Selectivity of the human sperm-zona pellucida binding process to sperm head morphometry

FERTILITY AND STERILITY@ Vol. 67, No. 2, February 1997

Copyright * 199’7 American Society for Reproductive Medicine Printed on ad-free paper m U. S. A.

Selectivity of the human sperm-zona pellucida binding process to sperm head morphometry*

Claire Garrett, Ph.D.t De Yi Liu, Ph.D. H. W. Gordon Baker, M.D., Ph.D.

University of Melbourne Department of Obstetrics and Gynecology, and the Reproductive Biology Unit, Royal Women$ Hospital,

Melbourne, Victoria, Australia

Objective: To obtain quantitative measures of morphometric selectivity of the human sperm- zona pellucida binding process as determined by light microscopy.

Design: Fully automated sperm head morphometry based on a 32-dimensional parameterization of images of Shorr-stained sperm. Zona pellucida selected sperm removed from reinsemi- nated oocytes that previously failed IVF.

Setting: Academic research group associated with a tertiary infertility service. Patient(s): Semen samples from 51 infertile patients. Intervention(s): None. Main Outcome Measure(s): Differences in morphometric parameter means observed before

and after swim-up and binding to zonae pellucidae. Result(s): Significant differences between insemination and bound sperm were observed in

1’7 parameter means and 21 standard deviations. The sperm-zona pellucida binding process preferentially selects sperm heads with a large anterior region with relatively low optical density, as well as high axial symmetry and minimal neck anomalies. Bias against sperm with pyriform morphology was not observed.

Conclusion(s): A causal link has been established between sperm head morphometry, partic- ularly within the acrosomal region, and the ability of sperm to bind to the human zona pellucida. As sperm-zona binding is necessary for fertility, it is possible to derive a physiologically ba.sed assessment for clinical diagnosis of male infertility using the “zona-preferred” morphometry results. Fertil Steril@ 1997;67:362-71

Key Words: Sperm-zona binding, sperm morphometry, automated image analysis, zona selectivity, acrosomal region

A recent review article reports various assessments of percent normal morphology of sperm in human semen that have shown a significant positive relationship with fertilization rates in vitro (1). How- ever, except in rare instances involving a specific and universal morphological defect, such as round- headed acrosomeless sperm (2), little is known about the mechanism underlying the relationship or the degree of causality. The finding that percent normal

Received June 3, 1996; revised and accepted September 12, 1996.

* Supported by the Sperm Research Fund, Melbourne IVF, Mel- bourne, Victoria, Australia.

+ Reprint requests: Claire Garrett, Ph.D., Professorial Unit, Royal Women’s Hospital, 132 Grattan Street, Carlton, Victoria, 3053. Australia (FAX: 61-3-347-1761).

362 Garrett et al. Morphometry of zona bound sperm Fertility and Sterility””

morphology in semen is a better predictor of IVF rate than percent normal morphology of the insemination medium (3) suggests that morphological abnormalities observed in individual sperm serve as a marker for the presence of more subtle defects common to the sperm population as a whole, thus identifying fertilizing potential as a uniform characteristic of a sample, rather than an average of the individual capabilities of spermatozoa in the insemination medium. In contrast, recent studies report that binding to both the human zona pellucida (ZP) and oolemma (4-6) demonstrate selectivity of spermatozoa with normal morphology, indicating causal elements in the relationship, at least between sperm morphology and these prerequisites of fertilization.

Failure of some studies to support a relationship between sperm morphology and M? rate (1) may be

attributable to patient selection or use of earlier, less stringent criteria for normality in the morphology assessment. In studies in which a positive relationship is observed between morphology and IVF suc- cess, there remain individual cases that show nota- ble departures from the general trend. Because oocyte quality and other semen characteristics are confounding factors, the occurrence of poor IVF suc- cess with semen possessing a high percentage of normal forms does not necessarily imply incorrect classification of morphological normality. However, the existence of IVF patients with percent normal morphology < lo’%, who achieve fertilization rates at or near 100% with substantial numbers of oocytes (7) or achieve fertilization with percent normal morphology = 07~ (81, does indicate some inadequacy in the definition of abnormality.

Because it is not possible to ascertain the fertilizing potential of individual human spermatozoa, physiological endpoints other than fertilization must be studied to obtain insight into the mechanisms by which sperm morphology influences the fertilization process. Study of the ZP binding process between human sperm and oocytes has the potential to provide this information because it is a necessary, though not sufficient, requirement of fertilization, both in vivo and in standard IVF procedures. It also is expected to be a highly selective process, given that only a relatively small number of sperm bind to the ZP despite high insemination concentrations (4, 9). Moreover, the correlation identified between sperm-zona pellucida binding rates and sperm morphology shows an implicit relationship between this selectivity and morphology (g-111, supporting other reports of explicit morphological selection of normal sperm by the ZP binding process (4-6). Finally, it is physically possible to isolate for morphology assessment those sperm that have bound to the ZP. Early methods, which involved crushing the oocytes, had the disadvantage that the presence of oocyte frag- ments prevented all sperm from lying in the focal plane with the maximal area orientation assumed for morphology assessment (4). This problem of orientation also is present with attempts to assess morphology of sperm whilst bound to the ZP (6, 12). However, with a new pipetting technique it is possible to remove bound sperm from the ZP and thus produce conventional slides for morphology assessment (5).

The predictive value of human sperm morphology assessment with respect to in vivo fertilization and IVF results generally has been established with application of strict qualitative limits of normality and particular attention to the differentially stained acrosomal region of the head (1). Our recently devel- oped fully automated image analysis system has

demonstrated accuracy and reproducibility for the detailed parameterization of both optical density contours and profiles of light microscopy images of human sperm heads (13). The sensitivity of this system to the staining heterogeneity of the head, as well as differentiation between anterior and posterior morphometry, makes it highly suitable for the quantitative analysis of the morphological differences observed between sperm bound to the ZP and those in the insemination medium. Provided these differences show a “ZP-preferred” morphometry, this analysis should yield unbiased criteria for the classification of spermatozoa with ZP bind:ing ability, which forms a subset of the ideal classification of spermatozoa with fertilizing ability.

MATERIALS AND METHODS

Sperm Samples and Preparation

Semen samples were obtained from 30 IVF patients and 21 patients under investigation for infertility. To obtain sufficient numbers of ZP bound sperm for morphometric analysis, samples were selected for normal sperm concentration (>20 M/mL) and motility (motility > 40% and forward progression > 25%) but covering a wide range of morphologies (1% c- percent normal morphology 5 51%, mean percent normal morphology = 18%).

In preparation for morphology assessment, semen samples were washed to remove the viscous seminal plasma and resuspended to a standard. concentration of approximately 80 x lO”/mL (n = ~301. Insemi- nation samples were prepared by standard swim-up technique (n = 51) (14).

Zona Pellucida Bound Sperm

Oocytes that had failed to fertilize in vitro after 48 hours were obtained from IVF patients, denuded of cumulus and previously bound sperm using a 120- pm diameter pipette, and then pooled for reinsemi- nation with an average of four oocytes per well. De- generate oocytes and oocytes with the ZP penetrated by > 10 sperm were excluded. The fresh oocytes were incubated with 2 x lo6 motile sperm in 1 mL of medium for 2 hours at 37°C in 5% CO, in air. After incubation, the oocytes were washed and aspirated with a micropipette (approximately 250-pm diameter) to dislodge spermatozoa loosely adhered to the surface of the ZP (14). Because insemination concentrations were chosen to provide sufficient numbers of ZP bound sperm for morphometric analysis, in most cases the number of sperm bound to the ZP exceeded 100 and could not be reliably counted. With subsequent and repeated aspiration using a pipette with a diameter slightly smaller than that of the

Vol. 67> No. 2, February 1997 Garrett et al. Morphometry of zona bound sperm 363

oocyte (approximately 120 pm), ZP bound sperm were removed in a small volume of buffer and dried on a microscope slide in a region labeled with a dia- mond-tipped pen for future location (14).

Acrosome Assessment

The percentage of intact acrosomes present in a sample was determined using fluorescein labelled Pisum satiuum agglutinin (15). Measurements were performed on samples of spermatozoa bound to the ZP, in the insemination medium before and after incubation, and in washed semen.

Morphometric Analysis

Spermatozoa were stained for manual morphology and automated morphometry assessment using Shorr stain (16) and all slides prepared from the same initial semen sample were stained in the same batch. Because the morphometry of ZP bound and postinsemination spermatozoa was assessed subsequent to labeling with Pisum satiuum agglutinin, the morphometry of 17 preinsemination samples was assessed before and after Pisum sativum agglutinin labeling as a control for the influence of the Pisum sativum agglutinin staining process.

The morphology assessment of a sample was performed manually by an experienced observer using World Health Organization criteria (171, with particular emphasis on a regular acrosome region. For ZP- bound samples, the percentage of normal forms was assessed for 100 spermatozoa, except in nine samples in which only 50 sperm were located for examination. All other assessments of morphology were performed on 200 sperm per sample.

The morphometry of each sample was assessed using our fully automated image analysis system (13). In all but the ZP-bound samples, 200 sperm were assessed using the fully automated image selection, sperm identification, and morphometry analysis capabilities of this system. The sparsity and scarcity of spermatozoa on the morphology slides prepared from 20 of the ZP-bound samples necessi- tated manual scanning of microscope fields to locate efficiently and to focus images of sperm that were in all other respects analyzed automatically. Because the morphology slides prepared from ZP-bound sperm are exceptionally clean with respect to back- ground and superposition of cells, the manual selection of images is not expected to compromise significantly the objectivity of the analysis. The mean number of ZP-bound sperm analyzed per sample was 146 (range 39 to 216).

The morphometric assessment of a sample is summarized by mean -C SD for each of 32 morphometric parameters, 13 of which are derived explicitly from

internal optical density considerations. With one ex- ception, the morphometric parameters used in this analysis are as described in Garrett and Baker (13). The area of the darkest 40% of the sperm head image &,) previously was given as a percfentage of the total head area, however, the absolute value of this area was found to be more sensitive with respect to ZP selectivity and subsequently was defined as such for this study. The morphometry of a sample is also assessed in terms of the percent conformity, repre- senting the percentage of sperm in $9 sample conforming to within 22.5 SD of a set of “normal” reference morphometric parameter means (13).

Statistical Analysis

The significance of differences observed between the various groups was determined for the morphometric parameter means and SDS u.sing the non- parametric paired Wilcoxon test. These differences include changes due to Pisum sativum agglutinin staining procedure, selectivity of swim-up, selectivity of ZP binding, and the sequential combined ef- fects of swim-up followed by ZP binding. Each difference is expressed as a percentage change to facilitate comparison of the influence of selectivity on each morphometric parameter.

For samples with Pisum sativum agglutinin results (n = 128), the relationship between the percentage of normal intact acrosomes in a sample and each morphometric parameter was analyzed by linear regression. Because Pisum sativum agglutinin results for the insemination samples were observed to have a restricted range (69% to 96% intact acrosomes), the sensitivity of the morpho!metry analysis to the acrosome reaction was enhanced by separate analysis of ZP (2% to 97% intact acrosomes) and washed semen (34% to 87% intact acrosomes) samples.

For certain parameters, the differences between sample means did not deviate significantly from zero, however, the corresponding SDS showed significant reduction after the ZP binding process or the swim-up procedure. Consequently, linear regression analysis was used for a more detailed examination of the morphometric selectivity of each process and enabled inclusion of the influence of change in acrosome status. Thus, for each parameter, percentage differences between sample means were analyzed by multiple linear regression, with the initial sample mean and the difference in Pisum sutiuum agglutinin results as covariates. The regression was weighted by the square root of the number of sperm in the ZP morphometry assessment. Although ZP sperm were physiologically selected from postswim- up insemination samples rather than from the gen-

364 Garrett et al. Morphometry of zona bound sperm Ferfility and SterilityE

era1 semen population, the regression analysis also was performed on the differences between morphometric parameter means of ZP and washed semen samples, as they may have clinical value in the future.

Differences in the manual percent normal (%N) and the morphometric percent conformity assessments (%C) for ZP and insemination samples were examined in terms of B, the ratio of the average ZP binding probabilities of sperm classified as “abnormal” and “normal” in each insemination sample. Thus,

%N

B*(l()O - %N) insemination

and therefore for %N B q,N = [%N/(lOO - %N)l,,,,.l[%N/(lOO - %N)lzp.

Similarly, for %C Bqc = [%C/(lOO - %C)]i,,,,,/[%C/(lOO - %C)]zp

where B is expected to vary between 0 and 1, re- flecting different responses of the ZP binding process to the various categories of sperm abnormality. B = 0 corresponds to exclusive ZP selection of normal forms and B = 1 indicates no differentiation by the ZP between sperm classified as normal and abnormal. Errors in Bnoc were calculated from standard errors in both %Czp and %Cinsem;

Unless otherwise specified, statistical significance refers to P < 0.005. This limit has been chosen in preference to the usual <0.05 to reduce the probability of type I error in the multiple comparisons performed in this work.

Theoretical Sperm-ZP Collision Frequency

The number of sperm that make physical contact with the ZP of a cumulus-denuded oocyte in an insemination well can be estimated theoretically. As- suming that the motile sperm in the insemination medium maintain a uniform density, d (xlO’/mL) and exhibit random direction of motion with constant speed, v (pm/s), then, at any instant in time, the average number of sperm located within a given spherical volume of radius r (pm) is 4/3&d X lo-’ and the average time taken for a spermatozoon to clear that volume is approximately r/v (seconds). Av- eraged over time, the number of sperm within the volume element must remain constant and, thus, the average rate at which sperm enter the volume is equal to the average rate at which they leave and is approximately 4/3&Dv x lo-’ sperm/s. Given that the human oocyte-ZP complex corresponds to a spherical volume with r approximately 60, then for typically normal sperm with v approximately 60, the

number of sperm making contact with the ZP per hour is 3,000 d. This corresponds to 6,000 possible sperm-ZP interactions per hour under the insemination conditions used in the ZP binding experiments described in this work. As an indication of the valid- ity of this simple model of sperm-ZP contact, the same calculation applied to equine data predicts a collision rate of approximately 75 sperm/h (r = 60; v = 75; d = 0.02), which is consistent with the concentration normalized (d = 0.02) average equine binding rate of 111% 32 sperm/h observed in the first hour of insemination of salt-stored oocytes retrieved from ovaries after slaughter (18).

Ethics

The project was approved by the Royal Women’s Hospital Research and Ethics Committee. Patients consented to the use of their sperm and oocytes for research.

RESULTS

Morphometric Influence of the Pisum satiuum Agglutinin Staining Process

No significant differences were observed in any morphometric parameter assessed before and after Pisum sativum agglutinin staining. The Pisum sativum agglutinin process was found on average to reduce the measured intensity of the axial optical density of sperm heads by only 1.5%, ranging from an 8% reduction to a 9% increase in intensity.

Morphometric Influence of Acrosome Status

In the linear regression analysis applied to all samples with Pisum sativum agglutinin results, the measured percentage of intact acrosomes in the sample was not related (r’ 5 0.1) to the sam:ple mean for any morphometric parameter. However, when the predominantly acrosome intact insemination samples were excluded from this analysis, seven parameters exhibited significant regression coefficients and r2 values > 0.1. The specific correlations observed between Pisum sativum agglutinin results and morphometric parameter sample means indicate a sensitivity of the image analysis system to the loss of sperm head length and, thus, area, and reduced mirror and elliptical symmetry of the anterior region of the head after spontaneous or zona- induced acrosome reaction.

Morphometric Selectivity of the Swim-up Procedure

Significant differences were observed between insemination and washed semen sample means for 12

Vol. 67, No. 2, February 1997 Garrett et al. Morphometry of zona bound sperm 365

morphometric parameters. Standard deviations for all parameters were smaller for insemination samples relative to those in washed semen, with significant differences in SDS obtained for 18 parameters. The observed differences and regression of the percentage differences on the initial washed semen values indicate a selectivity in the swim-up procedure of symmetric and regular head forms, with an ab- sence of neck anomalies. The two morphometric parameters most sensitive to pyriform head morphology (paw and pW) show no significant difference in sample means or SDS before and after the swim-up procedure.

Morphometric Selectivity of the Sperm-Zona Pellucida Binding Process

Percentage differences in morphometric parameter means and SDS observed between insemination

and ZP bound samples are summarized in Table 1. Ten of 17 parameters identified with significant differences are derived from either optical density profiles or contours. These densitometric differences are consistent with high zona selectivity of sperm heads exhibiting a large anterior region defined by relatively low optical density. More specifically, the parameter defining the area of the 40% optical density contour L&J is significantly reduced by ZP selection, the anterior percentage of the sperm head length associated with axial optical density below the mean density, referred to as the Bill length (Bill), is significantly higher, and the “ellilpticity” of the transverse optical density profiles (eCXL) is considerably thinner by ZP selection. Other significant differences indicate further preferential selection of symmetric (reduced aSym and pSym), oval anterior (reduced aRuff) head forms, no neck anomalies (re-

Table 1 Mean of ZP Sample Morphometric Parameter Means and SDS, Mean Percentage Differences Between ZP and Insemination Sample Means and SDS, and Coefficients of Determination Where There is a Strong Linear Relationship Between Percentage Differences in ZP and Insemination or Washed Semen Sample Parameter Means and Those of the Initial Sample+

Parameter namet Code

Mean of ZP

means

Mean Mean SD Mean percentage percentage

of ZP of ZP difference difference means SDS of mean& of SD& i-‘$$ 33

Head length (pm) L 4.46 0.26 Head width (pm) W 2.99 0.15 Perimeter (pm) P 12.65 0.62 Head area (p,rn’) A 10,24*:” 0.85 Neck area (pm”1 nA 1.25** 0.14 Ellipticity e 0.195 0.031 Anterior length (pm) aL 2.14 0.11 Anterior length (‘Ye) a 48.4 3.5 Anterior area (%j aA 49.6 3.1 Neck width (pm) nW 0.990** 0.051 Posterior width (%)+t PW 38.9 3.7 Posterior:anterior width ratio?+ paw 0.751 0.068 Normalized shape factor SF 1,o(p:i: 0.015 Anterior symmetry aSym 4,13*:,: 0.59 Posterior symmetry PSym 2.96 0.31 Anterior ruffle aRuff 4.00** 0.49 Posterior ruffleit pRuff 4.09*“’ 0.66 Curvature Curv 33.9”:’ 1.4 Contour gradient Grad 4.08 1.11 40% contour area (pm”) &II 5.45** 0.27 40% shape factor SF,, 1.12 0.04 Normalized axial density (Y&c) Z 68.9 2.7 Maximum density anterior length (%) ZaL 84.9”* 3.5 Axial density centroid (%c) ZCL 58.9 1.0 Bill length (%‘I Bill 38.8** 2.8 Anterior:posterior axial density ratio apZ 0.33*:* 0.03 20% L transverse ellipticity ezo 0.03 0.04 50% L transverse ellipticity es0 0.22** 0.05 70% L transverse ellipticity e:, 0.34”” 0.07 90% L transverse ellipticity es0 0.39”” 0.07 Bill shape factor BillSF -0.024 0.045

0.38 0.27 1.06 1.37 0.36 0.059 0.29 6.6 6.7 0.187 9.6 0.134 0.050 3.43 2.31 2.42 2.31 9.0 4.37 0.67 0.15 4.7 9.7 2.1 6.0 0.11 0.08 0.08 0.39 0.12 0.115

-1.4 --11.1/l 1.411 -2.1

-0.7 -11.2jl 0.3 -7.311

-3.7 --15.411 -5.811 -8.311 -0.8 ~~10.2ll

0.6 -5.2’1 1.0 -4.71’

-3.41, -- 14.711 -2.3 -0.6 ~0.6 -4.9

0.0 -5.3 -9.111 ~-12.61/ -8.211 ~11.011 -8.611 -3.0 -1.4

9.0 -8.511 -0.8

4.411 3.111 2.311 7.811 2.2

11.6, 27.8 29.711 32.611

-8O.O/(

-14.311 -4.3

-2::;11 -11.5// -10.2/l -28.211

-8.2ii ~21.611 -25.811

-0.6 -1.5 -3.8

-13.611 13.911

0.301 0.37 0.321 0.39 0.47

0.301 0.54

0.37

0.69 0.331

0.71

0.36 0.34 0.37

0.38 0.43

0.47

0.467 0.33 0.48

0.821 0.31

a.577

0.31 0.48 0.39 0.50

0.87

0.61 0.69

0.41 0.45 0.40 0.44 0.49 0.63

‘* Strong linear relationship is defined as P 5 0.001 and r2 2 1P c 0.005 for the regression coefficient associated with the 0.3. measured change in acrosome status.

t From Garrett and Baker (13). ** Zona-preferred parameter set established from sequential $ For ZP versus insemination. swim-up and ZP binding selectivity. 5 For difference between ZP and washed semen versus washed +t Parameters associated with pyriform morphology.

semen. $3 For difference between ZP and insemination versus insemi- I( P < 0.005 for the Wilcoxon test for zero percentage difference. nation.

366 Garrett et al. Morphometry of zona bound sperm Fertility and SterilityE

duced nW and nA), and shorter, wider head dimensions (increased W and e). As with swim-up selection, there was no significant difference observed in the parameters characterizing pyriform head morphology. Standard deviations generally were found to be considerably smaller for parameters measured in ZP-bound samples relative to insemination samples, with significant differences for 21 parameter SDS and >20% reduction in SDS for four of the densitometric parameters.

Percentage differences between ZP and insemination sample means for 21 morphometric parameters also were found to have regression coefficients significantly different from zero and r2 values 2 0.3 when analyzed with respect to the insemination sample means. The r2 values for these parameters are included in Table 1, together with indication of any significant contribution to the regression from differences in acrosome status between samples. Re- gression data are plotted for representative densitometric and morphometric parameters in Figure 1A and B, respectively.

-10 1 20 25 30 35 40 45% 0 0.1 0.2 0.3 0.4 0.5

SAMPLE MEAN (INS)

The posterior to anterior average width ratio, paw, is characterized by a low sample mean when the sample contains a high proportion of heads with pyriform morphology. Thus, the regression results displayed in Figure 1B indicate there is no obvious bias against ZP binding of pyriform heads. In fact, of the five samples with extremely low values of paw, three samples show less than a 4% change for ZP-bound sperm, and visual examination of sperm contours indicates that the approximately 8% change observed in the other two samples probably is related to the fact that the pyriform heads in these particular samples also exhibit large Ad0 values, which are associated with a very marked negative bias with respect to zona binding (Fig. 1A). Of the five samples with very large values of paw, the two showing significant change toward smaller paw for ZP-bound sperm can be identified with a substantial population of extremely large oval sperm heads, whereas the three showing little change all show a dominant acorn-like morphology. Figure 2 illustrates typical contours of ZP and insemination sperm observed in three samples identified with extreme values for the parameters paw and A in the insemination sample. Sperm contours for a fourth sample selected for minimal difference in morphometry between ZP and insemination sperm also are included in the figure.

0.5 0.6 0.7 0.8 0.9 1

SAMPLE MEAI; (INS;

5 6 7

Figure 1 (A), The percentage change observed between ZP and insemination sperm as a function of insemination sample mean for representative densitometrically derived morphometric parameters. Regression lines and coefficients of determination are indicated for weighted univariate linear regression analysis. INS, insemination; ho, area of 40% density contour (;lrn’); ZaL, percentage anterior head length at maximum axial optical density (5%); Bill, percentage head length associated with nuclear thinning (76); e,,, ellipticity of the anterior transverse optical density pro- file. Data points corresponding to patients identifed in Figure 2 as patients 1 to 4 are indicated. (BJ, The percent,age change observed between ZP and insemination sperm as a function of insemination sample mean for representative morphometric parameters. Regression lines and coefficients of determination are indicated for weighted univariate linear regression analysis. INS, insemination; A, head area (pm”); nW, neck width (pm); paw, posterior to anterior average head width area; aSym, axial symmetry of anterior head region (0 = perfect symmetry). Data points corresponding to patients identified in Figure 2 as patients 1 to 4 are indicated.

The net effect of swim-up followed by ZP selection on sperm morphometry is indicated by a significant difference observed in 22 morphometric parameters, with 15 displaying a strong linear relationship (r2 > 0.3) between percentage differences and the washed semen mean values. Mean and SDS for these

15 parameters evaluated for all ZP-bound sperm (n = 4,530) thus form a set of reference parameters for classification of sperm in washed semen with respect to ZP binding potential. This referencle parameter set is identified in Table 1 as a subset of the parameter means averaged over all ZP samples.


INSEMINATION ZONA PELLUCIDA

Figure 2 The 100% and 40% optical density sperm contours illustrating differences observed between sperm bound to the ZP and those in the corresponding insemination sample. The first 50 sperm contours are displayed for each sample. Patients 1 and 2 represent extreme low and high values of head area (A) in their respective insemination samples, patient 3 represents a predominantly pyriform insemination sample (minimum paw), and patient 4 corresponds to an insemination sample with minimal average difference between ZP and insemination sample morphometric parameter means. The ZP and insemination sample parameter means associated with these four patients are indicated by numerically labeled data points in the regression scatter plots of Figure 1A and B.

Relative Sperm-Zona Pellucida Binding Rates

The ratio of the ZP binding probability of abnormal sperm relative to normal sperm was calculated from both the automated (%C) and manual (%N) assessments for ZP and insemination samples. For %C, calculated values of B varied from 0.01 to 2.1, averaging 0.5 and differing significantly (P < 0.001) from both limiting values of 0 and 1. Similar calcula- tions for %N yielded a range of 0.09 to 1.2 for B, with an average of 0.35, and also differing significantly from 0 and 1 (P < 0.001). Figure 3 illustrates the strong relationship observed between B and %C of

the insemination medium, which is described using linear regression analysis by the expression

B %c = 0.05 + 0.03p<,1 + 1.08 k O.Ogp < ,ool

* [o/CC/(100 - %C)linseminati<,n (r2 = 0.76)

In general, the high insemination concentration of sperm used in these experiments prevented accurate evaluation of the numbers of ZP-bound sperm, pro- viding the limit of 2100 in many instances. In the 10 samples with countable numbers of bound sperm, the estimated probability of binding per theoretical sperm-ZP collision was < 1%. Previou;s experiments (5) using considerably lower insemination concentrations of lo5 sperm/ml report an average of 19 sperm boundlZP, which corresponds to a calculated binding per collision rate of (5%.

DISCUSSION

The detailed image analysis of differences between sperm bound to the ZP and those in the insemination medium indicates that the sperm-ZP binding process preferentially selects sperm with specific morphometric and densitometric characteristics. In particular, the differences show regression toNward a “zona- preferred” morphometry corresponding to a large symmetric anterior head region identified with relatively low optical density and no neck anomalies. Although preferential selection of sperm conforming

3

2

a

I3

1

0

r2 = 0.76

Figure 3 Plot of B, the ratio of the average ZP binding probabilities of sperm classified as “abnormal” and “normal,” against %C/ (100 - %C), where %C is the percentage of sperm in the insemination sample conforming to the “normal” classification. INS, insemination.

368 Garrett et al. Morphometry of zona bound sperm Fertility and Sterility@

to conventional classifications of normality also is demonstrated by the binding process, the loss of selectivity observed in Figure 3 for insemination samples with increasing percentages of normal forms needs further explanation. Although some variation in the abnormal:normal binding ratio is expected between samples with different categories or degrees of abnormality, the linearity of the relationship between B and %C or %N is unexpected. Several factors, including contamination of the ZP sample with loosely adhered sperm, damage to sperm during re- moval from the ZP surface, and misassignment of normality would cause the measured value of B to differ from the true binding ratio.

Contamination of the ZP bound sample with sperm not physiologically bound to the ZP results in

J&c meaS = (B + sY(1 + s)

where s is the ratio of the probability of a spermatozoa becoming loosely adhered to the ZP relative to the probability that a morphologically normal sperm would become bound. Because s is a maximum when B = 0, the mean result of 0.5 for Bcscmeas sets an upper estimate of such contamination at s = 1, corresponding to equal numbers of bound normal sperm and contaminant sperm present in the ZP sample. However, this correction for contamination fails to account for the apparent linear dependence of B4 meaS on [%C/(lOO - %C)l illustrated in Figure 3. Ozthe other hand, damage to zona-bound sperm during retrieval and also assignment of morphometric abnormality to sperm with full ZP binding capability result in a measured binding ratio of the form

* c

%C

(loo - “) ) insemination

where d represents the fraction of normal ZP-bound sperm inadvertently classified as abnormal, either because of damage during aspiration or application of incorrect classification criteria with respect to ZP binding capability. Equating the above expression with the linear regression results for Bl,o yields d/(1 - d) = 1.08 + 0.09 and hence d = 0.52 i 0.05 and (B + s)/[(l + s)(l - d)] = 0.05 2 0.03. Because B and s are both 10 by definition, upper limits can be set at B 5 0.05 and s 5 0.05 by applying a 2 SD limit on the first-order regression coefficient. This result indicates no significant contamination of the ZP-bound sample by loosely adhered sperm. More- over, because visual examination of the sperm contours for each sample shows little obvious evidence of damage to the sperm through manipulation, it would appear that the classification criteria for nor-

mality in %C is too stringent, at least with respect to the ZP binding process, This conclusion is not inconsistent with the outlying IVF results, in which good fertilization rates are achieved Twith sperm samples assessed to have very poor morphology.

There is an alternative interpretation of the results presented in Figure 3 suggested by the general observation that, where a sample has a high percentage of sperm for which all morphometric parameters conform to the reference “normal” data set, those sperm classified as “abnormal” have only a small number of parameters with values falling outside the limits defining conformity. Conversely, where %C is low, that is high nonconformity, the average degree of nonconformity is correspondingly high. Thus, if the ZP binding ability of an “abnormal” spermatozoon depends upon its degree of nonconformity, then the average binding ratio B would be expected to be an increasing function of %C, as observed in Figure 3. This interpretation of the data suggests that the best evaluation of the fertilizing potential of an individual spermatozoon or a semen sample should be based on a sliding scale of m,orphometric acceptability rather than the conventional approach of morphometric or morphological classification.

Irrespective of the interpretation placed on the abnormal:normal binding ratio results, it remains that morphometric selectivity has been demonstrated by the ZP binding process and thus indicates causality in the relationship between sperm head morphometry and ZP binding ability. It is to be nolted that the ZP selectivity displayed in the analysis of individual morphometric parameter differences provides no significant evidence to support the causal classification of abnormality to pyriform morphology that is indicated in an earlier manual morphology assessment of ZP bound sperm (4). However, without a direct comparison between ZP binding rates and the morphometry of the insemination sample, the possi- bility that pyriform morphology serves as a sample marker of reduced fertilizing potential cannot be ruled out. Further investigation also is required to establish the influence of pyriform morphology in subsequent steps toward fertilization an.d embryonic development. Recent studies using manual morphology assessments suggest that sperm-oolemma binding preferentially selects morphologic:ally normal sperm (5) and reduced embryo quality in IVF may be associated with postacrosomal abnormality (19).

Although the 1 to 56 range in %C assessments for insemination samples is of the same magnitude as the qualitative differences observed in numbers of sperm bound to the ZP, even with B = 0, %C alone cannot account for the apparent failure of >95% of sperm to bind upon contact with the ZP, according to the estimated collision frequency. It is perhaps


therefore not surprising that the current analysis identifies strong ZP selection of sperm with morphometric parameter values consistent with a large thin anterior region of the head, suggesting that the physical nature of the initial contact between sperm and ZP is a significant factor in the progression toward physiological binding. It is conceivable that sperm-oocyte interaction is conducive to binding when there is a maximum surface area of contact between the plasma membrane overlaying the acrosomal region of the sperm head and the surface of the ZP, a situation presumably influenced by the angle and speed of impact as well as the morphology of the head. It is also possible that finite numbers of oocyte binding sites and the beating tail action of ZP-bound sperm contribute to limiting the number of sperm-oocyte collisions that result in ZP binding. The role and influence of the cumulus, which was not present on oocytes used in these experiments, is also yet to be established.

By necessity, visual sperm morphology analysis is founded on subjective classification of cell type. A sample usually is assessed for percentage of normal forms, with different definitions of “normality” established by various groups, notably WHO and Tyg- erberg classifications (17, 20), which then are inter- preted nominally by individual observers. Given sufficient parameters, an image analysis system can be trained to identify sperm conforming to any pre- scribed classification system. However, the objectivity of automation is lost if the raw data from an image analysis system merely is used to assign with accuracy sperm to a subjective classification system. Thus, the set of “ZP-preferred” values defined by ZP means for those morphometric parameters showing selectivity in the binding process or sequential swim- up and binding processes can be used as a reference for a new objective classification system. Alterna- tively, these ZP-preferred parameter values can be used to devise a mathematical model quantifying the loss of fertilizing ability associated with deviation from the measured ZP preferred morphometry for an individual spermatozoon or the population of spermatozoa in a sample. A recent st.udy, which found that acrosomal morphology was superior to sperm morphology assessment (strict criteria) in the prediction of IVF outcome (21), supports the application of these approaches to the morphometric assessment of a sample, because the selectivity of the ZP demonstrated in the present study focuses on the differential staining and ellipsoidal symmetry of the acrosomal region of the sperm head. Support also is gained from a confocal scanning laser microscopy study of live sperm, in which the morphometric differences between insemination samples associated with zero and nonzero IVF rates were examined (22).

370 Garrett et al. Morphometry of zona bound sperm

The confocal microscopy study showed1 that the percentage of sperm head area identified by reduced optical density as the acrosomal region was reduced significantly in samples for which no fertilization was achieved. This finding is consistent with, although predictably not as pronounced., the selectivity shown by the ZP for sperm with low values for the parameter Lo.

In summary, the results of this work emphasize the importance of inclusion of abnormalities in the acrosomal region within current manual sperm morphology assessments and suggest that previous emphasis placed on sperm head dimensions in the classification of normality is justified only in cases of extreme deviation from the norm. In addition, the zona-preferred morphometric parameters deduced in this work provide the basis for derivation of new, objective, and physiologically based morphometric assessments for a semen sample. Because such assessments relate directly to the selectivity demonstrated by a prerequisite step in the fertilization process, they are expected to prove useful in the clinical diagnosis of male infertility. Further work quantifying ZP selectivity from insemination samples repre- senting a broader cross-section of morphologies, in addition to direct comparison of measured sperm-ZP binding rates with morphometrically deduced assessments of ZP binding ability, are suggested by the results presented in this paper.

Acknowledgments. The authors gratefully acknowledge the laboratory assistance of Ming Li Liu, B.Sc., and Peter Elliot, B.Sc., of the Royal Women’s Hospital, Melbourne, Victoria, Australia.

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Selectivity of the human sperm-zona pellucida binding process to sperm head morphometry