Modifiable and non-modifiable risk factors for poor sperm morphology.

STUDY QUESTION: Are common lifestyle factors associated with poor sperm morphology? SUMMARY ANSWER: Common lifestyle choices make little contribution to the risk of poor sperm morphology. WHAT IS KNOWN ALREADY: Although many studies have claimed that men's lifestyle can affect sperm morphology, the evidence is weak with studies often underpowered and poorly controlled. STUDY DESIGN, SIZE, DURATION: Unmatched case-referent study with 318 cases and 1652 referents. Cases had poor sperm morphology (<4% normal forms based on 200 sperm assessed). Exposures included self-reported exposures to alcohol, tobacco, recreational drugs as well as occupational and other factors. PARTICIPANTS/MATERIALS, SETTING, METHODS: Eligible men, aged 18 years or above, were part of a couple who had been attempting conception without success following at least 12 months of unprotected intercourse and also had no knowledge of any semen analysis before being enrolled. They were recruited from 14 fertility clinics across the UK during a 37-month period from 1 January 1999. MAIN RESULTS AND THE ROLE OF CHANCE: Risk factors for poor sperm morphology, after adjustment for centre and other risk factors, included: (i) sample production in summer [odds ratio (OR) = 1.99, 95% confidence interval (CI) 1.43-2.72]; and (ii) use of cannabis in the 3 months prior to sample collection in men aged ≤30 years (OR = 1.94, 95% CI 1.05-3.60). Men who produced a sample after 6 days abstinence were less likely to be a case (OR = 0.64, 95% CI 0.43-0.95). No significant association was found with body mass index, type of underwear, smoking or alcohol consumption or having a history of mumps. This suggests that an individual's lifestyle has very little impact on sperm morphology and that delaying assisted conception to make changes to lifestyle is unlikely to enhance conception. LIMITATIONS, REASONS FOR CAUTION: Data were collected blind to outcome and so exposure information should not have been subject to reporting bias. Less than half the men attending the various clinics met the study eligibility criteria and among those who did, two out of five did not participate. It is not known whether any of those who refused to take part did so because they had a lifestyle which they did not want subjected to investigation. Although the power of the study was sufficient to draw conclusions about common lifestyle choices, this is not the case for exposures that were rare or poorly reported. WIDER IMPLICATIONS OF THE FINDINGS: All participating clinics saw patients at no cost (under the UK National Health Service) and the study population may differ from those in countries without such provision. Even within the UK, low-income couples may choose not to undertake any investigation believing that they would subsequently be unable to afford treatment. Since a computer performed the measurements of sperm morphology, these results may not be comparable with studies where sperm morphology was assessed by other methods. STUDY FUNDING/COMPETING INTERESTS: The study was funded by the UK Health and Safety Executive, the UK Department of Environment, Transport and the Regions, the UK Department of Health (Grant Code DoH 1216760) and the European Chemical Industry Council (grant code EMSG19). No competing interests declared.

Modifiable and non-modifiable risk factors for poor semen quality: a case-referent study.

STUDY QUESTION: Are common lifestyle factors associated with low-motile sperm concentration (MSC)? SUMMARY ANSWER: Common lifestyle choices make little contribution to the risk of low MSC. WHAT IS KNOWN AND WHAT THIS PAPER ADDS: Reviews of male subfertility often highlight how aspects of men's adult lifestyle can significantly increase their risk of subfertility but the strength of supporting evidence is weak. In this study, although low MSC was associated with a history of testicular surgery, being in manual work, not wearing loose underwear and black ethnicity, no relation was found to consumption of alcohol, use of tobacco or recreational drugs or high body mass index (BMI). These results suggest that delaying assisted conception to make changes to lifestyle is unlikely to enhance conception. DESIGN: Unmatched case-referent study with 939 cases and 1310 referents. Cases had a low-MSC relative to the time since last ejaculation (<12 × 10(6) for 3 days of abstinence). Exposures included self-reported exposures to alcohol, tobacco, recreational drugs as well as occupational and other factors. PARTICIPANTS AND SETTING: Eligible men, aged 18 or above, were part of a couple who had been attempting conception without success following at least 12 months of unprotected intercourse and also had no knowledge of any semen analysis. They were recruited from 14 fertility clinics across the UK during a 37-month period from 1 January 1999. MAIN RESULTS AND THE ROLE OF CHANCE: Risk factors for low MSC, after adjustment for centre and confounding factors, included a history of testicular surgery [odds ratio = 2.39, 95% confidence interval (CI): 1.75, 3.28], being in manual work [odds ratio (OR) = 1.28, 95% CI: 1.07, 1.53] or not working (OR = 1.78, 95% CI: 1.22, 2.59) and having black ethnicity (OR = 1.99, 95% CI: 1.10, 3.63). Conversely, men who wore boxer shorts (OR = 0.76, 95% CI: 0.64, 0.92) or who had a previous conception (OR = 0.71, 95% CI: 0.60, 0.85) were less likely to be a case. No significant association was found with smoking and alcohol consumption, the use of recreational drugs, a high BMI or having a history of mumps or fever. BIAS, CONFOUNDING AND OTHER REASONS FOR CAUTION: Data were collected blind to outcome, and exposure information should not have been subject to reporting bias. Among men attending the various clinics less than half met the study eligibility criteria and among those who did, two out of five were not recruited. It is not known whether any of those who refused to take part did so because they had a lifestyle they did not want subjected to investigation. Although the power of the study was sufficient to draw conclusions about common lifestyle choices, it cannot comment on exposures that are perhaps rare and poorly reported: the finding that use of street drugs was unrelated to low MSC cannot be assumed to apply to all such drugs and all patterns of use. The case definition did not consider sperm morphology or sperm DNA integrity. GENERALIZABILITY TO OTHER POPULATIONS: All participating clinics saw patients at no cost (under the UK National Health Service) and the study population may differ from those in countries without such provision. Even within the UK, low-income couples may choose not to undertake any investigation believing that they would subsequently be unable to afford treatment.

Characterization of invasive trophoblasts generated from human embryonic stem cells.

BACKGROUND: Abnormal human embryo implantation leads to poor foetal development and miscarriage, or pre-eclampsia. Ethical and practical considerations concerning implantation limit its investigation, and it is often difficult to extrapolate findings in laboratory animals when implantation processes show diverse species differences. Therefore, it is important to develop new in vitro models to study the earliest events of human implantation. The aim of this study was to derive trophoblast cell lines from human embryonic stem cells (hESCs) by a robust protocol and co-culture of these cells with an established endometrial cell culture system to validate a model of trophoblast invasion at implantation. METHODS: Derivation of trophoblast cell lines from hESC lines was established by spontaneous and induced differentiation of embryoid bodies and by initial measurement of hCGß secretion by enzyme-linked immunosorbent assay and their phenotype investigated using gene- and protein-expression markers. Vesicles formed from an aggregating trophoblast were co-cultured with decidualized human endometrial stromal cells in hypoxic (2% oxygen) and normoxic (20% oxygen) environments. RESULTS: Derived villous cytotrophoblast cell (CTB) lines further differentiated to invasive, extra-villous CTBs. Eventually, cells lost their proliferative capacity, with some lines acquiring karyotypic changes, such as a gain in the X chromosome. Cell-invasion assays confirmed that the extra-villous CTBs were invasive and erosion of the endometrial stromal layer occurred, particularly under hypoxic conditions in vitro. CONCLUSIONS: Trophoblast cell lines derived from hESCs differentiate and adapt in vitro and can be used as a model to study the mechanisms of early trophoblast invasion.

In situ cryopreservation of human embryonic stem cells in gas-permeable membrane culture cassettes for high post-thaw yield and good manufacturing practice.

The development of efficient and robust methods for the cryopreservation of human embryonic stem cells (hESCs) is important for the production of master and working cell banks for future clinical applications. Such methods must meet requirements of good manufacturing practice (GMP) and maintain genetic stability of the cell line. We investigated the culture of four Shef hESC lines in gas permeable 'culture cassettes' which met GMP compliance. hESCs adhered rapidly to the membrane and colonies displayed good proliferation and expansion. After 5-7 days of culture, hESCs were cryopreserved in situ using 10% dimethyl sulphoxide in foetal calf serum at approximately 1 degrees C/min. This method was compared with a control of standard flask culture and cryopreservation in vials. Post-thaw cassette culture displayed relative proliferation ratios (fold increase above flask/cryovial culture) of 114 (Shef 4), 8.2 (Shef 5), 195 (shef 6) and 17.5 (Shef 7). The proportion of cells expressing pluripotency markers after cryopreservation was consistently greater in cassette culture than for the control with the markers SSEA3 and SSEA4 exhibiting a significant increase (P> or =0.05). The efficiency of cell line culture in cassette was associated with the overall passage number of the cell line. The procedure enables cryopreservation of relatively large quantities of hESCs in situ, whilst returning high yields of viable, undifferentiated stem cells, thereby increasing capacity to scale up with greater efficacy.

In vitro post-meiotic germ cell development from human embryonic stem cells.

BACKGROUND: Investigating the mechanisms of human primordial germ cell (PGC) and gamete development are important for understanding the causes of infertility and effects of environmental chemicals on reproductive development. However, there are practical and ethical difficulties associated with obtaining human tissue in early development. The aim of this study was to investigate whether human embryonic stem cell-hESC-generated germ cells could provide an in vitro model of gamete development. METHOD: Human ESCs were differentiated as embryoid bodies (EBs) in vitro. Gene and protein marker expression profiles of EBs in different periods of culture were analysed by quantitative polymerase chain reaction (Q-PCR) and immunolocalization to monitor germ cell development. Secretion of hormones involved in germ cell maturation was measured, to detect the existence of a germ cell niche within EBs. RESULTS: Q-PCR revealed gene expression profiles consistent with PGC formation and germ cell development. A small population of post-meiotic spermatid cells were identified using sperm-specific antibodies (Protamine 1 and 1.97). Although gene expression profiles characteristic of oocyte development and follicle-like structures were detected, a committed oocyte with extra-cellular zona pellucida was not recognized with zona pellucida-specific monoclonal antibody. CONCLUSIONS: hESCs can form PGCs and post-meiotic spermatids in vitro, however, there remains doubt about oocyte development. Levels of steroid hormones produced by EBs were significant when compared with known values for a similar quantity of human testis, suggesting that hESC may intrinsically create a favourable hormonal niche for spermatogenesis.

Interaction of a tyrosine kinase from human sperm with the zona pellucida at fertilization.

A 95-kilodalton mouse sperm protein with characteristics of a protein tyrosine kinase has been identified as a receptor for ZP3, a glycoprotein in the egg's extracellular matrix. The structure of the human homolog was determined by screening an expression library from human testis; a testis-specific complementary DNA was isolated that encodes a protein similar to receptor tyrosine kinases and appears to be expressed only in testicular germ cells. Antibodies against a synthetic peptide from the intracellular domain recognized a 95-kilodalton human sperm protein that contains phosphotyrosine; human ZP3 stimulates the kinase activity of this sperm protein. Synthetic peptides corresponding to regions of the predicted extracellular domain inhibited sperm binding to human zona pellucida. Availability of the primary sequence of a receptor for ZP3 provides a rational starting point for sperm-targeted contraceptive development.

Derivation of GMP raw materials for use in regenerative medicine: hESC-based therapies, progress toward clinical application.

The potential of somatic cell therapies from human embryonic stem cells (hESCs) as alternatives to traditional drug-based remedies for treating some of mankind's most debilitating diseases has resulted in the need to translate rapidly proof-of-principle and basic research into clinical application. Consequently, researchers and regulatory bodies are now facing one of the major obstacles of the field: the efficient and reproducible generation of clinical-grade cells suitable for producing therapeutic cell types to administer to patients in phase-I and phase-II clinical trials.

Production of activins by the human endosalpinx.

Uterine tubes from 11 premenopausal and 6 postmenopausal women were collected and examined for the presence of inhibin, activin, and follistatin in the endosalpinx. Immunocytochemistry of tissue from both the isthmic and ampullary regions demonstrated clear staining for the beta(A)- and beta(B)-subunits that increased in intensity from the isthmus to the ampulla. Staining for follistatin showed a similar pattern, but no staining for the alpha-subunit was observed. Although staining for the beta(A)-subunit was seen in almost every epithelial cell, staining for the beta(B)-subunit was more variable. Western blotting showed a band with an apparent molecular mass of 28 kDa (corresponding to the activin dimer) and a band of approximately 60 kDa (corresponding to the pro-protein of activin). In situ hybridization confirmed the presence of mRNA for the beta(A)- and beta(B)-subunits in the endosalpinx. These results indicate that the endosalpinx is able to synthesize activin, not inhibin, suggesting that in premenopausal women they may have an important role in the biology of the developing embryo. The role in postmenopausal women is less certain, but could lead to the stimulation of FSH secretion by the pituitary gland or other autocrine/paracrine function within the uterine tube.

No evidence for killer sperm or other selective interactions between human spermatozoa in ejaculates of different males in vitro.

This study examines one of the possible mechanisms of sperm competition, i.e. the kamikaze sperm hypothesis. This hypothesis states that sperm from different males interact to incapacitate each other in a variety of ways. We used ejaculates from human donors to compare mixes of semen in vitro from the same or different males. We measured the following parameters: (i) the degree of sperm aggregation, velocity and proportion of morphologically normal sperm after 1 and 3 h incubation in undiluted semen samples, (ii) the proportion of viable sperm plus the same parameters as in (i) in 'swim-up' sperm suspensions after 1 and 3 h incubation, (iii) the degree of self and non-self sperm aggregation using fluorescent dyes to distinguish the sperm of different males, and (iv) the extent of sperm capacitation and acrosome-reacted sperm in mixtures of sperm from the same and different males. We observed very few significant changes in sperm aggregation or performance in mixtures of sperm from different males compared with mixtures from the same male and none that were consistent with previously reported findings. The incapacitation of rival sperm therefore seems an unlikely mechanism of sperm competition in humans.

Maturation and function of the hamster spermatozoon probed with monoclonal antibodies.

To investigate the surface of the mammalian spermatozoon during its maturation in the epididymis, seven monoclonal antibodies were raised in mice against intact spermatozoa (and isolated sperm heads) recovered from the cauda epididymis of the golden hamster. These antibodies, which with one exception were species specific, recognized various membrane components over restricted regions of the sperm head and tail. During epididymal transit the localisation of four antigens bound by these antibodies was considerably altered. In two cases, antigens were first detected on spermatozoa in the distal corpus epididymis where sperm fertilising capacity is initially acquired. A monoclonal antibody which bound to the acrosome and midpiece specifically blocked fertilisation of intact oocytes of the hamster in vitro. Preliminary characterisation of the antigens was undertaken.

Monoclonal antibody against a sperm antigen Mr 95,000 inhibits attachment of human spermatozoa to the zona pellucida.

A murine monoclonal antibody raised against hamster spermatozoa was found to cross-react with human spermatozoa. By immunofluorescence, the antigen was visualized over the equatorial segment of human sperm heads. In the presence of antibody, sperm binding to the zona pellucida of salt-stored human oocytes was significantly inhibited (P less than or equal to 0.005) compared with other antibodies or control preparations. Using SDS-PAGE of whole spermatozoa and membrane preparations followed by Western blot analysis, the antigen was identified as a determinant with a relative molecular weight of 95,000.

Neuropeptide Y: behavioral effects in the golden hamster.

Neuropeptide Y (NPY) is found abundantly in nervous tissues of vertebrate species including the golden hamster. Centrally-administered NPY has been reported to elicit ingestive behaviors in the rat, squirrel, pig, mouse, and chick. To assess NPY's behavioral effects in a New World rodent that does not increase food intake after deprivation, NPY was injected intracerebroventricularly (10.0-0.04 micrograms/5 microliter) in home-caged golden hamsters with ad lib access to food, water and 5% w/v ethanol solution. Food and fluid intakes, and behavior displays were monitored after NPY injection. NPY promptly increased short-term food intake and observed feeding behaviors at 10.0, 3.3, 1.1, and 0.37 micrograms NPY, but there was no effect on 24 hr food intake. Water and ethanol intakes were increased only at 10.0 and 0.37 micrograms NPY, respectively. Resting behaviors decreased at NPY doses that increased feeding, but there were no consistent effects of NPY on any other category of behavior. Results demonstrate that NPY potently stimulates short-term food intake and decreases resting behavior in the golden hamster. The lack of compensatory food intake in deprived hamsters cannot be explained as an insensitivity to the putative orexigenic function of endogenous neuropeptide Y.

Computer-assisted measurement of sperm swimming speed in human semen: correlation of results with in vitro fertilization assays.

A semiautomatic computerized technique for the measurement of sperm swimming speed is presented. The equipment is easy to use and would be suitable for routine clinical laboratories. The value of the sperm speed measurements obtained from over 100 individuals in relation to fertility has been studied by the correlation of these results with human in vitro fertilization (IVF) data and sperm penetrating capability in the zona-free hamster egg assay. The results show that sperm speed measurements correlate very well with those of the IVF, both human and hamster, and can be used successfully, in conjunction with multivariate statistical methods, to predict the outcomes of such techniques with about 75% accuracy.

The culture of human epididymal epithelium and in vitro maturation of epididymal spermatozoa.

OBJECTIVE: To promote human sperm maturation in vitro. DESIGN: Spermatozoa from the proximal epididymis were coincubated with epididymal epithelial fragments. SETTING: Hospital and research institute. PATIENTS, PARTICIPANTS: Tissue samples were obtained from men undergoing epididymovasostomy procedures or vasectomy. INTERVENTIONS: Fragments of epididymal epithelium formed everted epithelial spheres that in the presence of androgen maintained cell integrity. Coincubation for up to 48 hours of caput epididymal spermatozoa with 3-day-old epithelial cultures from the cauda epididymis was undertaken. MAIN OUTCOME MEASURES: Morphology of epididymal epithelium was assessed by light and electron microscopy. Pulse labeling of tissue in vitro with 35S-methionine was performed with analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique and fluorography. Spermatozoa were assessed for progressive motility and their capacity to bind to salt-stored human zona pellucidae. RESULTS: Epididymal fragments formed everted epithelial spheres that maintained cell integrity and functional morphology for 5 to 7 days. Specific proteins were synthesized in culture, in particular, proteins of 20, 22, 40, and 66 kd. Coincubation of caput epididymal spermatozoa with cultures from the cauda epididymis induced a significant increase in progressive sperm motility and sperm binding to salt-stored human zona pellucidae compared with control cultures of epithelium incubated in the absence of androgens or overgrown with fibroblasts. CONCLUSIONS: Aspects of human sperm maturation processes can be mimicked in vitro using coculture techniques with epididymal epithelium. This method may be valuable for improving the fertilizing capacity of human spermatozoa retrieved from the proximal region of the excurrent ducts.

Fertilizing capacity of rat spermatozoa is correlated with decline in straight-line velocity measured by continuous computer-aided sperm analysis: epididymal rat spermatozoa from the proximal cauda have a greater fertilizing capacity in vitro than those from the distal cauda or vas deferens.

Rat spermatozoa recovered from different regions of the excurrent ducts of 10 adult males (proximal cauda epididymidis [PC], distal cauda epididymidis [DC], and vas deferens [VD]) were assessed by in vitro fertilization (LVF) using limited sperm numbers, and by continuous evaluation of motility parameters during 5 hours of incubation in vitro with automated computer-aided sperm analysis (CASA). Spermatozoa from the PC region fertilized (68 +/- 6%) a significantly greater (P < or = 0.005) number of oocytes than those from the DC (44 + 5%) or VD (47 +/- 7%). For pooled samples from all three regions, the mean fertilization rate (51 +/- 14%) was less tan for spermatozoa from the PC (P < 0.05) but was not significantly different from spermatozoa from the DC or VD. For each time point and sample, 1,592 +/- 428 sperm tracks were analyzed. CASA was verified by comparison with manual still-frame analysis of video recordings, by repeated analysis of the same or different samples of spermatozoa, and by examination of computer tracks. The coefficients of variation for various motion parameters suggested that the CASA obtained a high degree of precision. There were no significant differences in motility parameters for spermatozoa recovered from equivalent regions of the left or right tract or in motility parameters for spermatozoa from different regions of the tract immediately after recovery. However, during incubation in vitro, spermatozoa from the DC or VD regions exhibited a marked decline in straight-line velocity (VSL) compared with spermatozoa from the PC region. The reduction in VSL (combined values from right and left tract) for DC or VD spermatozoa compared with PC spermatozoa was significant at 2.5 hours of incubation (P < or = 0.05) and highly significant (P < or = 0.005) by the end of the incubation period. Differences in average path velocity (VAP) were also apparent after 4 hours (p < or = 0.05), but no significant differences were observed for measurements of curvilinear velocity (VCL) or lateral bead displacement (ALH). Overall, the decline in VSL over 5 hours was highly correlated (P < or = 0.001) with the outcome of fertilization in vitro. In contrast, initial VSL and changes in VCL of spermatozoa were not correlated with fertilization rate. These results indicate that the in vitro fertilizing capacity of rat spermatozoa is correlated with 1) the decline in straight-line velocity (VSL) as measured by repeated CASA during incubation in vitro and 2) with the site of recovery of mature rat spermatozoa from the distal excurrent duct. It is suggested that the deterioration of the quality of rat spermatozoa in the distal epididymidis and vas deferens during storage may occur sooner than previously realized, and therefore care must be taken when recovering samples for fertility assessment. In keeping with findings in other species, immediate "snapshot" analysis of rat motility was a poor predictor of sperm fertility. In contrast, continuous CASA provided significant information for determining sperm fertilizing capacity and will be a useful technique for reproductive toxicology.

Choice of operating conditions to minimize sperm subpopulation sampling bias in the assessment of boar semen by computer-assisted semen analysis.

The performance of a computer-assisted semen analysis system was evaluated for use with washed boar spermatozoa. Accuracy was tested using a computer graphics-generated series of spots moving along horizontal, vertical, and diagonal paths, with both straight and sinusoidal trajectories. Observed and expected values agreed to better than +/- 5%, and there was exact agreement in many cases. Reproducibility was tested by making 10 measurements of a single prerecorded sequence of boar spermatozoa. Coefficients of variation were < 3% for all sperm motion parameters tested. Setup conditions affecting the sample statistics of sperm populations were examined. Search radius (10 settings) and minimum track point (10 settings) were varied factorially to evaluate their biasing effects upon population sampling and accuracy. Low search radius (< 12 microns) or high minimum track point values (> 26 frames) precluded measurements of rapidly moving cells and thus led to selection of slow-moving cells. High search radius (> 16 microns) and low minimum track point settings (< 22 frames) led to erroneous tracking and poor data quality. Suitable settings for these setup parameters (search radius = 13 microns; minimum track points = 24) were chosen for use in subsequent fertility trials because they caused the least sampling bias.

Objectively measured boar sperm motility parameters correlate with the outcomes of on-farm inseminations: results of two fertility trials.

Two fertility trials were undertaken to evaluate the relationship between boar semen quality and fertility (conception rate and litter size) after on-farm artificial insemination (AI). Trial 1 included 98 ejaculates from 27 boars, and trial 2 included 72 ejaculates from 26 boars. The semen quality was measured by computer-assisted semen analysis (CASA) using the Hobson Sperm Tracker. Boar semen was diluted in a standard extender (Beltsville Thawing Solution, BTS), dispensed into 75 ml allquots each containing 1.5 x 10(9) spermatozoa and dispatched to farms by overnight mail for use by their normal AI procedures. Randomly selected 75 ml aliquots of semen from each boar were also sent to the institute of Zoology for CASA measurement. Prior to CASA analysis, the spermatozoa were recovered from the BTS using Percoll gradients, resuspended in trisbuffered saline media containing 40 mM Ca++, and incubated at 39 degrees C. Parameters of sperm motion were measured after 0, 2, 4, and 6 hours of incubation. Various multiple regression models based on measured motion parameters could account for up to 24% of the variation in litter size. Using logistic regression, highly significant (P < 0.0001) models explaining conception rate in terms of sperm motion were derived for trial 2 only. The change in sperm velocity during the first 2 hours of incubation and the magnitude of the velocity parameters after 2 hours were identified as the most consistent indicators of fertility. Other attributes of sperm quality, i.e., frequency of spontaneous acrosome reactions (AR) and ARs induced by ionophore A23187 or solubilized pig zona pellucida, were also examined. When the "within-trial" median litter size was used as a way of allocating ejaculates to "high" or "low" litter-size groups, higher litter size was associated with lower frequency of both spontaneous and induced AR. These results demonstrate that fertility information can be derived from the CASA analysis of boar semen provided it is combined with a period of incubation in capacitating conditions.

Continuous assessment of human spermatozoa viability during cryopreservation.

Cryomicroscopy has enabled direct observation of freezing and thawing of human spermatozoa. When used with a fluorescent viability kit, sperm membrane damage was not apparent down to temperatures of -5 degrees C, but significant damage occurred after thawing (55% of spermatozoa had damaged membranes). Semen samples were cooled or frozen to temperatures (at decrements of 10 degrees C) from 0 degree C to -110 degrees C. At all these temperatures the proportion of live to membrane-damaged cells remained constant. Samples held at temperatures above -30 degrees C were not adversely affected. Below -30 degrees C there was a gradual increase in the proportion of membrane-damaged cells on thaw and a decrease in the number of live cells recovering motility. At temperatures between -50 degrees C and -60 degrees C there was an equal proportion of live motile, immotile, and membrane-damaged cells. It is concluded that some irreversible damage to spermatozoa was a result of freezing processes in cells frozen to -30 degrees C or less, but most of the cryodamage was incurred during thawing, possibly due to recrystallization.

Measurement of intracellular calcium concentration and plasma membrane potential in human spermatozoa using flow cytometry.

We report 2 novel approaches using flow cytometry to measure intracellular calcium concentration and plasma membrane potential in human spermatozoa. Both approaches have the potential to measure different responses in subpopulations of cells, which is particularly useful when studying heterogeneous populations such as human spermatozoa. Intracellular calcium concentration ([Ca2+]i) was measured using the probe indo-1/AM. This allowed measurements to be made that were independent of variation in cell size and dye loading. It also enabled dead cells to be directly identified and excluded from the analyses without the need for counterstaining. Mean basal [Ca2+]i was determined as 50 nM (25-75 nM range) and, in response to the agonist progesterone (20 microM), this increased transiently to 195 nM (125-285 nM range) before declining to approximately half the maximal level within 2 minutes (values in parentheses correspond to the range of values typically found within a sperm population from 1 sample). These results are comparable with previously published data on whole sperm populations. Sperm membrane potential (VM) was assayed using the probe DiOC6(3). In carefully controlled experiments, a marked depolarization of the plasma membrane potential of capacitated spermatozoa was observed in response to progesterone (20 microM). Following in vitro capacitation, the sperm plasma membrane potential became hyperpolarized compared with the noncapacitated state. Therefore, this technique may be used to assay for sperm capacitation in vitro.