morphometric analysis of human sperm morphology

international journal of andrology, 17:248-255 (1 994)

Morphometric analysis of human sperm morphology

E PEREZ-SANCHEZ, J. J. de MONSERRAT and C. SOLER

Departament de Biologia Animal (Unitat de Fisiologia), Facultat de Ciencies Biolbgiques, Universitat de Valhcia, Pais Valencil, Spain

Summary Fourteen morphological forms of human spermatozoa were analysed morphometri-

cally using semi-automated image analysis techniques. Five basic (area, perimeter, length, width and mass) and five derived (ratio, length minus width, ellipticity, form and total mass) parameters were considered. Statistical analysis showed differences among all 14 types of human sperm heads. Basic parameters describing the size and shape were enough to distinguish most of the categories, whereas derived parameters as well as parameters dependent on stain intensity, were demonstrated to be useful for the discrimination of some morphological categories. The fact that statistical analysis showed differences among all 14 sperm types provides evidence for the reliability of our morphological classification. These results show that morphometry can be used for the fine study of sperm morphology and may serve as a database for future work dealing with sperm classification. As this is a pilot study to assess methodology, further studies will be required to validate the method in terms of its application and usefulness in assessing the fertilizing potential of human spermatozoa.

Keywords: human spermatozoa, image analysis, morphometry, sperm morphology

Introduction Sperm morphology is assessed routinely as part of stan-

dard laboratory analysis in the diagnosis of human male infedty. Ths practice has its origins in the work of MacLeod & Gold (1951), which showed that sperm morphology was significantly Merent in fertile men than in infertile men. Since then, Merent criteria have been. used in the evaluation of sperm morphology which have given rise to many type-classification systems and to a general lack of uniformity among laboratories (Freund, 1966; David er al., 1975; Fredricsson, 1979; Berenyi & Corradi, 1982).

The World Health Organization played an important role in hghlighting this problem, and established standards for the analysis of semen (WHO, 1987). Ths approach advanced the standadzation of semen analysis with respect

Correspondence: Professor Carles Soler i Vizquez, Departament de Biologia Animal (Unitat de Fisiologia), Facultat de Ciencies, Biolbgiques, Univenitat de Valencia, C/Dr. Moliner, 50; 46100 Burjassot, VaPncia, Spain.

to sperm motdity, concentration and biochemical aspects of semen analysis. However, despite this standardization, human semen evaluation continues to be influenced by considerable subjectiveness on the part of the investigator, and a lack of objective measurements for sperm morphology continues to present a problem. Whereas a good correlation between normal sperm morphology and their fertilizing ability has been reported by many authors (Rogers et al., 1983; Jeulin et al., 1986; Jouannet et al., 1988; Hinting et al., 1990), other investigators were unable to find any such correlation (Zaini et a l , 1985; Jeyendran et al., 1986; Check et al., 1992). The more recent so-called stricter criteria for the assessment of sperm morphology have been reported to improve the predictability of IVF outcome (Kruger et al., 1986, 1.988; Menkveld er al., 1990). However, this system requires careful training of observers and, even in this system, subjectivity cannot be avoided.

The intrinsic subjectivity of sperm morphology assessment calls for new approaches to tackle this potential prog-

Morphometry of human sperm 249

nostic tool for the developing techniques of assisted reproduction. In order to avoid subjectivity, over the past 15 years numerous studies that incorporate image analysis techniques in the assessment of sperm morphology have appeared (Schmassmann et al., 1982; Katz et al., 1986; Jagoe et al., 1986, 1987; Moruzzi et al., 1988; Schrader et a1 ., 1990; Wang et al., 1991a,b; Davis et al., 1992a,b; Kruger et al., 1993). These techniques allow objective characterization of the different sperm forms, some of whch may be indica- tors of dysfunction of spermatogenesis and whch nllght serve as markers of the fertilizing ability of spermatozoa (Jouannet et al., 1988).

The aim of the present work was to characterize the more frequent sperm head forms in semen morphometrically. The results of this work may serve as a database for the establishment of an adequate vector of characteristics in future works concentrating on building computer-assisted systems for the automatic classification of spermatozoa into different morphological categories.

Materials and methods

Staining procedure and classijcation categories Single ejaculates from four men attending an IVF pro-

gramme at ‘La Fe’ Hospital (Valhcia, Spain) were studied. Semen smears (1 pl) were prepared, air-dried, stained with Hemacolor (standard kit from Merck, Darmstadt, Germany, Cat. no. 11661), and mounted permanently. Stained smears were observed under brightfield illumination using a 1 0 0 ~ oil immersion objective.

Two hundred and sixteen spermatozoa were selected at random from the first patient by one observer (F.P.). The corresponding images were labelled and stored in the hard disc. Taking as reference the works of David e f al. (1975), WHO (1987) and Moruzzi et al. (1988), the spermatozoa were classified independently by three observers (F.P., C.S. and J.J.M.) from these images into the following categories:

normal, small, large, round, narrow, elongated, amorphous, pencil and double. Moreover, small, large and elongated forms were subdivided initially into subcategories (Fig. 1). Following the criteria defined by Moruzzi et al. (1988), only the 183 spermatozoa for whch all three observers agreed on their classification were used for the subsequent morphometric analysis. The same blinded schedule was used for the classification of the spermatozoa belonging to the other three patients but the number of spermatozoa per patient (183) and the proportions of spermatozoa in each category were made the same as in the first patient.

Morphometric analysis The system used for morphometric measurements

included a Compaq Deskpro 386/25e PC computer (Compaq Computer Corporation, Houston, Texas) with a PIP-1024 B video digitizer board (Matrox Electronic Systems Ltd., Quebec, Canada) - the array size of the video frame grabber was 512X512X8 bits - an Intel 387 DX Math CoProcessor, Olympus BH-S brightfield microscope with a 50 W bulb and a lOOx immersion objective (Olympus Optical Co., Ltd., Tokyo, Japan), connected to a Sony CCD AVC-D7CE video camera with a 3.3x photo-ocular (Sony Corporation, Tokyo, Japan), two monitors (Compaq Video Graphics Monitor, Compaq Computer Corporation, Houston, Texas; PVM-1443MD Sony Trinitron, Sony Corporation, Tokyo, Japan) and the image analysis software (Visilog, Noesis).

Cells were displayed live on the video monitor and each sperm head image was processed automatically using a spe- cific image analysis program created by us for image enhancement and thresholding. Analysis of the sperm midpiece and tad was not included in the program. The system detected the boundary of the sperm head and the outline was displayed as white overlays superimposed on the video image. The quality of the image analysis performance was tested visually by superimposing the digitized, binary sperm-

WOWL W L L

OVAL ROUND ANORPHOUS OTHERS 1 I I

7 1 T !’ ttt ELOlltATED

LARCE

PEAR OVAL ROUND

I I I 1 I I I I

Figure 1. Classification scheme used for the morphometric characterization of sperm head morphology.

250 F. P&az-S6nchez et a/.

head silhouettes on the original grey image, and exact fitting was required for approval between both images. Partially successful head boundary detections were traced manually by the operator using an edit facility provided by the system. All of the cases requiring manual editing in order to determine the boundary of the head were due to the fact that the base of the head of the spermatozoa and the midpiece presented the same grey level in the images. This was true for 7% of the total spermatozoa analysed, independent of the morphological class to which they had been assigned previously.

Computer software allowed us to make five basic measurements of sperm heads, and the system was interfaced with a database program to calculate five derived parameters (Table 1). All data were stored in computer files for further analysis.

Statistical analysis Data were analysed using one-way analysis of variance

(ANOVA), and differences between categories. computed using a posteriori Fisher’s PLSD test in the StatView statistical program (Apple Macintosh).

Results When measurements of spermatozoa in each category

were compared between patients, ANOVA did not show any significant difference (p0.05). Thus, measurements of spermatozoa in the same categories from the four patients were pooled for the analysis of inter-category differences.

Table 1. Parameters assessed for the morphometric characterization of sperm heads

Basic parameters Derived parameters

Parameter Parameter Formula

Area (A) Ratio w/1 Perimeter (P) Length-Width I- w Length (1)’ Ellipticity IL-Wl/(L + W ) Width (W)’ Form (4nA/P2) x lo3 Mass (M)3 Total Mass A x M

’I i s the largest value of the Feret diameter measured at angles of 0, 30, 60, 90, 120, 150 degrees. W i s the smallest value of the Feret diameter measured at angles of

0, 30, 60, 90, 120, 150 degrees. W is not necessarily orthogonal to I. ’A4 = 1 / N x 2 g (Xi, Yi); [Xi, Y.) E H, where N i s the number of pix- els and g (Xi, Yi) is the grey Ievei value of the pixel (Xi, Yi) of the head H.

Establishment ofsub-categories Statistical comparison by ANOVA of morphometric

parameters correspondmg to spermatozoa with elongated heads did not show significant differences among these groups (p0.05) . Subsequently, simple, tapering and peanut elongated forms were considered as a single morphological type (Table 2).

Table 2. Morphometric characterization of spermatozoa with elongated heads. No differences were found among parameters in each category after ANOVA (p0.05)

Categories Basic parameters

Name Code Area Perimeter Length Width Mass (Pm) (pixel) (PI (Pm) (grey level)’

~~ ~ ~~ ~~~ ~ ~

Single elongated (n=32) k l 16.13 i 1.31 109.50 i 11.40 6.47 i 0.31 3.67 * 0.20 66.41 * 7.8 Taper (n=28) k2 16.06 * 1.43 100.00 i 7.70 6.38 f 0.43 3.78 i 0.21 59.71 * 9.90 Peanut (n=2 8) k3 16.29 i 2.19 116.29 i 22.41 6.95 i 0.87 3.59 * 0.26 60.61 i 10.62

Categories Derived parameters

Name ~~ ~~

Code Ratio Length minus Width Ellipticity Form Total Mass (rm) (pm/pixeI)’ (pm2 x grey level)

Single elongated (11132) k l 0.57 i 0.04 2.80 i 0.33 0.28 * 0.03 1.73 * 0.36 1070.66 * 148.67 Taper (n=28) k2 0.59 i 0.05 2.60 i 0.47 0.26 i 0.04 2.04 * 0.27 962.31 * 207.9 Peanut (n=28) k3 0.52 i 0.07 3.36 i 0.98 0.32 * 0.07 1.61 * 0.44 987.71 * 207.60

Values are mean i SD. ‘Grey level range: 0-255.


When small head sub-categories were compared, significant differences were demonstrated among them @<0.01), which revealed the heterogeneity of the small category. Fisher’s test showed that differences in two parameters or more existed among all four sub-categories. Therefore, small oval heads, small round heads, s r d amorphous heads, and another category grouping the remaining small forms were maintained as different sub-categories in order to characterize them morphometrically. Data corresponding to these sub-categories are given in Tables 3 and 4.

Similar findings were obtained for spermatozoa with large heads in which differences were found among the three sub-categories (pcO.01). Ratio, length minus width and ellipticity were the most useful parameters in discriminating large round heads from each other, whereas perimeter and length contributed definitively to distinguishing large pear heads from large round and oval forms. Data corresponding to these sub-categories are given in Tables 3 and 4.

Inter-cutegory d$erences in sperm morphometry The values of the morphometric parameters which char-

acterize the form and size of each of the 14 morphological sperm types are given in Tables 3 and 4. These tables also show inter-category differences for each parameter as revealed by Fisher’s test after ANOVA. Basic morphometric parameters describing the size and form of sperm heads were sufficient to discriminate the majority of categories.

The parameters dependent on stain intensity (mass and total mass) were necessary for the discrimination of small oval heads from small (others) and pencil heads. These parameters were also very useful for the discrimination of other categories.

Moreover, derived parameters were essential for the separation of some groups. This was the case of large oval versus large round sub-categories and pencil head versus narrow head forms; these categories were only differentiat- ed by the parameters ratio, length minus width and ellipticity. Similarly, fom was the only parameter which distinguished between normal and pencil forms.

Discussion The effect of sperm morphology on fertilization is a con-

troversial issue (Ron-El et al., 1991; Simon et al., 1991). Some of the possible causes of these contradictory results may be the subjectiveness of the techniques used for morphology assessment, inaccurate or inappropriate semen measures, and inadequate definition and control of study populations (Baker & Clarke, 1987; Davis et al., 1992a; Davis & Gravance, 1994).

The use of different stains and their effect on sperm morphology has been discussed widely in the past, and different criteria exist throughout the world (Glover et at., 1990). In this work, Hemacolor stain was used because the staining procedure is very fast, about 15 s per smear, and allowed

adequate separation between sperm heads and the back- ground surrounding them. In a previous study of ours, Hemacolor gave similar results to Diff-Quik (unpublished results), which is employed widely in andrology laboratories (Harrison ef al., 1989; Enginsu el al., 1991). Moreover, Hemacolor was developed as a haematological stain and therefore it stains leucocytes with good definition and these cells can be distinguished easily from sperm precursors.

Alterations in head shape may decrease the total number of spermatozoa that potentially could reach and penetrate the oocyte to result in a normal pregnancy. Jouannet et al. (1988) found that the rate of pregnancy decreased when the percentage of microcephdc spermatozoa was >12% and also when that of duplicate heads exceeded 5%; these abnormalities, as discussed by the authors, may reflect general dysfunction of spermatogenesis leading to finctional alterations of the whole sperm population. In some extreme cases, all the spermatozoa present in semen correspond to a unique category of abnormal morphology, and reflect a genetic defect which correlates with irreversible infertility (Lalonde et al., 1988; Schmiady et al., 1992). Although there still needs to be a lot of work done to determine whether each and every one of the abnormal forms could have its accompanying physiological deficiency, we cannot ignore this evidence. In this respect the development of morphometry instruments to provide the percentages of each morphological type, in an objective and precise way, could shed further light on the problem.

The assignment of some abnormal spermatozoa to a ckstinct category based on visual assessment is extremely difficult, and in these cases morphometry is a usefil tool. In some studies a set of four to six morphometric parameters was estimated for the whole sperm population without distinguishing between normal and abnormal forms, and these parameters were used to distinguish between fertile and infertile men (Jagoe et al., 1986; Katz e f al., 1986; Turner ef al., 1989). In our opinion this kind of approxima- tion to the problem is prone to erroneous results because morphometric values apparently corresponding to the fertile group may mask the true situation when no categories are distinguished, i.e. a large amount of large sperm heads together with similar amounts of small forms may result in the apparent ‘normality’ of a semen sample based on the mean values of morphometric parameters. Therefore, we consider that morphometric measurements have to be used to define properly the largest number of different morphological categories in semen.

On the other hand, some multicentre studies have revealed a wide range of cksagreement by experienced observers in classlEying human sperm heads (Dunphy et al., 1989; Neuwinger et al., 1990). It has also been shown that there was good agreement for normal spermatozoa and poor agreement in categories of abnormal sperm shapes such as tapering and pyriform (Baker & Clarke, 1987; Neuwinger et al., 1990). As a result, routine reports on sperm morphology

252 F. Perez-Sanchez eta/.

Table 3. Morphometric characterization of different sperm morphological categories. For each basic parameter, differences among categories are presented as shown by ANOVA followed by Fisher’s PLSD test

Ca tegories Basic parameters

Name Code Area Perimeter Length Width Mass (P2) (pixel) I P 4 Cm) (grey level)’

Normal (n=68)

Small (oval) (n=20)

Small (round) (n=24)

Small (amorphous) (n=32)

Small (others) (n=28)

Large (pear) (11328)

large (oval) (n=52)

Large (round) (n=28)

Round (n= 104)

Narrow (n=64)

Elongated (n=88)

Amorphous (n= 1 1 2)

Pencil (n=28)

Double (n=56)

a

b

C

d

e

f

9

h

I

I

k

I

m

n

cdefghn 14.39 * 1.50

cfghkn 10.05 * 0.9

fghiiklmn 4.50 i 3.56

fghii klmn 5.25 i 2.54

fghkn 9.86 * 1.19

ghiiklm 26.15 i 7.55

iiklmn 20.54 * 3.84

ijlmn 19.94 * 4.64

kn 12.87 * 2.76

cdfghkn 90.06 * 6.96

cdfghkn 80.80 * 4.87

efghiiklmn 48.67 i 18.35


fghiklmn 76.86 * 7.54

ghijklm 128.14 * 13.75

ijlmn 109.08 * 1 1.91

ijln 107.14 * 12.25

kn 84.81 * 8.76

bcdefg i kn 5.31 * 0.28

cdfghikln 4.58 * 0.22

efghiiklmn 3.1 3 0.74

efghiiklmn 3.51 * 0.42

fghikln 4.62 * 0.33

ghijlm 7.10 i 0.71

ijlmn 6.25 * 0.54

ikn 5.69 i 0.48

iklmn 4.60 * 0.38

n kn kn 13.04 * 1.95 91.25 * 6.73 5.62 * 0.33

n Imn Im 16.16 * 1.61 108.64 * 15.76 6.60 * 0.60

n n n 13.40 i 2.85 90.36 i 10.02 5.30 * 0.71

n n n 14.15 i 3.23 94.57 i 13.77 5.32 * 0.69

27.39 * 14.60 132.07 i 40.72 6.88 i 1.69

cdefghin 3.92 * 0.17

cdfghin 3.52 i 0.06



fghin 3.49 i 0.20

ijklmn 4.67 i 0.96

ijklmn 4.48 i 0.44

ijklmn 4.72 * 0.53

jkln 4.10 i 0.31

In 3.47 * 0.31

n 3.68 * 0.23

n 3.84 * 0.36

n 3.88 * 0.32

5.23 * 1.10

bc 59.50 * 13.49

cefghiiklmn 78.82 * 9.77

defghijklmn 97.67 i 36.0

en 64.35 i 8.81

k 48.36 i 4.10

55.79 * 17.74

56.95 * 16.18

51.39 * 14.1 1

57.31 2 15.40

56.91 * 10.76

n 62.43 * 9.51

56.44 i 11.57

51.17 * 10.99

50.54 * 9.90

Values are mean i SD. Lowercase letters above numerical values indicate significant differences with other categories, peO.0 1. a Grey level range: 0-255.

are confined usually to the total percentage of nomial forms. Among the morphometry instruments documented, the Morphologyzer I1 enables automatic classification into five categories: normal, small, big, tapered and amorphous, although the value of manual assessment of sperm morphology in predicting the outcome of the zone-free hamster oocyte penetration test was not improved significantly by its use (Wang et al., 1991b). Other current morphologizers, the CellForm Human instrument and FERTECH for instance, allow the possibility of automatic sperm classification into

two types: normal and abnormal, with the former providing a data file of the metric measurements of each spermatozoon for the user’s information which could be used for any pos- terior reclassification @aVis et al., 1992b; Kruger et al., 1993).

The present study has conducted an exhaustive quantita- tive characterization of sperm head forms using computer- assisted image analysis techniques. The extensive categoriza- tion used here is based on the assumption that differences in morphological features of spermatozoa, even if small, may


Table 4. Morphometric characterization of different sperm morphological categories. For each derived parameter, differences among categories are presented as shown by ANOVA followed by Fisher‘s PLSD test

Categories Derived parameters

Name Code Ratio length minus width Ellipticity Form Total mass (vrn) (p/pixeI)2 (prn’xgrey tevet)

Normal (n=68)

Small (oval) (n=2O)

Small (round) (n=24)

Small (amorphous) (n=32)

Small (others) (n=28)

large (pear) (n-28)

large (oval) (n=52) .

large (round) (n=28)

Round (n= 104)

Narrow (n=64)

Elongated (n=88)

Amorphous (n= 1 1 2)

Pencil ( ~ 2 8 )

Double (n=56)

a

b

C

d

e

f

9

h

i

i

k

I

m

n

cdfhi i k 0.74 i 0.05

cfijk 0.77 i 0.03

defghiklmn 0.95 i 0.04

fggiklm 0.84 i 0.06

fiik 0.76 i 0.09

hikln 0.60 * 0.14

hiiklm 0.72 i 0.08

0.83 i 0.05 iklmn

0.89 i 0.04

klmn 0.62 i 0.05

Imn 0.56 i 0.06

0.74 i 0.1 1

0.74 i 0.08

0.77 * 0.13

ik

cdfiik 1.39 * 0.34

cfgjk 1.06 * 0.20

efghi klmn 0.18 i 0.18

fgiklmn 0.56 i 0.22

fgiik 1.13 i0 .46

ghilmn 2.43 * 1.10

hik 1.76 2 0.61

ikn 0.97 * 0.30

iklmn 0.50 i 0.21

klmn 2.15 * 0.31

Irnn 2.92 i 0.69

1.46 i 0.78

1.44 * 0.56

1.65 * 1.12

cdfhiik 0.15 i 0.03

cfiik 0.1 3 i 0.02

defghiklmn 0.03 i 0.02

fgiklm 0.09 i 0.04

fiik 0.14 i 0.06

hikln 0,.21 * 0.09

hiik 0.16 * 0.06

jlm 0.09 i 0.03

iklmn 0.06 * 0.02

klmn 0.24 * 0.04

Imn 0.28 i 0.05

0.16 * 0.08

0.15 i 0.05

0.13 i 0.08

fjkln cdefgn 2.24 * 0.23 859.20 * 227.06

2.04 i 0.26

k 2.07 i 0.32

k 2.09 i 0.27

k 2.12 0.27

1.98 I 0.29

k 2.18 i 0.27

k 2.16 i 0.18

I

cdefgn 827.61 i 102.56

fghiiklmn 339.20 * 139.73

fghiiklmn 339.09 i 166.10

fghikln 476.94 i 69.93

hiiklm 1391.40 * 315.83

ijlmn 1155.87 i 335.88

ijlmn 1034.81 * 393.87

ikln kn

k n

2.24 * 0.20 729.23 * 199.20

1.98 i 0.36 732.29 * 13 1.82

1.79 i 0.39 1009.79 * 185.36

n 2.06 * 0.23 758.81 * 233.79

n 2.01 * 0.35 713.91 i 166.1 1

In Irnn

2.01 * 0.27 1414.91 * 689.00

Values are mean * SD. Lowercase letters above numerical values indicate significant differences with other categories, p<O.O1.

imply different functional states. This hypothesis is supported by the fact that certain abnormahties of sperm heads are more common with certain functional or disease states such as varicocele (MacLeod, 1965; Panidis ef af., 1990). Finally, even if pregnancy is achieved by semen with a high propor- tion of morphologically abnormal spermatozoa, a relation- ship between sperm morphology and embryo quality has been established (Parinaud et ol., 1993). Moreover, the spontaneous abortion rate is correlated highly with abnormal morphology of the spermatozoa (Oehninger ef al., 1988; Rosenbusch & Sterzik, 1991). However, the diagnostic

benefit of such an exhaustive classification of sperm head morphology with respect to previous classifications must still be established.

The discrimination among the different types in the classification presented here is based on the measurement of a fairly large number of morphological features. Basic parameters except mass were enough to separate types which differ substantially in size, whereas they failed in discriminating other categories (see Table 3 and Results). Derived parameters of size basic features proved to be useful for the discrimination of morphological types similar in size but

254 F. Phz-Sanchez eta/.

with distinctive shape characteristics, i.e. narrow vs. amorphous, narrow vs. pencil, large (pear) vs. double, round vs. pencil (Table 4).

Parameters dependent on stain intensity (mass and total mass) have also been demonstrated to be important features for the discrimination of some abnornial categories (Tables 3 and 4). These parameters can only be assessed accurately using image analysis techniques, because differences i n stain uptake are more difficult to appreciate by visual assessment than are differences in size or shape.

It is our conclusion that the set of morphometric parameters used in this study constitutes a set of characteristics which is valid for characterization of the majority of nior- phological types of spermatozoa i n semen. T h e present work might be a contribution to the establishment of an accurate classification of different sperm forms, and highlight

the importance of using computer-assisted techniques in the evaluation of sperm morphology. In any case, and talung into account the methodological nature of this work, further studies will be required to validate the method i n terms of its application and usefulness in assessing the fertilizing potential of human spermatozoa.

Acknowledgments This work was supported partially by M I C R O P T I C ,

S C P (grant 683.6-674, ADEIT). Financial support was also obtained from the Direccib General d’Ensenyanients Universitaris i Investigacib (grant 683.3-130) and the Conselleria de Cultura, Educacib i Citncia de la Generalitat Valenciana (grant conceded to Dr Ptrez).

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989-993.

Received 3 April 1994; accepted 28ju ly 1994

morphometric analysis of human sperm morphology

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