Modern trends Edward E. Wallach, M.D., Associate Editor FERTILITY AND STERILITY
Vol. 58, No.3, September 1992
Copyright © 1992 The American Fertility Society
Printed on acid-free paper in U.S.A.
Tests of human sperm function and fertilization in vitro
De Yi Liu, Ph.D.* H. W. Gordon Baker, M.D., Ph.D. Department of Obstetrics and Gynaecology and Reproductive Biology Unit, University of Melbourne, Royal Women's Hospital, Melbourne, Victoria, Australia
Objective: To review recent studies on the development of new tests of human sperm function and evaluation of which sperm characteristics are most important for fertilization in vitro by logistic regression analysis. Study Selection: Recent studies on the relationship between putative and new tests of human sperm function and fertility in vitro or in vivo are discussed in this review. Some physiological and technical aspects are included. Main Outcome Measures: Fertilization rates in vitro and sperm tests including standard semen analysis, improved morphology assessment, objective assessment of sperm motility- and movement characteristics, nuclear maturity, hypo-osmotic swelling, the acrosome and the acrosome reaction, acrosin activity, human sperm-hamster OBGyte penetration assay, and sperm-zona pellucida (ZP) and sperm-oolemma binding. Results: The percentages of sperm with normal morphology and a normal intact acrosome, mean linearity, and the number of sperm binding to the ZP were highly significantly related to fertilization rates in vitro. Other sperm tests evaluated usually provided no additional information about fertilization rates. The human ZP is highly selective for binding of morphologically normal sperm. Acrosome-reacted human sperm have little or no ability to bind to the ZP. Conclusion: Results of in vitro fertilization can be used to evaluate tests of human sperm function. Logistic regression analysis is a powerful method for determining which groups of sperm characteristics are independently related to fertilization rates. Normal morphology, linearity, acrosome status, and sperm-ZP binding are the most important characteristics for fertilization in vitro. Fertil Steril 1992;58:465-83 Key Words: Sperm function tests, in vitro fertilization, fertilization rates
Standard semen analysis including sperm concentration' motility, and morphology is widely used as a fundamental indicator of male fertility. However, the results do not provide precise diagnostic or prognostic information for human fertility in vivo or in vitro (1-8). Men with absolute and persistently immotile sperm, azoospermia, or all sperm with morphological defects such as small round-headed sperm without acrosomes are sterile, but these are not common causes of male infertility (9, 10). Most men seen for infertility have reduced sperm concenReceived March 11, 1992.
* Reprint requests: De Yi Liu, Ph.D., Department of Obstetrics and Gynaecology, University of Melbourne, Royal Women's Hospital, Carlton, Melbourne, Victoria 3053, Australia. Vol. 58, No.3, September 1992
tration, motility, and morphology, often in combination. Other factors, known and unknown, and particularly those in the female partner, are also important in determining the ease of conception. Some couples conceive quickly, despite abnormal semen analysis results, and, conversely, rare men with normal semen analysis results are sterile. Because standard semen analysis has limited clinical value for predicting fertility, many other tests of human sperm function have been developed, such as objective assessment of motility, hypoosmotic swelling, tests for sperm nuclear maturity, measurements of acrosome status, acrosome reaction and acrosin activity, hamster zona-free oocyte penetration and human sperm-zona binding and penetration. Although some of these tests appear to Liu and Baker
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provide additional valuable information that may improve the prediction of fertility, for many of them it is still unclear if they will be useful clinically. So far, no single test of sperm function will predict fertility accurately except where there is an absolute disorder affecting all sperm such as azoospermia or totally absent acrosomes. In other situations, groups of tests are required to evaluate male fertility. The results of sperm tests such as concentration, motility, and morphology are correlated, and defects usually occur in combination (11, 12). It is therefore necessary to determine which groups of sperm tests give the most information about fertility. Then clinically useful sets of tests can be identified for predicting sperm fertilizing ability. In this article we will discuss tests of human sperm function and their predictive value for fertilization in vivo and in particular, preliminary studies of new tests of sperm function in relation to the results of in vitro fertilization (lVF). Technical and other factors influencing the results that may explain conflicting reports on the clinical value of some tests are covered. Because the new tests have not been widely used in clinical semen laboratories, the principles of the test and relevant procedural aspects are reviewed. Because of the concentration on clinical relevance of the sperm tests and their predictive ness for fertility, sperm function and fertilization are not discussed in detail. The physiology is dealt with first, followed by an overview of tests of human sperm function. PHYSIOLOGY AND TECHNICAL ASPECTS OF NEW TESTS OF HUMAN SPERM FUNCTION
In this section, we discuss selected physiological and technical aspects of new tests of human sperm function. These include sperm nuclear maturity, sperm membrane integrity, the acrosome and acrosome reaction, and sperm-oocyte interaction. Sperm Nuclear Maturity
During epididymal maturation, human sperm, like those of other mammals, undergo structural and biochemical changes that are considered to be prerequisites for optimal fertilizing ability. Among these maturational changes, formation of disulfide bonds between nuclear protamines results in an increase in the stability of the sperm head (13). This increase is concomitant with a decrease in free thiol groups in the nucleus (14). In humans, the transit time of sperm through the epididymis varies between individuals (15). Thus, ejaculated sperm have various 466
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degrees of nuclear stability (16). It is known that immature sperm in the epididymis have lower fertilizing ability than do mature sperm in the ejaculate. Thus assessment of the proportion of sperm with an immature nucleus in the ejaculate may predict male fertility. Several simple tests for assessing nuclear maturity are now available and will be briefly described. When sperm are exposed to the detergent, sodium dodecyl sulfate (SDS) or SDS in combination with the disulfide bond-reducing agent dithiothreitol, the sperm heads with immature nuclei with less disulfide cross-linking rapidly undergo decondensation and distend and may lyse (16). There is a large variation in rates of decondensation between normal fertile men (16, 17). It has been suggested that the proportion of immature sperm in semen may be related to male infertility (16). Colleu et al. (18) reported that asthenospermic semen contained more sperm with lower resistance to decondensation in SDS than did normospermic semen. Therefore, it is possible that the proportion of sperm with immature nuclei assessed with this method may relate to fertilizing ability. Immature sperm nuclei can also be detected by staining with acidic aniline blue (19). Because the immature sperm head contains lysine-rich histones, they stain dark blue with acidic aniline blue. It has been suggested that the persistence of lysine-rich nuclear proteins may be involved in the defects of chromatin condensation. Dadoune et al. (20) reported that the percentage of aniline blue-stained heads was significantly higher in the morphologically abnormal sperm population than in the normal population. However, 20% of morphologically normal sperm have partially or totally aniline bluestained heads (20). Defects in chromatin condensation are also observed by transmission electron microscope in some normally shaped sperm nuclei (21). Asthenospermic semen contains more sperm with immature nuclei strongly stained with aniline blue (18). Acridine orange stain has also been used to distinguish between sperm containing normal or abnormal nuclear chromatin (22-24). Acridine orange fluoresces green when bound to normal doublestranded deoxyribonucleic acid (DNA) and red when bound to denatured or single-stranded DNA (22). There is a significant correlation between the results of this test and those from the aniline blue stain (24). In general, the percentage of sperm with abnormal DNA, as indicated by red fluorescence, is Fertility and Sterility
correlated with percentage abnormal sperm morphology (23). Sperm Membrane Integrity
The integrity and functional activity of the sperm membrane are crucial for viability and, in addition, for the physiological changes that occur at the sperm surface during the fertilization process including capacitation' acrosome reaction, and binding to the zona pellucida (ZP) and oolemma. Therefore, assessment of sperm membrane function should be of value for predicting fertility. The traditional methods for assessing whether the membrane is intact or disrupted are based on examining the percentage of viable sperm by dye exclusion of Trypan Blue or eosin Y. The membrane of living sperm acts as a barrier to stain penetration; thus only damaged or dead sperm are stained. Although the clinical significance of this test in evaluating male fertility is unknown, it provides diagnostic information for necrospermia when sperm motility is very low (25). Therefore this test is still recommended as a part of standard semen analysis by World Health Organization (WHO) (26, 27). Jeyendran et a1. (28) developed another simple test called the hypo-osmotic swelling test. When live sperm with normal membrane function are exposed to a solution of low osmolarity (150 mOsm/L), the tails become swollen because of the osmotically driven influx of water. Hypo-osmotic swelling test results are correlated with sperm concentration, motility, normal morphology, and, particularly, with viability assessed by eosin Y exclusion (28, 29). It was suggested that eosin Y exclusion measures the structural integrity of the sperm membrane, whereas the hypo-osmotic swelling test evaluates the physiological integrity of the sperm membrane (30). Interestingly, Jeyendran et a1. (28) found that hypoosmotic swelling test was highly correlated (r = 0.9) with the hamster zona-free oocyte penetration, and they claimed that hypo-osmotic swelling is a simple and useful clinical test of human sperm function. The clinical value of this test in fertility evaluation will be discussed below. Acrosomes and the Acrosome Reaction
The acrosomal membrane closest to the nucleus is termed the inner acrosomal membrane, whereas that immediately beneath the sperm plasma membrane is termed the outer acrosomal membrane (31) (Fig. 1). The acrosome contains numerous hydrolytic enzymes, such as acrosin and hyaluronidase. The Vol. 58, No.3, September 1992
Intact
Reacting
Reacted
Figure 1 Diagram of the structure of the human acrosome and acrosome reaction. PM: plasma membrane; DAM: outer acrosomal membrane; AC: acrosome contents; lAM: inner acrosomal membrane; AR: acrosome region; ES: equatorial segment.
acrosome reaction involves fusion and vesiculation of sperm plasma and outer acrosomal membranes initially forming pores and finally resulting in the loss of both membranes anterior to the equatorial segment (Fig. 1). There is a release of acrosomal contents as well as exposure of the inner acrosomal membrane with its associated bound enzymes (31, 32). The acrosome is important for fertilization both in vivo and in vitro in humans. A normal acrosome reaction is essential for sperm penetration through the ZP and to prepare the sperm for fusion with the oolemma (31-34).Round-headed sperm without acrosomes will not bind to or penetrate through the human ZP (35). These sperm will also not bind to and penetrate the vitellus of the ZP-free hamster oocyte (35-37). Thus, men with only acrosomeless sperm are sterile. Although the condition is an uncommon cause of male infertility, high proportions of small round-headed sperm without an acrosome are observed in the ejaculates of some other infertile patients (38). Knowledge of the physiology of the human acrosome reaction is incomplete (39). It is reported that the human acrosome reaction can be induced by a number of potentially physiological components such as follicular fluid (FF) (40-43), cumulus complex (44, 45), whole oocytes (46), ZP (47,48), and chemicals such as the calcium ionophore A23187 (40). White et a1. (49) reported that the cumulus had little effect on induction of the acrosome reaction. Some preliminary studies have shown that the proportion of sperm undergoing the acrosome reaction induced by the calcium ionophore A23187 or human FF may predict fertility in men with oligoLiu and Baker
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spermia or normospermia (50-52). However, the biological and clinical significance of acrosome loss induced by these components is still poorly understood. In the mouse, the sperm receptor on the ZP is a glycoprotein (ZP 3). Sperm bind to the ZP of the mouse oocyte with the acrosome intact, and the acrosome reaction is subsequently induced by ZP 3 (53). The situation in humans is unclear because both acrosome intact and reacted sperm are observed on the ZP. Morales et al. (48) have reported that both acrosome intact and reacted sperm can initiate binding to the human ZP. However, in their study, no attempts were made to differentiate between adherence and real binding because the ZP was not washed to remove loosely attached sperm after incubation in a highly concentrated sperm suspension for 1 minute. In contrast, when Liu and Baker (54) washed oocytes to dislodge sperm loosely adherent to the ZP, sperm incubated with A23187 had decreased sperm-ZP binding and increased sperm-oolemma binding compared with sperm not exposed to A23187. This result suggests that acrosome-reacted human sperm do not bind to the ZP. Therefore, human sperm may be similar to mouse sperm in that for sperm to bind to the ZP they must have the acrosome intact, and the physiological acrosome reaction occurs on the ZP perhaps induced by one of the zona glycoproteins (55-57). It is reported that disaggregated zona material is a very efficient inducer of the human acrosome reaction (47). In contrast, the cumulus does not induce the acrosome reaction rapidly because electron microscopy of sperm-penetrating cumulus masses showed that the majority (48 of 53) were acrosome intact (49). Tesarik (56) concluded that the acrosome reaction of fertilizing sperm must occur on the ZP after showing there is a close relationship between the numbers of acrosomal ghosts on the surface of the ZP at the orifices of the penetration slits and the numbers of sperm penetrating the ZP. For a long time there was no simple technique for routine assessment of human acrosomes. The human acrosome is too small to be examined by light microscopy. Although electron microscopy is the most effective method for assessing acrosomes, it is impractical for routine analysis of many samples. Therefore, a number of other techniques have been developed such as the triple stain (58), monoclonal antibodies (mAbs) to acrosomal components (59), chlortetracycline fluorescence, and fluorescein isothiocyanate (FITC)-labeled lectins such as pisum sativum agglutinin (60) and peanut agglutinin (60468
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62). These methods can be performed easily in the clinical laboratory. In general, there is good agreement between the results obtained by these methods and those obtained by electron microscopy. The advantages and disadvantages of these methods have been discussed in detail by Cross and Meizel (63). Overall, pisum sativum agglutinin fluorescein stain is a simple and reproducible technique for routine assessment of human acrosomes (64). Use of a supravital stain such as Hoechst 33258 (Sigma Chemical Co., St Louis, MO) or hypo-osmotic swelling in conjunction with the pisum sativum agglutinin acrosome assessment allows differentiation of living and dead acrosomeless sperm (60). The acrosome contains numerous hydrolytic enzymes. Acrosin, the major and most important proteolytic enzyme, is believed to playa role in spermZP penetration (56, 65). It has been suggested that low acrosin activity is associated with male infertility (66-68) and low fertilization rates in vitro (69, 70). Therefore, assessment of acrosin activity may have prognostic value for fertility. A simple method for measuring total acrosomal proteinase activity of human sperm has been reported (71). This method is based on the measurement of the area about the sperm head digested by proteinase on a gelatin-coated slide. The diameter of the halo and the proportion of sperm that develop halos are measures of proteinase activity. Small round-headed sperm without acrosomes show no halo formation in the gelatin membrane (66, 67). Similarly, Hirayama et al. (72) developed a test for assessing hyaluronidase activity of human sperm by digesting hyaluronic acid substrate covering on a slide. They claim that hyaluronidase activity of human sperm assessed by this method correlated with the proportion of mature oocytes fertilized in vitro. Although both tests are simple, it is difficult to control preparation of the thickness of substrate membrane on the slides and this will affect measurement of the diameters of the halo. The clinical value of these tests is still unclear because only very small numbers of subjects have been investigated. Human Sperm-Oocyte Interaction
Human infertility may result from abnormalities of sperm-oocyte interaction involving sperm binding to and penetration of the ZP and fusion with the oolemma. Human sperm binding to the ZP is species-specific and an important prerequisite for fertilization (31-33, 73). The fertilizing sperm must be able to bind to and penetrate through the ZP and complete the acrosome reaction before fusing with Fertility and Sterility
oolemma (31, 32). In the mouse, acrosome-intact sperm bind to the ZP, specifically to ZP 3, one of the ZP glycoproteins. Subsequently, the acrosome reaction is induced by ZP 3, then acrosome-reacted sperm bind to ZP 2, another ZP glycoprotein, and sperm penetration through the ZP ensues (53, 74, 75). The possible events of the human fertilization process is summarized in Figure 2. Assessment of sperm-oocyte interaction should be a powerful method for evaluating human sperm fertilizing ability because only a small number of sperm bind to the ZP and only one or a few penetrate into the perivitelline space even when large numbers of sperm surround the oocyte. Therefore, a biological test for assessing the ability of human sperm to bind to the ZP may have substantial prognostic and diagnostic value. However, the use of mature human oocytes for tests is almost impossible both for ethical reasons and because of limited availability of material. Overstreet and Hembree (33) reported that the ZP of nonviable or immature human oocytes can support sperm binding and penetration. Failure of sperm-ZP binding may be associated with certain cases of male infertility (76). It has been shown that human oocytes fertilized in vitro have more sperm bound to the ZP than do unfertilized oocytes (77). In mouse IVF, the number of sperm bound to the ZP is directly related to sperm-ZP penetration and fertilization rates (78). Liu et al. (79) counted sperm bound to a large number of oocytes that had failed to fertilize in vitro and found a highly significant relationship between the number of sperm bound to the ZP and the fertilization rates in the clutches of oocytes collected from the patients. The number of sperm bound to the ZP could indicate the functional integrity of both sperm and oocytes. Although Mahadevan et al. (77) reported that oocyte quality influenced the number of sperm bound to the ZP, others (80) suggested that oocyte maturation was related to sperm-ZP penetration but did not influence the number of sperm bound to the ZP. However, Lopata and Leung (81) reported that human sperm could penetrate the ZP of oocytes at all stages of maturity from germinal vesicle to metaphase II. Liu et al. (79) found that there was no relationship between the number of sperm bound to the ZP and oocyte quality or maturity assessed morphologically. In clinical IVF, failure of sperm-ZP binding was mainly because of sperm defects rather than oocyte defects because most of the oocytes without husband's sperm bound to the ZP during IVF could bind donor sperm (79). The percentage of sperm with normal morphology and a normal inVol. 58, No.3, September 1992
Zona
Cortical reaction
Figure 2 The possible events of human fertilization: sperm bind to the zona pellucida with the acrosome intact, and the acrosome reaction occurs on the zona; after the acrosome reaction, the acrosomal enzymes are released to soften the zona. Motility is also important for sperm penetration through the zona. After penetration, the acrosome-reacted sperm fuse with the oolemma and induce the cortical granule reaction that prevents other sperm penetrating the zona and fusing with the oolemma.
tact acrosome were the most significant correlates of sperm-ZP binding. Because assessment of the ability of sperm binding to the ZP could predict sperm fertilizing ability, clinical tests for assessing sperm-ZP binding are emerging. Liu et al. (82) developed a sperm-ZP binding ratio test using oocytes that failed to fertilize in vitro. The oocytes are stored in concentrated salt (ammonium sulphate) solution. The majority (80%) of these oocytes, either fresh or salt-stored, can bind sperm (82, 83). This test is based on competitive binding of two sperm populations (test patient and fertile donor control) to several oocytes. Test and control sperm are labeled with different fluorochromes (FITC: green color; tetramethylrhodamine isothiocyanate: red color). Equal numbers of progressively motile test and control sperm are mixed and incubated with four to six salt-stored ZP. Then the ratio of test to control sperm bound to ZP is calculated and used as a measure of sperm-ZP binding ability. The sperm-ZP binding ratio was the most significant factor related to fertilization rate in vitro in logistic regression analysis of 106 patients studied. This test appears to have potential for evaluation of fertility. In IVF programs 20% to 40% oocytes fail to be fertilized, and thus there is a continuous supply of oocytes for this test. However, these 00cytes have been exposed to sperm in IVF and their quality and cortical granule reaction are unknown; Liu and Baker
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therefore, it is necessary to use donor sperm to control for variability between oocytes. Burkman et al. (84) established a similar spermzona binding test called the hemizona assay (HZA). The ZP of immature oocytes from ovarian surgery or IVF are cut into halves using a micromanipulator, normal fertile donor sperm are incubated with one half, and patient's sperm are incubated with the other half. The sperm-ZP binding index obtained by HZA is significantly correlated with fertilization rate in vitro (84-86). Therefore, it is suggested that the HZA is useful for assessing male fertility. However, immature human oocytes are in limited supply. Also, it is difficult to cut the hemizonae into equal sizes. Only one oocyte is used per test. Furthermore, it is unclear if there is a difference between sperm binding to the inside or to the outside of the ZP. Liu et al. (87) reported the development of a sperm-oolemma binding test using the oocytes that failed to fertilize in vitro. The ZP of the oocyte is dissolved in acidic (pH 2.5 to 3.0) saline. Four to six ZP-free oocytes are incubated with a mixture of test and control sperm labeled with different fluorochromes as for the sperm-ZP binding ratio test. Preliminary results showed that the sperm -oolemma binding ratio was significantly correlated with sperm-ZP binding ratio but less significantly related to IVF rates by logistic regression analysis (87). Because classical semen analysis often cannot predict fertility accurately, new sperm function tests are greatly needed, but detailed clinical evaluation is necessary to determine which of the tests will provide useful additional information about fertility. Physiological and technical aspects of new tests of human sperm function have been reviewed. The acrosome and acrosome reaction and sperm -oocyte interactions are important in fertilization. Tests for assessing each of these sperm functions have been developed, and these tests can be performed in a clinical semen laboratory. Although it is not clear if these tests will be useful clinically for assessing male fertility because they have not been widely evaluated, they are designed to measure important physiological functions of human sperm. Preliminary studies show that assessment of acrosomes and sperm-ZP binding have potential clinical value for predicting fertility. In the next section, studies of the clinical evaluation of tests of human sperm function and fertility are discussed. CLINICAL EVALUATION OF TESTS OF HUMAN SPERM FUNCTION AND FERTILITY
Factors influencing clinical evaluation of the usefulness of tests of human sperm function, including 470
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statistical methods and prognostic factors not directly related to semen quality, are briefly discussed together with the advantages of using the results of clinical IVF for evaluating male fertility. Then studies of the relationship between clinical tests of human sperm function and fertility in vivo and in vitro are discussed in order: sperm concentration, motility and movement characteristics, morphology, hamster oocyte penetration, and other tests of human sperm function. Statistical Aspects
Floating numerator pregnancy rates (PRs) (% pregnant) that ignore time are becoming infrequent in the literature, and life table estimates are now common. It is crucial that appropriate statistical methods are used and, particularly, that there are sufficient numbers of subjects for testing the significance of relationships between sperm tests and fertility. In general, if a variable lacks statistical significance, the variable is either not related or, alternatively, it could be related, but the relationship is not detected as statistically significant in the analysis. There may be insufficient statistical power, with the sample size being too small relative to the magnitude of the effect and its variability. Logistic regression analysis is the most appropriate method for binary data such as the occurrence, or failure of occurrence, of fertilization. It is used to examine the relationship between the probability of response and multiple explanatory variables that can be either continuous or nominal. The variables can be assumed to be independent or interactions can be explored. After adjusting for all other variables, only those with regression coefficients significantly different from zero are considered to influence the response. With PRs proportional hazards, Cox regression or multiple regression after normalizing time of follow-up can be used. Other Prognostic Factors for Fertility
A number of factors unrelated to semen analysis are known to affect the PRs in normal and sub fertile couples including age of the female, duration of infertility, previous fertility in the union, previous use of oral contraceptives, smoking and coffee intake in the female, and varicoceles and other abnormalities in the male and female partner (88-94). Baker et al. (92) were able to show that a number ofthese factors were significant predictors of PR by Cox regression analysis of 448 pregnancies in 1,367 subfertile couples seen for male infertility and who had at least Fertility and Sterility
some motile sperm in their semen and whose wives were not sterile. The PR was approximately 4 % per month for the first few months. Overall, 30% of the female partners conceived in 1 year and 45% by 2 years (92). Duration of infertility was inversely related to PR, and its statistical significance was about equal to that of average sperm concentration. These prognostic factors are valuable for advising subfertile patients. However, because the identified factors only explain a small proportion of the variance of PRs, the accuracy of prediction is limited. It should be possible to improve the prediction of potential fertility by discovery of new prognostic factors and improvements in tests of fertility such as semen analysis (12, 92). Use of Clinical IVF for Evaluating Sperm Function Tests
Relating results of tests of human sperm function to fertility in vivo poses great difficulties. Comparing semen analysis results in groups of men who have children or who are being examined for infertility is a poor approach for determining the normal range. Prospective studies of unselected couples attempting to conceive and efficacy trials of induced oligospermia for male contraception are in progress and should provide useful information. However, in groups of un selected couples, the average PR is 50% morphologically abnormal sperm) is classified as teratospermia and considered to be associated with subfertility (26). However, Poland et al. (130) reported 15 normal fertile men with an average of 45% sperm with normal morphology. It is probable that the value of 50% for normal morphology in the WHO manual is too high. In general, the percentage of sperm with normal morphology is lower in infertile men than in normal fertile men (131). Although semen quality varies widely within men (96), it has been claimed that sperm morphology is relatively stable between ejaculates from the same man (130). Clinical investigation of the relationship between semen quality and subsequent PRs in a large group of sub fertile couples indicated that sperm morphology assessed by the traditional method was not of significant predictive value for fertility (3, 92). In contrast, average morphology did differ in men with high and low PRs in a donor insemination program (132). Studies with the zona-free hamster oocyte sperm penetration assay (SPA) suggest that morphologically abnormal sperm have low fertilizing ability compared with that of morphologically normal sperm (133-135). There is a negative correlation beVol. 58, No.3, September 1992
tween the proportion of sperm with abnormal morphology and the results of SPA (134, 136). Liu and Baker (137) found that the human zona is highly selective for binding of sperm with normal morphology. The majority (80% to 100%) of sperm bound to the ZP had normal morphology (Fig. 3). Sperm with abnormal morphology had significantly lower binding rates than did those with normal morphology (137). Makler (138) claimed that it may be more valuable clinically if sperm morphology was assessed on the motile subpopulation. In the standard procedure, morphology is determined on a preparation of fixed (immobilized) sperm, and one cannot tell whether a morphologically abnormal sperm was previously motile or immotile. It is reported that sperm with abnormal morphology are twice as frequent in the immotile subpopulation as in the motile subpopulation (138). This is consistent with reports that motile sperm selected by swim-up or Percoll gradient centrifugation techniques have significantly better morphology compared with sperm in the original semen (139-141). Katz et al. (142) reported that morphologically normal sperm swim faster, straighter, and with higher tail beat frequencies than do morphologically abnormal sperm in semen. They also showed that the velocity of morphologically normal sperm swimming in human cervical mucus was higher than that of abnormal sperm (143). Despite this, percentage normal morphology in semen had better predictive value for fertilization rates in
Figure 3 Morphology of sperm bound to the zona pellucida of human oocytes that had failed to fertilize in vitro (Shorr stain). All the sperm bound to this zona have normal morphology (magnification X10,OOO). Liu and Baker Human sperm function and IVF
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100 90 80 70 "0 (])
~60 (])
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>. 0 840
* 30 20 10 59
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41-50
~51
0 21-3031-40
Normal Morphology Group
Figure 4 Relationship between the percentage of sperm in semen with normal morphology and IVF rates. (Mean + SEM, number of patients in each normal morphology group are shown in the lower part of the bars).
vitro than percentage normal morphology in the insemination medium (12). Thus it remains possible that the relationship between morphological defects of sperm and failure of fertilization is at least partly indirect; a low proportion of sperm with normal morphology may signify the presence of more subtle abnormalities in all the sperm. It is now clear that sperm morphology assessed with a modified improved method of strict scoring is one of the most significant predictors of fertilization rate in vitro (11, 12, 64, 79, 83, 127, 144, 145). Data from over 1,000 IVF treatments indicate normal morphology is consistently a very significant factor related to IVF rates (Fig. 4). Standardization of sperm morphology assessment is urgently needed to provide more consistent, reliable, and reproducible results in the clinical semen laboratory. In our modification of morphology assessment, sperm are washed with normal saline to remove seminal plasma and adjust sperm concentration. A smear is made and checked microscopically for density and evenness of spread of the spermatozoa before it is left to air dry. Then the slide is fixed in 90% ethanol and stained with the Shorr method (12, 127). This provides very clear images for technicians 474
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to count normal and abnormal sperm and avoids bias related to sperm density. The Shorr stain also gives a clear contrast between the acrosomal and postacrosomal regions. Sperm morphology is considered according to the WHO criteria for the silhouette, but, in addition, internal staining characteristics of the sperm head are also included; for normality the acrosomal region must be clearly seen, be regular in shape, and occupy at least half of the sperm head (Figs. 3 and 5). This modified morphology assessment is similar to the strict scoring method of Kruger et al. (144, 145). The importance of the acrosomal area is indicated in a study of sperm bound to the ZP (136, Fig. 3). The human ZP is highly selective for binding of morphologically normal sperm, but some abnormal sperm, mainly with normal acrosomal regions, pyriform, or small heads, bind at low rates. With the improved method for assessing sperm morphology, men with >30% of sperm with normal morphology are very unlikely to have complete failure of fertilization in vitro (11, 12,79). On the other hand, a few patients with poor morphology «10% to 20% normal) still fertilize most or all of the oocytes inseminated. Therefore, assessment of sperm morphology alone is not an accurate predictor of fertility. It is true that the subjective microscopic method for assessing sperm morphology has large variations within and between technicians (146). Each laboratory must have their own standards and quality control. A good technician should produce consistent results with a coefficient of variation for percentage
Figure 5 Shorr staining of sperm with normal morphology in semen (magnification XI,OOO).
Fertility and Sterility
normal morphology for different readings of the same slide within 10% to 15%. Although characteristics of morphologically normal and various abnormal forms have been defined (26, 27), the technician's experience is extremely important for producing consistent and reliable results. The quality of preparation of the sperm smear and staining of the slides are also important. The errors may be reduced by improving the methods for preparation of the slides and staining to produce high -quality images. In the future, the development of objective methods for sperm morphology holds the most promise for improving morphology assessment (147,148). The Human Sperm-Hamster Oocyte Penetration Assay
Since Yanagimachi et al. (149) first reported that human sperm can penetrate and undergo nuclear decondensation in zona-free hamster oocytes, the human sperm-hamster oocyte SPA has been used as a tool for evaluating human sperm fertilizing ability. The use of living human oocytes for testing human sperm function is almost impossible; therefore, the SPA is a powerful test for assessing the ability of human sperm to capacitate, acrosome react, fuse with the oolemma, and undergo decondensation in the cytoplasm. Only acrosome-reacted human sperm appear to be able to bind to and penetrate the zona-free oocyte (149, 150). Although the clinical significance of the SPAin predicting male fertility is still disputed, the SPA has been widely used as a clinical test of sperm function. Numerous clinical papers have claimed that poor SPA results are strongly correlated with male infertility (150, 151). In some reports, the SPA is positively correlated with most or all of the seminal parameters such as sperm concentration, motility, and morphology (124, 152). Sperm motility and particularly morphology are strongly correlated with the SPA (124, 135). Despite these reports, many others have shown no correlation between the SPA and sperm characteristics (150, 151). Some clinical investigations of the SPA and fertility have suggested that sperm from infertile men, regardless of their sperm characteristics, always penetrate
Vol. 58, No.3, September 1992
Copyright © 1992 The American Fertility Society
Printed on acid-free paper in U.S.A.
Tests of human sperm function and fertilization in vitro
De Yi Liu, Ph.D.* H. W. Gordon Baker, M.D., Ph.D. Department of Obstetrics and Gynaecology and Reproductive Biology Unit, University of Melbourne, Royal Women's Hospital, Melbourne, Victoria, Australia
Objective: To review recent studies on the development of new tests of human sperm function and evaluation of which sperm characteristics are most important for fertilization in vitro by logistic regression analysis. Study Selection: Recent studies on the relationship between putative and new tests of human sperm function and fertility in vitro or in vivo are discussed in this review. Some physiological and technical aspects are included. Main Outcome Measures: Fertilization rates in vitro and sperm tests including standard semen analysis, improved morphology assessment, objective assessment of sperm motility- and movement characteristics, nuclear maturity, hypo-osmotic swelling, the acrosome and the acrosome reaction, acrosin activity, human sperm-hamster OBGyte penetration assay, and sperm-zona pellucida (ZP) and sperm-oolemma binding. Results: The percentages of sperm with normal morphology and a normal intact acrosome, mean linearity, and the number of sperm binding to the ZP were highly significantly related to fertilization rates in vitro. Other sperm tests evaluated usually provided no additional information about fertilization rates. The human ZP is highly selective for binding of morphologically normal sperm. Acrosome-reacted human sperm have little or no ability to bind to the ZP. Conclusion: Results of in vitro fertilization can be used to evaluate tests of human sperm function. Logistic regression analysis is a powerful method for determining which groups of sperm characteristics are independently related to fertilization rates. Normal morphology, linearity, acrosome status, and sperm-ZP binding are the most important characteristics for fertilization in vitro. Fertil Steril 1992;58:465-83 Key Words: Sperm function tests, in vitro fertilization, fertilization rates
Standard semen analysis including sperm concentration' motility, and morphology is widely used as a fundamental indicator of male fertility. However, the results do not provide precise diagnostic or prognostic information for human fertility in vivo or in vitro (1-8). Men with absolute and persistently immotile sperm, azoospermia, or all sperm with morphological defects such as small round-headed sperm without acrosomes are sterile, but these are not common causes of male infertility (9, 10). Most men seen for infertility have reduced sperm concenReceived March 11, 1992.
* Reprint requests: De Yi Liu, Ph.D., Department of Obstetrics and Gynaecology, University of Melbourne, Royal Women's Hospital, Carlton, Melbourne, Victoria 3053, Australia. Vol. 58, No.3, September 1992
tration, motility, and morphology, often in combination. Other factors, known and unknown, and particularly those in the female partner, are also important in determining the ease of conception. Some couples conceive quickly, despite abnormal semen analysis results, and, conversely, rare men with normal semen analysis results are sterile. Because standard semen analysis has limited clinical value for predicting fertility, many other tests of human sperm function have been developed, such as objective assessment of motility, hypoosmotic swelling, tests for sperm nuclear maturity, measurements of acrosome status, acrosome reaction and acrosin activity, hamster zona-free oocyte penetration and human sperm-zona binding and penetration. Although some of these tests appear to Liu and Baker
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provide additional valuable information that may improve the prediction of fertility, for many of them it is still unclear if they will be useful clinically. So far, no single test of sperm function will predict fertility accurately except where there is an absolute disorder affecting all sperm such as azoospermia or totally absent acrosomes. In other situations, groups of tests are required to evaluate male fertility. The results of sperm tests such as concentration, motility, and morphology are correlated, and defects usually occur in combination (11, 12). It is therefore necessary to determine which groups of sperm tests give the most information about fertility. Then clinically useful sets of tests can be identified for predicting sperm fertilizing ability. In this article we will discuss tests of human sperm function and their predictive value for fertilization in vivo and in particular, preliminary studies of new tests of sperm function in relation to the results of in vitro fertilization (lVF). Technical and other factors influencing the results that may explain conflicting reports on the clinical value of some tests are covered. Because the new tests have not been widely used in clinical semen laboratories, the principles of the test and relevant procedural aspects are reviewed. Because of the concentration on clinical relevance of the sperm tests and their predictive ness for fertility, sperm function and fertilization are not discussed in detail. The physiology is dealt with first, followed by an overview of tests of human sperm function. PHYSIOLOGY AND TECHNICAL ASPECTS OF NEW TESTS OF HUMAN SPERM FUNCTION
In this section, we discuss selected physiological and technical aspects of new tests of human sperm function. These include sperm nuclear maturity, sperm membrane integrity, the acrosome and acrosome reaction, and sperm-oocyte interaction. Sperm Nuclear Maturity
During epididymal maturation, human sperm, like those of other mammals, undergo structural and biochemical changes that are considered to be prerequisites for optimal fertilizing ability. Among these maturational changes, formation of disulfide bonds between nuclear protamines results in an increase in the stability of the sperm head (13). This increase is concomitant with a decrease in free thiol groups in the nucleus (14). In humans, the transit time of sperm through the epididymis varies between individuals (15). Thus, ejaculated sperm have various 466
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degrees of nuclear stability (16). It is known that immature sperm in the epididymis have lower fertilizing ability than do mature sperm in the ejaculate. Thus assessment of the proportion of sperm with an immature nucleus in the ejaculate may predict male fertility. Several simple tests for assessing nuclear maturity are now available and will be briefly described. When sperm are exposed to the detergent, sodium dodecyl sulfate (SDS) or SDS in combination with the disulfide bond-reducing agent dithiothreitol, the sperm heads with immature nuclei with less disulfide cross-linking rapidly undergo decondensation and distend and may lyse (16). There is a large variation in rates of decondensation between normal fertile men (16, 17). It has been suggested that the proportion of immature sperm in semen may be related to male infertility (16). Colleu et al. (18) reported that asthenospermic semen contained more sperm with lower resistance to decondensation in SDS than did normospermic semen. Therefore, it is possible that the proportion of sperm with immature nuclei assessed with this method may relate to fertilizing ability. Immature sperm nuclei can also be detected by staining with acidic aniline blue (19). Because the immature sperm head contains lysine-rich histones, they stain dark blue with acidic aniline blue. It has been suggested that the persistence of lysine-rich nuclear proteins may be involved in the defects of chromatin condensation. Dadoune et al. (20) reported that the percentage of aniline blue-stained heads was significantly higher in the morphologically abnormal sperm population than in the normal population. However, 20% of morphologically normal sperm have partially or totally aniline bluestained heads (20). Defects in chromatin condensation are also observed by transmission electron microscope in some normally shaped sperm nuclei (21). Asthenospermic semen contains more sperm with immature nuclei strongly stained with aniline blue (18). Acridine orange stain has also been used to distinguish between sperm containing normal or abnormal nuclear chromatin (22-24). Acridine orange fluoresces green when bound to normal doublestranded deoxyribonucleic acid (DNA) and red when bound to denatured or single-stranded DNA (22). There is a significant correlation between the results of this test and those from the aniline blue stain (24). In general, the percentage of sperm with abnormal DNA, as indicated by red fluorescence, is Fertility and Sterility
correlated with percentage abnormal sperm morphology (23). Sperm Membrane Integrity
The integrity and functional activity of the sperm membrane are crucial for viability and, in addition, for the physiological changes that occur at the sperm surface during the fertilization process including capacitation' acrosome reaction, and binding to the zona pellucida (ZP) and oolemma. Therefore, assessment of sperm membrane function should be of value for predicting fertility. The traditional methods for assessing whether the membrane is intact or disrupted are based on examining the percentage of viable sperm by dye exclusion of Trypan Blue or eosin Y. The membrane of living sperm acts as a barrier to stain penetration; thus only damaged or dead sperm are stained. Although the clinical significance of this test in evaluating male fertility is unknown, it provides diagnostic information for necrospermia when sperm motility is very low (25). Therefore this test is still recommended as a part of standard semen analysis by World Health Organization (WHO) (26, 27). Jeyendran et a1. (28) developed another simple test called the hypo-osmotic swelling test. When live sperm with normal membrane function are exposed to a solution of low osmolarity (150 mOsm/L), the tails become swollen because of the osmotically driven influx of water. Hypo-osmotic swelling test results are correlated with sperm concentration, motility, normal morphology, and, particularly, with viability assessed by eosin Y exclusion (28, 29). It was suggested that eosin Y exclusion measures the structural integrity of the sperm membrane, whereas the hypo-osmotic swelling test evaluates the physiological integrity of the sperm membrane (30). Interestingly, Jeyendran et a1. (28) found that hypoosmotic swelling test was highly correlated (r = 0.9) with the hamster zona-free oocyte penetration, and they claimed that hypo-osmotic swelling is a simple and useful clinical test of human sperm function. The clinical value of this test in fertility evaluation will be discussed below. Acrosomes and the Acrosome Reaction
The acrosomal membrane closest to the nucleus is termed the inner acrosomal membrane, whereas that immediately beneath the sperm plasma membrane is termed the outer acrosomal membrane (31) (Fig. 1). The acrosome contains numerous hydrolytic enzymes, such as acrosin and hyaluronidase. The Vol. 58, No.3, September 1992
Intact
Reacting
Reacted
Figure 1 Diagram of the structure of the human acrosome and acrosome reaction. PM: plasma membrane; DAM: outer acrosomal membrane; AC: acrosome contents; lAM: inner acrosomal membrane; AR: acrosome region; ES: equatorial segment.
acrosome reaction involves fusion and vesiculation of sperm plasma and outer acrosomal membranes initially forming pores and finally resulting in the loss of both membranes anterior to the equatorial segment (Fig. 1). There is a release of acrosomal contents as well as exposure of the inner acrosomal membrane with its associated bound enzymes (31, 32). The acrosome is important for fertilization both in vivo and in vitro in humans. A normal acrosome reaction is essential for sperm penetration through the ZP and to prepare the sperm for fusion with the oolemma (31-34).Round-headed sperm without acrosomes will not bind to or penetrate through the human ZP (35). These sperm will also not bind to and penetrate the vitellus of the ZP-free hamster oocyte (35-37). Thus, men with only acrosomeless sperm are sterile. Although the condition is an uncommon cause of male infertility, high proportions of small round-headed sperm without an acrosome are observed in the ejaculates of some other infertile patients (38). Knowledge of the physiology of the human acrosome reaction is incomplete (39). It is reported that the human acrosome reaction can be induced by a number of potentially physiological components such as follicular fluid (FF) (40-43), cumulus complex (44, 45), whole oocytes (46), ZP (47,48), and chemicals such as the calcium ionophore A23187 (40). White et a1. (49) reported that the cumulus had little effect on induction of the acrosome reaction. Some preliminary studies have shown that the proportion of sperm undergoing the acrosome reaction induced by the calcium ionophore A23187 or human FF may predict fertility in men with oligoLiu and Baker
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spermia or normospermia (50-52). However, the biological and clinical significance of acrosome loss induced by these components is still poorly understood. In the mouse, the sperm receptor on the ZP is a glycoprotein (ZP 3). Sperm bind to the ZP of the mouse oocyte with the acrosome intact, and the acrosome reaction is subsequently induced by ZP 3 (53). The situation in humans is unclear because both acrosome intact and reacted sperm are observed on the ZP. Morales et al. (48) have reported that both acrosome intact and reacted sperm can initiate binding to the human ZP. However, in their study, no attempts were made to differentiate between adherence and real binding because the ZP was not washed to remove loosely attached sperm after incubation in a highly concentrated sperm suspension for 1 minute. In contrast, when Liu and Baker (54) washed oocytes to dislodge sperm loosely adherent to the ZP, sperm incubated with A23187 had decreased sperm-ZP binding and increased sperm-oolemma binding compared with sperm not exposed to A23187. This result suggests that acrosome-reacted human sperm do not bind to the ZP. Therefore, human sperm may be similar to mouse sperm in that for sperm to bind to the ZP they must have the acrosome intact, and the physiological acrosome reaction occurs on the ZP perhaps induced by one of the zona glycoproteins (55-57). It is reported that disaggregated zona material is a very efficient inducer of the human acrosome reaction (47). In contrast, the cumulus does not induce the acrosome reaction rapidly because electron microscopy of sperm-penetrating cumulus masses showed that the majority (48 of 53) were acrosome intact (49). Tesarik (56) concluded that the acrosome reaction of fertilizing sperm must occur on the ZP after showing there is a close relationship between the numbers of acrosomal ghosts on the surface of the ZP at the orifices of the penetration slits and the numbers of sperm penetrating the ZP. For a long time there was no simple technique for routine assessment of human acrosomes. The human acrosome is too small to be examined by light microscopy. Although electron microscopy is the most effective method for assessing acrosomes, it is impractical for routine analysis of many samples. Therefore, a number of other techniques have been developed such as the triple stain (58), monoclonal antibodies (mAbs) to acrosomal components (59), chlortetracycline fluorescence, and fluorescein isothiocyanate (FITC)-labeled lectins such as pisum sativum agglutinin (60) and peanut agglutinin (60468
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62). These methods can be performed easily in the clinical laboratory. In general, there is good agreement between the results obtained by these methods and those obtained by electron microscopy. The advantages and disadvantages of these methods have been discussed in detail by Cross and Meizel (63). Overall, pisum sativum agglutinin fluorescein stain is a simple and reproducible technique for routine assessment of human acrosomes (64). Use of a supravital stain such as Hoechst 33258 (Sigma Chemical Co., St Louis, MO) or hypo-osmotic swelling in conjunction with the pisum sativum agglutinin acrosome assessment allows differentiation of living and dead acrosomeless sperm (60). The acrosome contains numerous hydrolytic enzymes. Acrosin, the major and most important proteolytic enzyme, is believed to playa role in spermZP penetration (56, 65). It has been suggested that low acrosin activity is associated with male infertility (66-68) and low fertilization rates in vitro (69, 70). Therefore, assessment of acrosin activity may have prognostic value for fertility. A simple method for measuring total acrosomal proteinase activity of human sperm has been reported (71). This method is based on the measurement of the area about the sperm head digested by proteinase on a gelatin-coated slide. The diameter of the halo and the proportion of sperm that develop halos are measures of proteinase activity. Small round-headed sperm without acrosomes show no halo formation in the gelatin membrane (66, 67). Similarly, Hirayama et al. (72) developed a test for assessing hyaluronidase activity of human sperm by digesting hyaluronic acid substrate covering on a slide. They claim that hyaluronidase activity of human sperm assessed by this method correlated with the proportion of mature oocytes fertilized in vitro. Although both tests are simple, it is difficult to control preparation of the thickness of substrate membrane on the slides and this will affect measurement of the diameters of the halo. The clinical value of these tests is still unclear because only very small numbers of subjects have been investigated. Human Sperm-Oocyte Interaction
Human infertility may result from abnormalities of sperm-oocyte interaction involving sperm binding to and penetration of the ZP and fusion with the oolemma. Human sperm binding to the ZP is species-specific and an important prerequisite for fertilization (31-33, 73). The fertilizing sperm must be able to bind to and penetrate through the ZP and complete the acrosome reaction before fusing with Fertility and Sterility
oolemma (31, 32). In the mouse, acrosome-intact sperm bind to the ZP, specifically to ZP 3, one of the ZP glycoproteins. Subsequently, the acrosome reaction is induced by ZP 3, then acrosome-reacted sperm bind to ZP 2, another ZP glycoprotein, and sperm penetration through the ZP ensues (53, 74, 75). The possible events of the human fertilization process is summarized in Figure 2. Assessment of sperm-oocyte interaction should be a powerful method for evaluating human sperm fertilizing ability because only a small number of sperm bind to the ZP and only one or a few penetrate into the perivitelline space even when large numbers of sperm surround the oocyte. Therefore, a biological test for assessing the ability of human sperm to bind to the ZP may have substantial prognostic and diagnostic value. However, the use of mature human oocytes for tests is almost impossible both for ethical reasons and because of limited availability of material. Overstreet and Hembree (33) reported that the ZP of nonviable or immature human oocytes can support sperm binding and penetration. Failure of sperm-ZP binding may be associated with certain cases of male infertility (76). It has been shown that human oocytes fertilized in vitro have more sperm bound to the ZP than do unfertilized oocytes (77). In mouse IVF, the number of sperm bound to the ZP is directly related to sperm-ZP penetration and fertilization rates (78). Liu et al. (79) counted sperm bound to a large number of oocytes that had failed to fertilize in vitro and found a highly significant relationship between the number of sperm bound to the ZP and the fertilization rates in the clutches of oocytes collected from the patients. The number of sperm bound to the ZP could indicate the functional integrity of both sperm and oocytes. Although Mahadevan et al. (77) reported that oocyte quality influenced the number of sperm bound to the ZP, others (80) suggested that oocyte maturation was related to sperm-ZP penetration but did not influence the number of sperm bound to the ZP. However, Lopata and Leung (81) reported that human sperm could penetrate the ZP of oocytes at all stages of maturity from germinal vesicle to metaphase II. Liu et al. (79) found that there was no relationship between the number of sperm bound to the ZP and oocyte quality or maturity assessed morphologically. In clinical IVF, failure of sperm-ZP binding was mainly because of sperm defects rather than oocyte defects because most of the oocytes without husband's sperm bound to the ZP during IVF could bind donor sperm (79). The percentage of sperm with normal morphology and a normal inVol. 58, No.3, September 1992
Zona
Cortical reaction
Figure 2 The possible events of human fertilization: sperm bind to the zona pellucida with the acrosome intact, and the acrosome reaction occurs on the zona; after the acrosome reaction, the acrosomal enzymes are released to soften the zona. Motility is also important for sperm penetration through the zona. After penetration, the acrosome-reacted sperm fuse with the oolemma and induce the cortical granule reaction that prevents other sperm penetrating the zona and fusing with the oolemma.
tact acrosome were the most significant correlates of sperm-ZP binding. Because assessment of the ability of sperm binding to the ZP could predict sperm fertilizing ability, clinical tests for assessing sperm-ZP binding are emerging. Liu et al. (82) developed a sperm-ZP binding ratio test using oocytes that failed to fertilize in vitro. The oocytes are stored in concentrated salt (ammonium sulphate) solution. The majority (80%) of these oocytes, either fresh or salt-stored, can bind sperm (82, 83). This test is based on competitive binding of two sperm populations (test patient and fertile donor control) to several oocytes. Test and control sperm are labeled with different fluorochromes (FITC: green color; tetramethylrhodamine isothiocyanate: red color). Equal numbers of progressively motile test and control sperm are mixed and incubated with four to six salt-stored ZP. Then the ratio of test to control sperm bound to ZP is calculated and used as a measure of sperm-ZP binding ability. The sperm-ZP binding ratio was the most significant factor related to fertilization rate in vitro in logistic regression analysis of 106 patients studied. This test appears to have potential for evaluation of fertility. In IVF programs 20% to 40% oocytes fail to be fertilized, and thus there is a continuous supply of oocytes for this test. However, these 00cytes have been exposed to sperm in IVF and their quality and cortical granule reaction are unknown; Liu and Baker
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therefore, it is necessary to use donor sperm to control for variability between oocytes. Burkman et al. (84) established a similar spermzona binding test called the hemizona assay (HZA). The ZP of immature oocytes from ovarian surgery or IVF are cut into halves using a micromanipulator, normal fertile donor sperm are incubated with one half, and patient's sperm are incubated with the other half. The sperm-ZP binding index obtained by HZA is significantly correlated with fertilization rate in vitro (84-86). Therefore, it is suggested that the HZA is useful for assessing male fertility. However, immature human oocytes are in limited supply. Also, it is difficult to cut the hemizonae into equal sizes. Only one oocyte is used per test. Furthermore, it is unclear if there is a difference between sperm binding to the inside or to the outside of the ZP. Liu et al. (87) reported the development of a sperm-oolemma binding test using the oocytes that failed to fertilize in vitro. The ZP of the oocyte is dissolved in acidic (pH 2.5 to 3.0) saline. Four to six ZP-free oocytes are incubated with a mixture of test and control sperm labeled with different fluorochromes as for the sperm-ZP binding ratio test. Preliminary results showed that the sperm -oolemma binding ratio was significantly correlated with sperm-ZP binding ratio but less significantly related to IVF rates by logistic regression analysis (87). Because classical semen analysis often cannot predict fertility accurately, new sperm function tests are greatly needed, but detailed clinical evaluation is necessary to determine which of the tests will provide useful additional information about fertility. Physiological and technical aspects of new tests of human sperm function have been reviewed. The acrosome and acrosome reaction and sperm -oocyte interactions are important in fertilization. Tests for assessing each of these sperm functions have been developed, and these tests can be performed in a clinical semen laboratory. Although it is not clear if these tests will be useful clinically for assessing male fertility because they have not been widely evaluated, they are designed to measure important physiological functions of human sperm. Preliminary studies show that assessment of acrosomes and sperm-ZP binding have potential clinical value for predicting fertility. In the next section, studies of the clinical evaluation of tests of human sperm function and fertility are discussed. CLINICAL EVALUATION OF TESTS OF HUMAN SPERM FUNCTION AND FERTILITY
Factors influencing clinical evaluation of the usefulness of tests of human sperm function, including 470
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statistical methods and prognostic factors not directly related to semen quality, are briefly discussed together with the advantages of using the results of clinical IVF for evaluating male fertility. Then studies of the relationship between clinical tests of human sperm function and fertility in vivo and in vitro are discussed in order: sperm concentration, motility and movement characteristics, morphology, hamster oocyte penetration, and other tests of human sperm function. Statistical Aspects
Floating numerator pregnancy rates (PRs) (% pregnant) that ignore time are becoming infrequent in the literature, and life table estimates are now common. It is crucial that appropriate statistical methods are used and, particularly, that there are sufficient numbers of subjects for testing the significance of relationships between sperm tests and fertility. In general, if a variable lacks statistical significance, the variable is either not related or, alternatively, it could be related, but the relationship is not detected as statistically significant in the analysis. There may be insufficient statistical power, with the sample size being too small relative to the magnitude of the effect and its variability. Logistic regression analysis is the most appropriate method for binary data such as the occurrence, or failure of occurrence, of fertilization. It is used to examine the relationship between the probability of response and multiple explanatory variables that can be either continuous or nominal. The variables can be assumed to be independent or interactions can be explored. After adjusting for all other variables, only those with regression coefficients significantly different from zero are considered to influence the response. With PRs proportional hazards, Cox regression or multiple regression after normalizing time of follow-up can be used. Other Prognostic Factors for Fertility
A number of factors unrelated to semen analysis are known to affect the PRs in normal and sub fertile couples including age of the female, duration of infertility, previous fertility in the union, previous use of oral contraceptives, smoking and coffee intake in the female, and varicoceles and other abnormalities in the male and female partner (88-94). Baker et al. (92) were able to show that a number ofthese factors were significant predictors of PR by Cox regression analysis of 448 pregnancies in 1,367 subfertile couples seen for male infertility and who had at least Fertility and Sterility
some motile sperm in their semen and whose wives were not sterile. The PR was approximately 4 % per month for the first few months. Overall, 30% of the female partners conceived in 1 year and 45% by 2 years (92). Duration of infertility was inversely related to PR, and its statistical significance was about equal to that of average sperm concentration. These prognostic factors are valuable for advising subfertile patients. However, because the identified factors only explain a small proportion of the variance of PRs, the accuracy of prediction is limited. It should be possible to improve the prediction of potential fertility by discovery of new prognostic factors and improvements in tests of fertility such as semen analysis (12, 92). Use of Clinical IVF for Evaluating Sperm Function Tests
Relating results of tests of human sperm function to fertility in vivo poses great difficulties. Comparing semen analysis results in groups of men who have children or who are being examined for infertility is a poor approach for determining the normal range. Prospective studies of unselected couples attempting to conceive and efficacy trials of induced oligospermia for male contraception are in progress and should provide useful information. However, in groups of un selected couples, the average PR is 50% morphologically abnormal sperm) is classified as teratospermia and considered to be associated with subfertility (26). However, Poland et al. (130) reported 15 normal fertile men with an average of 45% sperm with normal morphology. It is probable that the value of 50% for normal morphology in the WHO manual is too high. In general, the percentage of sperm with normal morphology is lower in infertile men than in normal fertile men (131). Although semen quality varies widely within men (96), it has been claimed that sperm morphology is relatively stable between ejaculates from the same man (130). Clinical investigation of the relationship between semen quality and subsequent PRs in a large group of sub fertile couples indicated that sperm morphology assessed by the traditional method was not of significant predictive value for fertility (3, 92). In contrast, average morphology did differ in men with high and low PRs in a donor insemination program (132). Studies with the zona-free hamster oocyte sperm penetration assay (SPA) suggest that morphologically abnormal sperm have low fertilizing ability compared with that of morphologically normal sperm (133-135). There is a negative correlation beVol. 58, No.3, September 1992
tween the proportion of sperm with abnormal morphology and the results of SPA (134, 136). Liu and Baker (137) found that the human zona is highly selective for binding of sperm with normal morphology. The majority (80% to 100%) of sperm bound to the ZP had normal morphology (Fig. 3). Sperm with abnormal morphology had significantly lower binding rates than did those with normal morphology (137). Makler (138) claimed that it may be more valuable clinically if sperm morphology was assessed on the motile subpopulation. In the standard procedure, morphology is determined on a preparation of fixed (immobilized) sperm, and one cannot tell whether a morphologically abnormal sperm was previously motile or immotile. It is reported that sperm with abnormal morphology are twice as frequent in the immotile subpopulation as in the motile subpopulation (138). This is consistent with reports that motile sperm selected by swim-up or Percoll gradient centrifugation techniques have significantly better morphology compared with sperm in the original semen (139-141). Katz et al. (142) reported that morphologically normal sperm swim faster, straighter, and with higher tail beat frequencies than do morphologically abnormal sperm in semen. They also showed that the velocity of morphologically normal sperm swimming in human cervical mucus was higher than that of abnormal sperm (143). Despite this, percentage normal morphology in semen had better predictive value for fertilization rates in
Figure 3 Morphology of sperm bound to the zona pellucida of human oocytes that had failed to fertilize in vitro (Shorr stain). All the sperm bound to this zona have normal morphology (magnification X10,OOO). Liu and Baker Human sperm function and IVF
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100 90 80 70 "0 (])
~60 (])
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>. 0 840
* 30 20 10 59
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~51
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Figure 4 Relationship between the percentage of sperm in semen with normal morphology and IVF rates. (Mean + SEM, number of patients in each normal morphology group are shown in the lower part of the bars).
vitro than percentage normal morphology in the insemination medium (12). Thus it remains possible that the relationship between morphological defects of sperm and failure of fertilization is at least partly indirect; a low proportion of sperm with normal morphology may signify the presence of more subtle abnormalities in all the sperm. It is now clear that sperm morphology assessed with a modified improved method of strict scoring is one of the most significant predictors of fertilization rate in vitro (11, 12, 64, 79, 83, 127, 144, 145). Data from over 1,000 IVF treatments indicate normal morphology is consistently a very significant factor related to IVF rates (Fig. 4). Standardization of sperm morphology assessment is urgently needed to provide more consistent, reliable, and reproducible results in the clinical semen laboratory. In our modification of morphology assessment, sperm are washed with normal saline to remove seminal plasma and adjust sperm concentration. A smear is made and checked microscopically for density and evenness of spread of the spermatozoa before it is left to air dry. Then the slide is fixed in 90% ethanol and stained with the Shorr method (12, 127). This provides very clear images for technicians 474
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to count normal and abnormal sperm and avoids bias related to sperm density. The Shorr stain also gives a clear contrast between the acrosomal and postacrosomal regions. Sperm morphology is considered according to the WHO criteria for the silhouette, but, in addition, internal staining characteristics of the sperm head are also included; for normality the acrosomal region must be clearly seen, be regular in shape, and occupy at least half of the sperm head (Figs. 3 and 5). This modified morphology assessment is similar to the strict scoring method of Kruger et al. (144, 145). The importance of the acrosomal area is indicated in a study of sperm bound to the ZP (136, Fig. 3). The human ZP is highly selective for binding of morphologically normal sperm, but some abnormal sperm, mainly with normal acrosomal regions, pyriform, or small heads, bind at low rates. With the improved method for assessing sperm morphology, men with >30% of sperm with normal morphology are very unlikely to have complete failure of fertilization in vitro (11, 12,79). On the other hand, a few patients with poor morphology «10% to 20% normal) still fertilize most or all of the oocytes inseminated. Therefore, assessment of sperm morphology alone is not an accurate predictor of fertility. It is true that the subjective microscopic method for assessing sperm morphology has large variations within and between technicians (146). Each laboratory must have their own standards and quality control. A good technician should produce consistent results with a coefficient of variation for percentage
Figure 5 Shorr staining of sperm with normal morphology in semen (magnification XI,OOO).
Fertility and Sterility
normal morphology for different readings of the same slide within 10% to 15%. Although characteristics of morphologically normal and various abnormal forms have been defined (26, 27), the technician's experience is extremely important for producing consistent and reliable results. The quality of preparation of the sperm smear and staining of the slides are also important. The errors may be reduced by improving the methods for preparation of the slides and staining to produce high -quality images. In the future, the development of objective methods for sperm morphology holds the most promise for improving morphology assessment (147,148). The Human Sperm-Hamster Oocyte Penetration Assay
Since Yanagimachi et al. (149) first reported that human sperm can penetrate and undergo nuclear decondensation in zona-free hamster oocytes, the human sperm-hamster oocyte SPA has been used as a tool for evaluating human sperm fertilizing ability. The use of living human oocytes for testing human sperm function is almost impossible; therefore, the SPA is a powerful test for assessing the ability of human sperm to capacitate, acrosome react, fuse with the oolemma, and undergo decondensation in the cytoplasm. Only acrosome-reacted human sperm appear to be able to bind to and penetrate the zona-free oocyte (149, 150). Although the clinical significance of the SPAin predicting male fertility is still disputed, the SPA has been widely used as a clinical test of sperm function. Numerous clinical papers have claimed that poor SPA results are strongly correlated with male infertility (150, 151). In some reports, the SPA is positively correlated with most or all of the seminal parameters such as sperm concentration, motility, and morphology (124, 152). Sperm motility and particularly morphology are strongly correlated with the SPA (124, 135). Despite these reports, many others have shown no correlation between the SPA and sperm characteristics (150, 151). Some clinical investigations of the SPA and fertility have suggested that sperm from infertile men, regardless of their sperm characteristics, always penetrate
