Long-term study of vasectomy in Macaca radiata--histological and ultrasonographic analysis of testis and duct system.

This study was aimed to investigate the long-term effect of vasectomy using the bonnet monkey (Macaca radiata) as a primate animal model. Animals weighing around 6 to 8 kg were randomly chosen for bilateral, unilateral vasectomy and sham-control. The postoperative periods of six months and two years were considered as short and long-term, respectively. Sperm were collected and subjected to analysis before euthanasia. The testes and epididymides were excised from euthanized animals then embedded in paraffin. Normal histological changes were observed in sham-operated animals and short-term contralateral testes. In contrast, marked alterations were observed in the testes and epididymides of both short and long-term groups. Seminiferous epithelium was thinned out showing marked depletion of germ cells in long-term; only a thin layer of Sertoli cells, spermatogonia, and fewer spermatocytes were seen. Exfoliation of germ cells and the occurrence of multinucleated giant cells were common features in these tubules. The epididymal tubular lumens were greatly dilated with accumulated spermatozoa in short and long-term animals; significant defects were observed in the epithelium of the long-term animals. Microscopic spermatic granulomas were noticed in epididymides and the vas deferens. Large granulomas were seen in long-term vasectomized monkeys, frequently compressing the surrounding structures. These granulomas could be visualized in ultrasound, however, only at the late stage of its occurrence. Sperm collected from the unilateral vasectomized animals showed a poor motility score in the capillary mucus penetration test (CMPT). Results indicate that the changes observed after vasectomy might be due to pressure initially, whereas in the long-term the damage was supplemented by autoimmune attack. With immunoglobulin (IgG) deposition in contra-lateral unoperated testis of unilateral vasectomized animals it also showed degenerative changes and a concomitant drop in sperm quality. Although, granulomatous reactions were observed in the epididymis and vas deferens but testes were spared from such reactions even in the long-term.

Persistence of spermatozoa on decomposing human skin: a scanning electron microscopy study.

Finding spermatozoa is of the utmost importance in judicial cases involving both the living and the dead; however, most of literature actually deals with inner genitalia and does not take into consideration the chance of external deposition of semen on skin, which is not rare. In addition, the most advanced microscopic technologies such as scanning electron microscopy (SEM) have not been thoroughly investigated within this specific field of research. This study aims at applying SEM analysis to samples of decomposed skin in order to test its potential in detecting spermatozoa particularly in decomposed cadavers. A sample of skin was obtained at autopsy and divided into two thin strips; one of the samples was used as a negative control. Semen was then taken from a "donor" (with a normal spermiogram) and was spread onto the other skin sample. Every 3 days for the first 15 days (for a total of six samples), a standard slide was prepared from swabs on the treated and control skin and analyzed by standard light microscopy. In addition, every 7 days up to 91 days (3 months circa), a skin sample was taken from the positive and negative control and examined by SEM for a total of 14 samples. Results show that after 12 days, light microscopy failed in detecting spermatozoa, whereas they were still visible up to 84 days by SEM analysis. This study therefore suggests the persistence of sperm structures in time and in decomposing material as well as the possible application of SEM technology to decomposed skin in order to detect semen.

Morphology and ultrastructure of the freshwater rotifer Brachionus bidentatus (Monogononta: Brachionidae) using scanning and transmission electron microscopy.

The study of sexual reproductive behavior supported by ultrastructural evidence is important in rotifers to describe differences among potential cryptic species. In this research, the morphology of the rotifer Brachionus bidentatus is described at the ultrastructural level, using electronic microscopy, together with a brief description and discussion of its sexual reproductive behavior. The characteristics of the (a) male, (b) the female, (c) the sexual egg or cyst, (d) the partenogenic egg, (e) the no-fecundated sexual egg (male egg), and (f) the trophi, were described. Another part of this research is dedicated to the ultrastructure of the sex cells of the male rotifer B. bidentatus. Samples were obtained from La Punta pond in Cosio, Aguascalientes, Mexico (22 degrees 08' N - 102 degrees 24' W), and a culture was maintained in the laboratory. Fifty organisms, from different stages of the rotifer Brachionus bidentatus, were fixed in Formol at 4% and then prepared; besides, for the trophi, 25 female rotifer Brachionus bidentatus were prepared for observation in a JEOL 5900 LV scanning electronic microscope. In addition, for the observation of male sex cells, 500 males of Brachionus bidentatus were isolated, fixed and observed in a JEOL 1010 transmission microscope. Females of B. bidentatus in laboratory cultures had a lifespan of five days (mean+one SD = 4.69 +/- 0.48; N=13), and produced 4.5 +/- 3.67 (N=6) parthenogenetic eggs during such lifespan. In the case of non-fertilized sexual eggs, they produced up to 18 eggs (mean+one SD = 13 +/- 4.93; N=7). Sexual females produced a single cyst on average (mean +/- one SD = I +/- 0; N=20). For the sexual cycle, the time of copulation between male and female ranged from 10 to 40 seconds (mean +/- one SD = 17.33 +/- 10.55, N=7). The spermatozoa are composed of a celular body and a flagellum, the size of the body is of 300 nm while the flagellum measures 1 700nm. The rods have a double membrane. Their mean length is almost 2.45 microm +/- 0.74, N=6; and their mean wide is 0.773 microm +/- 0.241, N=11. The evidence on the specific ultrastructural characteristics of the rotifer B. bidentatus is notorious, even more in the male and in the cyst cell. Regarding the ultrastructure of the spermatozoa and the rods, compared to other species they only differ in size, despite their structural resemblance. Our study of the ultraestructure of this species adds useful information that along with molecular data will help clarify the taxonomy of brachionid rotifers.

3D multi-isotope imaging mass spectrometry reveals penetration of 18O-trehalose in mouse sperm nucleus.

The prevalence of genetically engineered mice in medical research has led to ever increasing storage costs. Trehalose has a significant beneficial effect in preserving the developmental potential of mouse sperm following partial desiccation and storage at temperatures above freezing. Using multi-isotope imaging mass spectrometry, we are able to image and measure trehalose in individual spermatozoa. We provide the first evidence that trehalose penetrates the nucleus of a mammalian cell, permitting tolerance to desiccation. These results have broad implications for long-term storage of mammalian cells.

The efficacy of ultrasound treatment as a reversible male contraceptive in the rhesus monkey.

BACKGROUND: The use of therapeutic ultrasound as a contraceptive approach has involved nonhuman primates as well as rats and dogs. The current study was undertaken to determine whether this treatment could be a method for reversible contraception, using a model with testes size similar to adult humans. METHODS: Two methods of ultrasound exposure were used, either the transducer probe at the bottom of a cup filled with saline (Cup) or direct application to the surface of the scrotum (Direct). Four adult rhesus (Macaca mulatta) males with normal semen parameters were treated with therapeutic ultrasound at 2.5 W/cm(2) for 30 min. Treatment was given 3 times, one every other day on a Monday-Wednesday-Friday schedule. For each male, semen quality was evaluated a minimum of three times over several months prior to ultrasound exposure and weekly for two months following ultrasound treatment. RESULTS: Semen samples from all males, regardless of exposure method, exhibited a decrease in the percentage of motile sperm following ultrasound treatment. There was an average reduction in motility of 40% the week following treatment. Similarly, curvilinear velocity and the percentage of sperm with a normally shaped flagellum were also reduced in all males following ultrasound treatment. A significant reduction in the total number of sperm in an ejaculate (total sperm count) was only observed in males that received ultrasound via the cup method. Following treatment via the cup method, males exhibited up to a 91.7% decrease in average total sperm count (n = 2). Sperm count did not approach pre-treatment levels until 8 weeks following ultrasound exposure. CONCLUSIONS: The sustained reduction in sperm count, percent motility, normal morphology, and sperm vigor with the cup exposure method provides proof of principle that testicular treatment with ultrasound can be an effective contraceptive approach in humans.

In search of an efficient injection technique for future clinical application of spermatogonial stem cell transplantation: infusion of contrast dyes in isolated cadaveric human testes.

OBJECTIVE: To develop an efficient infusion technique for human spermatogonial stem cell transplantation. DESIGN: A mixture with ultrasonic contrast, computerized tomography (CT) contrast, and Chinese ink was injected into isolated human testes through different sites (the rete testis, the head of the epididymis, the deferent duct, and blind testicular infusion). Ultrasound transducer was used to visualize the injection site and to observe the flow of the mixture injected in the testes. Then, micro-CT scan was used to construct three-dimensional images, allowing the calculation of the testicular volume filled by the mixture. Finally the efficiency of the infusion was evaluated on histologic sections. SETTING: Research laboratory. PATIENT(S): Cadaver testes obtained from autopsied bodies at the department of pathology. INTERVENTION(S): Ultrasound-guided infusion of contrast liquid. MAIN OUTCOME MEASURE(S): Contrast liquid-filled testis volume and presence of ink in seminiferous tubules. RESULT(S): Ultrasonography clearly visualized the flow when seminiferous tubules were injected from the rete testis. No flow was observed when infusions were made either blindly, into the deferent duct, or into the head of the epididymis. On micro-CT no significant differences were observed between the different volumes. After rete testis infusion, ink particles were found in the lumen of the rete testis and in tubules, close and distant from the rete testis. CONCLUSION(S): A single ultrasound-guided injection of 800 µL in the rete testis may provide a promising method to transplant human spermatogonial stem cells in a clinical setting.

Functional testicular evaluation using PET/CT with 18F-fluorodeoxyglucose.

PURPOSE: PET/CT using (18)F-FDG is a well-established diagnostic examination in oncology, cardiology and neurology. The clinical significance of nontumoral testicular uptake of FDG is unknown. Functional testicular imaging may have important clinical applications in the diagnosis and prognosis of male infertility. The aim of this study was to determine the andrological value of a FDG PET/CT in analysing testicular function, by correlating the PET/CT data with the sperm parameters. METHODS: Retrospective analysis of FDG PET/CT in 20 consecutive cancer patients without testicular pathology in whom two semen samples had been obtained for analysis before any chemotherapy. FDG PET/CT parameters were the mean standardized uptake value (SUVmean), used for measuring the intensity of uptake, and the functional testicular volume (FV). For statistical analysis, a Spearman's rank correlation test and a Mann-Whitney test were used. RESULTS: Of 20 patients (mean age 22 years), 18 had provided two sperm samples for cryopreservation. Sperm concentration was above 20 × 10(6)/ml in 55% of the patients. The intensity of uptake and the FV were correlated with the total sperm count, the sperm concentration and motility (p < 0.05). The difference in SUVmean between the two testes showed an inverse correlation with sperm concentration (p = 0.036). Normospermic and oligospermic men had significant differences in: (1) mean SUVmean, (2) mean FV, and (3) the difference in intensity of uptake between the testes (p < 0.05). CONCLUSION: This is the first report on the andrological value of FDG PET/CT in analysing nontumoral testicular function. This pilot study showed a significant correlation between intensity of uptake of FDG and testicular FV with the main sperm parameters. PET/CT with FDG could become a useful new tool in assisted reproductive technologies and other andrological or urological applications.

High incidence of multiple aster formation in vitrified-warmed bovine oocytes after in vitro fertilization.

In vitro-matured bovine oocytes do not tolerate vitrification as well as mature murine or human oocytes. Delayed first cleavage in vitrified and in vitro-fertilized bovine oocytes may be responsible for the decreased yield of blastocysts in vitro. Because formation of sperm-aster and the subsequent assembly of microtubule network play an important role for migration and fusion of both pronuclei, aster formation in vitrified-warmed oocytes was analyzed by confocal laser-scanning microscopy. At 10 h post-insemination (hpi), proportions of oocytes fertilized normally were comparable between the vitrified and fresh control groups (67 and 70%, respectively). Proportions of oocytes that exhibited microtubule assembly were similar between the two groups (95% each), but the proportion of oocytes with multiple asters was higher in the vitrified group when compared with the fresh control group (68 vs 29%, P < 0.05). Both migration and development of two pronuclei were adversely affected by multiple aster formation. In the next experiment, multiple asters observed in 5.5 vs 8 hpi pronuclear zygotes were located near the male pronucleus, suggesting that those multiple asters were not the cytoplasmic asters of maternal origin. In conclusion, multiple aster formation frequently observed in vitrified-warmed bovine oocytes may be related to loss of ooplasmic function responsible for normal microtubule assembly from the sperm-aster.

Male accessory gland infection frequency in infertile patients with chronic microbial prostatitis and irritable bowel syndrome: transrectal ultrasound examination helps to understand the links.

The aim of this study was to evaluate the frequency of male accessory gland infection (MAGI) in patients with chronic bacterial prostatitis (CBP) plus irritable bowel syndrome (IBS) and to compare the sperm parameters of patients with or without MAGI. In addition, another objective of this study was to evaluate the ultrasound characterization of the anatomical space between the posterior wall of the prostate and the anterior wall of the rectum using transrectal ultrasonography. Fifty consecutive patients with the following criteria were enrolled: 1) infertility, 2) diagnosis of CBP, and 3) diagnosis of IBS according to the Rome III criteria. The following 2 age-matched control groups were also studied: infertile patients with CBP alone (n = 56) and fertile men (n = 30) who had fathered a child within the previous 3 months. Patients and controls underwent an accurate patient history; administration of the National Institutes of Health-Chronic Prostatitis Symptom Index and the Rome III questionnaires for prostatitis and IBS, respectively; physical examination; semen analysis; and transrectal ultrasound evaluation (limited to patients with CBP and IBS or CBP alone). A significantly higher frequency of MAGI was found in patients with CBP plus IBS (82.0%) compared with patients with CBP alone (53.6%) or fertile men (0%). The presence of MAGI in patients with CBP plus IBS was associated with a significantly lower sperm concentration, total number, and forward motility, and with a higher seminal leukocyte concentration compared with patients with CBP alone and MAGI. Sperm normal morphology was similar in the groups of patients. All sperm parameters did not differ significantly in both groups of patients without MAGI. With ultrasound evaluation, a significantly higher frequency of dilatation of prostatic venous plexus was found in patients with CBP plus IBS (75%) compared with patients with CBP alone (10%). Patients with CBP plus IBS had a significantly higher frequency of MAGI compared with patients with CBP alone. This was associated with worse sperm parameters and, hence, poorer reproductive prognosis. We suggest searching for the presence of IBS in patients with prostatitis syndrome, in particular when CBP and/or worse sperm parameters are present. Finally, this is the first observation on ultrasound examination of the anatomical space between the posterior wall of the prostate and the anterior wall of the rectum reported in patients with CBP and IBS. Further studies should clarify the meaning of the ultrasound findings.

Effect of butylated hydroxytoluene on cryopreservation of Boer goat semen in Tris egg yolk extender.

The aim of this study was to determine the effect of butylated hydroxytoluene (BHT), a lipid-soluble anti-oxidant added in different concentrations to the Tris egg yolk extenders on semen cytological parameters pre freezing and post thawing (motility, morphology, viability, acrosome integrity and membrane integrity) of Boer goat spermatozoa. A total of 40 ejaculates from four Boer goat bucks were collected using an artificial vagina. Ten replicates of the ejaculates were diluted with a Tris egg yolk based extender which contained various concentrations (0.5mM, 1.0mM, 2.0mM and 3.0mM) of butylated hydroxytoluene while one sample was processed without supplementation of antioxidant and served as control. The diluted semen was cooled at 4°C and loaded into the straw and then stored in liquid nitrogen. It was evident that supplementation of BHT produces positive effect in terms of motility, membrane integrity and acrosome integrity in comparison with the control group in cooled and frozen Boer goat semen. Results showed significant differences in motility, membrane integrity, acrosome integrity and viability of cooled and frozen Boer goat spermatozoa at different concentrations. Motility, membrane integrity, acrosome integrity and viability was significantly higher in all treated groups than the control group (P<0.05) while there was no significant differences (P>0.05) in morphology trait between all group in cooled semen. However, improvement (P<0.05) was observed only in terms of the membrane integrity and acrosome integrity compared to the control and other treated groups in frozen semen. In conclusion, BHT can be used in cryopreservation of Boer goat semen in order to reduce the oxidative stress on spermatozoa.

Reconstitution of amoeboid motility in vitro identifies a motor-independent mechanism for cell body retraction.

Crawling movement in eukaryotic cells requires coordination of leading-edge protrusion with cell body retraction [1-3]. Protrusion is driven by actin polymerization along the leading edge [4]. The mechanism of retraction is less clear; myosin contractility may be involved in some cells [5] but is not essential in others [6-9]. In Ascaris sperm, protrusion and retraction are powered by the major sperm protein (MSP) motility system instead of the conventional actin apparatus [10, 11]. These cells lack motor proteins [12] and so are well suited to explore motor-independent mechanisms of retraction. We reconstituted protrusion and retraction simultaneously in MSP filament meshworks, called fibers, that assemble behind plasma membrane-derived vesicles. Retraction is triggered by depolymerization of complete filaments in the rear of the fiber [13]. The surviving filaments reorganize to maintain their packing density. By packing fewer filaments into a smaller volume, the depolymerizing network shrinks and thereby generates sufficient force to move an attached load. Our work provides direct evidence for motor-independent retraction in the reconstituted MSP motility system of nematode sperm. This mechanism could also apply to actin-based cells and may explain reports of cells that crawl even when their myosin activity is compromised.

Semen analysis by electron and fluorescence microscopy in a case of partial hydatidiform mole reveals a high incidence of abnormal morphology, diploidy, and tetraploidy.

OBJECTIVE: To perform a highly detailed semen analysis in a man whose wife had a partial mole. DESIGN: Case report. SETTING: Gynecology departments of two university hospitals and a laboratory of histology/embryology. PATIENT(S): A 32-year-old man whose wife had a partial mole. INTERVENTION(S): Sperm characteristics were examined by light microscopy, morphology was analysed by electron microscopy (TEM), DNA fragmentation was evaluated by TUNEL using fluorescence microscopy, and chromosomal abnormalities were assessed by fluorescence in situ hybridization using probes for chromosomes 13, 15, 16, 18, 21, 22, X, and Y. MAIN OUTCOME MEASURE(S): Sperm count, motility, morphology, DNA fragmentation, and incidence of diploidy, tetraploidy, and aneuploidy. RESULT(S): Sperm concentration was 61 million/mL, with 31% progressive motility and 4% normal morphology. TEM revealed a high incidence of head, neck, and tail abnormalities as well as the presence of phagocytes. DNA fragmentation was within normal limits (11.6%). Aneuploidy levels were low for all chromosomes tested. However, there was a high level of diploidy, with XY, XX, and YY constitution. Tetraploid sperm (XXYY) were also noted. CONCLUSION(S): Semen analysis revealed a high incidence of abnormal morphology and increased diploidy. It may be important to perform FISH testing, to verify increased diploidy in sperm, in men whose wives have had partial moles. These couples could be informed of the option to have preimplantation genetic diagnosis as a means to distinguish between diploid and triploid embryos arising from fertilization of a haploid egg by diploid sperm.

Sperm chromatin structure is associated with the quality of spermatogenesis in infertile patients.

OBJECTIVE: To establish the diagnostic value of sperm chromatin structure assessment for the evaluation of male factor infertility, in addition to conventional andrological workup. DESIGN: Cross-sectional controlled study. SETTING: A tertiary referral andrology clinic. PATIENT(S): Two hundred seventy-nine male partners of infertile couples. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The DNA fragmentation index (DFI) determined by the sperm chromatin structure assay (SCSA), semen parameters, serum levels of reproductive hormones, and World Health Organization (WHO) classification of male factor subfertility. RESULT(S): In all patient categories, except those including patients with hypogonadotrophic hypogonadism, sperm antibodies, or normospermia, DFI was significantly higher compared with in proven fertile controls. After classification of the quality of spermatogenesis based on mean testicular volume (<10 ml vs. >15 ml), follicle stimulating hormone (FSH; > 10 U/L vs. <5 U/L), and inhibin-B (<100 nmol/L vs. >150 nmol/L), the DFI was significantly higher in patients with poor spermatogenesis (35.9%) than in patients with normal spermatogenesis (25.9%). In a multiple regression analysis, the teratozoospermia index, sperm vitality, and FSH were significant determinants of the DFI level. Male age was associated with DFI, but leukocytospermia, body mass index, and smoking were not confounders of DFI. CONCLUSION(S): Impaired spermatogenesis, irrespective of the WHO classification of male factor subfertility, is generally associated with an increase of sperm DNA damage.

Targeted disruption of Nphp1 causes male infertility due to defects in the later steps of sperm morphogenesis in mice.

Juvenile nephronophthisis type I is the most common genetic disorder causing end-stage renal failure in children and young adults. The defective gene responsible has been identified as NPHP1. Its gene product, nephrocystin-1, is a novel protein of uncertain function that is widely expressed in many tissues and not just confined to the kidney. To gain insight into the physiological function of nephrocystin, Nphp1-targeted mutant mice were generated by homologous recombination. Interestingly, homozygous Nphp1 mutant mice were viable without renal manifestations of nephronophthisis. They appeared normal, but males were infertile with oligoteratozoospermia. Histological analysis of the seminiferous tubules showed that spermatogenesis was blocked at the early stages of spermatid elongation, with degenerating spermatids sloughing off into the lumen. Electron microscopic analysis revealed detachment of early elongating spermatids from Sertoli cells, and a failure of sperm head and tail morphogenesis. However, a few mature spermatozoa were still deposited in the epididymis, though they were frequently dead, immotile, or malformed. These novel findings indicate that nephrocystin is critically required for the differentiation of early elongating spermatids into spermatozoa in mice. The possible roles of nephrocystin in the formation and maintenance of Sertoli-spermatid junctions are still under investigation.

Sperm suspension is a highly ultrasonically visible material: a novel model to study uterine activity.

BACKGROUND: The aims of this study were to find an ultrasonically echogenic material to study the uterine activity, and to test whether closing the vaginal speculum on the cervix prevents the displacement of the injected material. METHODS: A concentrated sperm suspension was used as an ultrasonically visible material. Forty-five women undergoing intrauterine insemination were randomized into: open speculum group (n=23) and closed speculum group (n=22). Mimicking embryo transfer, 50 ul of concentrated sperm suspension was injected intrauterine while the vaginal speculum was open in 23 patients. In the other group, the two blades of the vaginal speculum were closed on the cervix, then 50 ul of concentrated sperm suspension was injected. The ultrasonically visible material was observed in the uterine cavity for 10 min during which the procedure was video-recorded. RESULTS: The injected sperm suspension was clearly visible in all cases. In the closed speculum group, the echogenic droplet remained in the upper uterine segment in 18 cases (82%) and moved towards the lower uterine segment in six cases (18%). In the open speculum group, the echogenic droplet remained in the upper uterine segment in only six cases (26%) and it moved towards the lower uterine segment and passed through the cervical canal in 17 cases (74%). CONCLUSIONS: For the first time in the medical literature, a concentrated sperm suspension was used as an ultrasonographically visible material to study uterine activity. Closing the portio-vaginalis of the cervix prevents the displacement of the injected material.

Bovine epididymal sperm morphology obtained from caput, corpus and cauda epididymides.

To investigate the proportion of normal sperm cells in bovine epididymis, bovine testicles (n = 50), obtained from a local slaughterhouses, epididymides were incised and sperm cells were transferred into slide glasses where eosin nigrosin stain was applied either in the place or in laboratory. When sperm were stained in slaughterhouse, 88% of caput epididymal sperm were alive; 9% without protoplasmic droplet (NPD), 10.2 and 68.8% had distal (DD) and Proximal Droplets (PD), respectively. Of dead sperm, 10.4% were NPD and 0.33 and 1.27% had DD and PD, respectively. Of corpus epididymal sperm, 77.2% were alive of which 14.7% were NPD, 58.3 and 4.2% had DD and PD, respectively. Of dead sperm, 20.4% were NPD and 2.2 and 0.2% had DD and PD, respectively. When spermatozoa were stained in laboratory, 71.7% were alive of which 17.4% were NPD, 49.7 and 4.6% had DD and PD, respectively. Of dead sperm, 23.1% had no droplet and 4.21 and 0.99% were DD and PD, respectively. The proportion of live spermatozoa from caudal epididymis was 86.1%, of which 9.9% were NPD, 68.3 and 7.9% had DD and PD, respectively. Of dead spermatozoa, 10.1% had no droplet and 3.3 and 0.5% had DD and PD, respectively. No significant difference observed between different parts of epididymis and also between slaughter house staining and laboratory staining of sperm cells. Data showed that approximately all parts of epididymis contained similar status of live sperm cells and the sperm cells containing protoplasmic droplets.

A stable isotope-mass spectrometric method for measuring human spermatogenesis kinetics in vivo.

PURPOSE: Currently it is thought to take 60 to 70 days to produce and ejaculate human sperm. This estimate is derived mainly from a single older, descriptive, kinetic analysis of spermatogenesis. We developed a noninvasive method to assess germ cell turnover time accurately in vivo using stable isotope labeling and gas chromatography/mass spectrometry analyses. We confirmed the postulated length of a normal cycle of spermatogenesis. MATERIALS AND METHODS: A total of 11 men with normal sperm concentrations ingested (2)H(2)O daily for 3 weeks. Semen samples were collected every 2 weeks for up to 90 days. Label incorporation into sperm DNA was quantified by gas chromatography/mass spectrometry, allowing calculation of the percent of new cells present. A cycle of sperm production was determined as the lag time until labeled sperm appeared in the ejaculate. RESULTS: Labeled sperm were detected after a mean +/- SD of 64 +/- 8 days (range 42 to 76). In 1 subject the time lag was 42 days but it was at least 60 in all other subjects. In most subjects plateau labeling in sperm was not attained. In 2 subjects the rise and fall of the labeling curve was steep and reached greater than 85% new cells, suggesting rapid washout of old sperm in the epididymal reservoir. CONCLUSIONS: This direct kinetic assessment confirms a course of spermatogenesis that is on the shorter side of traditional estimates based on prior analyses. In addition, the variability observed in healthy men suggests that characteristics such as the epididymal reservoir effect may influence the modeling of in vivo spermatogenesis.

Fertilization failure from a sperm chromatin defect in couples with unexplained infertility.

OBJECTIVE: To investigate the relationship between unexplained infertility and fertilization failure from nucleoprotein defects in ejaculated human sperm and to study the usefulness of sperm chromatin assays, using AO fluorescence dye, to evaluate patients with unexplained infertility before treatment. STUDY DESIGN: From January 1999 to January 2000, 513 infertile couples had the clinical causes of their infertility assessed. During the next investigative period (February 2000-February 2001), 137 cases of unexplained infertility (n = 80) were chosen for this study, as were cases of tubal factor infertility (n = 57) as controls. The status of nuclear chromatin in ejaculated sperm was examined using acridine orange staining, followed by a conventional in vitro fertilization procedure. RESULTS: The number of patients with immature ejaculated sperm was 16 of 30 (53.3%) unexplained infertility cases involving fertilization failure, 8 of 50 (16.0%) unexplained infertility cases without fertilization failure and 5 of 57 (8.8%) tubal factor infertility cases. A significant difference was observed between unexplained infertility cases with fertilization failure and the other groups (P < .0001). CONCLUSION: These results suggest that the nuclear immaturity of ejaculated human sperm may be 1 of the primary factors underlying unexplained infertility.

Experimentally induced orchitis associated with Arcanobacterium pyogenes: clinical, ultrasonographic, seminological and pathological features.

The objectives of this study were to describe the features of experimentally induced orchitis associated with Arcanobacterium pyogenes and confirm the pathogenicity of the organism for the ovine testicle. One testicle of each of nine rams was inoculated with 1.3 +/- 10(4) colony-forming-units of an A. pyogenes isolate and regular clinical, ultrasonographic, bacteriological and seminological examinations were carried out up to 204 days after challenge. The rams were sequentially euthanatized 3, 6, 9, 18, 30, 50, 71, 113 and 204 days after challenge and a gross- and histopathological examination of their testicles was performed. All rams developed clinical orchitis and general signs. The initial ultrasonographic findings were changes of size and echogenicity of the genitalia, whilst in the long-standing phase they were wider appearance of the mediastinum testis, presence of hyperechogenic foci, changes of echogenicity of the genitalia and increased echogenicity of the scrotum and tunics. The following changes in semen evaluation parametres were recorded: the pH, the percentage of dead sperms, the percentage of abnormal sperms and the number of nonsperm round cells increased, whilst the mass motility, the individual motility and the sperm concentration decreased; the following sperm defects were observed: misshapen or piriform heads, sperms with coiled tails, sperms without tail and sperms with proximal cytoplasmic droplet; at the early stages neutrophils were the prevailing nonsperm round cell type, later the proportion of immature germ cells increased and in the long-standing phase there were enlogated spermatids and leucocytes; it is noteworthy that semen evaluation parametres were restored to normal at the late stages of the disease. A. pyogenes was consistently isolated from the semen samples after challenge, as well as from the dissected genitalia. The salient post-mortem findings were: initially, subcutaneous oedema, fluid into the vaginal cavity, congested and distended vessels, increased size of the genitalia and a hard dark area inside the testicles; subsequently, there were changes of size of the genitalia, thickening of scrotum and tunics and presence of fibrin on the testicular surface; in the long-standing phase of the disorder, there were induration of scrotum and tunics with adhesion between the tunics and discolouration of the surface of the genitalia. The prominent histopathological changes were observed in the inoculated testicles; milder changes were seen in the respective epididymides; interstitial oedema, diffuse neutrophilic infiltration and extravasation were observed in the early stages after challenge; lymphocytic infiltration with concurrent fibrosis, mineralization and inspissation of the tubular elements of the seminiferous tubules and presence of vacuolated Sertoli cells were seen later; finally, regeneration of the epithelium and presence of Sertoli cells and spermatogonia with various degrees of spermatogenic activity were evident. These findings, allied to the isolation of A. pyogenes from field cases of ovine orchitis, provide clear evidence that A. pyogenes is pathogenic for the ovine genitalia; however, the mechanisms of transition of the organism from commensal to pathogenic state are not clear. It is also noteworthy that some degree of fertility was restored in the late stages of the disorder. Ultrasonography appeared to be useful for the diagnosis of intra-scrotal abnormalities, especially during investigation of the long-standing stage of the disease, after clinical findings have subsided.

An increased incidence of autosomal aneuploidies in spermatozoa from a patient with Klinefelter's syndrome.

OBJECTIVE: To determine whether there is an increased incidence of autosomal aneuploidies in spermatozoa from a subject with nonmosaic Klinefelter's syndrome. DESIGN: Analysis of sperm nuclei by fluorescence in situ hybridization. SETTINGS: Hospital-based laboratory for reproductive biology. PATIENT(S): A patient with Klinefelter's syndrome and a 46,XY fertile man were analyzed. INTERVENTION(S): The sperm samples were prepared for fluorescence in situ hybridization. MAIN OUTCOME MEASURE(S): The disomy frequencies for chromosomes 7, 9, 13, 18, and 21 and sex chromosomes were determined using fluorescence in situ hybridization. RESULT(S): Significant differences were found in the frequency of disomy for chromosomes 13, 18, and 21 between the patient and a normospermic control. No significant differences were observed for chromosomes 7 and 9. The frequencies of gonosomal abnormalities and diploid spermatozoa were also significantly increased in the patient. CONCLUSION(S): Our results indicate that there is an increased incidence of autosomal aneuploidies for chromosomes 13, 18, and 21 in the spermatozoa from the patient with Klinefelter's syndrome than in the general population. Furthermore, most of the studies have also shown an increased frequency of autosomal aneuploidy in the spermatozoa from 46,XY males with oligozoospermia or oligoasthenoteratozoospermia. Thus, the offspring of the patients with Klinefelter's born by intracytoplasmic sperm injection may be at higher risk of autosomal aneuploidy due to oligoasthenoteratozoospermia.