Comparison between chromatin condensation and morphology from testis biopsy extracted and ejaculated spermatozoa and their relationship to ICSI outcome

A significant association between male subfertility, imperfect spermiation and abnormal nuclear condensation has been suggested. The DNA content of spermatozoa might be responsible for inducing alterations in sperm morphology. The final nuclear shape, which is species-specific, depends on chromatin condensation during spermatogenesis as well as a precise organization of DNA within the nucleus. Many reports have described the association between disturbances in sperm chromatin condensation, morphology and male infertility. Chromatin condensation is achieved by gradual substitution of lysinerich somatic histones by testis-specific histone and finally by protamine. In this study two groups of patients were compared: the first consisted of 63 patients who had undergone intracytoplasmic sperm injection (ICSI) with freshly ejaculated spermatozoa whereas the second included 47 patients assigned to ICSI with testes biopsy-extracted spermatozoa. In both groups chromatin condensation was assessed by aniline blue staining and morphology evaluated according to strict criteria. The condensed chromatin and morphology of spermatozoa were significantly (P < 0.0001) less in the second group compared to the first. However the fertilization, cleavage, implantation and pregnancy rates were almost the same in both investigated groups. There was no significant difference between the two groups with respect to ICSI outcome. The percentage of chromatin condensation (nuclear maturity) and morphologically-normal spermatozoa were significantly higher (P < 0.0001) in the ejaculated spermatozoa than in those from testis biopsy but the ICSI outcome (fertilization, cleavage, implantation and pregnancy rates) was the same. In view of these results the fertilization capability and the embryo quality obtained using testis biopsy extracted spermatozoa is not influenced by chromatin condensation and sperm morphology in testicular sperm extraction (TESE)-ICSI programmes. Therefore, it could be said that neither chromatin condensation nor morphology of testis extracted sperm could predict the fertilization, implantation and pregnancy rate in TESE-ICSI programmes.     

Comparative evaluation of two density gradient preparations for sperm separation for medically assisted conception

To evaluate and optimize the sperm separation efficiency of a novel silane-coated silica bead (Puresperm), serial studies were carried out to compare the various sperm parameters between: (i) three-layer (90%-70%-40%) Puresperm and three-layer (90%-70%-40%) conventional polyvinylpyrrolidone (PVP)-coated silica bead (Percoll) gradients; (ii) three-layer (90%-70%-40%) and two-layer (90%-45%) Puresperm gradients and separately the same for Percoll; and (iii) large (3.0 ml) and small (0.75 ml) semen loading volumes on three-layer Puresperm gradients. Normozoospermic semen samples were treated and analysed in 12 replicates for each experiment. Manual evaluation of concentration, percentage motility, percentage vitality, percentage normal morphology; computer-assisted semen analysis evaluation of concentration, percentage motility, grade of motility, motion characteristics (curvilinear velocity, linearity, amplitude of lateral head velocity, beat cross frequency, percentage hyperactivation); and yields from the initial semen samples were compared. Percoll was found to be superior to Puresperm in concentration, percentage motility, percentage vitality and yields after three-layer density gradient centrifugation. There were no significant differences in sperm parameters between two- and three-layer Percoll gradients, but three-layer Puresperm gradients behaved significantly better than two-layer gradients. Large semen volume loads on three-layer Puresperm gradients resulted in greater sperm concentrations, percentage motility, percentage vitality and percentage normal morphology, but small semen volume loads produced greater yields of good-quality spermatozoa. In the light of Percoll being withdrawn from the shelf for the use of assisted reproduction because of the presence of PVP, three-layer Puresperm gradients with large semen loading volumes appear to be an attractive alternative for sperm separation in medically assisted conception.     

Use of aniline blue to assess chromatin condensation in morphologically normal spermatozoa in normal and infertile men.

Chromatin condensation is vital for the function of the spermatozoon as the motile carrier of the paternal genome. The degree of condensation can be shown with the aid of acidic aniline blue staining, which is able to discriminate between lysine-rich histones and arginine- and cysteine-rich protamines. Using this technique and employing the Düsseldorf classification of sperm morphology in cases of disturbance of spermatogenesis, it was demonstrated that chromatin condensation is impaired not only in malformed but also in morphologically normal spermatozoa. Among morphologically normal spermatozoa, the percentages of spermatozoa with chromatin condensation disturbances increase in patients with different patterns of sperm malformation, if compared with patients with normozoospermia.     

The use of silane-coated silica particles for density gradient centrifugation in in-vitro fertilization

Silane-coated silica particles (PureSperm) were evaluated as an alternative to Percoll for gradient separation of spermatozoa, for use in assisted reproduction. Recovery of motile and morphologically normal spermatozoa after using a four-layer Percoll and a two- and four-layer PureSperm gradient respectively was recorded. In-vitro fertilization (IVF) results after using PureSperm for the sperm preparation were also evaluated. No difference in sperm recovery or sperm motility was found when comparing the use of Percoll and the four-layer gradient of PureSperm. When using a two-layer PureSperm gradient, motility was significantly decreased (P < 0.05) compared to Percoll. Normal sperm morphology increased from 8-17.2% after using Percoll and to 12.7% and 11.4% after using a four-layer and a two-layer PureSperm gradient respectively. All gradient preparations showed a significant decrease in the teratozoospermia index compared to the ejaculate (P < 0.01). No significant differences in IVF results regarding fertilization and pregnancy rates were found when PureSperm or the swim-up technique were used for the sperm preparation. PureSperm seems to be an acceptable alternative to Percoll but although the percentage of sperm recovery was higher after PureSperm we still recommend the swim-up technique to be the first choice, as a higher percentage of progressive motile spermatozoa is obtained without using other chemicals than IVF culture medium.     

Predictive value of classical and automated sperm analysis for in-vitro fertilization.

The fertilization rates observed in 122 attempts at in-vitro fertilization were examined in relation to sperm characteristics assessed by visual and automated screening. Using linear regression analysis, a significant correlation was found between the fertilization rate and (i) evaluations in fresh semen sperm concentration, percentages of sperm motility, vitality and normal morphology and velocity, (ii) measurements in swim-up preparations of percentages of sperm motility, vitality and morphology, velocity and amplitude of lateral head displacement. No significant correlation was found between the fertilization rate and any of the parameters studied in 24-h-old swim-up suspensions. Analysis by multiple variable stepwise linear regression showed an optimal correlation (R6 = 0.62) between the observed fertilization rate and theoretical calculation obtained from the following predictive function: fertilization rate = -0.3 + (0.008 x swim-up motility) + (0.004 x normal sperm morphology in fresh semen). Introduction of kinematic characteristics studied by automated screening improved the multiple correlation between the calculated and observed fertilization rate in cases of normal or mildly defective semen. Because of the limited availability of motile spermatozoa, automated analysis could not supersede classical sperm analysis in cases of more severe sperm defects.     

Chromatin packaging and morphology in ejaculated human spermatozoa: evidence of hidden anomalies in normal spermatozoa

This study aimed to investigate the association between anomaliesin sperm chromatin packaging, morphology and fertilization inpatients undergoing routine in-vitro fertilization (IVF) orsubzonal insemination (SUZI). Sperm chromatin packaging wasassessed using chromomycin A3 (CMA3), a fluorochrome specificfor guanine-cytosine rich sequences of DNA. One hundred to 150sperm cells were assessed in 55 patients to compare sperm chromatinpackaging and morphology to fertilization after IVF or SUZI.When the morphology and CMA3 fluorescence of individual spermatozoawas assessed, >75% of the macrocephalic sperm fluorescedin all patients. In contrast a mean of 37% of the spermatozoawith normal morphology fluoresced in IVF patients compared with58% of the normal spermatozoa in male factor patients treatedby SUZI. SUZI patients displaying a high fluorescence (>70%)in their spermatozoa also had a significantly lower fertilizationrate. Lower packaging quality in morphologically normal spermatozoamay represent a major limiting factor in the fertilizing abilityof male factor patients. This study confirms that a high percentageof CMA3 positivity is present in certain forms of male factorinfertility and that such a test may be used to distinguishseparate populations in morphologically normal spermatozoa. chromatin/chromomycin A₃/in-vitro fertilization/male infertility/spermatozoa     

Density gradient centrifugation and glass wool filtration of semen remove spermatozoa with damaged chromatin structure.

The ability of double-layered density gradient centrifugation (DGC) or glass wool filtration (GWF) of semen to remove spermatozoa with damaged chromatin structure was assessed by the flow cytometric sperm chromatin structure assay (SCSA), which measures the susceptibility to sperm nuclear denaturation in situ. Ejaculates from 26 men attending a university-affiliated assisted reproduction laboratory were processed by DGC and GWF. Unprocessed, DGC- and GWF-processed specimens were assessed by the SCSA and by conventional semen parameters. Changes in chromatin structure were compared with conventional semen parameters. Both sperm preparation techniques yielded sperm suspensions with improved sperm chromatin structure as well as motility (%), forward progression (1-4) and viability (%). DGC was superior to GWF in the efficiency of recovering motile, morphologically normal, mature sperm suspensions. However, GWF produced improved chromatin integrity (SDalpha(t)) and viability. Moderate correlations between SCSA and conventional sperm parameters were observed. Nevertheless, the SCSA provides additional information about the biochemical integrity of sperm DNA and may be used in future studies to provide insight into assisted reproduction technology outcomes not explained by conventional sperm parameters.     

Fertility of ejaculated and testicular megalohead spermatozoa with intracytoplasmic sperm injection

In this study the fertility and outcome of intracytoplasmic sperm injection (ICSI) using megalohead spermatozoa from the ejaculates and testicles was evaluated. Seventeen males with megalohead and pinhead sperm forms in their ejaculate were studied in 22 cycles. A high number of sperm heads without tails and abundant round spermatid forms were commonly observed. Round-headed spermatozoa were seldom accompanied by these severely abnormal spermatozoa. The majority of megalohead spermatozoa were observed to have multiple tails, were predominant in the sample, and were used for ICSI. Ejaculated megalohead spermatozoa were used for ICSI in 15 cycles, while testicular spermatozoa were used in seven cycles where there were no vital spermatozoa or spermatozoa of low vitality in the ejaculate. The same abnormal morphology was observed in the testicles as in the ejaculated spermatozoa in the same males. Mean (+/- SD) low motility 4.7 +/- 5.6% and sperm count (3.8 +/- 4.19 x 10(6)) were common findings in these severely teratozoospermic patients. A low fertilization rate (43.2%) was achieved by using megalohead sperm forms (group I, n = 17) in comparison with the control group (60.2%) which had zero normal sperm morphology according to strict criteria (group II, n = 30) (P <0.01). Furthermore, a low pregnancy rate (9.1%) was obtained in the megalohead sperm group in comparison with the control group (40%) (P <0.05). Low fertilization and pregnancy rates may be due to a high incidence of chromosomal abnormalities from severely defective spermatozoa in the ejaculate. Couples should be counselled and warned about possible low fertilization and pregnancy rates with ICSI when only pinhead and megalohead forms with a high number of sperm heads without tails are present in the ejaculate.     

Evaluation of chromatin condensation in human spermatozoa: a flow cytometric assay using acridine orange staining

The quality of sperm chromatin is an important factor in fertilization and is especially critical where one spermatozoon is artificially selected for fertilizing an egg (as in intracytoplasmic sperm injection). In this study, flow cytometry after staining of human spermatozoa with Acridine Orange was used to study chromatin structure. A method is described for estimating the percentage of cells in a human sperm sample that have completed epididymal maturation in regard to chromatin condensation. Of the 121 samples of the semen that were examined, nine contained a higher percentage of hypocondensed spermatozoa and six samples contained elevated amounts of hypercondensed spermatozoa. In addition to aberrancies in chromatin condensation other defects showed up as satellite populations of spermatozoa with higher than normal ratios of red/green fluorescence after Acridine Orange staining. Such defects were found in 15 semen samples. The use of swim-up and Percoll gradient centrifugation methods was shown to improve the percentage of spermatozoa with normal chromatin structure in some samples with poor initial quality.      

Comparison of different hypo-osmotic swelling solutions to select viable immotile spermatozoa for potential use in intracytoplasmic sperm injection

The hypo-osmotic swelling test, originally developed as a diagnosticsperm test, is used to discriminate viable from non-viable spermatozoafor intracytoplasmic sperm injection (ICSI) in cases of completeasthenozoospermia. In the present study, three hypo-osmoticsolutions were compared, i.e. (A) Jeyendran solution containingsodium citrate and fructose; (B) a mixture of 50% culture mediumand 50% milli-Q water; and milli-Q water. While both the percentageof swelling and vitality assessed by eosin Y remained unchangedafter 5-30 min of sperm exposure to solutions A and B, incubationin water for only 5 min was in itself detrimental. Ten frozen-thaweddonor samples and 10 asthenozoospermic patient samples wereexposed to the three solutions for B and C, but only weaklycorrelated for solution A. Percentage viability was furtherassessed by eosin Y and motility of spermatozoa after 2 h and24 h exposure to the three solutions was compared with unexposedcontrol spermatozoa. While a significant decrease in both parameterswas observed for all three solutions in comparison with thecontrol, sperm quality was significantly higher after exposureto solution B than after exposure to solutions A and C. It maybe concluded that solution B (composed of 50% culture mediumand 50% water) is to be preferred for the selection of viableimmotile spermatozoa for ICSI.     

The use of two density gradient centrifugation techniques and the swim-up method to separate spermatozoa with chromatin and nuclear DNA anomalies

Human semen is heterogeneous in quality, not only between males but also within a single ejaculate. Differences in quality are evident, both when examining the classical parameters of sperm number, motility and morphology and in the integrity of the sperm nucleus. The aim of this study was to determine the efficiency of the PureSperm((R)), Percoll((R)) and swim-up preparation techniques to eliminate spermatozoa with nuclear anomalies. Semen samples were collected, washed and one part of the semen spread on a slide, the remainder was prepared using the swim-up, PureSperm((R)) or Percoll((R)) techniques. Spermatozoa from different fractions were fixed on slides and assessed. Sperm samples (n) from different men were stained using the chromomycin A(3) (CMA(3)) fluorochrome, which indirectly demonstrates a decreased presence of protamine (n = 31 for swim-up; n = 45 for PureSperm((R)); n = 39 for Percoll((R))). Spermatozoa prepared using PureSperm((R)) (n = 35) and Percoll((R)) (n = 37) were also examined for the presence of endogenous DNA nicks. Good quality spermatozoa should not possess DNA nicks and not stain (i.e. fluoresce) with CMA(3). When prepared using the swim-up technique the spermatozoa recovered showed no significant improvement with the CMA(3) staining. When spermatozoa were prepared using the PureSperm((R)) and Percoll((R)) techniques, a significant (P < 0.001) decrease in both CMA(3) positivity and DNA strand breakage was observed. These results indicate that both the PureSperm((R)) and Percoll((R)) techniques can enrich the sperm population by separating out those with nicked DNA and with poorly condensed chromatin.     

Evaluation of in vivo reproductive toxicity of potassium chromate in male mice

To evaluate the effects of potassium chromate on mice sperm cells after a short-term exposure, male ICR-CD1 mice were administered with 5 or 10 mg K₂CrO₄/bw for 4 consecutive days. One group of mice was sacrificed at day 5, starting from the beginning of the experiment and another group was sacrificed at day 35. Testis and epididymis histology was evaluated by light microscopy and testicular cells populations were evaluated by flow cytometry (FCM). Spermatozoa were collected from the epididymis and their morphology and several functional parameters (density, motility, viability, mitochondrial function, acrosome integrity) were evaluated. Furthermore, DNA fragmentation and chromatin status of sperm cells were assessed at both experimental periods.Besides a reduction in seminiferous tubules diameter, exposure to potassium chromate did not induce further histopathological changes in mice testis or epididymis. These results were supported by the analysis of testicular cellular subpopulations by FCM. Concerning spermatozoa morphology, an increase in the percentage of multiple abnormalities and a decrease in the percentage of normal spermatozoa were found at days 5 and 35, respectively. Although spermatozoa mitochondrial function or viability was not affected, its motility was significantly reduced by potassium chromate exposure at both experimental periods. A decrease in acrosome integrity was found in mice injected with 10 mg K₂CrO₄/bw after 35 days. Exposure to potassium chromate did not affect either DNA fragmentation or chromatin susceptibility to acid denaturation of sperm cells. In this work, we were able to show the effects of potassium chromate on spermatozoa physiological parameters such as motility, morphology and acrosome status and also demonstrate that the doses tested did not induce DNA damage to sperm cells after one spermatogenic cycle.     

Hyaluronic acid substantially increases the retention of motility in cryopreserved/thawed human spermatozoa

We have demonstrated previously that hyaluronic acid (HA) improves the velocity and the retention of motility in freshly ejaculated human spermatozoa. In the present work, we examined the effect of HA on cryopreserved/ thawed spermatozoa in four paradigms: (i) effect of HA on sperm motility and velocity in semen; (ii) stabilizing effect of HA after 4 h of incubation when the decline of sperm motility is already detectable; (iii) the duration of improved motility after the separation of spermatozoa from HA by Percoll gradient centrifugation; and (iv) motility of sperm cryopreserved in the presence of HA. HA improved the retention of sperm motility in thawed spermatozoa. Indeed, the motility values after 30 h were approximately 100% higher in the HA compared with the control samples. This effect of HA was also evident in the stabilization of spermatozoa with already declining motility. After removal of the HA from the incubation medium, significantly increased motility in the HA-exposed spermatozoa was still detectable for at least 4 h. Cryopreservation of spermatozoa in the presence of HA did not improve the recovery of motility. The data indicate that HA improves the retention of motility of cryopreserved/thawed spermatozoa, even after the removal of HA from the incubation medium. The utilization of HA will probably prove beneficial in assisted reproduction: in intrauterine insemination and in in-vitro fertilization (IVF), the extended sperm motility and velocity will enhance the fertilizing efficiency; in intracytoplasmic sperm injection (ICSI), the improved motility will facilitate the identification of viable spermatozoa. Because HA is a physiological component of the cumulus and of the female and male reproductive tracts, administration of HA should not cause ethical concerns.      

Comparison of Percoll, mini-Percoll and swim-up methods for sperm preparation from abnormal semen samples.

Two methods of density gradient centrifugation, Percoll (P) and mini-Percoll (MP), were compared with the swim-up technique for preparing spermatozoa from each of 40 abnormal semen samples. P and MP produced similar results with a mean recovery of spermatozoa with progressive motility which was significantly higher (18-19%) than that achieved with swim-up (5%). However, the swim-up method resulted in the recovery of spermatozoa with a higher mean motility (89 versus 58%), velocity (69 versus 56 microns/s), percentage with normal morphology (22 versus 16%) and intact acrosomes (61 versus 36%) than P and MP. The mean amplitude of lateral head displacement was the only characteristic of spermatozoa in semen which correlated with the recoveries of motile spermatozoa. Combining MP and swim-up methods for 10 samples produced a higher recovery (11 versus 6.9%) of spermatozoa with significantly better mean motility (94 versus 87%) than did swim-up alone. Although P and MP resulted in greater yields of motile spermatozoa than the swim-up preparation, the latter procedure selected higher proportions of spermatozoa with improved characteristics (velocity, intact acrosomes and normal morphology) which correlate with fertilization rates in vitro. It is concluded that P and MP are not superior to swim-up. However, sequential MP and swim-up preparation improves yields of high quality spermatozoa from some abnormal semen samples and therefore has potential for improving fertilization rates.     

Outcome of in-vitro culture of fresh and frozen-thawed human testicular spermatozoa

The effect of in-vitro culture on the motility and morphology of fresh and frozen-thawed human testicular spermatozoa obtained from obstructive azoospermic patients and on the motility of testicular spermatozoa obtained from non-obstructive azoospermic patients was evaluated. The outcome of intracytoplasmic sperm injection (ICSI) with fresh and frozen-thawed human testicular spermatozoa was studied. The results showed that significant improvement of sperm morphology and motility was observed in culture of fresh (n = 17) and frozen-thawed (n = 15) testicular sperm samples obtained from patients with obstructive azoospermia. The motility of cultured testicular spermatozoa reached a peak at 72 h without the need for special media. In six of 20 samples obtained from patients with non-obstructive azoospermia, improvement of sperm motility was observed. When only non-motile testicular spermatozoa were cultured, they all remained non-motile (n = 9). In patients with obstructive azoospermia, fertilization rates of 80 and 81% were obtained using ICSI with fresh and frozen-thawed testicular spermatozoa respectively. Clinical pregnancies were observed in four out of nine patients with fresh testicular spermatozoa and two out of five patients after using frozen-thawed spermatozoa. When fresh testicular spermatozoa obtained from patients with non-obstructive azoospermia were used for ICSI, the fertilization rate was 68% and two out of seven patients achieved clinical pregnancies. In conclusion, the morphology and motility of fresh and frozen-thawed testicular spermatozoa in patients with obstructive azoospermia can be significantly improved after in-vitro culture. The outcome of in-vitro culture of testicular spermatozoa in patients with non-obstructive azoospermia is unpredictable. In-vitro culture of non-motile testicular spermatozoa is not successful so far. The outcome of ICSI with fresh and with frozen-thawed testicular spermatozoa was similar.      

Critical evaluation of methylcellulose as an alternative medium in sperm migration tests.

BACKGROUND: The aim of this study was to evaluate the ability of human spermatozoa to penetrate methylcellulose (MC) and to compare this with penetration in hyaluronic acid. METHODS: Spermatozoa from normal (>or=20 x 10(6) sperm/ml, >or=50% progressive motility, >or=5% normal forms) and oligozoospermic (<20 x 10(6) sperm/ml) semen samples were allowed to swim into glass capillary tubes containing methylcellulose with a viscosity of 15 centipoise (cp) (MC15) and 4000 cp (MC4000), hyaluronic acid (rooster comb) or Sperm Select. Penetration of the spermatozoa at 1, 2, 3 and 4 cm were correlated with basic semen parameters (concentration, motility and morphology). The effects of temperature on penetration into MC4000 were explored at 17-37 degrees C. RESULTS: Higher numbers of spermatozoa penetrated MC4000 (10 mg/ml) compared with MC15 and the hyaluronic acid preparations. There was a highly significant correlation between the number of spermatozoa at all migration distances in MC4000 (10 mg/ml) and semen parameters. Increases in temperature from 17-37 degrees C were accompanied by significantly higher numbers of spermatozoa at each penetration distance. MC4000 at 10 mg/ml was at least as favourable to sperm penetration as human cervical mucus. Effective discrimination between normal and abnormal samples was achieved using MC4000 (10 mg/ml). CONCLUSION: Our results suggest the potential use of methylcellulose (MC4000, 10 mg/ml) as a reproducible and effective alternative to hyaluronic acid in sperm migration tests.     

A new test for the assessment of sperm- zona pellucida penetration: relationship with results of other sperm tests and fertilization in vitro

The spermatozoa of some patients attending for in-vitro fertilization(IVF) fail to penetrate the zona pellucida in vitro. A testhas been devised to identify these cases. It is based on thenumber of spermatozoa penetrating into the zona pellucida, whichwere counted after removing spermatozoa bound to the zona surfaceby vigorous aspiration of each oocyte through a narrow gauge(120 µm) glass pipette. The oocytes were collected from197 patients undergoing IVF treatment with their own gametes;79 with no oocytes fertilized and 118 with some oocytes fertilized.Sperm motility, morphology and DNA normality (acridine orangestain) were also measured. The relationships between sperm testresults and IVF rate were examined by logistic regression. Theproportions of penetrated zonae, normal sperm morphology andnormal DNA were the most significant factors related to IVFrate in the whole group. Also, in patients with 30 spermatozoabound per zona pellucida or with normal sperm morphology 30%,the proportion of penetrated zonae and normal DNA were mostsignificant. Oocytes from 42 patients who had zero fertilizationand low sperm-zona binding (average, 2.2 spermatozoa/zona pellucida)were re-incubated with normal donor spermatozoa: large numbersof spermatozoa bound (average, 88 spermatozoa/zona pellucida)and each zona was penetrated by at least one spermatozoon. Inconclusion, the percentage of zonae penetrated was the variablemost significantly correlated with IVF rate. Penetration ofthe zona was also strongly related to fertilization rates inpatients without defects of sperm morphology and sperm-zonabinding. In patients where all zonae were penetrated, poor fertilizationmay be due to sperm morphology and DNA abnormalities. Failureof sperm-zona binding and penetration in vitro in patients withfailure of fertilization was mainly due to sperm defects andnot oocyte defects     

Calcium ionophore-induced acrosome reaction correlates with fertilization rates in vitro in patients with teratozoospermic semen

The aim of this study was to determine the relationship between calcium ionophore A23187-induced acrosome reaction (AR) and sperm fertilizing ability. Semen samples remaining after preparation for standard IVF were studied in 109 patients who had sperm concentrations > or =20 x 10(6)/ml. Ionophore-induced AR was performed on motile spermatozoa selected by centrifugation on a Percoll gradient. Semen analysis was performed using standard methods. Patients with higher (>50%, n = 76) fertilization rates had significantly higher ionophore-induced AR than patients with lower (<50%, n = 33) fertilization rates (49 +/- 14 versus 38 +/- 21%, P < 0.05). When the data from all patients were analysed by logistic regression, only the percentage sperm motility in insemination medium and ionophore-induced AR were significantly related to fertilization rates. Similar results were also obtained when the data from a subgroup of patients with poor (<15% normal) sperm morphology were analysed. However, when patients with normal sperm morphology > or =15% were analysed separately, only sperm count and the percentage of spermatozoa with progressive motility in semen were significantly related to fertilization rates. In conclusion, ionophore- induced AR was significantly related to fertilization rates in vitro mainly in patients with teratozoospermic semen. Tests for ionophore- induced AR may provide additional information about sperm fertilizing ability but may not indicate specific defects of the physiological AR.      

Linear and non-linear relationships between the 'swelling test' and conventional semen variables in men suspected of primary infertility

The relationship and degree of association between the percentage of sperm swelling (HOS-test) and conventional semen variables was investigated in 263 consecutive ejaculates. The semen samples were exclusively obtained from men suspected of primary infertility. It was found that the correlation coefficients (Spearman's rho) followed the order: percentage of progressive motility at 3 h greater than count/ml greater than percentage of total motility at 3 h greater than percentage of normal spermatozoa. Of the three morphology sub-classes considered (sperm head, mid-piece and tail abnormalities), only mid-piece abnormalities correlated with the outcome of the HOS-test (rho = -0.409). Linear relationships between HOS-test results and sperm motility and morphology, but not sperm count, were indicated by LOWESS-smoothing. However, a linear relationship between the HOS-test, sperm count and a 'functional index' combining the conventional semen variables could be demonstrated after normalization of the data. Our findings suggest that the HOS-test may be of value in assessing the functional integrity and viability of spermatozoa; however, its prognostic power for fertility is probably not different from that of conventional semen variables.     

Occurrence of GABA and GABA receptors in human spermatozoa

Gamma-aminobutyric acid (GABA) concentrations in seminal plasma and washed spermatozoa from normal donors were assessed by a sensitive radioreceptor assay, and were detectable in both fractions. Specific binding of [3H]-muscimol was shown to be dependent on protein concentration, temperature and incubation time. [3H]-muscimol specific binding to human sperm membranes was significantly inhibited by the GABA type A receptor (GABA(A)) antagonist, bicuculline, and by the GABA(A) agonists, muscimol and isoguvacine, but not by the GABA type B receptor (GABA(B)) agonist baclofen. Scatchard analysis of [3H]- muscimol binding yielded a linear plot consistent with a single population of binding sites with a dissociation constant in the low nanomolar range. Incubation with GABA at a high micromolar concentration for 3 h under capacitating conditions resulted in an increase in the percentage of spermatozoa showing hyperactivated motility as assessed by computerized motility analyser. However, low micromolar concentrations of the GABA(A) agonist, muscimol, were sufficient to significantly increase sperm hyperactivity. These results suggest that the effect of GABA on human sperm motility might be mediated through a specific GABA(A) receptor.