Correlation between the DNA fragmentation index (DFI) and sperm morphology of infertile patients.

PURPOSE: To evaluate the correlation between the DNA Fragmentation Index (DFI) and sperm morphology in patients undergoing ICSI, as a predictive parameter in reproductive outcomes. METHODS: A retrospective study was conducted on 125 infertile patients enrolled in a fertility clinic. Seminal characteristics were measured following the WHO guidelines (2010) for the examination of the seminal fluid. After collecting motile sperm population by pellet swim up, DFI was calculated and simultaneously associated with sperm morphology using in situ TUNEL assay and an image analyzer software in at least 250 spermatozoa for each patient. RESULTS: All subjects were divided into two groups according to a cutoff established, by choice, of the sperm DFI (15%): group A (< 15%) consisting of 65 patients and group B (≥ 15%) of 60 patients. Data were analyzed using non-parametric statistical methods. The results demonstrate that there is no statistical difference between the two groups in seminal characteristics. The collective data show a high significant correlation, suggesting that spermatozoa with abnormal morphology are the best candidates to contain DNA damage (p < 0.001). Also, when group A is compared with group B, an increased percentage of morphologically normal spermatozoa with fragmented DNA was observed in patients, with DFI values ≥ 15% (p < 0.001). CONCLUSION: These results are aimed at providing an exact value of DFI in morphologically normal spermatozoa, which will be helpful to the embryologist in evaluating the risk of transferring, during the ICSI procedure, a spermatozoon whit normal morphology but fragmented DNA.

Evaluation of the relationship between the detectability of seminal stains on laundered fabric and stain age.

In some cases of sexual assault that are not reported to judicial authorities within a certain time, it is important to detect and identify seminal stains on laundered fabrics. In this study, we aimed to reveal the effect of the time from staining to laundering on the detectability and identification of seminal stains on laundered clothes. A total of 180 pieces of fabric (four different colours and five different types) were stained with seminal fluids, and three different lag times (12 hours, 1 week and 1 month) from staining to laundering were used. Three different laundering protocols were applied to these fabrics after staining. The built-in camera of the Mobile Multispectral UV-VIS-IR Imaging System® was used to take photos (1260 in total) of the stains with seven different wavelength and filter options, and the obtained images were evaluated. The Seratec® PSA Semiquant test was used to analyse the presence of prostate-specific antigen (PSA) in the seminal stains laundered after different lag times. We observed that in examining with the forensic light source (FLS) system, the time from staining to laundering affected the detectability of seminal stains on pieces of cloth. The best fluorescence was obtained in the examination of semen-stained fabric with FLS, particularly when the fabric was not laundered for one month after staining. On the other hand, the time from staining to laundering had a more limited effect on PSA test positivity than on the results of the examination with FLS.

Classification of the sperm chromatin compaction alterations in bulls (Bos taurus) and its correlation with the efficiency of the in vitro production of embryos

This paper proposes to classify the sperm chromatin compaction alterations in bulls, according to the affected area location and its objective is evaluating the correlation of the intensity, the heterogeneity and these kinds of chromatin decompaction with the rates of cleavage and the formation of blastocysts of in vitro production of embryos (IVPE). It was used several subfertile animals sperm samples, which were evaluated using the toluidine blue staining and computer image analysis, making possible the categorization of the chromatin decompaction according to their location. The percentages of chromatin decompaction and heterogeneity were also evaluated. IVPEs were done and the rates of cleavage and of blastocysts were correlated with the chromatin characteristics. It made possible the classification of the chromatin decompaction according to the head affected part in at least four types: base decompaction, basal half decompaction, central axis decompaction, total decompaction. Based on the correlation, it can be implicated that each type of classification has different influences on the bull fertility. It made possible understanding that sperms amount with 5% or more of chromatin decompaction intensity interferes in the bull fertility and this condition can be featured as an uncompensable defect, while the heterogeneity of chromatin is not an important factor in the IVPE results.

Pattern of varicocele vein blood gases in patients undergoing microsurgical Varicocelectomy.

BACKGROUND: Varicocele is known to be associated with infertility and sperm disorders. The exact cause of this ailment is not fully understood. There are limited numbers of studies where venous blood gases (VBGs) of varicocele veins were determined with conflicting results. Therefore, we have investigated the pattern of VBGs in both internal spermatic and external spermatic varicocele veins and correlation with semen quality parameters in infertile individuals who underwent left microsurgical varicocelectomy. METHODS: Patients (n = 27) undergoing left microsurgical varicocelectomy at a tertiary care hospital, were included in the study. Before surgery, semen parameters and scrotal color Doppler ultrasonography was performed. During surgery, blood sample was drawn from varicocele veins (internal spermatic and external spermatic veins) and a peripheral arm vein of the same patient as a control. The VBGs of all veins under study were estimated and compared with each other. The VBGs were also correlated with various semen quality parameters. Data, expressed as Mean ± SD, regarding VBGs in three veins were analyzed using one-way ANOVA. The correlation between VBGs and semen quality parameters was determined using Pearson's correlation. Differences were considered significant at p < 0.05. RESULTS: The pH was found to be higher (p < 0.01) in the internal spermatic vein compared with the external spermatic and the peripheral veins. Partial pressure of oxygen (pO2) and oxygen saturation (sO2) were higher (p < 0.01) in the internal spermatic vein compared with the peripheral vein. However, concentration of bicarbonate (HCO3) was lower (p < 0.01) in both veins compared with the peripheral vein. Partial pressure of carbon dioxide (pCO2) was also lower (p < 0.01) in the varicocele veins compared with the control vein. CONCLUSION: The internal spermatic veins had higher pH and oxygen tension, but lower HCO3 and pCO2 levels compared with the control peripheral veins. External spermatic veins had lower pCO2 and HCO3 but other VBGs were similar to the peripheral veins. The shift of VBGs of internal spermatic vein toward arterial blood pattern may be a missing link to understand the pathophysiology of varicocele.

A Novel Method for Investigating the Role of Reflux Pattern in Color Doppler Ultrasound for Grading of Varicocele.

Varicocele is the most common correctable cause of infertility. Color Doppler Ultrasound (CDUS) has a sensitivity of 97% and specificity of 94% for diagnosing this condition. This study aimed to propose a new pattern of scrotal Doppler for predicting the severity of varicocele. An observational study was conducted from January 2016 to January 2017 on 120 testes units in 60 patients. Scrotal CDUS and semen analysis were done in all participants. Patients were evaluated for reflux pattern, pampiniform venous plexus diameter, and venous reflux time. The ultrasonography parameters and semen analysis data were compared to assess the correlations between the results. The reflux pattern and vein diameters had a significant correlation. Also, a significant correlation was detected between the reflux pattern and reflux time. There was a significant correlation between the reflux pattern and two parameters of semen analysis namely sperm count and its motility. In conclusion, the reflux pattern classification suggested in this study can be used as a useful predictor of varicocele severity and sperm parameters in patients with varicocele.

Multi-Target Tracking of Human Spermatozoa in Phase-Contrast Microscopy Image Sequences using a Hybrid Dynamic Bayesian Network.

Male infertility is mostly related to semen and spermatozoa, and any diagnosis or treatment requires the investigation of the motility patterns of spermatozoa. The movements of spermatozoa are fast and involve collision and occlusion with each other. In order to extract the motility patterns of spermatozoa, multi-target tracking (MTT) of spermatozoa is necessary. One of the most important steps of MTT is data association, in which the newly arrived observations are used to update the previous tracks. Dynamic Bayesian network (DBN) is a powerful tool for modeling and solving various types of problems such as tracking and classification. There can also be a hybrid-DBN (HDBN), in which both continuous and discrete nodes are present. HDBN has a suitable structure for modeling problems that have both discrete and continuous parameters like MTT. In this research, the data association for MTT of human spermatozoa has been studied. The proposed algorithm was tested over hundreds of manually extracted spermatozoa tracks and evaluated using several standard measures. The superior results of the proposed algorithm in comparison to the other well-known algorithms, show that it could be considered as an improved alternative to traditional computer assisted sperm analysis (CASA) algorithms.

Diffusion-Weighted and Magnetization Transfer Imaging in Testicular Spermatogenic Function Evaluation: Preliminary Results.

PURPOSE: To assess the use of functional magnetic resonance imaging (MRI), including diffusion-weighted MRI (DWI) and magnetization transfer MRI (MTI) in evaluating male infertility. MATERIALS AND METHODS: Sixteen men with testicular spermatogenesis hypofunction confirmed by percutaneous testis biopsy and 31 volunteers (control group B, age range: 20-40 years) with normal semen analysis including younger (By, n = 15, age range: 20-30 years) and older (Bo, n = 16, age range: 31-40 years) men underwent pelvic 3T MRI, including DWI and MTI. Apparent diffusion coefficient (ADC) and magnetization transfer ratio (MTR) were compared. RESULTS: The ADCs in 32 testes of 16 patients (0.497 ± 0.037 × 10-3 mm2 /s) were significantly (P < 0.001) higher than that of control group B (0.460 ± 0.031 × 10-3 mm2 /s), group By (0.453 ± 0.018 × 10-3 mm2 /s), and group Bo (0.461 ± 0.034 × 10-3 mm2 /s), whereas the MTRs were significantly lower than that of group B (16.14 ± 4.20), group By (17.88 ± 2.00), and group Bo (15.09 ± 4.28) (P < 0.001). CONCLUSION: Functional MRI, including DWI and MTI, appears promising for evaluating male infertility with higher ADC and lower MTR in testicular spermatogenesis hypofunction. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2018;47:186-190.

Large area imaging of forensic evidence with MA-XRF.

This study introduces the use of macroscopic X-ray fluorescence (MA-XRF) for the detection, classification and imaging of forensic traces over large object areas such as entire pieces of clothing and wall paneling. MA-XRF was sufficiently sensitive and selective to detect human biological traces like blood, semen, saliva, sweat and urine on fabric on the basis of Fe, Zn, K, Cl and Ca elemental signatures. With MA-XRF a new chemical contrast is introduced for human stain detection and this can provide a valuable alternative when the evidence item is challenging for conventional techniques. MA-XRF was also successfully employed for the chemical imaging and classification of gunshot residues (GSR). The full and non-invasive elemental mapping (Pb, Ba, Sr, K and Cl) of intact pieces of clothing allows for a detailed shooting incident reconstruction linking firearms and ammunition to point of impact and providing information on the shooting angle. In high resolution mode MA-XRF can even be used to provide information on the shooting order of different ammunition types. Finally, by using the surface penetration of X-rays we demonstrate that the lead signature of a bullet impact can be easily detected even if covered by multiple layers of wall paint or human blood.

Role of Preoperative Testicular Shear Wave Elastography in Predicting Improvement of Semen Parameters After Varicocelectomy for Male Patients With Primary Infertility.

OBJECTIVE: To evaluate the role of preoperative testicular shear wave elastography (SWE) in the prediction of improvement of semen analysis parameters after subinguinal microsurgical varicocele ligation in patients with primary infertility and clinically detectable varicocele. PATIENTS AND METHODS: Testicular SWE before the surgical intervention was done. Forty-eight patients were scheduled for subinguinal microsurgical varicocele ligation as a treatment option. Computer-assisted semen analysis was repeated 6 months after the intervention. RESULTS: At a cutoff value of 4.5 kPa, the stiffness index had a sensitivity of 86.4% and a specificity of 84.2% for semen parameter improvement after varicocelectomy. Correlation between different parameters of semen analysis and SWE showed a statistically significant negative correlation between SWE stiffness index and both sperm count (million/mL) and total motility. On the other hand, a nonsignificant negative correlation was found between SWE stiffness index and percentage of normal form. CONCLUSION: Testicular SWE is a good test that can be used in the assessment of male infertility with clinically detectable varicocele, and its results may predict semen parameter improvement after varicocelectomy. Further studies on a larger number of patients are needed to verify our results.

Effect of prolonged freezing of semen on exosome recovery and biologic activity.

Exosomes are important vehicles of intercellular communication that shape host responses to physiologic, tumorigenic, and pathogenic conditions. The composition and function of exosomes are dynamic and depends on the state and condition of the cellular source. In prior work, we found that semen exosomes (SE) from healthy donors who do not use illicit drugs potently inhibit HIV-1. Following semen donation, specimens are either used immediately or frozen for use at a later time. It has been shown that short-term freezing of semen has no effect on SE-mediated HIV-1 inhibition. However, the effect of illicit drugs and prolonged freezing on SE bioactivity is unknown. Here, we show preservation of SE physical properties, (morphology, concentration, intensity/size) irrespective of illicit drug use or duration of semen freezing. Interestingly, illicit drugs and prolonged freezing decreased the levels of SE-bound CD63/CD9 and acetylcholinesterase activity respectively. Furthermore, we show differential effects of illicit drug use and prolonged freezing on SE-mediated HIV-1 inhibition. Our results highlight the importance of the source of SE and condition of semen storage on SE content and function. In-depth evaluation of donor drug-use and duration of semen storage on SE cargo and bioactivity will advance our understanding of SE composition and function.

Giant panda (Ailuropoda melanoleuca) sperm morphometry and function after repeated freezing and thawing.

This work examines the effects of subsequent cycles of freezing-thawing on giant panda (Ailuropoda melanoleuca) sperm morphometry and function, and assesses whether density-gradient centrifugation (DGC) can increase the number of freezing-thawing cycles this sperm can withstand. A sperm sample was collected by electroejaculation from a mature giant panda and subjected to five freezing-thawing cycles. Although repeated freezing-thawing negatively affected (P < 0.05) sperm motility and membrane integrity, in both nonselected and DCG-selected sperm samples, >60% of the sperm cells in both treatments showed acrosome integrity even after the fifth freezing cycle. In fresh semen, the sperm head length was 4.7 µm, the head width 3.6 µm, area 14.3 µm(2) and perimeter length 14.1 µm. The present results suggest that giant panda sperm trends to be resistant to repeated freezing-thawing, even without DGC selection.

Semen apoptotic M540 body levels correlate with testis abnormalities: a study in a cohort of infertile subjects.

STUDY QUESTION: What are the associations between semen apoptotic M540 bodies and other parameters of semen quality and sonographic alterations of the male genital tract in a cohort of infertile subjects? SUMMARY ANSWER: In infertile subjects, semen M450 bodies are highly correlated with ultrasound and clinical signs of testis abnormalities but not with alterations of other parts of the male genital tract, suggesting a testicular origin of M540 bodies. WHAT IS KNOWN ALREADY: We have reported the presence in semen of round anucleate elements, named 'M540 bodies', resembling apoptotic bodies as they contain several apoptotic markers. STUDY DESIGN AND SIZE: A consecutive series of 130 males with couple infertility were evaluated, during the same day session, for clinical, scrotal and transrectal color-Doppler ultrasound characteristics, and hormonal and semen parameters, including interleukin 8 (sIL-8) and M540 body levels. PARTICIPANTS/MATERIALS, SETTING METHODS: Semen parameters were analyzed by WHO recommended procedures. CDU was performed using the ultrasonographic console Hitachi H21. sIL-8 and serum hormones were evaluated by ELISA methods. MAIN RESULTS AND THE ROLE OF CHANCE: The average percentage value of M540 bodies was 24.6 ± 18.3. After adjusting for possible confounders (age, waist, calculated free testosterone and smoking habit), M450 body levels negatively correlated with sperm number/ejaculate, progressive motility, normal morphology and sIL-8 levels (adj.r = -0.455, P < 0.0001; adj.r = -0.464, P < 0.0001; adj.r = -0.430, P < 0.001; adj.r = -0.236, P < 0.05, respectively). In a subset of patients with a history of cryptorchidism (n = 8), M540 bodies were higher than in non-cryptorchid men (40.5 ± 14.8 versus 23.6 ± 18.2%; P < 0.02). A negative correlation was found between M540 and ultrasound testis volume (adj.r = -0.241, P < 0.05), whereas a positive association was found with testis inhomogeneity [HR = 1.06 (1.02-1.09); P = 0.002], hypoechogenicity [HR = 1.05 (1.01-1.08); P < 0.02] and FSH levels (adj.r = 0.309, P < 0.01). No relationships were found with CDU characteristic of the prostate, seminal vesicles, epididymis and vas deferens. In a multivariate model, testis inhomogeneity and history of cryptorchidism were independently associated with M540 body levels (adj.r = 0.355, P < 0.01 and adj.r = 0.223, P < 0.05, respectively). Receiver operating characteristic analysis demonstrated that at the threshold of 27%, M540 bodies discriminate subjects with testis inhomogeneity with a sensitivity of 72% and specificity of 73%. LIMITATIONS, REASONS FOR CAUTION: The increased M540 body semen levels in men with a history of cryptorchidism should be confirmed in a larger number of patients. WIDER IMPLICATIONS OF THE FINDINGS: M540 bodies may be considered a semen marker of altered testis function and thus their evaluation may be helpful in the diagnosis of male infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from Ministry of University and Scientific Research (Prin project to E.B. and FIRB project to S.M.) and Regione Toscana (to G.F.).

Ultrasonography for monitoring reproductive function in the bull.

Diagnostic ultrasonography has been widely used for examination of the reproductive tract of female cattle, but more sparingly in bulls. Typical clinical ultrasonographic examinations of bull testes are unlikely to affect semen quality or sperm production. The ultrasonographic anatomy of bull testes and accessory sex glands has been reported. Although testicular echogenicity increased (i.e. the parenchyma appeared more white) as a bull approached puberty, echogenicity was not superior to scrotal circumference as a predictor of puberty. Ultrasonography can be used to detect and characterize testicular pathology. It is noteworthy that areas of increased echogenicity (testicular fibrosis) are common, especially in young bulls, but are not associated with decreased semen quality (e.g. percentage of morphologically abnormal sperm). Neither visual evaluation nor computerized pixel analysis of testicular ultrasonic echotexture was consistently predictive of semen quality in bulls. Therefore, we concluded that the primary clinical use of ultrasonography in assessment of reproductive function in the bull is characterization of grossly detectable lesions in the testes and scrotum.

Distribution of cell-free and cell-associated HIV surrogates in the female genital tract after simulated vaginal intercourse.

BACKGROUND: Rational development of drugs to prevent human immunodeficiency virus (HIV) transmission benefits from an understanding HIV distribution in the female genital tract after intercourse. This study describes HIV distribution using surrogates of cell-free and cell-associated HIV and semen. METHODS: Apheresis-derived, autologous, lymphocyte-rich cells radiolabeled with 3.7-MBq (100-µCi) indium 111 ((111)In)-oxine (cell-associated HIV surrogate) and 18.5-MBq (500-µCi) technetium 99m ((99m)Tc)-sulfur colloid (HIV-sized 100-nm particle, cell-free HIV surrogate) were resuspended in 3 mL of hydroxyethylcellulose gel (semen simulant) with gadoteridol and dosed via artificial phallus after simulated intercourse. Postdosing dual-isotope single photon emission computed tomography with computed tomography (SPECT/CT) and magnetic resonance (MR) images were acquired to determine the surrogates' distribution. Seven hours after dosing, vaginal biopsy and luminal samples were collected at discrete locations in 8 subjects. RESULTS: SPECT/CT and MR analysis showed HIV and semen surrogate distribution with highest signal intensity in the vaginal pericervical area, without detectable signal in the uterus. One-third of the administered dose was retained in the female genital tract after 4 hours. Cell-free and cell-associated surrogate distribution coincided. CONCLUSIONS: We demonstrate the feasibility of dual-isotope SPECT/CT and MR imaging to determine the distribution of HIV and semen surrogates after simulated intercourse without disrupting vaginal contents. Surrogate distribution suggests topical microbicides do not need to reach the uterus for efficacy.

Factors affecting the distribution of copulatory plugs in rhesus monkeys (Macaca mulatta) on Cayo Santiago.

Although the proximate mechanisms behind the formation of copulatory plugs are well understood, their distribution and function among primates remain largely unstudied. During a study of female rhesus macaque (Macaca mulatta) mating behaviour on Cayo Santiago, we examined the pattern of the distribution of visible copulatory plugs among females and their potential relationship to female reproductive traits and sexual skin colouration. Our results indicate that non-pregnant females were observed more frequently with visible copulatory plugs than were pregnant females. Female parity status, age and rank were unrelated to copulatory plug observation. Genital colouration components were higher among females observed with copulatory plugs. We suggest that although copulatory plugs are generally thought of in terms of male physiology, plug observation may also be affected by female reproductive status.

Ultrasound as a mechanical method for male dog contraception.

Therapeutic ultrasound (1.5 W/cm(2)) was investigated for its effect upon testicular size and consistency and semen quality in five dogs. Dogs were treated every other day with a total of three treatments and were examined up to 2 weeks after treatment. There was some tenderness after treatment and a significant decrease in testicular size and semen quality. This method may provide a suitable contraception technique in dogs and requires further investigation.

Cryptorchidism and semen quality: a TEM and molecular study.

Cryptorchidism is a pathological condition defined as the failure of the testis to descend into the scrotum, the location of the cryptorchid testis can be in the inguinal canal or in the prescrotal and abdominal area, sometimes resulting in atrophic seminiferous tubules. The aim of this study was to analyze semen quality of men who underwent orchidopexy for unilateral or bilateral cryptorchidism during childhood. Semen quality was investigated by light microscopy to evaluate sperm concentration and motility. Sperm morphology was performed by transmission electron microscope (TEM), and the data were mathematically elaborated. The presence of Y microdeletions was investigated by polymerase chain reaction. The effect of cryptorchidism on meiosis was explored by fluorescence in situ hybridization (FISH). The incidence of azoospermia was higher in the group with bilateral compared with unilateral cryptorchidism, and semen parameters were better in the unilateral group. Sperm pathologies detected by TEM indicated a severe deterioration of sperm quality in both groups. Necrosis and apoptosis appeared to be the most frequent pathologies, and their values reached statistical significance compared with those from fertile controls. The presence of chromosome Y microdeletions in patients with cryptorchidism and severe spermatogenetic defects is controversial. No microdeletions were found in this study. FISH values indicated that the mean percentage of gonosome disomies and diploidies were generally out of normal range, indicating a severe disturbance of meiotic segregation. The effects induced by cryptorchidism resolved in childhood seem to include a spermatogenetic impairment, leading to recommendation of detailed ultrastructural and chromosomal sperm analyses before undertaking assisted reproductive techniques.

High-resolution X-ray computed tomography scanning of primate copulatory plugs.

In this study, high-resolution computed tomography X-ray scanning was used to scan ring-tailed lemur (Lemur catta) copulatory plugs. This method produced accurate measures of plug volume and surface area, but was not useful for visualizing plug internal structure. Copulatory plug size was of interest because it may relate to male fertilization success. Copulatory plugs form from coagulated ejaculate, and are routinely displaced in this species by the penis of a subsequent mate during copulation (Parga [2003] Int. J. Primatol. 24:889-899). Because one potential function of these plugs may be to preclude or delay other males' successful insemination of females, we tested the hypothesis that larger plugs are more difficult for subsequent males to displace. Plugs were collected opportunistically upon displacement during data collection on L. catta mating behavior on St. Catherines Island, Georgia (USA) during two subsequent breeding seasons. Copulatory plugs exhibited a wide range of volumes: 1,758-5,013.6 mm3 (n = 9). Intraindividual differences in plug volume were sometimes greater than interindividual differences. Contrary to predictions, larger plugs were not more time-consuming for males to displace via penile intromission during copulation. Nor were plugs with longer vaginal residence times notably smaller than plugs with shorter residence times, as might be expected if plugs disintegrate while releasing sperm (Asdell [1946] Patterns of Mammalian Reproduction; Ithaca: Comstock). We found a significant inverse correlation between number of copulatory mounts leading to ejaculation and copulatory plug volume. This may indicate that if males are sufficiently sexually aroused to reach ejaculation in fewer mounts, they tend to produce ejaculates of greater volume.

Testicle hemodynamics in patients after laparoscopic varicocelectomy evaluated using color Doppler sonography.

OBJECTIVES: Testicular blood supply and semen quality were compared in two groups of patients undergoing laparoscopic varicocelectomy: group I (n = 27) with spermatic artery ligation and group II (n = 48) with spermatic artery preservation. METHODS: Laparoscopic varicocelectomy was performed on 75 patients divided on the basis of whether ligation took place during surgery or not. The blood flow parameter (resistance index-RI) measured using color Doppler sonography was the index of vascular efficiency selected. RESULTS: Mean RI was 0.69 in group I and 0.65 in group II. The difference was not significant. Both groups showed improvements in sperm density and motility but there was no significant difference between groups for either parameter. CONCLUSION: Using color Doppler sonography confirms that ligation of the testicular artery during varicocelectomy does not lead to major changes in testicular blood supply or sperm quality.