glutathione peroxidase 5 (GPX5), knockout mice and restored pregnancy rates to near-normal levels in mice subjected to scrotal heat stress. Animal and human studies have documented the adverse effect of sperm DNA damage on fertilization rates, embryo quality, miscarriage rates and the transfer of de novo mutations to offspring. Semen samples of infertile men are known to be deficient in several key antioxidants relative to their fertile counterparts. Antioxidants alone or in combination have demonstrated limited efficacy against sperm oxidative stress and DNA damage in numerous human clinical trials, however these studies have not been definitive and an optimum combination has remained elusive. The efficacy of the antioxidant formulation was evaluated in two well-established mouse models of oxidative stress, scrotal heating and Gpx5 knockout (KO) mice, (n = 12 per experimental group), by two independent laboratories. Mice were provided the antioxidant product in their drinking water for 2-8 weeks and compared with control groups for sperm DNA damage and pregnancy rates. In the Gpx5 KO model, oxidative DNA damage was monitored in spermatozoa by immunocytochemical detection of 8-hydroxy-2'-deoxyguanosine (8OHdG). In the scrotal heat stress model, male fertility was tested by partnering with three females for 5 days. The percentage of pregnant females, number of vaginal plugs, resorptions per litter, and litter size were recorded. Using immunocytochemical detection of 8OHdG as a biomarker of DNA oxidation, analysis of control mice revealed that around 30% of the sperm population was positively stained. This level increased to about 60% in transgenic mice deficient in the antioxidant enzyme, GPX5. Our results indicate that an 8 week pretreatment of Gpx5 KO mice with the antioxidant formulation provided complete protection of sperm DNA against oxidative damage. In mouse models of scrotal heat stress, only 35% (19/54) of female mice became pregnant resulting in 169 fetuses with 18% fetal resorption (30/169). This is in contrast to the antioxidant pretreated group where 74% (42/57) of female mice became pregnant, resulting in 427 fetuses with 9% fetal resorption (38/427). In both animal models the protection provided by the novel antioxidant was statistically significant (P < 0.01 for the reduction of 8OHdG in the spermatozoa of Gpx5 KO mice and P < 0.05 for increase in fertility in the scrotal heat stress model). It was not possible to determine the exact level of antioxidant consumption for each mouse during the treatment period. Recent clinical studies confirm moderate to severe sperm DNA damage in about 60% of all men visiting IVF centers and in about 80% of men diagnosed with idiopathic male infertility. Our results, if confirmed in humans, will impact clinical fertility practice because they support the concept of using an efficacious antioxidant supplementation as a preconception therapy, in order to optimize fertilization rates, help to maintain a healthy pregnancy and limit the mutational load carried by children. The study was funded by the Clermont Université and the University of Madrid. P.G. is the Managing Director of CellOxess LLC, which has a commercial interest in the detection and resolution of oxidative stress. A.M. and A.P. are employees of CellOxess, LLC. J.R.D., A.G.-A. and R.J.A. are honorary members of the CellOxess advisory board.

Changes in Levels of Seminal Nitric Oxide Synthase, Macrophage Migration Inhibitory Factor, Sperm DNA Integrity and Caspase-3 in Fertile Men after Scrotal Heat Stress.

BackgroundThis study observes changes in levels of seminal nitric oxide (NO), nitric oxide synthase (NOS), macrophage migration inhibitory factor (MIF), sperm DNA integrity, chromatin condensation and Caspase-3in adult healthy men after scrotal heat stress (SHS).MethodsExposure of the scrotum of 25 healthy male volunteers locally at 40–43°C SHS belt warming 40 min each day for successive 2 d per week. The course of SHS was continuously 3 months. Routine semen analysis, hypo-osmotic swelling (HOS) test, Aniline blue (AB) staining, HOS/AB and terminal deoxynucleotidyl transferase-mediated d UDP nick-end labeling (TUNEL) were carried out before, during and after SHS. Seminal NO and NOS contents were determined by nitrate reduction method. The activated Caspase-3 levels of spermatozoa and MIF in seminal plasma were measured by the enzyme-linked immunosorbent assay (ELISA) method. Statistical significance between mean values was determined using statistical ANOVA tests.ResultsThe mean parameters of sperm concentration, motile and progressive motile sperm and normal morphological sperm were significantly decreased in groups during SHS 1, 2 and 3 months compared with those in groups of pre-SHS (P<0.001). Statistically significant differences of sperm DNA fragmentation, normal sperm membrane, and Caspase-3 activity as well as the level of NO, NOS and MIF in semen were observed between the groups before SHS and after SHS 3 months and the groups during SHS 1, 2 and 3 months (P<0.001). After three months of the SHS, various parameters recovered to the level before SHS. WBC in semen showed a positively significant correlation with the levels of NO, NOS, MIF and Caspase-3 activity. The percentage of abnormal sperm by using the test of HOS showed a positively significant correlation with that of HOS/AB.ConclusionsThe continuously constant SHS can impact the semen quality and sperm DNA and chromatin, which may be contributed to the high level of NO, NOS, MIF and Caspase-3 by SHS.

Evaluation of a mouse model for scrotal heat stress

Evaluation of a mouse model for scrotal heat stress

Genetic factors derived from the MRL/MpJ mouse function to maintain the integrity of spermatogenesis after heat exposure.

MRL/MpJ mice possess highly heat-shock-resistant spermatocytes (HRS) in comparison with C57BL/6 mice. This resistance depends on the MRL/MpJ-type loci at the 81cM region of Chromosome (Chr) 1 and the 40cM region of Chr 11. To evaluate the functions of these loci in detail, we examined the histopathological changes resulting from experimental cryptorchidism or transient scrotal heat stress (SHS) in the testes of C57BL/6-based congenic strains (B6.MRLc1, B6.MRLc11, and B6.MRLc1c11) carrying the MRL/MpJ-derived loci responsible for HRS. Among cryptorchid testes from congenic strains, those in B6.MRLc1c11 mice showed the highest heat resistance, indicating that the genetic interactions between MRL/MpJ-derived HRS loci on Chrs 1 and 11 may be important for maintaining spermatogenesis under continuous testicular hyperthermia. In contrast, immediately after SHS induction, germ cell loss via apoptosis was inhibited in B6.MRLc11 and B6.MRLc1c11 mice, similar to that in MRL/MpJ mice. However, this HRS phenotype was not observed in C57BL/6 or B6.MRLc1 mice after SHS induction. Furthermore, testicular calcification owing to long-term damage by SHS induction was inhibited in all congenic strains in comparison with that in C57BL/6 mice, indicating that each MRL/MpJ-derived locus on Chrs 1 and 11 acted independently to facilitate the recovery of heat-induced testicular damage by inhibiting calcification. B6.MRLc11 and B6.MRLc1c11 mice showed greater recovery in spermatogenesis than B6.MRLc1 mice 60days after SHS induction. Therefore, the MRL/MpJ-derived HRS locus on Chr 11 might play an important role in recovery from heat stress damage. On the basis of these results, we concluded that MRL/MpJ-derived loci on Chrs 1 and 11 cooperatively or independently regulate testicular heat sensitivity depending on the various heat stresses.

Curcumin dose-dependently improves spermatogenic disorders induced by scrotal heat stress in mice.

Approximately 20% of couples worldwide are infertile and about half of these couples have male infertility. Therefore, it is important to develop effective strategies for preventing male infertility. In this study, we examined the effects and regulatory mechanisms of curcumin, an active ingredient in the traditional herbal treatment derived from the dietary spice turmeric (Curcuma longa), on exogenous scrotal heat stress-induced testicular injuries in mice. Adult mice were orally administered three different doses of curcumin (20, 40, or 80 mg per kg per day) for 14 consecutive days and then subjected to transient scrotal heat stress at 43 °C for 20 min on day 7. The testes and blood of the mice were collected on day 14. Mice exposed to heat stress showed low testicular weight, severe vacuolization of seminiferous tubules followed by loss of spermatogenic cells, and the appearance of multinucleated giant cells and degenerative Leydig cells. In addition, great changes in oxidative stress (lipid peroxidation, superoxide dismutase (SOD) activity, cytoplasmic SOD, mitochondrial SOD, and phospholipid hydroperoxide glutathione peroxidase mRNAs), apoptosis (B-cell lymphoma-extra large and caspase 3 mRNAs), heat shock reaction (heat shock transcription factor-1 and transforming growth factor-β1 mRNAs) and androgen biosynthesis (testosterone concentration and 3β-hydroxysteroid dehydrogenase mRNA) were observed. However, all these testicular injuries induced by the scrotal hyperthermia were significantly improved by curcumin treatment (20, 40 and 80 mg kg(-1)) in a dose-dependent manner via its antioxidative, anti-apoptotic and androgen synthesis effects, indicating that it has the potential to prevent male infertility.

Effect of a Traditional Herbal Prescription, Kyung-Ok-Ko, on Male Mouse Spermatogenic Ability after Heat-Induced Damage.

Kyung-Ok-Ko (KOK), a well-known traditional Korean medicinal formula, has long been used to invigorate the essential qi. This use of KOK may be associated with reproductive ability as a more modern concept. The protective effect of KOK was evaluated against deterioration of testicular function induced by heat exposure in male mice. Male fertility was disrupted by scrotal heat stress at 43°C for 5 weeks. KOK (0.25, 0.50, and 2.00 g/kg/day) was administered orally at 3 h after the stress. To evaluate the protective effect of KOK, body weight, testicular weight, sperm count, sperm motility, and histopathological changes in the testes were evaluated. KOK-treated mice significantly recovered their general health, as evidenced by body weight. KOK-treated mice also showed significantly higher testes weights, sperm counts, and sperm motility than did the heat stress group. KOK-treated mice significantly recovered the morphological appearance of the seminiferous tubules and seminiferous epithelium. Furthermore, KOK-treated mice significantly increased antioxidant enzyme activities and reduced the protein expressions of apoptosis in the testes. KOK significantly protects against heat-induced damage to testicular function in male mice by inhibiting oxidative stress and apoptosis, indicating that KOK may be an effective agent for treatment of heat-induced male infertility.

The Onset of Heat-Induced Testicular Calcification in Mice: Involvement of the Telomeric Locus on Chromosome 1

Spermatocytes of MRL/MpJ mice are more heat resistant than those of C57BL/6 mice in experimental cryptorchidism. This phenotype depends in part on the locus at the 81-cM region of MRL/MpJ-type chromosome 1 (Chr1). To evaluate the function of this locus, we examined pathological changes in mouse testes resulting from transient scrotal heat stress. Immediately after scrotal heat stress, meiosis progression and blood-testis barrier integrity were preserved in MRL/MpJ but not in C57BL/6 mice, nor in a C57BL/6-based congenic strain carrying the MRL/MpJ-derived Chr1 locus (B6.MRLc1). Testicular damage was severe in the weeks after scrotal heat stress in all three strains; however, testicular calcification was observed only in C57BL/6 and MRL/MpJ mice (initially as nanocrystals in mitochondria of degenerating germ cells). In testes, expression of gremlin 2, a bone morphogenetic protein antagonist encoded on Chr1, was markedly higher in B6.MRLc1 than in C57BL/6 or MRL/MpJ mice. Furthermore, gremlin-2 and bone morphogenetic protein 2 mRNA levels in heated testes correlated negatively and positively, respectively, with calcification. Thus, although the MRL/MpJ-derived locus on Chr1 may play a pivotal role in recovery from heat-induced testicular damage, especially via inhibition of calcification, MRL/MpJ mice have a precipitating factor for testicular calcification and heat shock-resistant factors that reside outside the 81-cM region of Chr1.

Scrotal heat stress causes a transient alteration in tight junctions and induction of TGF-β expression

Specialized junctions, which occur at sites of Sertoli-Sertoli and Sertoli-germ cell contact of seminiferous epithelium, play pivotal roles in spermatogenesis. Slight increase in scrotal temperature can induce oligospermia or azoospermia via increasing germ cell apoptosis. In this study, we demonstrated that the expression of tight junction (TJ) components, such as occludin, claudin-3 and zonula occludens-1 (ZO-1), was reduced 24-48h after a single mild scrotal heat exposure (43°C for 30min), whereas mRNA levels of claudin-11 were increased. Moreover, the protein localization of occludin and ZO-1 was lost from the blood-testis barrier (BTB) site, whereas claudin-11 immunostaining became diffuse and cytoplasmic 2days following heat exposure. Electron microscopic analysis showed that 2days after the heat treatment, the intercellular space between the two adjacent Sertoli cells was expanded, coupled with defragmentation of actin bundles and the endoplasmic reticulum. In addition, the TJ permeability increased significantly 2days after the heat exposure and recovered approximately 10days later. Heat-induced reversible BTB disruption was associated with a transient induction of transforming growth factor (TGF)-β2, -3 and p38 mitogen-activated protein kinase activation. However, the TGF-β antagonist only partially prevented the heat-induced BTB disruption. In conclusion, the expression of TJ-associated molecules and BTB were reversibly perturbed after mild testicular hyperthermia, and the induction of TGF-β expression may be partially involved in heat-induced BTB damage.

A single, mild, transient scrotal heat stress causes hypoxia and oxidative stress in mouse testes, which induces germ cell death.

Spermatogenesis is a temperature-dependent process, and increases in scrotal temperature can disrupt its progression. We previously showed that heat stress causes DNA damage in germ cells, an increase in germ cell death (as seen on TUNEL staining), and subfertility. The present study evaluated the stress response in mouse testes following a single mild transient scrotal heat exposure (40 degrees C or 42 degrees C for 30 min). We investigated markers of three types of stress response, namely, hypoxia, oxidative stress, and apoptosis. Heat stress caused an increase in expression of hypoxia-inducible factor 1 alpha (Hif1a) mRNA expression and translocation of HIF1A protein to the germ cell nucleus, consistent with hypoxic stress. Increased expression of heme oxygenase 1 (Hmox1) and the antioxidant enzymes glutathione peroxidase 1 (GPX1) and glutathione S-transferase alpha (GSTA) was consistent with a robust oxidative stress response. Germ cell death was associated with an increase in expression of the effector caspase cleaved caspase 3 and a decrease in expression of the protein inhibitor of caspase-activated DNase (ICAD). Reduced expression of ICAD contributes to increased activity of caspase-activated DNase and is consistent with the increased rates of DNA fragmentation that have been detected previously using TUNEL staining. These studies confirmed that transient mild testicular hyperthermia results in temperature-dependent germ cell death and demonstrated that elevated temperature results in a complex stress response, including induction of genes associated with oxidative stress and hypoxia.

A single, mild, transient scrotal heat stress causes DNA damage, subfertility and impairs formation of blastocysts in mice

Infertility represents a major clinical problem and 50% of cases are attributable to the male partner. Testicular function is temperature dependent, and in both man and mouse the position of the testes in the scrotum ensures that they are kept at between 2 and 8 degrees C below core body temperature. We used a mouse model to investigate the impact of a single, transient, mild, scrotal heat stress (38, 40 or 42 degrees C for 30 min) on testicular function, sperm DNA integrity and embryo survival. We detected temperature-dependent changes in testicular architecture, number of apoptotic cells and a significant reduction in testis weight 7 and 14 days after heat stress at 42 degrees C. We report for the first time that DNA strand breaks (gamma-H2AX-positive foci) were present in spermatocytes recovered from testes subjected to 40 or 42 degrees C. Fertility of heat-stressed males was tested 23-28 d after treatment (sperm at this time would have been spermatocytes at time of heating). Paternal heat stress at 42 degrees C resulted in reduced pregnancy rate, placental weight and litter size; pregnancies from the 40 degrees C group had increased resorptions at e14.5. Abnormalities in embryonic development were detected at e3.5 and in vitro fertilisation with sperm recovered 16 h or 23 d after scrotal stress at 42 degrees C revealed a block in development between the 4-cell and blastocyst stages. This study has provided evidence of temperature-dependent effects on germ cell DNA integrity and highlighted the importance of an intact paternal genome for normal embryo development.

Sperm DNA Integrity From Sperm to Egg

Sperm DNA Integrity From Sperm to Egg

Scrotal heat stress effects on sperm viability, sperm DNA integrity, and the offspring sex ratio in mice

Evidence exists to suggest detrimental effects of heat stress on male fertility. This study was designed to assess the effects of scrotal heat stress on mature and developing sperm in a mouse model. After receiving shock heat treatment (42 degrees C for 30 min), mature spermatozoa were recovered from the epididymis hours (6) or Days (7, 14, 21, 28, 60) later, to determine the variables: number of spermatozoa, sperm viability, motility and progressive motility, sperm DNA integrity as established by the TUNEL method, embryo implantation rate, and sex ratio of the fetuses conceived using the heat-exposed spermatozoa. Our results indicate that transient mild heat treatment does not affect in the same way the different types of male germ cells. Spermatocytes present within the testis at the time of heat stress resulted into a lower concentration of spermatozoa with reduced viability and low motility. Even though, DNA integrity of spermatozoa resulting from spermatocytes was also compromised by heat stress, the higher degree of DNA damage was found among spermatozoa resulting from spermatids present within the testis at the time of heat stress. At last, heat shock effect on spermatozoa present in the epididymis at the time of thermal stress resulted into a sex ratio distortion. These findings point to a higher sensitivity of spermatocytes to heat exposure and also suggest a different response of X and Y chromosome-bearing spermatozoa to heat stress that warrants further investigation.

OC6 Scrotal Heat Stress in Mice Affects Viability and DNA Integrity of Sperm, and Sex Ratio of the Offspring

Heat stress causes infertility in farm animals and represents a major source of economic loss. The objective of this work was to examine the impact of scrotal heat stress (42°C for 30 min) on viability of spermatozoa, integrity of their DNA, and the in vivo reproductive performance of CD1 mice. These parameters were assessed in the spermatozoa recovered from epididymis at 0, 7, 14, 21 and 28 days after heat treatment. Heat stressed males were mated with CD1 females, and the number and the sex ratio of Day14 fetuses was analyzed by PCR. Heat stress increases the percentage of TUNEL positive sperm present in the epididymis at the time of heating (Day 0 and 7). Also the damage was significantly higher than the control at Day 14, 21 and 28 indicating that some germ cells, which were present within the testis at the time of heat stress, were not removed by apoptosis and completed their development producing sperm with damaged DNA. Heat stress also affects viability and motility at Day 14, 21 and 28. When the sex ratio of the offspring of the males mated the Day 0 or 7 after the heat stress was assessed, we observed that the percentage of males was significantly lower than expected (p &lt; 0.05). No differences were observed in the sex ratio of the offspring of the other groups. In conclusion, these data demonstrate that the increase in epididymal temperature can compromise the DNA integrity of spermatozoa and the sex ratio of offspring generated with this sperm, and suggest that spermatocytes are most vulnerable than spermatids to DNA damage induced by heat stress.

Impact of a mild scrotal heat stress on DNA integrity in murine spermatozoa

An increase in scrotal temperature can lead to the production of poor quality spermatozoa and infertility. In the present study we have used mice to examine the impact of mild, scrotal heat stress (42 degrees C for 30 min) on numbers of spermatozoa as well as on the integrity of their DNA. Spermatozoa recovered from the epididymides hours (1 to 24) or days (7 to 32) after treatment were analysed using COMET and sperm chromatin structure (SCSA) assays. The treatment induced a stress response in both the testis and the epididymis that was associated with reduced expression of the cold inducible RNA binding protein (Cirp) and an increase in germ cell apoptosis (Apotag positive cells). Although spermatozoa present in the epididymis at the time of heating contained correctly packaged DNA, its integrity was compromised by heat stress. In addition, although some germ cells, which were present within the testis at the time of heat stress, were removed by apoptosis, many germ cells completed their development and were recovered as motile spermatozoa with damaged DNA. In conclusion, these data demonstrate that scrotal heat stress can compromise the DNA integrity of spermatozoa and this may have clinical implications for patients undergoing IVF and intra-cytoplasmic sperm injection (ICSI).

Improvement of semen quality by nocturnal scrotal cooling and moderate behavioural change to reduce genital heat stress in men with oligoasthenoteratozoospermia

A questionnaire assessing factors that might cause an increase in scrotal temperature was completed by patients with reproducible oligoasthenoteratozoospermia of idiopathic nature or caused by varicocele. Evaluation by means of a grading scale revealed increased scrotal heat stress in oligoasthenoteratozoospermic patients compared with normozoospermic men (P < 0.01). In addition, long-term determination of 24 h scrotal temperature profiles showed that compared with semen donors, oligoasthenoteratozoospermic patients frequently had scrotal temperatures above 35.5 degrees C despite the same environmental temperatures (P < 0.05). In 88% of cases, maximum scrotal temperatures were measured during rest or sleep phases, whereas minimum values were recorded during physical activity or frequent change of position. Nocturnal scrotal cooling by means of an air stream resulted in a decrease in scrotal temperature of approximately 1 degrees C. Furthermore, a highly significant increase in sperm concentration (P < 0.0001) and total sperm output (P < 0.0001) was achieved after nocturnal scrotal cooling for 12 weeks together with a moderate decrease in factors leading to genital heat stress. A significant improvement in sperm motility (P < 0.05) and sperm morphology (P < 0.05) was also observed, but this improvement was markedly less pronounced than the changes in sperm concentration. This study shows the importance of genital heat stress as a cofactor in fertility impairment in men and indicates nocturnal scrotal cooling as a therapeutic option.

Improvement of semen quality by nocturnal scrotal cooling and moderate behavioural change to reduce genital heat stress in men with oligoasthenoteratozoospermia.

A questionnaire assessing factors that might cause an increase in scrotal temperature was completed by patients with reproducible oligoasthenoteratozoospermia of idiopathic nature or caused by varicocele. Evaluation by means of a grading scale revealed increased scrotal heat stress in oligoasthenoteratozoospermic patients compared with normozoospermic men (P < 0.01). In addition, long-term determination of 24 h scrotal temperature profiles showed that compared with semen donors, oligoasthenoteratozoospermic patients frequently had scrotal temperatures above 35.5 degrees C despite the same environmental temperatures (P < 0.05). In 88% of cases, maximum scrotal temperatures were measured during rest or sleep phases, whereas minimum values were recorded during physical activity or frequent change of position. Nocturnal scrotal cooling by means of an air stream resulted in a decrease in scrotal temperature of approximately 1 degrees C. Furthermore, a highly significant increase in sperm concentration (P < 0.0001) and total sperm output (P < 0.0001) was achieved after nocturnal scrotal cooling for 12 weeks together with a moderate decrease in factors leading to genital heat stress. A significant improvement in sperm motility (P < 0.05) and sperm morphology (P < 0.05) was also observed, but this improvement was markedly less pronounced than the changes in sperm concentration. This study shows the importance of genital heat stress as a cofactor in fertility impairment in men and indicates nocturnal scrotal cooling as a therapeutic option.

Scrotal heat stress induces altered sperm chromatin structure associated with a decrease in protamine disulfide bonding in the stallion.

A variety of testicular insults can induce changes in the structure of spermatozoal chromatin, resulting in spermatozoal DNA that is more susceptible to acid-induced denaturation. The degree of change in the DNA can be measured using the sperm chromatin structure assay (SCSA). The SCSA measures the relative amounts of single- and double-stranded DNA after staining with the metachromatic dye, acridine orange. Here we used a stallion model (n = 4) to study the effects of scrotal heat stress on spermatozoal DNA. This model was created by insulating stallion testes for 48 h and collecting sperm daily thereafter for 60 days. Changes in the SCSA were then correlated with protamine disulfide content and protamine types and levels. Results of the SCSA indicated that the susceptibility of spermatozoal DNA to denaturation was dependent on the spermatogenic cell stage that the ejaculated sperm was in at the time of the heat stress. Spermatozoa with altered DNA had a decrease in the extent of disulfide bonding that was associated with an increase in the susceptibility of DNA to denaturation. However, there were no detectable changes in either the protamine type or level. Thus, in this model, decreased disulfide bonding is associated with an increased susceptibility of spermatozoal DNA to denaturation in the absence of protamine changes.