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Keywords:

  • Teratospermia;
  • reactive oxygen;
  • fertilization;
  • semen analysis

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

ABSTRACT: A body of evidence indicates that morphologically abnormal human spermatozoa may exhibit impaired ability to fertilize. Yet teratospermia has widely varying etiologies, including associations with varicoceles, following fever, cigarette smoking, and exposure to polychlorinated biphenyls. Abnormalities of sperm shape in mice have also been shown to be associated with autosomal gene mutations. These varying causes of teratospermia could have different molecular consequences reflected in altered sperm function. We studied the ability of morphologically abnormal human sperm to penetrate zona-free hamster eggs as a measure of their ability to undergo an acrosome reaction and gamete membrane fusion. Motile sperm from ejaculates containing 15% normal sperm or less, as judged by World Health Organization (1999) criteria, were recovered by ISolate density centrifugation and capacitated by overnight incubation. Zona-free hamster eggs were inseminated with 1 × 106 motile capacitated cells and scored for sperm penetration after 3 hours of coincubation. A significant trend was found between the percent of abnormal spermatozoa within the ejaculate and impaired egg-penetrating ability, reflected in the percent of eggs penetrated, the number of penetrating sperm per egg, and the number of sperm adherent to the oolemma. Because only acrosome-reacted human spermatozoa adhere to the oolemma, these results support the notion that abnormally shaped sperm may exhibit an impaired ability to undergo an acrosome reaction. A correlation was also noted between the loss of motility of sperm following overnight incubation and impairment of their ability to undergo gamete membrane fusion. These results confirm prior findings at the level of the zona pellucida that abnormally shaped sperm exhibit functional abnormalities. However, a wide variation was observed between men in the behavior of such sperm, including occasionally high rates of egg penetration. These observations suggest that assessment of morphology may be an unreliable measure, for the individual, of sperm fertilizing ability and emphasize that sperm function testing is an important part of the evaluation of teratospermia.

A current paradigm suggests that alterations in human sperm morphology deviating from the “normal” oval head are associated with impairment of the ability of sperm to fertilize eggs. A body of evidence supports this view. An association has been found between the morphology of a population of spermatozoa, as judged by strict criteria, and fertilization rates in vitro (Kruger and Coetzee, 1999). The ability of morphologically abnormal sperm to undergo an acrosome reaction following their binding to the zona pellucida has been shown to be impaired (Liu and Baker, 1994; Liu et al, 2004). The increase in cytosolic Ca++ promoted by progesterone appears to be reduced in morphologically abnormal sperm (Oehninger et al, 1994), as has their ability to undergo an acrosome reaction in response to a calcium ionophore (Liu and Baker, 1998).

The causes of teratospermia are varied. Evidence has been presented that testis and epididymal functions are both temperature sensitive (Bedford, 1991). Abnormalities noted in sperm morphology have been observed following fever (Carlson et al, 2003) and exogenous heat exposure (Thonneau et al, 1998) as well as in association with varicoceles (Marmar, 2001; Templeton, 2003). Cigarette smoking has also been associated with abnormalities of sperm shape and motility (Vine, 1996; Zitzmann et al, 2003), possibly on the basis of smoke by-products that promote an increased production of reactive oxygen species (ROSs) (Saleh et al, 2002). A group of men exposed to polychlorinated biphenyls and dibenzofurans has been found to exhibit abnormal sperm morphology (Hsu et al, 2003)

There is increasing evidence of a genetic basis of altered spermatogenesis as well, including effects on sperm morphology. Male carriers of Robertsonian chromosome translocations have recently been shown to exhibit abnormalities of sperm motility and morphology within their ejaculates (Ogur et al, 2006). While evidence has accumulated regarding the role of mutations of Y chromosome-associated genes in normal spermatogenesis in humans (Foresta et al, 2001), attention has recently focused on identifying in mice specific autosomal genes likely to have human homologs that may also be critical in sperm formation. Targeted deletion of the gene for transition nuclear protein TNP1 results in altered chromatin structure observed by transmission electron microscopy. Sperm motility was severely reduced, and 60% of TNP1-null mice were infertile (Zhao et al, 2001). TP1 is highly conserved in mammals, and its reduced expression in human spermatozoa exhibiting spermatid arrest suggests a requirement for successful spermiogenesis (Steger et al, 1999). The nectin-2 receptor, a member of a highly conserved class of adhesion molecules of the immunoglobulin superfamily, is expressed on Sertoli cells, in mice, sperm-associated nectin-3 playing a role in the formation of sperm-Sertoli junctions. Males lacking this receptor produce sperm with severe malformations of the head and neck and a diminished ability to adhere to the zona pellucida and undergo sperm-oolemmal fusion (Bouchard et al, 2000; Mueller et al, 2003).

Prior research using the penetration of zona-free hamster eggs by human spermatozoa, attempting to study the effect of abnormal sperm morphology on egg penetrating ability at the level of the oolemma, has yielded conflicting results. Kruger et al (1987, 1988) demonstrated, using an assay dependent upon the spontaneous acrosome reaction of spermatozoa, a significant positive correlation between the percent of sperm with normal morphology, as judged by strict criteria, and egg penetration rates. In contrast, the study of Zahalsky et al (2003) did not confirm these results. Using an “optimized” sperm penetration assay in which the acrosome reaction was promoted by prior exposure of sperm to egg yolk-containing medium at low temperature, there was no correlation observed by multivariate analysis between sperm morphology and number of penetrating sperm.

In this study, we retrospectively reviewed the results of sperm penetration assays performed as part of an evaluation of men from infertile couples whose sperm exhibited abnormal morphology, as an indirect indicator of their ability to undergo an acrosome reaction and subsequent gamete membrane fusion. All tests were performed in conjunction with 1 of 2 known fertile controls. The ability of sperm to fuse with the oolemma was scored both by percent inseminated eggs penetrated and the number of penetrating sperm per inseminated egg. Because only acrosome-reacted spermatozoa adhere to the oolemma of hamster (Talbot and Chacon, 1982) and human eggs (Bronson et al, 1999a), their enumeration on the egg surface provided an indirect measure of the proportion of acrosome-reacted sperm capable of undergoing this event. In addition, the ability of such oolemmal-adherent sperm to undergo gamete membrane fusion was judged as reflected in the percentage of these sperm entering the cortical ooplasm.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

Semen Analysis

The morphology of Spermac-stained (Cooper Scientific, Trumble, Conn) sperm was assessed using criteria established in the 4th edition of the WHO Laboratory Manual for the Examination of Semen and Semen-Cervical Mucus Interaction (WHO, 1999). To validate our assessment of sperm morphology, which is subjective, freshly prepared smears from 21 consecutive ejaculates submitted to our laboratory for analysis were sent to the laboratory of Dr James Overstreet, University of California, Davis, which had been the reference laboratory for a multicenter study establishing semen parameters of fertile and infertile men (Guzick et al, 2001). These slides were analyzed independently and the results between laboratories then compared.

Based on this comparison as well as the recommendations of Guzick et al (2001) and McLachlan et al (2003), we chose to study the relationships between postincubation sperm motility and abnormal sperm morphology on egg-penetrating ability in a subsequent series of 64 ejaculates submitted to our laboratory, which exhibited up to 15% normal sperm. These cases were further subdivided for analysis into 3 groups (11%–15%, 6%–10%, and 5% or less) of increasing morphologic abnormalities.

Sperm Recovery

Ejaculates from infertile men exhibiting 15% normal sperm or less were submitted for study of their ability to penetrate zona-free hamster eggs. A population of highly motile cells was recovered from the neat semen by ISolate (Irvine Scientific, Santa Ana, Calif) density centrifugation using 3 layers: 90% ISolate bottom layer (1.5 mL), 70% middle layer (1.0 mL), and 40% upper layer (1.5 mL). Gradients were made up in Enhance Human Tubal Fluid (HTF) medium with HEPES (Conception Technologies, San Diego, Calif), in a 15-mL centrifuge tube. Depending upon the sperm concentration, 0.5–2.0 mL of semen was placed above the upper layer of the gradient column and centrifuged at 200 × g for 15–25 minutes. The 3 layers were removed and the pellet resuspended in 4 mL of HTF and centrifuged at 300 × g for 8 minutes. The process was repeated and the final pellet resuspended in 350–400 μL of HTF.

Sperm Penetration Assay

As suggested by the ESHRE Consensus Workshop on Advanced Diagnostic Andrology Tests (ESHRE Andrology Special Interest Group, 1996), the sperm penetration assay performed in our laboratory did not rely upon the spontaneous acrosome reaction (which is associated with a high incidence of false negative results). Spermatozoa were incubated in the presence of both progesterone and mannosylated bovine serum albumin (BSA) to promote the acrosome reaction in vitro, the former to mimic the exposure of sperm to progesterone during their passage through the cumulus oophorous (Laufer et al, 1985), the latter as a surrogate for the zona pellucida (Chen et al, 1995; Benoff et al, 1997). Both agents promote, through receptor-mediated events, the rapid influx of calcium into spermatozoa that leads to an acrosome reaction (Bronson et al, 1999a; Brewis et al, 1996) and were used in lieu of a calcium ionophore, such as A23187. This approach allowed us to detect any defective sperm receptor-mediated events that promote sperm calcium uptake, which would be bypassed through the use of a calcium ionophore.

Motile sperm (20 × 106/mL) were incubated 18 hours overnight at 37°C in 5% CO2 in Biggers-Whitten-Whittingham (BWW) medium containing human serum albumin (HSA) 30 mg/mL (Sigma A 9500, lot no. 126H 9322; Sigma Chemical, St Louis, Mo) to promote their capacitation (Bronson and Fusi, 1990). Capacitated sperm were then preincubated for 1 hour in BWW containing progesterone 15 ug/mL, mannosylated BSA 5 mg/mL, and D-mannose 75 mM (Benoff et al, 1997; Bronson et al, 1999a) prior to their insemination of zona-free eggs. Initial sperm motility following recovery out of semen and post-overnight capacitating incubation motility were noted. The number of sperm inseminated was adjusted so that 1 × 106 motile cells were placed within a 200-μL droplet of BWW/HSA 30 mg/mL containing 20–25 hamster eggs removed of their vestments with hyaluronidase and trypsin. Following a 3-hour coincubation, eggs were washed free of sperm suspension by serial passage through 3 100-μL droplets of sperm-free medium using pipettes of 90–100 μm diameter (Bronson et al, 1999a), stained with acridine orange, and whole mounts prepared and scored using fluorescent microscopy (Bronson and Fusi, 1990). Penetrating sperm heads within the cortical ooplasm appeared expanded and green as contrasted with unexpanded sperm adherent to the oolemma. Eggs were scored for both the number of sperm adherent to the oolemma and the number of penetrating sperm. To further assess the ability of spermatozoa to undergo gamete membrane fusion, we calculated the ratio of the number of sperm penetrating eggs as a percent of those adherent to the oolemma (ie, the total number of penetrating the egg plus those sperm adherent to the oolemma: %Pen = [Pen/Pen + Adh] × 100). All tests were performed in conjunction with 1 of 2 known fertile controls whose ejaculates exhibited normal semen parameters and results invalidated if sperm penetration frequency of the control was more than 1 SD from the mean penetration rate.

This research was approved by the Committee on Research in Human Subjects at the State University of New York, Stony Brook (project no. 20070295).

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

Comparison of sperm morphology (Table 1) assessed in 21 consecutive ejaculates sent to the laboratory of Dr James Overstreet at the University of California, Davis, and our own laboratory indicated that results were not significantly different between laboratories (P > .35, Mann-Whitney U test) for those samples possessing less than 20% normal sperm in the neat ejaculate but diverged in samples containing 20% or more normal sperm, as assessed at Stony Brook (P < .001).

Table 1. . Comparison of sperm morphology (% normal sperm*) in 21 consecutive semen specimens as assessed at State University of New York (SUNY), Stony Brook, and the University of California, Davis
SUNY, Stony BrookUniversity of California, Davis
  1. *Spermatozoa were scored by World Health Organization criteria (1999) at SUNY, Stony Brook, and by strict criteria (Guzick et al, 2001) at University of California, Davis. There was no difference between the results of the 2 laboratories for samples with sperm morphology less than 20% normal (P > .35, 2-tailed Mann-Whitney U test), whereas the results diverged for those cases judged to have 20% or more normal forms (P < .001) by SUNY, Stony Brook, when compared with results at the University of California, Davis

1.02.0
2.02.5
5.51.0
10.016.0
13.09.5
15.012.5
15.012.5
15.77.0
17.018.0
17.39.5
18.023.0
20.011.5
24.012.0
26.011.0
27.011.0
28.023.5
30.317.0
31.013.0
32.316.0
34.018.5
38.320.0

Most spermatozoa in ejaculates studied exhibited amorphous heads, the next most common abnormality being small oval heads. Tail defects were uncommon. In specimens (n = 23) with the least abnormal morphology (13.2% ± 1.4% normal sperm, mean ± SD), 3.5% ± 6.6% exhibited tapered heads, 52.6% ± 11.5% amorphous heads, 0.3% ± 0.1% large oval heads, 22.8% ± 12.6% small oval heads, 2.4% ± 4.6% acrosomal defects, 4.5% ± 5.6% midpiece defects, and 4.4% ± 6.1% tail defects. In specimens (n = 22) with the greatest abnormalities of morphology (4.8% ± 6.7% normal sperm), 5.2% ± 4.6% exhibited tapered heads, 70.5% ± 7.0% amorphous heads, 0.1% ± 0.3% large oval heads, 16.6% ± 7.9% small oval heads, 1.5% ± 1.6% acrosomal defects, 1.5% ± 12.1% midpiece defects, and 2.2% ± 2.7% tail defects.

Spermatozoa that exhibited decreased motility following their overnight capacitating incubation demonstrated a diminished ability to penetrate zona-free hamster eggs, as judged both by the percent of inseminated eggs penetrated as well as the number of penetrating sperm per egg (Table 2). A correlation was observed between the percent motile sperm following overnight capacitating incubation (Spearman rank-order correlation) and the percent of eggs penetrated (rs = 0.584, P < .001) as well as the number of penetrating sperm per egg (rs = 0.571, P < .001). The likelihood of gamete membrane fusion, as assessed by the percent of oolemmal-adherent sperm that had entered the ooplasm, was also noted to diminish as postincubation sperm motility decreased below 50% when compared with those spermatozoa that maintained motility above 80% (%Pen = 8.8 ± 4.2 vs %Pen = 3.2 ± 5.2; P < .01, Mann-Whitney U test).

Table 2. . Correlation between sperm motility following capacitation and the ability of sperm to penetrate zona-free hamster eggs*
% Motile Sperm After IncubationNo.% Eggs Penetrated (Mean ± SD)No. of Penetrating Sperm Per Egg (Mean ± SD)No. of Adherent Sperm Per Egg (Mean ± SD)
  1. *The difference in percent eggs penetrated (80% or more vs 70% to less than 80%) was not significant (P > .5), as judged by the Mann-Whitney U test, whereas the difference was significant for comparison with the other groups (80 or more vs 50% to less than 70%, P .05; and 80 or more vs 50% or less, P < .001). Comparison of the number of penetrating sperm per egg and the number of oolemmal-adherent sperm per egg were also significantly different between the later groups (P < .001 and P < .01, respectively)

≥801585.5 ± 22.94.2 ± 4.144.9 ± 22.5
70–<801659.1 ± 29.61.7 ± 2.322.1 ± 12.6
50–<702152.3 ± 31.52.9 ± 5.318.6 ± 13.0
≤501228.6 ± 32.70.67 ± 1.219.8 ± 19.3
Fertile controls1095.6 ± 4.33.5 ± 1.157.0 ± 9.7
 893.9 ± 4.93.4 ± 1.156.1 ± 9.8

Increasing teratospermia was associated with deterioration in the ability of abnormally shaped sperm to fuse with the oolemma, as reflected in the percent of eggs penetrated and number of penetrating sperm per egg (Table 3). A correlation was observed (Spearman rank-order correlation) between the percent normal sperm and percent eggs penetrated (rs = 0.409, P < .001) as well as the number of penetrating sperm per egg (rs = 0.407, P < .001). Despite this observed trend, a wide range in sperm function was noted within each group. This is best illustrated by an enumeration of the sperm morphologies noted in 19 cases in which the percent of eggs penetrated was 95% or more. In 11 instances, the percent sperm with normal morphology was 11%–15%, in 5 cases 6%–10%, and in 3 cases 5% or less.

Table 3. . Comparison of the ability of morphologically abnormal sperm to adhere to and penetrate zona-free hamster eggs*
% Normal SpermNo.% Eggs Penetrated (Mean ± SD)No. Penetrating Sperm Per Egg (Mean ± SD)No. Adherent Sperm Per Egg (Mean ± SD)
  1. *Comparison of the percent eggs penetrated, number of penetrating sperm per egg, and number of sperm adherent to the oolemma revealed significant differences between ejaculates containing 11%–15% normal sperm and up to 5% normal sperm (% eggs penetrated, P < .001; number penetrating sperm per egg, P < .001; number adherent sperm per egg, P < .05; Mann-Whitney U test)

11 — 152375.4 ± 32.83.8 ± 4.135.4 ± 24.1
6 — 101951.2 ± 31.81.5 ± 1.832.0 ± 15.6
≤52240.8 ± 33.50.9 ± 1.119.0 ± 14.3
Fertile controls1095.6 ± 4.33.5 ± 1.157.0 ± 9.7
 893.9 ± 4.93.34 ± 1.156.1 ± 9.8

Because only acrosome-reacted human sperm adhere to zona-free hamster eggs, we also counted the number of oolemmal-adherent sperm following 3-hour coincubation of gametes, at the time of scoring the other parameters, to assess indirectly the ability of populations of sperm to undergo an acrosome reaction in response to progesterone and mannose. The number of oolemmal-adherent sperm was found to diminish with increasingly abnormal sperm shape (Table 3), as it had done when comparing sperm with more than 80% motility versus 50% or less motility following their overnight incubation (Table 2). There was also a trend toward diminishing values of %Pen (percent oolemmal-adherent sperm that entered the ooplasm) as the percent normal spermatozoa diminished, although the differences between ejaculates containing 11%–15% normal sperm versus 5% or less just failed to reach statistical significance (%Pen = 8.25 ± 5.4 vs 5.56 ± 6.8; P = .051, Mann-Whitney U test).

Table 4 compares sperm motility before and following overnight incubation with sperm morphology. Whereas motility was comparable following initial recovery of sperm out of the neat semen, loss of motility after incubation was significantly greater as the percent of normal sperm decreased (72.1% ± 15.3% vs 53.6% ± 20.7% for ejaculates containing 11%–15% normal sperm vs 5% or less; P < .01, Mann-Whitney U test).

Table 4. . Correlation between sperm morphology and postincubation motility*
% Normal SpermNo.% Motile Sperm Initially (Mean ± SD)% Motile After Overnight Incubation (Mean ± SD)
  1. *The difference in percent motile sperm was not significant initially (P > .37 for 11%–15% vs 5% or less; Mann-Whitney U test) but was significant after overnight incubation (P < .01)

11–152388.6 ± 7.672.1 ± 15.3
6–101984.2 ± 7.766.0 ± 12.2
≤52276.0 ± 19.653.6 ± 20.7

Because the assessment of morphology was made on sperm within the neat ejaculates, before ISolate density gradient centrifugation, we determined how this was related to the morphology of the spermatozoa to which oocytes were exposed by performing comparisons of the 2 groups in 29 samples exhibiting varying sperm head abnormalities. The percent normal sperm in each group doubled approximately following ISolate density gradient centrifugation (Table 5). This resulted in only a small difference in percent sperm with normal morphology, in the most severely abnormal samples (up to 5% normal). For these samples (n = 8), 3.0% ± 1.3% sperm were normal (mean ± SD) in the neat sample vs 6.3% ± 2.3% following gradient centrifugation.

Table 5. . Comparison of sperm morphology in neat ejaculate and following ISolate density centrifugation
% NormalNo.% Normal in Neat Semen (Mean ± SD)% Normal After Gradient Centrifugation (Mean ± SD)
>15821.1 ± 4.331.1 ± 8.6
10–15812.0 ± 1.021.8 ± 3.2
>5–<1057.6 ± 1.015.4 ± 3.1
≤583.0 ± 1.36.3 ± 2.3

Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

Successful fertilization occurs through a complex series of steps during which the spermatozoon undergoes capacitation, penetrates the cumulus oophorous, binds to the zona pellucida, acrosome reacts, penetrates the zona, adheres to the oolemma, undergoes gamete membrane fusion, and enters the ooplasm (Yanagimachi, 1994; Stein et al, 2004; Rubinstein et al, 2006). We studied the interaction of capacitated human spermatozoa with zona-free hamsters eggs as a measure of the ability of morphologically abnormal sperm to adhere to the oolemma and undergo gamete membrane fusion. That these eggs can act as a surrogate for the human oocyte has been suggested by the fact that only acrosome-reacted sperm are capable of entering them (Yanagimachi, 1994). There also does not appear to be a major block to polyspermy at the level of the oolemma of hamster eggs (Binor et al, 1982; Stewart-Savage and Bavister, 1988), as occurs in the mouse (Maluchnik and Borsuk, 1994).

Sperm populations obtained from ejaculates containing a high proportion of abnormally shaped spermatozoa exhibited increasingly impaired egg-penetrating ability, whether judged by the percent of eggs penetrated or number of penetrating sperm per egg, as the percent normal sperm in the ejaculate decreased. However, the wide variation observed made it difficult to predict by morphologic description of his spermatozoa the degree of impaired sperm function for the individual. In some cases, very abnormally shaped spermatozoa penetrated eggs at normal frequencies. The number of sperm adherent to the oolemma also decreased with their increasingly abnormal morphology (Table 3). While both acrosome-intact and acrosome-reacted mouse sperm bind to the oolemma of mouse eggs (Phillips and Yanagimachi, 1982), only acrosome-reacted human sperm bind to zona-free hamster and human eggs (Talbot and Chacon, 1982; Bronson et al, 1999a). These results suggest that the ability of such abnormally shaped spermatozoa to undergo an acrosome reaction in vitro, as promoted by a combination of progesterone and mannosylated BSA, was also impaired. Again, even in the most severely abnormal group, large numbers of oolemmal-adherent sperm were sometimes observed, with a wide range noted between men.

Morphologically abnormal spermatozoa have previously been shown to exhibit an impairment in their ability to bind to the zona pellucida and to undergo a subsequent acrosome reaction (Liu and Baker, 1994; Liu et al, 2004). Our results confirm these findings at the level of the oolemma. Of note in these prior studies is the wide range observed of zona pellucida-induced acrosome reaction in sperm exhibiting teratospermia, including overlap with the fertile, normospermic group.

A strong correlation was found between the motility of spermatozoa observed after their overnight incubation and their subsequent egg-penetrating ability. These results could not be explained on the basis of impaired gamete contact in vitro, because the number of sperm added to the culture dishes was normalized so that 1 000 000 motile cells were present within the 200-μL droplet of medium containing eggs at the time of gamete mixing. Variation in production of endogenous ROSs leading to sperm damage could account for the observed differences. ROS production plays both physiologic and pathologic roles in human spermatozoa (de Lamirande and Gagnon, 1995; Aitken, 1999). The physiologic effects of endogenous ROS generation appear to involve elevation of intracellular cyclic adenosine monophosphate (cAMP) and are mediated via protein kinase A. In contrast, human spermatozoa contain a high concentration of polyunsaturated fatty acids within their plasma membranes (Jones et al, 1979) and are susceptible to free radical attack. Increased pathologic degrees of ROS generation are associated with deterioration of sperm motility and compromise the ability of sperm to fertilize eggs.

ROSs may affect motility of human sperm via 2 mechanisms: by damaging sperm axonemes (de Lamirande and Gagnon, 1992a) and through a rapid decrease in tail beating associated with loss of intracellular adenosine triphosphate (ATP; de Lamirande and Gagnon, 1992b). ROSs may also directly lead to loss of gamete membrane fusion through the peroxidation of fatty acids or denaturation of proteins within the sperm plasma membranes. Aitken and Clarkson (1987) correlated the excess production of ROSs by spermatozoa with an impaired ability to fuse with the oolemma of zona-free hamster eggs. When less than 10% of zona-free eggs were penetrated, sperm exhibited high basal production of ROSs 40-fold above that seen in sperm exhibiting high egg penetrating ability. Very high rates of ROS generation observed experimentally following the addition of NADPH to medium were associated with sperm membrane damage, as measured in a malonaldehyde assay. The deterioration of sperm motility that we observed following overnight capacitating incubation could be reflective of oxidative damage to spermatozoa that was also manifest in the impaired ability of such oolemmal-adherent sperm to penetrate the oocyte.

To assess the ability of spermatozoa to undergo gamete membrane fusion, we calculated the percent of the total number of sperm adherent to the oolemma that had penetrated the egg: %Pen = [Pen/Pen + Adh] × 100. In previous experiments using this measure to investigate the interval between sperm oolemmal adherence and their subsequent entrance into the cortical ooplasm (Bronson et al, 1990, 1999b), eggs were washed out of sperm suspension shortly after gamete contact and further incubated in sperm-free medium. In this manner, only the limited number of sperm initially adherent to the oolemma was observed. We found that only a minority of sperm adherent to the oolemma was capable of being incorporated by the oocyte, suggesting that the acrosome reaction, while a prerequisite necessary for gamete membrane fusion, was not sufficient in itself. In the present experiments, the likelihood of gamete membrane fusion of oolemmal-adherent sperm was noted to diminish as postincubation sperm motility decreased below 50% when compared with those spermatozoa that maintained motility above 80%. Evidence has been presented (Talbot and Chacon, 1982; Fukuda et al, 1989) relevant to these findings suggesting that spermatozoa that have undergone a degenerative acrosome reaction inconsistent with gamete membrane fusion (as might be observed within those sperm populations exhibiting decreased motility following overnight incubation) can adhere to the oolemma of zona-free hamster eggs. Observations of Tesarik and Mendoza (1994) support this hypothesis. They studied the ability of human spermatozoa placed within the perivitelline space by subzonal sperm injection (SUZI) to fertilize eggs. The frequency of sperm-oocyte fusion was consistently lower than the frequency of the acrosome reaction irrespective of the number of sperm injected per egg.

Our results reemphasize that sperm morphology, whether assessed by World Health Organization criteria or “strict” criteria, does not predict sperm function for the individual. The etiology of teratospermia in different men may play a significant role in how these sperm function. Spermatozoa may undergo commonly observed alterations in shape due to varying etiologies that result in different molecular consequences. These considerations emphasize that teratospermia is a histologic description of cells and should not be considered a diagnosis. One would not accept a clinical diagnosis of anemia based solely on an assessment of red blood cell morphology. Microcytic anemia may have several different etiologies, including varying genetic bases, as seen in sickle cell anemia or thalassemia, as well as nutritional causes such as iron deficiency. It is reasonable to assume that the constellation of receptors expressed by spermatozoa required for successful gamete interactions leading to fertilization (Bronson, 1998; Bronson et al, 1999a) might be altered in different ways depending upon the etiology of teratospermia, and this could determine the ability of sperm to penetrate eggs. Our results would argue the need for a molecular diagnosis of teratospermia and that the performance of sperm function testing be a necessary part of the evaluation performed by an andrology laboratory.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

We thank Charlene Brazil in the laboratory of Dr James Overstreet for her assistance in providing the analysis of sperm morphologies performed at the University of California, Davis.

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  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
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