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L J Burkman

Publications and source records attributed to L J Burkman.

15 recordsLinked to original sources
PubMed1991

Defining the valid hemizona assay: accounting for binding variability within zonae pellucidae and within semen samples from fertile males.

OBJECTIVE: To achieve a better understanding of the variability in sperm and oocyte binding capacities will optimize use of the hemizona assay (HZA) as a predictor of sperm function. DESIGN: Limitations of the HZA were more clearly delineated by current studies: (1) variability of sperm binding capacity of men over a 90-day interval; (2) variability of sperm binding using different oocytes; and (3) lower limits of the number of sperm bound from the fertile control in two laboratories. PATIENTS: Semen was obtained from proven fertile men and one subfertile individual. MAIN OUTCOME MEASURE: The number of sperm tightly bound to the hemizona were measured and compared. RESULTS: In the initial study, 6 fertile control men exhibited a similar degree of variability in zona binding when studied over a 90-day interval. Average sperm binding for individuals ranged from 68 to 127. Second, 3 of the 15 simultaneous assays showed very low numbers of sperm bound, indicating that 20% of the zonae had poor binding. Third, from 18 men who had 0% fertilization in an in vitro fertilization system using mature oocytes, evaluation of their sperm by HZA was performed. The sperm bound poorly and the 95% confidence interval was 20 sperm bound. Thus, the fertile controls should bind greater than 20 sperm to distinguish them from the infertile group in the HZA system resulting in a valid assay. CONCLUSIONS: With these guidelines, applications of the HZA may be made with greater reassurance of a valid bioassay of sperm fertilizing potential.

Biological Assay↗
PubMed1991

Discrimination between nonhyperactivated and classical hyperactivated motility patterns in human spermatozoa using computerized analysis.

The objective identification of human hyperactivated (HA) motility has been controversial. The present study defines new criteria for automatic sorting of all HA patterns that are consistent with the classical descriptions. Sperm from fertile men were prepared in Ham's F-10 medium. Based on the author's previously published slow-motion method, 342 sperm were selected that represented the non-HA, circling high-curvature, thrashing, star, and helical patterns. Automatic sorting for HA was achieved with the use of three criteria: linearity less than or equal to 65 and velocity greater than or equal to 100 microns/s and head displacement greater than or equal to 7.5 microns. Additional criteria were determined for identifying each HA class separately. Whether using the new or old method, the incidence of HA is significantly associated with multiple fertilization endpoints.

Autoanalysis↗
PubMed1990

Human follicular fluid stimulates hyperactivated motility in human sperm.

Since human follicular fluid (FF) is known to enhance the acrosome reaction and capacitation, we investigated whether hyperactivated motility is stimulated by FF. Follicular fluid-treated sperm exhibited a threefold increase in hyperactivation compared with the controls. The use of fetal cord serum in the medium, instead of bovine serum albumin, supported the same high levels of hyperactivation, although the peak occurred at 3 hours rather than 5 hours of capacitation. When sperm were treated with a steroid-rich fraction of the FF, hyperactivation was stimulated to the same degree as with whole FF. In contrast, no stimulation occurred when sperm were treated with a FF fraction stripped of steroids. The FF enhancement of hyperactivation in vitro could augment the fertilizing capacity of subfertile sperm samples, providing also a glimpse of possible in vivo events as sperm traverse the FF-laden cumulus oophorus.

Body Fluids↗
PubMed1989

Hemizona assay using salt-stored human oocytes: evaluation of zona pellucida capacity for binding human spermatozoa.

Human oocytes were stored (25 degrees C) in 1.5 M MgCl2 for 6-30 days, then utilized in the new hemizona assay (HZA) for tight binding of human spermatozoa [Burkman et al.: Fertil Steril 49:688-697, 1988]. We have compared 1) the ability of matching salt-treated hemizonae or dimethylsulfoxide (DMSO)-treated hemizonae to distinguish between sperm from semen having normal versus subnormal characteristics and, 2) the kinetics of fertile sperm binding to salt-treated or DMSO-treated hemizonae. After sperm preparation one salt-treated hemizona was incubated with normal spermatozoa and the matching hemizona was placed with sperm from the subnormal group. As a control, DMSO-treated hemizonae were incubated in additional sperm droplets. After 4 hours, the number of sperm tightly bound to each hemizona was counted. Within the normal semen group, there was equivalent binding to salt- or DMSO-treated hemizonae (54.0 +/- 12 and 49 +/- 14, respectively, mean +/- SEM). Similarly, tight binding of sperm from the subnormal group was not affected by the zona storage method (21 +/- 8 and 17 +/- 5, respectively). For either storage approach, binding of subnormal sperm was significantly less (P less than 0.01) compared with the number of normal sperm attached to the matching hemizona. For the kinetics study, the hemizona binding of proven fertile spermatozoa was followed throughout 8.5 hours. The shape of the binding curve was the same for zonae stored by either method and was consistent with our published kinetics data. Salt storage offers a simple and inexpensive means for accumulating and transporting human zonae pellucida; the resulting hemizonae function effectively in the HZA for estimating sperm binding potential.

Dimethyl Sulfoxide↗
PubMed1989

The hemizona assay (HZA): a predictor of human sperm fertilizing potential in in vitro fertilization (IVF) treatment.

The hemizona assay (HZA) was developed to assess human sperm fertilizing potential. This blinded study investigated the relationship between sperm binding to the hemizona and in vitro fertilization (IVF) success (36 patients). Nonliving human oocytes were recovered from excised ovaries and stored. Each zona pellucida was cut into equal hemispheres by micromanipulation. For the HZA, one droplet exposed a hemizona to abnormal spermatozoa, while the control droplet contained the matching hemizona and spermatozoa from normal semen. After 4 hr, the number of tightly bound spermatozoa was counted. Binding to the hemizona was significantly higher for those having IVF success (mean of 36.1 +/- 7, versus 10.4 +/- 4 from the failure group; P less than 0.05). Fewer sperm from the failure group had a strictly normal morphology (3.2 versus 12.7%; P less than 0.05, Kruger method). Tight zona binding was significantly correlated with the percentage motile sperm, percentage normal morphology, and seminal sperm concentration. These results enhanced our confidence that the HZA is diagnostic for identification of patients at high risk of failing to achieve fertilization in vitro.

Female↗
PubMed1989

Hemizona assay: assessment of sperm dysfunction and prediction of in vitro fertilization outcome.

The hemizona assay (HZA) was used in a prospective, blinded study to assess the relationship between tight sperm binding in the HZA and sperm fertilizing ability in in vitro fertilization (IVF). In each controlled assay, the authors compared sperm binding of proven fertile men with that of patients undergoing IVF. Human oocytes stored in a salt solution were used in the study, and binding results were correlated with the fertilization rate of preovulatory oocytes during IVF. Patients with poor fertilization rates in IVF had significantly lower binding than those cases with successful fertilization (7.3 +/- 1.4 versus 62.1 +/- 10.9, respectively; mean +/- standard error, P less than 0.02). Based on current standards, the HZA was able to predict fertilization accurately in 26 of 28 cases (sensitivity of 83%, specificity of 95%, positive predictive value of 83%). The authors conclude that the HZA is a valuable tool for evaluating dysfunctional sperm-zona pellucida binding, with good predictive value for fertilization in vitro.

Adult↗
PubMed1988

The hemizona assay (HZA): development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential.

The authors present their initial results with the hemizona assay (HZA), which was developed to predict the fertilizing potential of spermatozoa. The HZA uses the matching halves of a human zona pellucida from a nonfertilizable and nonliving oocyte, providing an internal control on zona-to-zona variability. Maximal binding of human sperm to the hemizona usually occurred after 4 to 5 hours of coincubation. Sperm from fertile men exhibited significantly higher binding capacity to hemizonae compared with sperm from men who had fertilization failure during in vitro fertilization (IVF) treatment. The HZA index is calculated as follows: (bound sperm from subfertile male) divided by (bound sperm from fertile male) X 100. These findings demonstrate that the HZA may be a useful diagnostic tool in male infertility evaluations.

Female↗
PubMed1985

Male factor evaluation in in vitro fertilization: Norfolk experience.

Thirty-three patients from the in vitro fertilization (IVF) program at Norfolk are critically reviewed. A battery of tests was designed and an endocrine investigation was carried out on these patients. The fertilization rate for preovulatory oocytes was lower than in the normal male population (39.6% versus 88.6%). When total concentration of sperm with rapidly progressive motility was less than 6 X 10(5), to fertilize several eggs together the fertilization rate was zero. No fertilization was obtained when the number of sperm with rapidly progressive motility recovered after the separation was less than 1.5 X 10(6). The hamster zona-free oocyte penetration test correlated well with the human IVF system. The other parameters investigated did not show good correlation. When fertilization was achieved, the results of the IVF procedure in the series reviewed rendered a 30.8% pregnancy rate per transfer in 26 transfers. Fifty percent of the pregnancies were normal (either ongoing or delivered). Thirty-seven percent were preclinical miscarriages, and 12.5% were clinical abortions. In the abnormal male population, higher concentrations of sperm per egg should be used for insemination for achievement of optimum fertilization rates. Once fertilization is obtained, the results do not differ substantially from the IVF population at large.

Animals↗
PubMed1984

A possible role for potassium and pyruvate in the modulation of sperm motility in the rabbit oviducal isthmus.

Spermatozoa were recovered from the isthmus of the rabbit oviduct at 4 and 11 h post coitum using several defined flushing media. The motility of spermatozoa in the isthmic flushings was subsequently analysed from video recordings. There was little sperm movement in the native isthmic fluid, but vigorous flagellar activity and hyperactivated movement were induced by flushing the isthmus with 0.25 M-sucrose, apparently an effect of dilution. Flushing with a complex culture medium resulted in similar stimulation of sperm movement. When pyruvate was present in the medium, hyperactivated flagellar bending was stimulated, whereas these movements were virtually absent when glucose alone was present. The stimulating effect of dilution was less pronounced when the flushing medium contained 50 mM-potassium. When the isthmus was flushed with media containing 50 mM-K+ and the K+ concentration was lowered to 5 mM during a washing procedure, large-amplitude flagellar movements were sequentially suppressed and restored. The restoration of large-amplitude movements was enhanced when pyruvate was present in the 5 mM-K+ washing medium. These results suggest that alterations in the concentration of both K+ and pyruvate may have a role in regulating the motility of rabbit spermatozoa in the oviducal isthmus, K+ being inhibitory and pyruvate stimulatory.

Animals↗
PubMed1984

Characterization of hyperactivated motility by human spermatozoa during capacitation: comparison of fertile and oligozoospermic sperm populations.

Suspensions of capacitating human spermatozoa were analyzed for potential hyperactivated movements using videomicrographic methods. Analysis was carried out on aliquots of 22 sperm suspensions, which were proved fertile several hours later during human in vitro fertilization. After approximately 3 h of capacitation, 22.1% of the fertile spermatozoa displayed motility patterns designated as hyperactivated. Over 80% of these hyperactivated spermatozoa moved with a wide-amplitude, two-dimensional whiplash pattern, displaying marked lateral displacement of the head. Only 8.4% of capacitating spermatozoa from oligozoospermic patients showed these hyperactivated movements. The incidence of hyperactivated movements by fertile and oligozoospermic spermatozoa could be significantly increased after exposure to various motility stimulants. The clinical significance of hyperactivation as a functional assay of fertilizing capacity is discussed.

Humans↗
PubMedDate not supplied

A microperfusion chamber for study of mammalian spermatozoa.

The design of a microperfusion chamber is presented for use with spermatozoa or other cell suspensions. This chamber allows perfusion of a small number of spermatozoa during simultaneous observation of cell behavior at the microscope. The chamber is made from a flat glass capillary tube that is fitted at both ends with a filter unit containing Millipore filter discs. The entire assembly is designed to fit the stage of an inverted microscope. A population containing as few as several hundred sperm cells may be observed in the chamber during successive changes of the suspending medium as controlled by a perfusion pump. Several experiments are presented demonstrating sperm survival in the sealed chamber and the response of rabbit and human sperm motility after the washing process. For these manipulations, the percentage of motile cells, linear swimming speed and incidence of hyperactivated motility are reported. Simple incubation in the chamber for 1 hour was not deleterious to the motility of rabbit spermatozoa. Human seminal spermatozoa showed no decline in vigorous motility after the washing procedure. Compared with in vitro capacitated spermatozoa, however, washing of rabbit seminal spermatozoa showed a variable response. Finally, partially capacitated human spermatozoa were examined for any alteration of motility during chamber incubation with a subsequent wash. When small numbers of spermatozoa or other cell types must be manipulated, the methodology can be effectively substituted for the standard washing procedure that uses repeated centrifugation and resuspension.

Animals↗
PubMedDate not supplied

Electron microscopic evidence on the acrosomal status of bound sperm and their penetration into human hemizonae pellucida after storage in a buffered salt solution.

The hemizona assay (HZA) was developed to evaluate sperm binding potential using microbisected human zona pellucida. In this study, eight human oocytes stored in a buffered salt solution for 60 days were bisected into two identical hemispheres (hemizonae) and coincubated with the spermatozoa from a fertile man. All evaluated spermatozoa were tightly bound to the outer surface or had begun penetration into the zona pellucida. The hemizonae with bound spermatozoa were prepared and fixed for transmission electron microscopy (TEM) using standard techniques. Among the 108 sperm bound to the zone we were able to evaluate 25 by TEM. Twenty (80%) of the zona bound spermatozoa were partially or completely acrosome reacted, while six (20%) of the zona bound sperm had intact acrosomes. Acrosome intact, partially acrosome reacted and completely reacted spermatozoa were observed within the zona. Penetration pathways or tunnels were seen within the zona matrix. The results illustrate, that typically spermatozoa tightly bound the human zona pellucida show induction of the acrosome reaction. Importantly, following storage of human eggs in salt solution (buffered to 7.4), the zona pellucida retain their biological and functional characteristics for at least 90 days.

Acrosome↗
PubMedDate not supplied

Functional aspects of human sperm binding to the zona pellucida using the hemizona assay.

The hemizona assay (HZA) has facilitated investigations of sperm function in relation to zona pellucida binding. In this study, the authors examined: 1) the association between hyperactivated sperm motility and HZA binding; 2) the binding kinetics and efficiency of sperm from subfertile men; and 3) the influence of sperm freezing and thawing on binding capacity. For each HZA, a nonviable human oocyte was cut into equal zona hemispheres. The mean number of bound sperm and the incidence of hyperactivation were significantly greater for samples of sperm from fertile men compared with sperm from subfertile men (P less than 0.05). Subfertile sperm had a binding curve that paralleled the curve for fertile sperm, although the magnitude of binding was markedly reduced. Freezing and thawing of sperm from fertile samples impaired their capacity to bind to the zona pellucida. The HZA binding efficiency was reduced by 30%, although the binding curves for fresh versus frozen samples remained parallel.

Biological Assay↗