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Sperm motility analysis using multi-exposure photography (MEP): validity of the method with normal and abnormal patterns.

Spermatozoon motility is an important semen parameter that can be correlated with fertilizing capacity. It can be evaluated by subjective or objective methods such as the multiple exposure photography technique (MEP). This technique was: (1) validated in terms of accuracy and sensitivity, and (2) used to investigate the possibility of differentiating patient subgroups on the basis of sperm motility. The method was shown to be accurate. The within and between assay variation coefficients were less than 6% and 17% respectively. Maximal sensitivity required 100 spermatozoa, since coefficients of variation were high when counting smaller numbers. A significant difference between fertile, normozoospermic and asthenozoospermic groups on the basis of the population pattern was found in a retrospective study. Moreover, the asthenozoospermic group could be divided into three subgroups, depending on whether one, two, or three motility variables were altered.

Humans↗

A biomarker approach to endocrine disruption in flounder--estrogen receptors, hepatocyte proliferation, and sperm motility.

Three different physiological parameters were assessed to determine their potential for serving as biomarkers to predict abnormally elevated vitellogenin (VTG) production in male and immature flounder. Whereas abnormally elevated mean VTG plasma concentrations clearly distinguished the Mersey and Dee flounder studied, the results showed no significant differences in estrogen receptor binding capacity or binding affinity between the two groups. Hepatocyte proliferation was not found to be a "biomarker of effect" that could specifically be used to assess an increase in VTG-related proliferation. Nevertheless, immunohistochemical staining for proliferating cell nuclear antigen did show a significantly higher proliferative activity in the livers of Mersey flounder than that in Dee flounder. Sperm motility also was not found to be a biomarker of effect linked to an abnormal elevation of VTG plasma concentration. The results (higher sperm quality in terms of motility in Mersey flounder) were unexpected but interesting.

Animals↗

Differences in osmolality, pH, buffering capacity, superoxide dismutase and maintenance of sperm motility in human ejaculates according to the degree of coagulation.

The purpose of this study was to compare various seminal plasma parameters in fresh human ejaculates exhibiting different amounts of coagulum. Poorly coagulating samples demonstrated significantly lower osmolality and buffering capacity but had a higher pH than did samples with good coagulation. However, no correlation was obtained between the activity of superoxide dismutase and the amount of coagulum. Eight hours after ejaculation, sperm motility had decreased by 15 and 80% in samples with good and poor coagulation, respectively. It is suggested that subfertility may be associated with poor coagulation of ejaculates.

Adult↗

Extraction of sp18 family membrane proteins on posterior head of bull sperm and the effect of anti-sp18 IgG on sperm motility and murine in vitro fertilization.

Bovine sperm heads were separated via ultrasonic treatment and centrifugation. Anti-bull sperm IgG was produced by immunizing rabbits with acrosome-reacted bull sperm heads. SDS PAGE patterns revealed that the main membrane proteins on acrosome-reacted bull sperm head were sp18 family, including 18, 16, and 14 kD, which represented about 64% of the total membrane proteins in bull sperm. Indirect immunofluorescence shown sp18 antigens primarily distributed in postacrosomal and proximal tail regions. Western blot analysis revealed that the anti-bull sperm IgG reacted with sp18 antigens in acrosome-reacted bull sperm head and bull seminal plasma. Anti-bull sperm IgG also reacted with 14, 16, 18, 42, 57 and 60 kD proteins in fresh bull, mouse and rabbit sperm. Anti-sp18 IgG caused agglutination of bull and rabbit sperm, but had no effect on murine sperm. In murine in vitro fertilization trials, preincubating capacitated sperm with 0.364 mg/ml of anti-sp18 IgG resulted in a decrease in the fertilization rate from 75.6% in the controls to 50.8% in the experimental groups (p < 0.001).

Acrosome↗

Dynamic aspects concerned with the mechanism of separating motile sperm from nonmotile sperm, leukocytes, and debris with the use of high-density Percoll gradients.

OBJECTIVE: To investigate the dynamics and the efficacy of separating motile from nonmotile sperm with the use of Percoll gradients as a function of centrifugation time. DESIGN: In vitro laboratory experiments on human sperm. SETTING: Male Infertility Institute. PATIENT(S): Fresh sperm specimens collected from donors and patients. INTERVENTION(S): Aliquots of sperm specimens, spread on top of 40% and 80% Percoll layers in test tubes, were centrifuged for 5-60 minutes. MAIN OUTCOME MEASURE(S): After centrifugation, samples were aspirated from the bottom, interface, and top layers for microscopic analysis. RESULT(S): Within 5 minutes, only fast-moving sperm reached the bottom as a result of both centrifugation force and their own tail movements. From 10 minutes onward, nonmotile sperm, slowed down by Percoll, joined them and gradually reversed the separation process. Leukocytes, debris, and abnormal sperm remained in the upper levels and never reached the bottom. CONCLUSION(S): Separation of motile sperm with the use of Percoll gradients is strongly dependent on centrifugation time. The separation is the gap formed between down-swimming sperm, accelerated by centrifugation, and nonmotile sperm, decelerated by Percoll. This gap gradually disappears with extended spinning.

Cell Separation↗

Laser light-scattering study of the effect of progesterone on sperm motility.

It is known that progestins can induce in the secretory cells of the cervix the excretion of a mucus that is highly viscuos, scanty, and impenetrable to spermatozoa. Mucus of this type is similar to that excreted during the luteal phase of the normal human menstrual cycle and the cow estrous cycle. It is a natural sequence to ask the question, do progestins also have a direct effect on sperm motility? With dynamic laser light-scattering we measured the motility of freshly washed human spermatozoa and of spermatozoa in the presence of a progesterone, both in terms of their swimming speed distribution as expressed in the spectrum of scattered light. The swimming speed was significantly reduced when the concentration of progesterone was three orders of magnitude greater than that of the physiologic level. This finding confirms the finding in earlier biochemical studies that progesterone has a distinct spermiostatic effect. We suggest this answer to the above question: progestin-releasing contraceptive devices may act on spermatozoa directly as well as in the secretory cells of the cervix.

Animals↗

Mice deficient for soluble adenylyl cyclase are infertile because of a severe sperm-motility defect.

To acquire the ability to fertilize, spermatozoa undergo complex, but at present poorly understood, activation processes. The intracellular rise of cAMP produced by the bicarbonate-dependent soluble adenylyl cyclase (sAC) has been suggested to play a central role in initiating the cascade of the events that culminates in spermatozoa maturation. Here, we show that targeted disruption of the sAC gene does not affect spermatogenesis but dramatically impairs sperm motility, leading to male sterility. sAC mutant spermatozoa are characterized by a total loss of forward motility and are unable to fertilize oocytes in vitro. Interestingly, motility in sAC mutant spermatozoa can be restored on cAMP loading, indicating that the motility defect observed is not caused by a structural defect. We, therefore, conclude that sAC plays an essential and nonredundant role in the activation of the signaling cascade controlling motility and, therefore, in fertility. The crucial role of sAC in fertility and the absence of any other obvious pathological abnormalities in sAC-deficient mice may provide a rationale for developing inhibitors that can be applied as a human male contraceptive.

Adenylyl Cyclases↗

Cryopreservation of tench, Tinca tinca, sperm: Sperm motility and hatching success of embryos.

The aim of the present study was to elaborate cryopreservation methods for ex situ conservation of tench. Success of cryopreservation was tested during two series of experiments. The first set of experiments studied the effects of two types of cryoprotectants (DMSO and a combination of DMSO with propanediol at ratio 1:1) at concentrations of 8 and 10% and three different equilibration times in two different immobilization solutions (IS) (Kurokura 180 and Kurokura) before freezing (0.0, 2.0 and 4.0h after T(0)). The K4 cooling programme was used to freeze 1ml of cryoextended sperm using 1.8ml cryotubes. Main monitored parameter was hatching rate after using of cryopreserved sperm. The second set of experiments studied the volume effect of 0.5, 1 and 5ml straws and compared these with 1.8ml cryotubes as well as the effect of the cooling programme (K4 and L1). Following the results of the first study, a combination of DMSO and propanediol (ratio 1:1) at concentration of 10% was added to extended sperm in Kurokura 180 IS. Main monitored parameter was hatching rate after using cryopreserved sperm, supplementary parameters were sperm velocity and motility percentage assessed at 10s post-activation. Sperm was collected directly into IS and stored at 4 degrees C for 2.5h. Thereafter were sperm samples pooled, equlibred in IS (first set of experiments) or directly mixed with cryoprotectants (DMSO or a mixture of DMSO with propanediol at ratio 1:1) and transferred to 1.8ml cryotubes or straws (0.5, 1 and 5ml). Then the cryotubes/straws were directly transferred to pre-programmed PLANER Kryo 10 series III and cooled using two different cooling programmes including a slow cooling programme (a) named K4 (from +4 to -9 degrees C at a rate of 4 degrees Cmin(-1) and then from -9 to -80 degrees C at a rate of 11 degrees Cmin(-1)) and a rapid cooling programme (b) named L1 (directly from +4 to -80 degrees C at a rate of 20 degrees Cmin(-1)). Both slow (K4) and rapid (L1) cooled samples were held 6min at -80 degrees C. Finally, samples were transferred into liquid N(2). The frozen spermatozoa were thawed in a water bath (40 degrees C) according to the frozen volume and checked for fertilization and hatching rates. Percentage of sperm motility and sperm velocity were measured using video recorded frames. ANOVA showed a significant influence of frozen and fresh sperm in all treatments. The hatching rates of 33.8% were obtained when sperm was equilibrated for 0h before freezing in IS of Kurokura 180 and frozen with a 10% of mixture 1:1 of DMSO and propanediol into straws of 5ml and cooled using program L1. The velocity of frozen-thawed spermatozoa ranged from 31 to 46microms(-1) and in post-thawed sperm was not significantly different according to frozen sperm volume, but a higher velocity was obtained when sperm was fast frozen using programme L1. A large volume of frozen sperm could reveal the best procedure for freezing, but also for simulating methods of artificial propagation for future practical use of frozen tench sperm at a large scale.

Animals↗

Effects of lipid peroxide formation in fowl semen on sperm motility, ATP content and fertilizing ability.

The ability of samples of semen from individual male fowl to form the products of lipid peroxidation during 5 h aerobic incubation at 40 degrees C varied between 0 and 8 nmol malonaldehyde/10(9) spermatozoa. Formation of higher concentrations of malonaldehyde was associated with a partial or complete loss of fertilizing ability whilst the fertilizing ability of samples producing low or negligible concentrations of malonaldehyde remained unimpaired. The semen of birds which showed a tendency to form high concentrations of malonaldehyde was not readily identifiable as abnormal by assessment of sperm motility, morphology or ATP content. Nor was the loss of fertilizing ability during aerobic incubation associated with an obvious change in these characteristics.

Adenosine Triphosphate↗

Computer assisted sperm motility analysis at the moment of induced pregnancy during gonadotropin treatment for hypogonadotropic hypogonadism.

Two azoospermic patients with isolated hypogonadotropic hypogonadism (IHH) no. 1,24-year-old, and no. 2,31-year-old), never treated before, were given gonadotropin therapy (GT), (FSH, 225 IU/week, and HCG, 4000 IU/week) and followed up monthly for sperm characteristics. After 6 (no. 1) and 9 (no. 2) months of treatment, immotile spermatozoa appeared in the semen. Two months later motile spermatozoa also appeared. A motile sperm concentration sufficiently high for computer motion analysis (CASA) was achieved 1 month later 9th and 12th month, respectively. At the 13th (no. 1) and 17th (no. 2) month of GT, CASA showed a small population of spermatozoa with a very good motility pattern. In the next ovulatory cycle the patients' partners became pregnant.

Adult↗

The postwash total progressively motile sperm cell count is a reliable predictor of total fertilization failure during in vitro fertilization treatment.

OBJECTIVE: To predict the chance of total fertilization failure (TFF) before the day of ovum pickup with known semen and female variables. DESIGN: A statistical model was constructed to predict TFF by retrospective analysis (2,366 couples) and subsequently tested on a new IVF population (917 couples). SETTING: Academic tertiary referral center. PATIENT(S): Three thousand three hundred eighty-three couples who underwent an IVF-ET treatment. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The ability to predict the probability of TFF in IVF. RESULT(S): Two variables-postwash total progressively motile sperm cell count (postwash TPMC) and number of follicles-were found to be significant. Taking a probability of 25% as an acceptable risk of TFF, we calculated that a postwash TPMC of <1.1 x 10(6) cells results in a risk of TFF of >25%. Low responders (<4 follicles) needed a postwash TPMC of >2.2 x 10(6) cells to reduce the risk of TFF to <25%. High responders (>15 follicles) needed only 0.35 x 10(6) postwash progressively motile spermatozoa. CONCLUSION(S): When postwash TPMC and number of follicles are known and an unacceptable TFF outcome is expected, one can propose an ICSI procedure a few days before the day of ovum pickup.

Female↗

The activating effects of bicarbonate on sperm motility and respiration at ejaculation.

Mature porcine sperm preserved in the cauda epididymis are quiescent. At ejaculation, they are mixed with the seminal vesicle fluid containing HCO3- and are rapidly activated. The role of HCO3- on the sperm activation process at ejaculation was studied in vitro. HCO3- quickly increased the motility, respiration rate and cAMP content of the porcine epididymal sperm. The extent of activation was proportional to the pCO2 in the medium. The activating effect of HCO3- on the motility was observed even in the absence of fructose as well as in the presence of KCN. 8-Bromoadenosine 3',5'-cyclic monophosphate and theophylline showed similar activating effects to that of HCO3-. However, HCO3(-)-free seminal plasma, Ca2+, amino acids, intermediates of the Krebs cycle, substrates of respiration and increases in the intracellular pH, extracellular pH or ionic strength of the medium had no effect. Fructose sustained the active state of the sperm and gradually increased both the motility and respiration rate when the dose of HCO3- was low. The anion channel blocker enhanced the activating effect of HCO3-. These results suggest that, upon ejaculation, HCO3- is a unique activator in vivo which makes the quiescent sperm motile via the HCO3(-)-adenylate cyclase-cAMP system, to which an endogenous HCO3- derived from metabolic CO2 may be related.

Animals↗

Fowl (Gallus domesticus) sperm motility depends upon mitochondrial calcium cycling driven by extracellular sodium.

A relationship between extracellular Ca(+2), fowl sperm phospholipase A2 activity, long-chain acylcarnitine content, and motility was demonstrated in previous work. Sperm motility appeared to depend upon Na+-dependent Ca(+2) cycling when sperm were incubated at body temperature without glucose. In the present work, motility decreased as a function of time when sperm were incubated in 2 mM Ca(+2) prepared with either buffered isotonic sucrose or LiCl. However, this effect was less pronounced in the case of LiCl. The sparing effect of Li+ was attributed to the mitochondrial Na+/Ca(+2) exchanger. Motile concentration decreased exponentially in response to micromolar concentrations of CGP 37157, a specific inhibitor of the mitochondrial Na+/Ca(+2) exchanger. KB-R7943 mesylate, an inhibitor of the reverse mode of the Na+/Ca(+2) exchanger, prevented re-initiation of motility when exogenous Ca(+2) was added to sperm rendered immotile by incubation with 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, a high-affinity Ca(+2) chelator. The presence of voltage-gated Ca(+2) channels was confirmed by the effect of nifedipine on motile concentration. Neither motile concentration nor straight line velocity was affected by either ouabain or orthovanadate, which inhibit Na+-K+ ATPase and Ca(+2)-ATPase, respectively. In summary, we infer that 1) fowl sperm motility is dependent upon extracellular Ca(+2) cycling through mitochondria; 2) such cycling is dependent upon extracellular Na+; and 3) fowl sperm conserve ATP by moving neither Na+ nor Ca(+2) by active transport. Understanding the relationship between mitochondrial Ca(+2) cycling and ATP production may be applicable to long-term semen storage.

Animals↗

Zona pellucida binding improvement effect of different sperm preparation techniques is not related to changes in sperm motility characterizations.

OBJECTIVE: To study the relation between changes in sperm motion characteristics and sperm binding capacity to the zona pellucida (ZP) after different sperm preparation techniques. DESIGN: Prospective study. SETTING: Andrology laboratory at the Lis Maternity Hospital. PATIENT(S): Sixty-three infertile men. INTERVENTION(S): In vitro preparation of 57 sperm samples by pentoxifylline (PTX), 38 samples by SpermPrep I (SP1), and 27 samples by mini-Percoll (mPER). Sperm preparation was evaluated in comparison with the control washing technique. MAIN OUTCOME MEASURE(S): Motility variables and binding capacity. RESULT(S): Binding capacity after PTX, SP1, and mPER preparation methods was significantly higher compared with the control medium preparation. Computer-assisted sperm analysis (CASA) of sperm motility revealed a significant increase in a number of variables after the different treatments. A statistically significant but low correlation was observed between sperm binding capacity after control swim-up preparation and VAP (r = 0.27) as well as with VSL (r = 0.30). No significant correlations were found between the changes in the CASA and the improved sperm binding to the ZP. CONCLUSION(S): The two biologic effects of binding capacity enhancement and motility features improvement are not related. Furthermore, because the binding assay is highly correlated with fertilization capacity, these alterations in motility, as evidenced by CASA, probably have a minor effect on fertilization in vitro.

Electronic Data Processing↗

A comparison of subjective measurements of human sperm motility and viability with two live-dead staining techniques.

Two commonly used live-dead stains (eosin-nigrosin (EN) and eosin-opal blue (EOB)) were compared with the estimated active spermatozoa in semen samples from patients attending an infertility service. Twenty-eight semen samples were analyzed throughout the day of their collection by estimating the number of active spermatozoa and by staining a portion of the incubated sample (37 degrees C) with each stain. The samples were analyzed 30, 60, 120, 240, and 360 minutes after the initial collection. At 30 minutes there were no significant differences between the estimated values and those of either stain. The slope of the EN stain closely paralleled that of the estimated measurements throughout the remainder of the time periods, while the EOB slope was somewhat steeper. Repeatability of values for semen samples obtained on different days was generally good for each stain. A comparison of semen samples from 85 patients, 30 minutes after collection, showed no significant differences between the numbers of estimated active sperm and the percentage live using the EN stain. Studies of 132 semen samples using the EN stain showed a positive correlation with the over-all quality of sperm motility. The results indicate that there is a role for live-dead staining in assessing semen quality.

Cell Biology↗

The effects of human skin fibroblast monolayers on human sperm motility and mouse zygote development.

A new system for co-culture in human in-vitro fertilization (IVF), using human skin fibroblasts, is described and tested pre-clinically. The first test involved the development of 1-cell mouse embryos which exhibit the 2-cell developmental block in vitro. Passage through this block (pb1-ratio) was determined by the ratio of compacted morula stages on day 4 of incubation. For nine human skin cell lines tested (fetal, neonatal and adult), the pb1-ratio was approximately 0.45 (0.07 in culture medium alone; P less than 0.0005). At the compacted morula stage, a second developmental block was observed. The ratio of passing this block (pb2-ratio) was 0.70 +/- 0.09 on skin fibroblasts obtained from fetal or neonatal tissue. On fibroblasts from adult patients the pb2-ratio was 0.30 +/- 0.04 (P less than 0.0005). The second test examined the influence of skin fibroblasts from fetal or neonatal tissue on human sperm motility. After 24 h of incubation, all skin cell lines had a positive influence (P less than 0.01) on the percentage motility compared to culture medium alone. The curvilinear velocity was not significantly increased. From the results we conclude that (i) human skin fibroblasts (especially from fetal tissue) have a positive influence on the development of mouse embryos in vitro, (ii) there is a positive influence of human skin fibroblasts on the percentage motility of human spermatozoa, and (iii) a clinical trial of co-culture with human skin fibroblasts can be justified.

Animals↗

Validation of a single-step procedure for the objective assessment of sperm motility characteristics.

A relatively cheap method is described for the objective assessment of sperm concentration and motility characteristics. The method uses a digitizing tablet with cursor, a micro-computer and a phase-contrast or dark-field microscope equipped with a drawing tube. With this technique the following are accurately assessed: sperm concentration, percentage motility, motility grading, concentration of grade a motile spermatozoa, sperm velocity, linear velocity, linearity and angularity. The data are acquired in less than 5 min. Validation studies reveal this method to be accurate, reproducible (coefficient of variation of motility characteristics = less than 7%) and clinically useful.

Algorithms↗