Calcium requirements for human sperm function in vitro

OBJECTIVE: To determine extracellular calcium (Ca(2+)) requirements for the maintenance of human sperm function in vitro. DESIGN: Prospective study. SETTING: Basic research laboratory. PATIENT(S): Normozoospermic volunteers provided fresh semen samples; follicular fluid (human FF) and oocytes were collected from women undergoing IVF-ET. INTERVENTION(S): Spermatozoa were incubated for /=0.1 mM of Ca(2+) were able to undergo the AR when exposed to human FF in the presence of 2.5 mM of Ca(2+). Calcium concentrations of >/=0.22 mM were sufficient to reach protein tyrosine phosphorylation levels and hyperactivated motility values similar to those of controls. Higher Ca(2+) concentrations (>/=0.58 mM) were required to produce maximum human FF-induced AR in previously capacitated cells and to obtain an adequate sperm-ZP binding. CONCLUSION(S): Different steps of the fertilization process have distinctive Ca(2+) requirements. Whereas 0.22 mM of Ca(2+) is sufficient for the development of some capacitation-related events, human FF-induced AR and sperm-ZP interaction require 0.58 mM of this cation.     

Modulation of human sperm function by peritoneal fluid

OBJECTIVE: To examine the effect of peritoneal fluid on various parameters of sperm function in vitro. DESIGN: Prospective study. SETTING: Basic research laboratory. PATIENT(S): Semen samples were obtained from normozoospermic volunteers (n = 43). Peritoneal fluids were aspirated laparoscopically from women with unexplained infertility (n = 14). Follicular fluid and oocytes were collected from patients undergoing IVF-ET. INTERVENTION(S): Sperm incubated under capacitating conditions were exposed to peritoneal fluid, and functional variables were evaluated in vitro. MAIN OUTCOME MEASURE(S): Sperm viability and motility, follicular fluid and calcium ionophore-induced acrosome reactions, protein tyrosine phosphorylation, expression of D-mannose binding sites, and ability of sperm to interact with zona pellucida. RESULT(S): Exposure of sperm to peritoneal fluid for up to 6 hours did not affect sperm viability or motility. Unlike follicular fluid, peritoneal fluid did not induce the acrosome reaction. Moreover, incubation of sperm with > or =20% v/v peritoneal fluid for 1 hour prevented the follicular fluid and the ionophore-induced acrosome reaction. Although treatment with peritoneal fluid allowed protein tyrosine phosphorylation during capacitation, it resulted in a significant decrease in the expression of D-mannose binding sites and sperm-zona pellucida binding. CONCLUSION(S): Peritoneal fluid maintains sperm survival and decreases sperm ability to respond to inducers of the acrosome reaction and bind to the zona pellucida in vitro, indicating that this fluid might modulate sperm function in vivo.     

Follicular fluid enhances sperm attraction and its motility in human

Purpose: Follicular fluid has a pivotal effect on motility and chemotaxis of spermatozoa for successful fertilization. The effect of human follicular fluid (hFF) and progesterone on attraction and motility of spermatozoa were investigated using simplified capillary assays. Methods: Capillary tubes loaded with hFF, modified human tubal fluid (m-hTF), or m-hTF supplemented with progesterone, respectively, were used for assessments of attraction and motility of spermatozoa following culture at various time intervals. Results: Number and motile ratio of spermatozoa in the tubes loaded with hFF were significantly (P < .05) higher than those with m-hTF. In the tubes loaded with m-hTF, m-hTF supplemented with progesterone, and hFF, the attracted number of spermatozoa were 34 × 10⁵, 131 × 10⁵, and 108 × 10⁵, and motile ratio of spermatozoa was 37, 48, and 82%, respectively. Conclusions: We conclude that hFF clearly plays a crucial role in enhancing attraction and motility of spermatozoa, and progesterone has strong effect on attraction of spermatozoa.     

Spontaneous and follicular fluid-induced acrosome reaction in sperm samples from in vitro fertilizing and nonfertilizing normozoospermic patients

Purpose: One of the most challenging and intriguing groups of infertile patients is that of normozoospermic men who have repeatedly not achieved fertilization in vitro. These cases probably present a wide range of gamete disorders manifested at different stages of the fertilization process. The occurrence of spontaneous and follicular fluid (FF)-induced acrosome reactions (ARs) in in vitro fertilizing and nonfertilizing specimens from normozoospermic IVF patients was assessed, and the effect of Percoll and cryopreservation on the incidence of ARs was evaluated. Methods: Semen samples from 62 normozoospermic (15 in vitro nonfertilizing and 47 fertilizing) patients were analyzed. Spermatozoa were double-stained with FITC-conjugated Pissum sativumlectin, to assess their acrosomal status, and propidium iodide, to evaluate their vitality, using flow cytometry analysis. Results: A lower average of AR incidence was observed in the nonfertilizing than in the fertilizing group with all treatments. Both groups exhibited an increase in the proportion of ARs following incubation with FF. This rise was most prominent when Percoll-separated fractions were used (15.8 and 25.6% AR in the nonfertilizing and fertilizing groups, respectively). Thawed cryopreserved and fresh fertilizing samples exhibited similar AR rates. Conclusions: That normozoospermic recurrently nonfertilizing compared to fertilizing semen samples have a lower capacity to undergo ARs is suggested.     

Sperm immobilized before intracytoplasmic sperm injection undergo ultrastructural damage and acrosomal disruption

The aim of this study was to describe morphologic sperm changes observed via scanning electron microscopy and the acrosomal status resulting from immobilization and micromanipulation. The manipulations made on sperm before intracytoplasmic sperm injection were the possible cause of sperm ultrastructural damages and could trigger an acrosome reaction.     

Effect of paternal age on human sperm chromosomes

Objective: To investigate whether increased age alters the frequency and type of chromosomal anomalies in human spermatozoa.

Design: Semen specimens were collected from donors via masturbation; cytogenetic studies were performed on sperm chromosomes after heterologous (human-hamster) in vitro fertilization.

Setting: Cytogenetics Laboratory, Genetics Department, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil.

Patients(s): Seven men ages 59–74 (older group) and five men ages 23–39 (control group).

Main Outcome Measure(s): Frequency and types of chromosomal anomalies in older and control group donors.

Result(s): The frequency of numerical and structural aberrations (acentric fragments and complex radial figures) was significantly greater in chromosomes of older donors when compared with those of the control group.

Conclusion(s): The higher frequency of sperm chromosome aberrations in older men was mainly a result of increased nondisjunction, acentric fragments, and complex radial figures.     

Seasonal changes in human sperm chromatin condensation

Purpose: The aim of this study was to investigate possible seasonal changes in human sperm parameters, especially chromatin condensation. Method: In a first run, 3155 patients attending the andrological outpatient clinic at the Centre of Dermatology and Andrology at Justus Liebig University, Giessen, Germany, from January 1992 to October 1995 were examined for sperm count, motility, vitality, and chromatin condensation. Results: The respective results were correlated according to season. Significant seasonal changes were observed in chromatin condensation and sperm count, with mean maximum values (for chromatin condensation and sperm count) of 86.24% aniline blue-negative spermatozoa in January and 68.75 × 10⁶mL^–1 in April. To confirm the observation of seasonal changes in sperm chromatin condensation in Germany on the Southern Hemisphere, 179 patients attending the Reproductive Biology Unit at Tygerberg Hospital, Tygerberg, South Africa, were examined by means of the aniline blue stain from April 1999 to April 2000. For chromatin condensation, a significant seasonal change shifted by 4–5 months was observed on the Southern Hemisphere. However, no seasonal variations could be found for the sperm count. Conclusions: Our results clearly demonstrate seasonal changes in sperm count and chromatin condensation. In contrast, no circannual relation was observed for motility and vitality.     

Effect of fatty acids on boar sperm motility viability and acrosome reaction

Aim  The present study was undertaken to determine which fatty acids improve motility viability and increase acrosome reaction (AR) in boar spermatozoa. Methods  Boar spermatozoa were washed, swum-up and incubated at 37°C for 4 h in TALP medium supplemented with myristic, palmitic, stearic, lignoceric, oleic, linoleic, arachidonic, docosahexaenoic and palmitoleic acid. Sperm motility, viability and AR were evaluated during 4 h of incubation. Results  Results show that oleic and linoleic acid significantly improved (P < 0.05) the motility and viability of boar spermatozoa. The AR was significantly improved (P < 0.05) by oleic and arachidonic acid in almost all incubation periods. When combinations of oleic, linoleic and arachidonic acid were studied for motility, viability and AR, it was found that oleic plus linoleic acid significantly increased (P < 0.05) motility, whereas arachidonic plus oleic acid significantly increased (P < 0.05) AR. Conclusion  Unsaturated fatty acids, especially arachidonic acid, can improve boar sperm motility and AR. A combination of arachidonic and oleic acid is important for inducing boar sperm AR.     

Effect of vaginal probiotic lactobacilli on in vitro-induced sperm lipid peroxidation and its impact on sperm motility and viability

A combination of three selected strains of lactobacilli (Lactobacillus brevis [CD2], L. salivarius [FV2], and L. plantarum [FV9]), whose effectiveness in treating bacterial vaginosis in the form of vaginal tablets has been reported recently, prevented sperm lipid peroxidation that was induced in vitro by a ferrous ion promoter, thus preserving sperm motility and viability. This finding suggests the potential of vaginal probiotic lactobacilli for protecting human spermatozoa from radical oxygen species in the presence of vaginal disorders, thereby improving the fertilization potential of the female host.     

Effect of storage temperature on sperm cryopreservation

Objective: To evaluate the influence of cryopreservation temperature on human sperm motility and morphology.

Design: Controlled study, investigator was blinded to the type of cryopreservation.

Setting: University-based andrology laboratory.

Patient(s): Sixteen semen samples with normal motility and sperm count from men after a fertility work up.

Intervention(s): Semen aliquots were either stored in a mechanical freezer at −70°C or in liquid nitrogen at −196°C for 7 days or 3 months. Test yolk buffer was used as a cryoprotectant. With use of a programmable freezing unit, all samples were cooled at a controlled rate.

Main Outcome Measure(s): Sperm motility and morphology.

Result(s): After 7 days of cryopreservation, there was a greater decrease in sperm motility among specimens maintained at −70°C than among those maintained at −196°C (47% versus 39% decrease). The difference in sperm motility was even greater after 3 months of cryopreservation (72% versus 39% decrease). No difference in postthaw sperm morphology was detected among sperm preserved at −70°C versus −196°C.

Conclusion(s): Sperm cryopreservation at −196°C is superior to cryopreservation at −70°C. Sperm can be stored at −70°C for a short period of time with a relatively modest loss of motility.     

Fusion of prostasomes to human spermatozoa stimulates the acrosome reaction

OBJECTIVE: To determine the effect of the fusion of prostasomes to spermatozoa on the acrosome reaction. DESIGN: In vitro study of human spermatozoa. SETTING: Healthy volunteers in an academic research environment. PATIENT(S): Healthy volunteer men, 25 to 35 years old. INTERVENTION(S): Human semen was fractionated into spermatozoa and prostasomes. Fusion of prostasome to spermatozoa was performed at pH 5.5. Progesterone (1 microM) was added when required. MAIN OUTCOME MEASURE(S): Evaluation of the acrosome reaction by fluorescence microscopy. RESULTS(S): The percentage of spontaneously acrosome-reacted cells was very low unless the Ca(2+)-ionophore A 23187 was added. The treatment of spermatozoa with 1 microM of progesterone scarcely affected the acrosome reaction; a pretreatment in conditions permitting fusion increased it. The addition of progesterone to prostasome-fused spermatozoa further increased the extent of the acrosome reaction. CONCLUSION(S): The H(+)-dependent fusion with prostasomes makes spermatozoa more sensitive to the effect of progesterone on acrosome-reaction induction.     

Specificity of human sperm antigens solubilized by N-cetylpyridinium chloride

To test the specificity of sperm fractions solubilized by N-acetylpyridinium chloride, human spermatozoa were washed 3 times with phosphate-buffered saline and resuspended in 5 ml of the solubilizing solution. The mixture was ultrasonated for 5 minutes in ice water and then centrifuged for 20 minutes. The supernatant was used for specificity tests. Details of the techniques used are given. Using N-acetylpyridinium chloride, a medium polar cationic detergent, 8 antigenic sperm fractions were extracted from ultrasonated spermatozoa run against human sperm-agglutinating sera in 2-dimensional immuno-electrophoresis. In runs against sperm-immobilizing sera, 9 sperm fractions were visualized. In runs against rabbit antihuman spermatozoa serum 11 fractions were found. Most of the antigenic fractions that were separated showed cross-reactivity with allogenic or xenogenic tissues. Cross-reactivity with human and animal organs was observed in many instances. Sperm as well as species specificities were found in some fractions. None of the fractions found could be detected on the surface membranes of intact human spermatozoa. Specific reactivity against a sperm-immobilizing serum was detected but no specific activity against a sperm-agglutinating serum was found. The fraction designated Gi is considered to be the "immobilizing" antigen. The origin of the antigens detectable on the surface membranes of intact spermatozoa was not determined.     

Effect of reactive oxygen species produced by spermatozoa and leukocytes on sperm functions in non-leukocytospermic patients

OBJECTIVE: To investigate whether there is an impact of different sources of reactive oxygen species (ROS) on sperm functions. DESIGN: Prospective study. SETTING: Patients at the Center for Dermatology and Andrology, Giessen, Germany. PATIENT(S): Semen collected from 63 randomly collected patients attending the IVF unit of the University of Giessen, Germany. INTERVENTION(S): Only patients with nonleukocytospermia were included in this study. MAIN OUTCOME MEASURE(S): Sperm count and motility before and after sperm separation by swim-up, morphology, DNA fragmentation, and extrinsic (by leukocytes) and intrinsic ROS production (by spermatozoa) were evaluated. RESULT(S): Leukocytes correlated significantly with extrinsic ROS production (r = 0.576), but markedly less with intrinsic ROS production (r = 0.296). Sperm count, morphology, and motility in the ejaculate were markedly more affected by extrinsic than by intrinsic ROS. The DNA fragmentation was strongly positively correlated with intrinsic ROS production, whereas this correlation was weaker for extrinsic ROS production. No correlation was found between DNA fragmentation and the number of leukocytes, whereas the correlations with motility in the ejaculate and the motile sperm count after swim-up were highly significant. Moreover, significant differences were observed for extrinsic and intrinsic ROS production between groups of patients having a high (> or = 1 x 10(6)/mL) and a low number (<1 x 10(6)/mL) of leukocytes in the ejaculate. CONCLUSION(S): The origin of ROS seems to have an influence on the site of the damage. Because leukocyte counts <1 x 10(6)/mL caused a significant decrease of motility and DNA integrity, the threshold given by the World Health Organization (WHO) should be re-evaluated.     

Comparative genomic hybridization analysis of sperm DNA apoptosis after exposure to heat shock

Purpose: A DNA disc chip assay based on comparative genomic hybridization (CGH) was developed to measure sperm DNA integrity. The objective was to correlate DNA integrity of heat-treated sperm with the sperm capacitation index (CI) determined from the sperm penetration assay. Methods: Basic semen and kinematic parameters were measured (N = 6). Sperm were washed in two-layer colloid suspensions and split portions incubated at either 37°C (control) or 40°C for 4 h. Single-stranded DNA of heated sperm were stained in SYBR Gold and hybridized to bisbenzimide (Hoechst 33342) stained control DNA in a membrane disc. Fluorescent intensities of the discs were measured and correlation analyses with sperm parameters performed. Results: Sperm CI was positively correlated (R = 0.737) with sperm DNA integrity. Two populations of sperm could be discerned: low capacitating sperm that initiated apoptosis and high capacitating sperm unaffected by heat shock treatment. The remaining parameters were not related to sperm DNA stability. Conclusions: Fragile DNA were found in a population of sperm associated with poor capacitation characteristics and apoptosis was observed after heat treatment. The results suggested that sperm dysfunction might be due to apoptotic sperm DNA resulting from an elevated temperature in the surroundings. The data suggested that the second population of high capacitating sperm induced chaperones such as heat shock proteins hsp 70 to protect against apoptosis.     

The effects of lignocaine on human sperm motility

Objective The purpose of this study was to assess the motility of human sperm incubated with various concentrations of lignocaine.Methods Eleven semen samples with a sperm density 20 ×10 ⁶ ml and progressive motility 40% were prepared using a swim-up technique. Aliquots from each sample were incubated for 4 hr under capacitating conditions with lignocaine concentrations of 100, 10, 1, and 0.1 µg/ml and without additional lignocaine as a control. Digital computerized motion analysis was performed on all samples at 1, 2, and 4 hr after the addition of lignocaine. Results After 2 hr of incubation a significant (P <0.05) increase in the percentage of sperm with a curvilinear velocity >100 µm/sec was observed in those samples incubated with 100 µg/ml lignocaine. This stimulatory effect was no longer apparent after a further 2 hr of incubation. No other significant changes were identified in any of the motility parameters examined. Conclusions No adverse effects on human sperm motility were identified during incubation with low concentrations of lignocaine. A transient stimulatory effect was observed at a lignocaine concentration of 100 µg/ml.     

Effects of human follicular fluid on spermatozoa that have been cocultured with human oviductal cells

Objective: To investigate the sequential effects of human oviductal cells and human follicular fluid (hFF) on various sperm functions.Design: Laboratory experimental study.Setting: University gynecology unit.Patient(s): Fallopian tubes were from patients undergoing tubal ligation or hysterectomy. Semen was from men attending the subfertility clinics.Intervention(s): Spermatozoa were treated with [1] 6 hours in Earle’s balanced salt solution (EBSS-BSA; control); [2] 5 hours in EBSS-BSA and 1 hour with hFF (hFF); [3] 5 hours with oviductal cells and 1 hour in EBSS-BSA (coculture); and [4] 5 hours with oviductal cells and 1 hour with hFF (sequential).Main Outcome Measure(s): Motility, acrosome reaction, zona binding, and oocyte fusion.Result(s): Groups II and III spermatozoa had similar motility and were better than that of group I. Group IV displayed higher motility parameters than the other groups. Human follicular fluid induced acrosome reaction. The incidence of acrosome reaction in group IV was significantly lower than that in group II. Group III did not affect the acrosome reaction. Spermatozoa in groups II–IV had lower zona binding capacity than those in group I. Human follicular fluid stimulated oocyte penetration, whereas oviductal cells suppressed this effect of hFF.Conclusion(s): Oviductal cells maintained the fertilizing capacity of spermatozoa, whereas hFF facilitated the fertilization process of oviductal spermatozoa.