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PubMed1981

Penetration of human ejaculated spermatozoa into human and bovine cervical mucus. I. Correlation between penetration values.

Midcycle bovine cervical mucus (BCM), fresh (FBCM) or frozen at -20 degrees C (BCMF), and human cervical mucus (HCM) were collected and tested in in vitro penetration tests, using human ejaculated spermatozoa of good quality. Penetration tests were performed at 34 degrees C for 1 hour, and the penetration value (PV) was calculated. Duplicates of each run were in a narrow deviation. Correlations between PV rates of the same semen samples in the 3 sources were found to be significantly high (HCM versus FBCM, r = 0.958; HCM versus BCMF, r = 0.982, and FBCM versus BCMF, r = 0.985), suggesting that human ejaculated spermatozoa penetrate the cervical mucus of all three sources at the same rate.

Animals↗
PubMed1985

Seminal parameters of ejaculates collected from oligospermic and normospermic patients via masturbation and at intercourse with the use of a Silastic seminal fluid collection device.

Seminal fluid parameters of ejaculates collected via intercourse with the use of a new Silastic seminal fluid collection device (SCD; HDC Corporation, Mountain View, CA) and via masturbation were compared. Thirty couples participated in this study. All males produced two specimens within 8 days with exactly 4 days of sexual abstinence each time. Thirteen patients were classified as oligospermic and the remaining 17 as normospermic on the basis of sperm numbers per milliliter. Each male patient subjectively rated (scale, 0 to 10) his sexual stimulation elicited during production of specimens with the use of the two methods. Specimens were evaluated and compared. Significant increases (P less than 0.05) were observed in all parameters observed within each group of patients when the SCD and masturbation methods were compared. The improvements in sperm parameters because of the method of collection were greater (P less than 0.05) in the oligospermic group than in the normospermic group of patients. The results point out that the SCD, as applied in this study, can assist in the improvement of the collected specimens and produce a specimen that closely resembles the ejaculate obtained during intercourse. In addition, the SCD, as shown in this study, can be of greater assistance to male infertility patients with oligospermia and other spermatogenic dysfunctions.

Adult↗
PubMed1989

Semen analysis data from fresh and cryopreserved donor ejaculates: comparison of cryoprotectants and pregnancy rates.

Patients (155) were selected at random for fresh or cryopreserved semen and inseminated on the predicted day of ovulation. Semen analysis was performed using a microcomputerized multiple-exposure photography system. Frozen semen was used with either glycerol or TEST-yolk (TEST-buffered 20% egg yolk with 10% glycerol) as the cryoprotectant. Cryopreservation resulted in significant decreases in all semen parameters measured. Of these, velocity appeared to be the least effected. TEST-yolk provided significantly more protection against a reduction in velocity compared with glycerol. A total of 18, 17, and 27 patients conceived using fresh, glycerol, or TEST-yolk-preserved semen, respectively. For these same groups, a cumulative pregnancy rate of 52.9%, 27.1%, and 68.5%, respectively, was observed (not significant). The total number of motile sperm per insemination used for fresh artificial inseminations resulting in conception (132.4 X 10(6] was significantly greater than the number used for successful glycerol- and TEST-yolk-preserved semen (approximately 24 X 10(6]. These results demonstrate that although the number of motile sperm of cryopreserved ejaculates are dramatically reduced compared with the fresh counterparts, if a minimum criteria for ejaculate quality is established, the use of cryopreserved semen can offer a viable, effective, and relatively safe alternative to artificial insemination by donor with fresh semen.

Cell Survival↗
PubMed2003

Sephadex and sephadex ion-exchange filtration improves the quality and freezability of low-grade buffalo semen ejaculates.

The effect of sephadex and sephadex ion-exchange filtration on the improvement in quality and freezability of low-grade buffalo semen ejaculates was assessed. Two types of filtration columns were used: one containing only sephadex G-10 (FS) and the other sephadex G-10 along with ion-exchangers (diethyl amino ethane-52 (DEAE-52) cellulose and carboxy methyl-52 (CM-52) cellulose; FS+IE). Unfiltered samples served as controls. Semen ejaculates extended in Tris-citric acid (1:4) (n=16; initial motility 40-50%) were filtered at the rate of 1.5 ml/min under negative pressure at room temperature (28-30 degrees C). The mean recovery rate (%) of motile spermatozoa in the FS (85.9+/-1.51) and FS+IE (77.10+/-2.28) filtrates did not differ significantly. Percentages of sperm motility, normal acrosomes, and intact plasma membranes were highest (P<0.05) in FS+IE, intermediate (P<0.05) in FS and lowest (P<0.05) in controls at the three stages of cryopreservation (postfiltration final dilution, after equilibration, and after freezing). Mean sperm abnormalities were lowest (P<0.05) in the filtrates of FS+IE, moderate (P<0.05) in FS and highest in controls at all stages of freezing. Compared to dilution and equilibration, freezing greatly reduced (P<0.05) the overall percent motility, normal acrosomes and intact plasma membranes. The spermatozoa eluted through FS+IE columns proved more resistant (P<0.05) in bearing dilution, equilibration, freezing and thawing stresses than the spermatozoa from FS and control samples. It is concluded that filtration systems containing an FS+IE column can effectively enhance the quality and freezability of extended, low quality buffalo semen.

Acrosome↗
PubMedDate not supplied

Abnormal spermatozoa in the ejaculate: abortive apoptosis and faulty nuclear remodelling during spermatogenesis.

The mechanisms responsible for producing abnormal spermatozoa in the ejaculate are relatively unknown. Numerous studies have now shown the presence of nuclear DNA strand breaks in human ejaculated spermatozoa and the abnormal persistence of apoptotic marker proteins. The reason why human spermatozoa, in particular from men with abnormal semen parameters, possess these abnormalities is still not clear. Two processes that have been linked to the presence of nuclear DNA strand breaks in spermatozoa are anomalies in apoptosis during spermatogenesis or problems in the replacement of histones with protamines during spermiogenesis. Understanding the mechanisms responsible for producing abnormal spermatozoa in the human will improve knowledge about certain causes of male infertility.

Apoptosis↗
PubMed2003

Isolation of ejaculated spermatozoa six hours after unsuccessful testicular biopsy in nonobstructive azoospermia.

Azoospermia, the absence of sperm in ejaculated semen, is the most severe form of male factor infertility and is present in approximately 5% of all investigated infertile couples. The condition is currently classified as obstructive and nonobstructive subgroups. In nonobstructive azoospermia, testicular sperm extraction (TESE) is usually necessary for sperm recovery. This is a case report of pregnancy and subsequent birth of healthy babies following intracytoplasmic sperm injection (ICSI) of ejaculated motile spermatozoa presented 6 h after unsuccessful testicular biopsies in four subsequent in vitro fertilization (IVF) cycles with infertility due to nonobstructive azoospermia.

Adult↗
PubMed2003

Relationship between Fertilizing Ability of Ejaculated Human Spermatozoa and Its Chromatin Heterogeneity.

OBJECTIVE: The aim of this study was to examine the relationship between the fertilizing ability of ejaculated human sperm and its chromatin heterogeneity. METHOD: We used the D-AO staining method (acridine orange epifluorescence accompanied by diamide, a thiol oxidizing agent) to analyze the sperm chromatin structure of infertile patients with IVF-ET treatment. SDS-PAGE was performed to analyze the sperm nuclear proteins collected from patients with immature sperm (stained red with D-AO staining) and proven fertile men. RESULTS: 1. It was suggested that D-AO staining allowed the immature sperm to be divided into two groups. One was immature sperm, which had disturbance of S-S formation in the epididymides, and the other was sperm that which had an abnormal exchange process of nuclear proteins in the testes. 2. The stainability after D-AO staining showed no correlation with the findings of semen analysis. 3. The fertilization rate of IVF-ET was significantly correlated to the percentage of green sperm staining with D-AO staining. 4. In the pregnant group after IVF-ET, it was noticed that sperm of the green type with D-AO staining was increased in comparison with the non-pregnancy group. 5. The fertilization rate in the group of the sperm stained red with D-AO staining was increased to 73.5% by ICSI. 6. Definite differences were noticed between the protein components of the patients with immature sperm and those of the proven fertile men by analysing with SDS-PAGE. CONCLUSION: D-AO staining was an efficient method for evaluating the fertilizing ability of human ejaculated sperm, and to determine an appropriate ART tool such as ICSI.

Journal Article↗
PubMed1998

A prospective clinical trial investigating the efficacy of a method of preparing subpopulations of antibody-free spermatozoa from the ejaculates of antibody-positive patients.

Couples undergoing in vitro fertilization treatment (IVF) were invited to take part in a controlled prospective clinical trial. The aim was to determine the effect on the fertilization rate of a technique devised to obtain an antibody-free preparation of spermatozoa from an antibody-positive ejaculate. Oocytes collected during IVF were allocated into one of two groups, ensuring that quality and maturity were comparable in each. One group, the control, was inseminated with Percoll-processed spermatozoa. The experimental group was inseminated with identical numbers of Percoll-processed spermatozoa which had been treated to obtain an antibody-free preparation. The treatment was found to have no beneficial effect on the fertilization rate at IVF. Laboratory studies were also performed on the ejaculates of antibody-positive volunteers to determine whether this treatment led to any effects, whether beneficial or detrimental, on sperm function. Membrane integrity was found to be unaffected, as was the percentage of spermatozoa undergoing the spontaneous acrosome reaction following overnight incubation. The percentage of spermatozoa undergoing the ionophore-induced acrosome reaction following treatment, however, was higher than that of the controls. The results of sperm-zona pellucida binding studies were equivocal. The findings indicated that the treatment procedure could not be justified for use in IVF, but may be beneficial for intrauterine insemination.

Autoantibodies↗
PubMed1979

Ethanol extraction of basic proteins from ejaculated human spermatozoa.

A method for the extraction of basic proteins from human ejaculated spermatozoa has been developed. It relies on the previously unreported observation that such basic protein is soluble in a solution containing 60% (v/v) ethanol. This unconventional method yields a high percentage of arginine-rich basic protein which is then able to be characterized on the basis of its amino acid composition. This method also allows comparisons to be made between single ejaculates by the banding pattern each displays when subjected to polyacrylamide gel electrophoresis.

Amino Acids↗
PubMed2000

Only low levels of spermadhesin AWN are detectable on the surface of live ejaculated boar spermatozoa.

The zona-binding protein family of spermadhesins are constituents of boar seminal plasma that are generally believed to attach to the acrosomal region of spermatozoa and thereby assist sperm interaction with the zona pellucida at fertilization. However, previous studies have yielded conflicting results with respect to amounts of adhesin bound to ejaculated cells, to the distribution of bound adhesin within the sperm population, and the regionalization of binding on the sperm surface. In the present study, spermadhesin AWN in unfixed living suspensions of boar spermatozoa was assessed by means of flow cytometry and immunocytochemistry, using a polyclonal antibody raised in chicken. Direct probing with an Oregon Green conjugate of the antibody was compared with indirect probing using Alexa Fluor-conjugated goat anti-chicken IgG as second antibody. Regardless of staining procedure, the live sub-population showed homogeneously low levels of staining, whereas the dead sub-population showed high (more than 5-fold greater) levels of staining. The live cells were stained about 2-fold more intensely by anti-AWN than by preimmune immunoglobulin, indicating the presence of small amounts of AWN. Immunocytochemistry showed the live cells to be faintly stained all over their surface, whereas staining of the dead cells was largely localized to the acrosomal region. This latter staining was non-specific, preimmune immmunoglobulin resulting in as much bound fluorescence as anti-AWN. Attempts to block non-specific staining with appropriate pretreatment with chicken or goat serum (as compared with routine use of BSA) met with variable and incomplete success, and did not increase staining by anti-AWN relative to preimmune serum in either live or dead cells. It is concluded that limited amounts of spermadhesin AWN bind tightly over the whole surface of live ejaculated boar sperm. However, the acrosomal region of disrupted sperm has an alarming tendency to bind fluoro-conjugates of immunoglobulins non-specifically.

Animals↗
PubMedDate not supplied

Correlation of the bovine cervical mucus penetration test with human sperm characterisitics in 1,406 ejaculates.

The bovine cervical mucus penetration test (BCMPT) was performed to determine its usefulness in screening the ability of sperm to successfully penetrate mucus in vitro. Ejaculates were obtained by masturbation from patients attending an infertility clinic. Routine semen analysis was performed using a microcomputerized multiple-exposure photography system. The BCMPT was performed. Overall, the average penetration of the mucus was 38 +/- 0.46 mm. Of the 1,406 ejaculates analyzed, 244 (17%) displayed a negative result (0-20 mm), 291 (21%) a questionable result (21-30 mm), and 871 (62%) a positive result (>30 mm). A highly significant (p < .001) correlation between mucus penetration distance and sperm MD (r = 0.541), MI (r = 0.484), count (r = 0.475), motility (r = 0.448), velocity (r = 0.400) and morphology (r = 0.369) was observed. Overall, the finding of an abnormal semen parameter resulted in a 34 +/- 5% accurate prediction of a negative or questionable BCMPT (<30 mm), while a normal semen parameter resulted in a 90 +/- 4% accurate prediction of a positive BCMPT (>30 mm). Sperm MD showed the strongest positive predictive value (98%), while morphology showed the greatest negative predictive value (50%). Of the 1,406 samples, 25 +/- 2% of the samples with normal semen parameters displayed a negative BCMPT. Conversely, 6 +/- 2% of samples with abnormal parameters showed a positive BCMPT. The BCMPT successfully identifies a significant subpopulation of patients as having an inadequate penetration of mucus with otherwise normal semen characteristics.

Animals↗
PubMed1980

A collagen-binding protein on the surface of ejaculated rabbit spermatozoa.

A rabbit antibody to mouse 3T3 cell fibronectin was used in conjunction with a fluorescein-tagged second antibody to detect fibronectin-like activity on the surface of rabbit spermatozoa. Only ejaculated sperm displayed an intense and highly localized fluorescence over the acrosomal region. Cauda epididymal sperm of the rabbit as well as several other species did not exhibit any reaction. The fluorescent activity could be eliminated by trypsin treatment but was re-established by incubation in cell-free seminal fluid. Sperm recovered from females 10-12 h after mating showed a reduction or absence of antifibronectin fluorescence, suggesting that this component's loss could be a factor in sperm capacitation. Because fibronectins show strong binding to collagen, mixtures of ejaculated sperm and collagen were examined in the light and electron microscope. Living sperm appear to have a strong affinity for collagen and quickly adhere to the filaments by their heads, while continuing vigorous flagellations. Surface labeling of sperm with the galactose-oxidase-NaB[3H]4 technique, extraction with urea-detergent mixtures and affinity chromatography of extracts on gelatin-Sepharose revealed a single radioactive band of mot wt approximately 40,000 after SDS polyacrylamide gel electrophoresis and fluorography.

Animals↗
PubMed1998

Correlation of testicular pathology and sperm extraction in azoospermic men with ejaculated spermatids detected by immunofluorescent localization.

Limiting testicular biopsy for intracytoplasmic sperm injection (ICSI) to those with a high chance of having testicular spermatozoa has not been possible because of the poor predictive value of current clinical and laboratory methods. In order to predict testicular pathology and sperm extraction, we characterised the semen of 28 men with azoospermia due to gonadal failure in terms of the presence of spermatids using an immunological method. The results were compared with the assessment of testicular biopsies by histology and the extraction of spermatozoa into culture medium. Washed cellular elements in the ejaculate were smeared on microscope slides and fixed in 100% methanol, before incubation with acrosome-specific monoclonal antibody (18.6), fluorescein isothiocyanate-labelled anti-mouse goat IgG, and examination by epifluorescent microscopy. Semen from men with oligozoospermia and obstructive azoospermia served as positive and negative controls, respectively. Twelve patients who had positive immunofluorescence (one or more spermatids present) had spermatozoa retrieved from their testes (five hypospermatogenesis, seven focal spermatogenesis), and 16 patients with negative immunofluorescence (spermatids absent) had apparent Sertoli cell-only syndrome (12) or maturation arrest histological pattern (four). However, four patients with apparent Sertoli cell-only syndrome had testicular spermatozoa present after extraction from the biopsy. Plasma follicle stimulating hormone concentration and testicular volume did not predict retrieval of seminal spermatids or testicular spermatozoa. We conclude that the immunofluorescent localization of one or more spermatids in the ejaculate can be used to predict the likelihood of obtaining testicular spermatozoa for ICSI. However, in some patients with Sertoli cell-only syndrome, spermatozoa could still be recovered in the absence of apparent seminal spermatids.

Acrosome↗
PubMed1998

Increase of large granular lymphocytes in human ejaculate containing antisperm antibodies.

Using flow cytometry, we studied the expression of the CD16 antigen by lymphocytes present in human semen samples from three groups of patients: 60 fertile men attending for vasectomy, 60 sterile patients without antisperm antibodies (ASA) and 18 immunological sterile patients with ASA in their ejaculate. No significant difference was found in the concentration of leukocytes or subpopulations of these cells (monocytes, lymphocytes and granulocytes) between fertile, sterile without ASA and immunological sterile groups. However, we detected a predominance of macrophages/monocytes within the population of seminal leukocytes. No statistically significant difference was found in the absolute number of T and B lymphocytes between the three groups studied. However, a significant increase in the number of CD16+ lymphocytes was observed in the ejaculate of sterile patients with ASA as compared to the other groups. This finding might establish an important parameter in the follow-up and prognosis of patients with immunological sterility.

Adult↗
PubMed1998

Comparison of the fertilizing capability of spermatozoa from ejaculates, epididymal aspirates and testicular biopsies using intracytoplasmic sperm injection.

A prospective study was carried out to compare the fertilizing capability and pregnancy outcome following intracytoplasmic sperm injection (ICSI) using spermatozoa obtained from ejaculates, or surgically from epididymis or seminiferous tubules. A total of 77 ICSI cycles (one per patient) was included. In all, 28 patients had severe oligoasthenoteratozoospermia, 19 patients had obstructive azoospermia and 30 patients had non-obstructive azoospermia. The main outcome measures were fertilization rate per injected metaphase II oocyte and the clinical pregnancy rate per embryo transferred back to the female recipients. In patients with severe oligoasthenoteratozoospermia, the fertilization and pregnancy rates were 79 and 25 %. In patients with obstructive azoospermia, for whom epididymal spermatozoa were used, these were 75 and 28%, and in the non-obstructive group for which testicular spermatozoa were used for injection, they were 69 and 21% respectively. These rates were not significantly different in the three groups (P = 0.85 and P = 0.14 respectively), suggesting that spermatozoa from the ejaculates and epididymal or testicular biopsies are able to fertilize equally by using ICSI. Live birth per embryo transfer was significantly reduced in patients with non-obstructive azoospermia compared to the other two groups. The high abortion rate (50%) in the group in which testicular spermatozoa were used raises doubts about the developmental competence of such embryos.

Biopsy↗
PubMed2006

Characterization and quantification of mRNA transcripts in ejaculated spermatozoa of fertile men by serial analysis of gene expression.

BACKGROUND: Accumulated evidence proves that mature spermatozoa contain a complex yet specific array of mRNA, which could provide information on the past events of spermatogenesis. OBJECTIVE: To quantitatively microdissect these mRNA transcripts by a digital approach. METHODS: Serial analysis of gene expression (SAGE) was used to study the mRNA transcripts from ejaculate of a fertile individual and of a pool of 10 fertile men. Online DAVID software suite was also utilized to cluster the UniGene data. RESULTS: A SAGE library from the individual produced 20,237 raw tags representing 2459 unique tags and that from pooled 10 men generated 21,052 raw tags representing 2712 unique tags. When the unique tags occurring > or = 4 times were analysed, 564 overlapping tags were produced by 638 unique tags from the individual and 682 from the pooled library. Fifty-four of these overlapped tags were considered to be novel genes. Online analysis of the overlapping tags revealed 25 functional gene groups, with the dominant one comprising 96 nuclear protein genes involving transcription and transcription regulation and also a group with 84 ribosomal subunit genes involving protein synthesis. CONCLUSION: A SAGE analysis of ejaculate from fertile men has revealed a large number of transcripts, which occur in steady frequencies and probably have important roles in spermatogenesis and fertilization.

Adult↗
PubMed1995

Anaerobes in ejaculates of subfertile men.

The clinical significance of micro-organisms in semen samples of asymptomatic subfertile patients is a matter of constant debate. Usually little attention is paid to anaerobic bacteria as they are sensitive to transportation and culturing, and differentiation is difficult, costly and time-consuming. In the present study, special screening was carried out for anaerobes in ejaculates in addition to the routine microbial cultures of genital secretions of both partners. In addition to standard semen analysis and evaluation of sperm ability to penetrate cervical mucus (CM) in vivo (post-coital testing) and in vitro using a standardized test system, semen samples from 126 randomly chosen males of couples with a median duration of infertility of 4 years were examined for colonization with anaerobic bacteria. All couples were without clinical signs or symptoms of genital tract infection. The special care taken for anaerobic growth in semen samples gave a high rate of positive cultures and showed that nearly all ejaculates (99%) were colonized with anaerobic micro-organisms, and potentially pathogenic species were found in 71% of men. This rate was more than four times higher than that obtained with routine cultures and standard transportation (16%). Anaerobic bacterial growth of > or = 10(6) colony forming units (CFU)/ml was seen in 42% (total range 10(3)-10(8) CFU/ ml). In addition, aerobic growth was found in 96% (> or = 10(6) CFU/ml in 21%), potentially pathogenic species in 61% of semen specimens. There were no marked differences in the prevalence of anaerobic micro-organisms in patients with reduced or normal sperm count, motility or morphology. Nor was there any significant difference in anaerobic colonization between samples with impaired or good ability to penetrate CM of female partners (in vivo or in vitro), or the CM of fertile donors in the in-vitro sperm-cervical mucus penetration test (SCMPT) in this asymptomatic group of patients. There was no clear association between microbial colonization and subsequent fertility in vivo within an observation period of 6 months. The results of this study suggest that anaerobic bacteria are often not detected when routine methods for microbial evaluation are used. This should be considered during assisted reproduction and in patients with symptoms of genital tract infection and should lead to further studies in infertile patients where subclinical infection or inflammation is indicated by specific markers in semen samples.

Adult↗