Morphology comparison of individually selected hyperactivated and non-hyperactivated human spermatozoa

Steven Green1 and Simon Fishel

Centres for Assisted Reproduction (CARE), The Park Hospital, Sherwood Lodge Drive, Burntstump Country Park, Arnold, Nottingham, NG5 8RX, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The objective of this study was to compare the morphology of human spermatozoa undergoing hyperactivated motility in vitro with those that were non-hyperactivated (non-hyp). Hyperactivation criteria were established by the Hobson Sperm Tracker (HST), sampling at 25 Hz, as curvilinear velocity (VCL) >=70 µm/s, amplitude of lateral head displacement (ALH) >=7 µm, linearity (LIN) <=30% and straight-line velocity (VSL) <=30 µm/s. Specially developed software incorporated in the HST produced a white computer-generated overlay for spermatozoa satisfying hyperactivation criteria. These spermatozoa, visually identified on a tracking monitor, were individually removed with micromanipulation equipment using a 12 µm-diameter needle. Fifty-six patient ejaculates were examined comprising a total morphological analysis of 1886 non-hyp spermatozoa and 1051 hyperactivated spermatozoa. Hyperactivated spermatozoa had a significantly higher mean percentage of normal heads and small acrosomes (P < 0.0001 and < 0.0001 respectively) and a significantly lower percentage of large and round heads, midpieces and tail defects (P = 0.002, < 0.0001, 0.02 and < 0.0001 respectively) when compared with non-hyp spermatozoa. These data demonstrate, for the first time, that a homogeneous live population of human hyperactivated spermatozoa, selected in vitro from patients with highly variable degrees of teratozoospermia, is comprised predominantly of cells with normal morphology (P < 0.0001).

Key words: capacitation/hyperactivation/image analysis/morphology


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The first report of hyperactivated motility in capacitating spermatozoa was in 1970, with the observation that the flagellar movements of hamster spermatozoa became extremely active at the time that they completed capacitation (Yanagimachi, 1970Go). Since this report, similar phenomena have been observed in capacitated spermatozoa of many other species, including guinea pig (Yanagimachi, 1972Go; Barros et al., 1973Go; Yanagimachi and Usui, 1974Go), mouse (Fraser, 1977Go), dog (Mahi and Yanagimachi, 1976Go), rabbit (Cooper et al., 1979Go; Johnson et al., 1981Go), bat (Lambert, 1981Go), dolphin (Fleming et al., 1982Go), sheep (Cummins, 1982Go) and man (Burkman, 1984Go; Mortimer et al., 1984Go).

Hyperactivation is a physiological event, closely associated with ovulation and fertilization in those species studied. Detailed movement characteristics of hyperactivation have been examined, especially in man (Katz and Overstreet, 1979Go, 1981Go; Overstreet et al., 1979Go; David et al., 1981Go; Burkman, 1984Go; Mortimer et al., 1984Go; Aitken et al., 1985Go; Robertson et al., 1988Go; Mack et al., 1989Go; Mortimer and Mortimer, 1990Go; Burkman, 1991Go; Mortimer, 1997Go), as it is regarded as an important marker for capacitation and an essential prerequisite for penetration and fusion of a spermatozoon with an oocyte.

The extent of hyperactivation in human sperm samples has been reported to be associated with fertilization rates in vitro, and samples showing good fertilization rates in vitro undergo a significant change in hyperactivation during capacitation, whereas those showing poor fertilization rates in vitro demonstrate no change in hyperactivation (Coddington et al., 1991Go; Mbizvo and Alexander, 1991Go; Pilikian et al., 1991Go; Wang et al., 1993Go; Peedicayil et al., 1997Go; Chan et al., 1998Go). However, other authors have failed to show conclusively a correlation between the extent of hyperactivation and fertilization rates in vitro (Johnston et al., 1994Go; Sukcharoen et al., 1996Go).

The percentage of normal-shaped spermatozoa in the ejaculate, determined after strict morphological assessment, is also correlated with fertility (Kruger et al., 1988Go; Chavarria and Reyes, 1991Go; Bielsa et al., 1994Go), with morphologically abnormal spermatozoa having impaired functional ability (Carrell et al., 1994Go).

Hence, morphology and motility characteristics of human sperm populations have both been implicated in sperm fertility potential (Overstreet et al., 1981Go; Morales et al., 1988Go). In this study, carried out on patients undergoing in-vitro fertilization (IVF), we examined the relationship of the morphology of individually isolated spermatozoa to their hyperactivation status, using Computer Image Sperm Selection (CISS) (Green, 1995Go; Green et al., 1995Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Determination of hyperactivation
There is no single unified agreement on a defined criterion for hyperactivated motility. It is universally agreed, however, that the distinguishing characteristics of hyperactivated spermatozoa is a curvilinear velocity (VCL) >70 µm/s, irrespective of the frame rate (Robertson et al., 1988Go; Grunert et al., 1990Go; Mortimer and Mortimer, 1990Go; Burkman, 1991Go; Zhu et al., 1994Go) and a low linearity (LIN), although the threshold values for LIN vary considerably across independent studies, ranging from 19% (Robertson et al., 1988Go) to 80% (Zhu et al., 1994Go). These variations in LIN are related to the fundamental differences in the way in which hyperactivation has been classified. In general, those studies that regarded the non-progressive starspin and thrashing description of the trajectory as the dominant hyperactivated cell quoted thresholds for LIN as approximately 20% (Robertson et al., 1988Go; Burkman, 1991Go; Sukcharoen et al., 1995Go) to 30% (Grunert et al., 1990Go). Where higher LIN values have been quoted, for example the figures of 65% (Burkman, 1991Go) and 80% (Zhu et al., 1994Go), the definition of hyperactivation has been expanded to include a population of more progressive spermatozoa which have been described by Robertson et al. (1988) as transitional hyperactivated, also characterized by high VCL values, but which are now believed to be a false hyperactivation classification (Mortimer et al., 1997Go). A similar population of spermatozoa have been classified by Burkman (1991) as circling high curvature and helical, according to whether the flagellar beat was planar or three-dimensional. Therefore, the various LIN values selected in different classification systems reflect fundamental differences in the underlying nature of the sperm populations classified as hyperactivated.

In addition to VCL and LIN, a third component—amplitude of lateral head displacement (ALH)—has been widely used to identify hyperactivated motion, and the values for this parameter are >=7 µm in all published studies (Robertson et al., 1988Go; Grunert et al., 1990Go; Mortimer and Mortimer, 1990Go; Burkman, 1991Go; Zhu et al., 1994Go).

In addition to the various classifications for hyperactivation, the effects of pixellation of the image and sampling frequency also alter the measured values for parameters used to describe hyperactivated trajectories. For any image-processing system, measurements can only be to the accuracy of 1 pixel, or picture element. Since ALH requires the measurement of two turning points, the inherent error in the ALH measurement is approximately the square root of 2 pixels. If, for example a system were calibrated so that 1 pixel was equivalent to 1.1 µm, then the error in the ALH measurement would be approximately 1.5 µm. VCL is similarly affected, but the inherent error is not as significant, since the actual distance measured to obtain a value for VCL is greater than that for ALH.

The values for VCL and ALH are also affected by the framing rate, which refers to the video-sampling rate, used to obtain the measurements (Mortimer et al., 1988Go; Mortimer and Swan, 1995bGo). For the present study, the video sampling rate used for the Hobson Sperm Tracker (HST) was 25 Hz, which was the European standard (Sukcharoen et al., 1995Go), although recent ESHRE guidelines for the use of computer-aided sperm analysis (CASA) (ESHRE Andrology Special Interest Group, 1998Go), state that a video sampling rate of 50 Hz should be adopted as the standard.

Before establishing hyperactivation thresholds for the HST, the measurement of amplitude, frequency and velocity were validated by obtaining measurements of simulated waveforms of known values and at varying angles of trajectory, from a calibration videotape especially prepared by Hobson Tracking Systems Ltd (Sheffield, UK). The values obtained for amplitude and frequency measurements only differed from the expected values within limits that could be explained as a consequence of the pixel effect and the measured values for velocity were consistently below 5% from the expected values.

Although CISS incorporates a unique application for CASA, the conditions for the measurement of hyperactivation still conformed to the guidelines recently recommended by the ESHRE Andrology Special Interest Group (1998).

Hyperactivation criteria were established for the HST from measurements of reference hyperactivated spermatozoa trajectories, adopting the method of Burkman (1991). Hyperactivated spermatozoa trajectories were identified as displaying a wide lateral head amplitude, little forward progression, low frequency of flagella beating, and a high degree of flagella curvature (Mack et al., 1989Go), and recorded onto video tape after positive phase-contrast microscopy. An image threshold value of +78 /–13 was used for the HST in order to obtain complete coverage of the computer-generated colour overlay over the sperm head and a search radius of 18 µm, i.e. the distance the HST computer searched to link up the trajectory of the spermatozoon, was used to provide uniform computer-generated trails of the trajectory.

The thresholds for hyperactivated trajectories sampled for CISS were VCL >=70 µm/s, straight-line velocity (VSL) <=30 µm, LIN <=30% and ALH >=7 µm, values corresponding to those previously reported from 30 Hz measurements describing starspin and thrashing hyperactivated trajectories (Burkman, 1991Go).

Manually drawn trajectories for all the reference spermatozoa also corresponded to the description of starspin and thrashing hyperactivation previously reported by Burkman (1991). In order to ensure that only spermatozoa satisfying the hyperactivated thresholds were included for analysis, a specially developed HST computer program changed the colour of the computer-generated overlay on the spermatozoon, from yellow to white for those trajectories that reached all the parameter thresholds. This facility demonstrated that the HST could reproducibly identify all the reference hyperactivated spermatozoa by attributing a white computer-generated colour to the trajectory in real time, which was seen on the tracking monitor.

Criteria for non-hyperactivated spermatozoa
Our criteria for non-hyp spermatozoa were those displaying uniformly progressive trajectories with no agitation that would indicate a transition to the hyperactivated state. The CASA parameters corresponded to the description of linear trajectories, which were highlighted with yellow computer-generated trails, confirming visually that hyperactivation parameters for these spermatozoa had not been reached.

Patient selection
A total of 67 patients were recruited into the study and grouped according to the percentage of normal spermatozoa in the original ejaculate. This analysis was performed using the modified classification after Kruger et al. (1987), specific for the Diff Quick stain (Hall et al., 1995Go). Eleven patients were excluded, as their spermatozoa did not hyperactivate during the incubation time, although only two of these patients failed to fertilize with IVF. This supported the view that measurement of spontaneous hyperactivation in populations of human spermatozoa in vitro does not give a good correlation with IVF outcome (Johnston et al., 1994Go; Sukcharoen et al., 1996Go).

Group A patients (n = 14) had spermatozoa morphology scores of >=14% normal forms. Group B patients (n = 21) had percentage normal forms ranging from 6–13%. Group C patients (n = 21) had percentage normal forms of <=5%. Group D comprised the combined data for all 56 patients. The clinical treatment for all patients was IVF but, to compensate for the poorer morphology, high insemination concentration (HIC) IVF was used for patients in groups B and C (Fishel et al., 1995Go), with all patients achieving fertilization.

Sperm preparation
Sperm samples used for this study were from those produced routinely from male partners of couples attending for fertility treatment. Couples signed consent forms relating to the use of spermatozoa for research purposes.

Semen samples were produced by masturbation and carefully layered over a 45% and 90% discontinuous Percoll® (Sigma, Poole, Dorset, UK) gradient. This was centrifuged at 500 g for 10 min. The motile spermatozoa at the bottom of the 90% fraction were removed with a glass pipette, mixed with Earle's balanced salt solution (EBSS) 10% and warmed to 37°C. The spermatozoa were centrifuged at 250 g for 5 min, after which the supernatant was removed. The spermatozoa were resuspended in fresh EBSS 10% and centrifuged at 250 g for 5 min. After the second washing step, the spermatozoa were diluted to a concentration of approximately 200 000 motile cells per ml, a concentration that would facilitate CISS and spermatozoa retrieval. All samples were incubated at 37{infty}C in 5% CO2 in air at a relative humidity of 95%.

Computer image sperm selection (CISS)
Details of the CISS technique used for the identification and removal of spermatozoa in hyperactivation have been published previously (Green, 1995Go; Green et al., 1995Go). Briefly, selection of spermatozoa was performed from a 200 µl volume of Earle's Balanced Salt Solution containing 10% v/v maternal serum (EBSS 10%) under light mineral oil (Sigma), held within a temperature-conserving stage (Research Instruments Ltd, Penryn, Cornwall, UK), on an inverted IMT2 microscope (Olympus UK Ltd, London, UK) equipped with micromanipulation tools (Research Instruments Ltd). The HST computer uses real-time analyses of sperm motion via a Sony camera (model DXC 101P) attached to the microscope, providing on-line data analysis after continuous tracking. This is an essential requirement for CISS. A computer-generated colour overlay highlighted spermatozoa that satisfied hyperactivation thresholds. These spermatozoa were removed with a 12 µm microneedle under the visual control of the tracker monitor, situated at eye level to the left of the microscope. The aspiration was controlled by air-filled, screw-actuated syringes (Research Instruments Ltd). Once the spermatozoa were retrieved they were transferred to a microwell slide (C.A.Hendley Ltd, Loughton, Essex, UK) under microscope visualization, and fixed and stained for morphological assessment using the Diff Quick procedure (Hall et al., 1995Go)

Preparation of samples
Diluted aliquots of 200 000 spermatozoa per ml concentration were incubated for up to 6 h, and as many hyperactivated spermatozoa as possible—highlighted by white trails—were aspirated from the samples and transferred to separate microwells on slides (C.A.Hendley Ltd). At the same sampling times, a collection of non-hyperactivated spermatozoa, highlighted by yellow trails were aspirated and transferred to microwell slides in the same way. The microwells relating to hyperactivated and non-hyperactivated spermatozoa were labelled with a non-identifying code. The slides were fixed and stained in the manner described by Hall et al. (1995) and analysed independently by seminologists experienced in strict criteria morphological analysis, but who had no knowledge of the code.

Morphology preparation and evaluation
The slides were placed in the Diff Quick fixative solution (1.8 mg/l triarylmethane in methyl alcohol) for 15 s before staining with Diff Quick solution 1 (1g/l xanthene in sodium azide-preserved buffer) for 5 s, allowed to drain, followed by dipping for 5 s in Diff Quick solution 2 (0.625 g/l azure A and 0.625 g/l methylene blue in buffer). The slides were rinsed thoroughly with distilled water, dried and mounted using DPX mountant with a coverslip.

For each patient, all the selected spermatozoa still present on the microwells after fixing and staining were evaluated using light microscopy under oil immersion at a magnification of 1000x using an Olympus BH2 compound microscope (Olympus UK Ltd) for seven categories of morphology. A diagrammatic representation of these criteria is shown in Figure 1Go and described below.



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Figure 1. Schematic representation of sperm morphological criteria, as described in the text.

 
Sperm morphological criteria
Normal spermatozoa (N):
defined as those with a smooth oval head, 5–6 µm in length and 2.5–3.5 µm in diameter. A well-defined acrosome, occupying 40–70% of the sperm head. A midpiece without defect, slender, axially attached width <1 µm. The tail uniform, free from kinks, uncoiled width, thinner than midpiece, length approximately 45 µm. Remnants of cytoplasmic droplets which comprised less than half the head area were acceptable if retained in the midpiece region only.

Small acrosome (SA):
defined as accounting for <40% of the head size.

Small (SH) and large head (LH):
identified those spermatozoa with measured parameters outside the normal range.

Round head (RH):
identified as those spermatozoa with a round, rather than ovoid, head shape independent of size.

Mid piece (MP) and tail abnormalities (T):
defined as any structural defect.

Statistical analysis
The chi-square statistic equation {sigma} (O–E)2/E with 1 degree of freedom was used to compare the frequency of each morphology category between hyperactivated and non-hyperactivated sperm types, where O = observed frequencies and E = expected frequencies. P values are tabulated in Table IIGo. Data are given ± SD.


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Table II. {chi}2 P values for the distribution of seven morphological criteria to differentiate between hyperactivated and non-hyperactivated (non-hyp) human spermatozoa selected after computer image sperm selection.
 

    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
All semen samples in the study achieved fertilization, with mean fertilization rates per oocyte of 78.1 ± 37.5% in group A, 70.6 ± 31.6% in group B, and 63.8 ± 28.6% in group C patients. Eleven patients were excluded from the study as their spermatozoa did not reach hyperactivation thresholds during incubation.

The total number of spermatozoa assessed for morphology in the non-hyperactivated and hyperactivated spermatozoa for each patient group is shown in Table IGo.


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Table I. Number of hyperactivated and non-hyperactivated (non-hyp) spermatozoa studied in patient groups A, B, C and D
 
The mean percentage distribution of the seven morphological criteria for all groups is shown in Figures 2–5GoGoGoGo.



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Figure 2. Mean frequency (%) of seven morphological criteria (detailed in Figure 1Go) for hyperactivated (n = 247) and non-hyp (n = 484) spermatozoa from group A patients (>=14% normal-morphology spermatozoa). Error bars represent the SD. Values in parentheses are the sperm numbers for each group.

 


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Figure 3. Mean frequency (%) of seven morphological criteria (detailed in Figure 1Go) for hyperactivated (n = 483) and non-hyp (n = 723) spermatozoa from group B patients (6–13% normal-morphology spermatozoa). Error bars represent the SD. Values in parentheses are the sperm numbers for each group.

 


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Figure 4. Mean frequency (%) of seven morphological criteria (detailed in Figure 1Go) for hyperactivated (n = 321) and non-hyp (n = 679) spermatozoa from group C patients (<=5% normal-morphology spermatozoa). Error bars represent the SD. Values in parentheses are the sperm numbers for each group.

 


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Figure 5. Mean frequency (%) of seven morphological criteria (detailed in Figure 1Go) for all hyperactivated (n = 1051) and non-hyp (n = 1886) spermatozoa from groups A, B and C patients (see Figures 2, 3 and 4GoGoGo). Error bars represent the SD. Values in parentheses are the sperm numbers for each group.

 
Morphology comparison between selected hyperactivated and selected non-hyp spermatozoa
Group A
A statistical comparison between spermatozoa selected in hyperactivation and those selected as non-hyp, collected from fertile non-teratozoospermic patients, defined as >=14% normal spermatozoa in the originating ejaculates (Kruger et al., 1988Go), showed no significant difference in the mean percentage of small and round-headed spermatozoa and spermatozoa with midpiece defects. There was a significantly higher mean percentage of normal forms (P < 0.0001) and significantly lower percentage with small acrosome, large head and tail defects (P = 0.001, 0.012 and 0.001 respectively; Figure 2Go, Table IIGo) in hyperactivated spermatozoa.

Group B
When the same morphological comparisons were made between selected hyperactivated and non-hyp spermatozoa, collected from patients with moderate teratozoospermia, defined as 6–13% normal forms in the originating ejaculates (Kruger et al., 1988Go), but proven fertile after HIC, there was no significant difference in the mean percentage of spermatozoa with small head and midpiece defects, a significantly higher mean percentage of normal and small acrosome (P = 0.0002 and < 0.0001) and significantly lower percentage with large head, round head and tail defects (P = 0.03, < 0.0001 and 0.03 respectively; Figure 3Go, Table IIGo) in the hyperactivated spermatozoa.

Group C
Morphological comparisons between selected hyperactivated and non-hyp spermatozoa, collected from patients with severe teratozoospermia, defined as <=5% normal forms in the originating ejaculates (Kruger et al., 1988Go), but proven fertile after HIC, demonstrated no significant difference in the mean percentage of small head spermatozoa, significantly higher mean percentage of those with normal head and small acrosome (P = 0.021 and < 0.0001 respectively), and a significantly lower mean percentage of large and round head, midpiece and tail defects (P = 0.04, < 0.0001, 0.0008 and 0.007 respectively; Figure 4Go, Table IIGo) in the hyperactivated spermatozoa.

Group D
When the total number of selected hyperactivated and selected non-hyp spermatozoa were compared, there was no significant difference in the mean percentage of small head, a significantly higher mean percentage of spermatozoa with normal head and small acrosome, (P < 0.0001 and < 0.0001 respectively), and a significantly lower mean percentage of spermatozoa with large and round head, midpiece and tail defects, (P = 0.002, < 0.0001, 0.02 and < 0.0001 respectively; Figure 5Go, Table IIGo) in hyperactivated spermatozoa.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The HST acquires motility data continuously and provides on-line analysis of any length of trajectory in real time. Its adaptation to facilitate CISS was developed after a collaborative effort with Hobson Tracking Ltd and has been described previously (Green et al., 1995Go). For some non-human species, >80% of spermatozoa show hyperactivated motility (Yanagimachi, 1970Go; Katz and Yanagimachi, 1980Go), but in the human approximately 20% of spermatozoa washed and incubated in synthetic media develop hyperactivated motility (Burkman, 1984Go), which prohibits routine morphological analysis of a homogeneous population of these cells. The CISS technique, although time-consuming and requiring manipulation skills, can provide a homogeneous collection of hyperactivated human spermatozoa for analysis, with each individual spermatozoon being selected after satisfying the thresholds determined for hyperactivation. In this respect, the CISS approach is a novel tool for the study of hyperactivation.

Although overall we found that preparing the ejaculates through Percoll® changed the morphological distribution, with a significant increase in the mean percentage of normal spermatozoa, coincident with significant decreases in those with midpiece and tail defects, in agreement with previous studies (Brandeis and Manuel, 1993Go; Hall et al., 1995Go; Yao et al., 1996Go), this did not affect the comparative nature of the study. An objective analysis of the total data demonstrated that spermatozoa selected in hyperactivation comprised a significantly higher mean percentage of normal forms compared with those spermatozoa selected as non-hyp, a feature that was not affected by the severity of the teratozoospermia, as shown in the analysis of the individual patient groups. This suggests that spermatozoa with normal morphology may be more predisposed to undergo hyperactivation after capacitation, the latter conferring upon the spermatozoon the ability to undergo fertilization (Fraser, 1992Go; Benoff, 1993Go). One aspect of capacitation is the transition to hyperactivated motility (Boatman and Robbins, 1991Go; Tournaye et al., 1994Go).

The role of hyperactivation is complex. Suarez (1996) proposed that this pattern of motility might confer a mechanical advantage to the spermatozoon and, near the time of ovulation, a change to a `hyperactivated' motility pattern may help spermatozoa detach from the epithelium, escape mucosal pockets and move through the oviductal mucus. As the spermatozoa reach the ampulla, frequent changes in direction may enable them finally to reach the cumulus matrix. Factors in the periovulatory oviduct or follicular fluid, such as free calcium, sodium, potassium and energy substrates (Fraser, 1992Go), may induce a hyperactivated state in capacitated spermatozoa, providing the forces required for penetration of the oocyte investments. Our morphological analysis of hyperactivated spermatozoa in vitro after CISS suggests that hyperactivated spermatozoa are significantly of normal morphology, irrespective of the originating teratozoospermia of the ejaculate. If, as we suggest, spermatozoa with normal morphology may be more predisposed to undergo hyperactivation after capacitation, then the selection process that occurs in vivo, starting from penetration of the cervical mucus, which is known to exclude many abnormal spermatozoa (Hanson and Overstreet, 1981Go; Katz et al., 1990Go), may result in spermatozoa arriving at the site of fertilization with morphological characteristics that may confer a greater propensity for hyperactivation and a better chance of interacting with the oocyte.

Spermatozoa displaying different patterns of movement have been reported as being non-equivalent in relation to fertility potential, with the non-progressive pattern of movement consistently selected as that component of motility that had the greatest relevance to the fertilizing capacity (Sukcharoen et al., 1995Go). However, it has been noted in the human that hyperactivation phases are not stable and spermatozoa do switch between them (Mortimer and Swan, 1995aGo). Manual drawings of spermatozoa trajectories used to establish our hyperactivated thresholds for the HST were in agreement with those previously described as starspin and thrashing (Burkman, 1991Go), with both types of trajectory displaying a characteristic non-progressive form which was confirmed by HST tracking over a period of several seconds. The data presented here support the view of an association between a non-progressive pattern of spermatozoa movement with fertilization capacity, and this may be related to a significantly increased normal morphology of spermatozoa displaying this pattern of movement.

Biological selection of normal spermatozoa has been implicated at the level of the oocyte. Tesarik et al. (1990) demonstrated that the intercellular matrix of the human cumulus oophorus exerts a specific effect on human spermatozoa motility, thought to act preferentially on the hyperactivated sperm subpopulation, while Carrell et al. (1993) reported that the cumulus oophorus may play a role in the selection of morphologically normal spermatozoa that ultimately reach the zona pellucida. Both these reports are consistent with that of Liu and Baker (1994), who found that 84% of acrosome-reacted spermatozoa bound to the zona pellucida were morphologically normal, compared with 38% in the culture medium.

Carrell et al. (1994) reported that spermatozoa with head abnormalities have a lower rate of spontaneous acrosome reactions, while Moutaffian and Parinaud (1995) noted that spermatozoa able to undergo the acrosome reaction had fewer head and tail abnormalities. The mean percentage of other abnormalities recorded in our study—with the exception of spermatozoa with small acrosomes—were either not changed or significantly lowered in groups A, B and C. When data from all the teratozoospermic groups were combined, group D—the hyperactivated spermatozoa—showed a significantly higher mean percentage of spermatozoa with small acrosomes, no significant difference in the mean number of spermatozoa with small heads, and significantly reduced numbers of spermatozoa with large and round heads, midpiece and tail defects, compared with the selected non-hyp. This distribution pattern could suggest that spermatozoa predisposed to undergo hyperactivation have significantly less abnormal forms than non-hyp spermatozoa, and in addition small-head spermatozoa may be functionally equivalent to normal spermatozoa in this respect. Recent data also suggest that spermatozoa with heads categorized as small by strict morphological analysis should be regarded as functionally normal spermatozoa (Rashid et al., 1998Go).

Although the occurrence of small acrosomes was significantly higher in the selected hyperactivated spermatozoa examined in this study, there was no evidence that spermatozoa categorized as small-acrosome after Diff Quick staining had undergone acrosomal loss. However, the frequency of spontaneous acrosomal loss in hyperactivated human spermatozoa, using the same selection criteria, is currently under investigation using a fluorescent staining procedure.

A positive association between increased VCL and average path velocity (VAP) with percentage normal morphology, has been proposed by Claassens et al. (1996) which is in support of our hypothesis, since the motility thresholds defining hyperactivation for CISS included a high VCL of >70 µm/s.

For the first time, this paper describes the isolation of homogeneous populations of spermatozoa displaying hyperactivated criteria in real time. These populations demonstrated a significant positive correlation between normal morphology and the spermatozoa undergoing hyperactivation.


    Acknowledgments
 
The authors would like to thank Jayne Thomas and Tom Newton for their expertise in reading the morphology slides, and Jamie Smith for producing the computer-generated schematic drawing.


    Notes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on July 7, 1998; accepted on October 14, 1998.