5. Page 4
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6. Page 5
Contents
Preface 7
List of contributors 9
1
The Tygerberg strict criteria: what are the clinical thresholds for in vitro fertilization, intrauterine insemination, and in vivo fertilization?
T.F.Kruger, F.Van der Merwe and J.Van Waart
13
2
The role of sperm cell morphology in intracytoplasmic sperm injection (ICSI)
M.L.Windt and T.F.Kruger
19
3
Relationship between sperm morphology and binding capacity to the zona pellucida: a critical step leading to fertilization
S.C.Oehninger
27
4
The use of the acrosome index in assisted reproduction
R.Menkveld
35
5
Quality assurance for sperm morphology assessment
D.R.Franken
41
6
What is a normal spermatozoon?
D.R.Franken and T.F.Kruger
49
Appendices
1 The Tygerberg strict criteria for morphologically normal spermatozoa 75
2 Papanicolaou staining method 75
3 DiffQuik staining method 77
4 Shorr staining 77
5 Spermac staining 77
Index 79
14. Page 13
1
The Tygerberg strict criteria: what are the clinical thresholds for in vitro
fertilization, intrauterine insemination, and in vivo fertilization?
T.F.Kruger, F.Van der Merwe and J.Van Waart
Clinical thresholds to distinguish between fertile and infertile or subfertile patients were first attempted by our unit in the early 1970s1. Normal forms were defined as
follows, based on criteria laid down by cervical mucus selection.
A spermatozoon is considered normal when the head has a smooth, oval configuration with a welldefined acrosome comprising about 40–70% of the sperm head. In
addition, there must be no neck, midpiece, or tail defects and no cytoplasmic droplets of more than onehalf the size of the sperm head. We consider borderline forms
abnormal. At least 100, but preferably 200, spermatozoa with tails were classified into one of seven groups: normal (head and tail normal), normal head but with
another abnormality present, large heads, small heads, tapering heads, duplicated heads, or amorphous heads all with or without tail, neck or midpiece defects. Tail,
neck, and midpiece defects, loose head, immature germinal cells, and unknown cells were recorded separately and reported per 100 spermatozoa. The size of the
spermatozoa was evaluated in five different areas to ensure a more randomized evaluation.
In vitro fertilization (IVF) led to the development of a model where certain variables could be studied in more detail. In 1986, a study was performed correlating
normal sperm morphology with IVF2.
In this study all male patients had a sperm concentration of >20×106/ml and motility parameters of >30% to negate the possible impact of the other parameters. One
hundred and ninetynine cycles were studied using logistic regression analysis. A threshold of 14% was calculated with a 37% fertilization rate in the <14% normal
morphology group and >82% fertilization rate in the group >14% normal forms. A second prospective study followed, evaluating the fertilization rate in the sperm
morphology group with <14% normal forms3. In this study it was shown that, with a sperm morphology of <5% normal forms, the fertilization rate was only 7.6%
(previously defined as the 0–4% group, i.e. Ppattern or poorprognosis group). For the sake of convenience, we refer to this group as the 5% threshold group. The
fertilization rate in the 5–14% normal forms group was 63% (Gpattern or goodprognosis group). The difference was highly significant. Three morphologic patterns
were thus defined, namely the Ppattern group, or poorprognosis group (0–4% normal forms); the Gpattern group, or goodprognosis group (5–14% normal forms);
and the Npattern group (those with normal forms >14%).
Structured literature reviews on sperm morphology (strict criteria)
This was an attempt to evaluate the results of different authors studying the impact of sperm morphology on pregnancy outcome in IVF and intrauterine insemination
(IUI), and to record clinical thresholds of semen parameters for in vivo fertilization.
15. Page 14
In vitro fertilization
To study the impact of strict criteria on IVF outcome, a structured literature review was undertaken in 19984. Published literature in which normal sperm morphology
was used to predict fertilization and pregnancy during the period 1978 to 1996 was reviewed. The statistical outcomes and conclusions of the studies were tabulated,
and where sufficient data were available, the odds ratios for fertilization (per oocyte) and pregnancy (per cycle) were calculated. A total of 216 articles were identified
by the sourcing methodology, but only 49 provided data that could be tabulated and analyzed. The majority (82%) of the analyzed studies concluded that normal sperm
morphology (including acrosomal morphology) had a role to play in the determination of male fertility potential. Eighteen of the analyzed studies provided sufficient data
for statistical analysis. Fifteen studies used the strict criteria to evaluate sperm morphology, two used WHO guidelines, and one used both the strict criteria and the
WHO guidelines.
Using a 5% threshold (strict criteria), ten studies provided data that could be analyzed for the prediction of fertilization5–14 (Figure 1.1) and eleven studies for the
prediction of pregnancy (Figure 1.2). All the studies showed a positive predictive value for fertilization in vitro, with only one8 (odds ratio 1.42 (CI: 0.9–2.25)) not
reaching significance (Figure 1.1). In the prediction of pregnancy (per CyCle)2,6,9–13,15,16, nine studies obtained a positive predictive value. The predictive value of the
studies by Oehninger et al.6, Enginsu et al.9, and Grow et al.12 reached significance (Figure 1.2).
Using a 14% threshold (strict criteria), five studies provided data that could be analyzed for the prediction of fertilization2,6–8,14 (Figure 1.1), and eight studies for the
prediction of pregnancy (Figure 1.2) Similar to the 5% analyses, all these studies showed positive and significant predictive value with regard to fertilization in vitro
(Figure 1.1). In the prediction of pregnancy, two studies7,8 did not obtain a positive predictive value, while two5,6 studies were positive and significant. This proved the
value of the IVF model to study spermoocyte interaction and fertilization in vitro.
Not only did the structured review prove the value of strict criteria, but it also set a new threshold for the patient with poorer prognosis in IVF, not only for
fertilization rate in vitro but also for pregnancy rate per embryo transfer. The ≤5% threshold clearly
Figure 1.1 Odds ratio and confidence intervals for the predictive value of normal sperm morphology (strict criteria) for fertilization in
vitro
17. Page 16
tified by the sourcing methodology, but only four articles provided data that could be tabulated and analyzed. Using ROC (receiver operating characteristic) curves,
morphology (strict criteria) proved to be the best predictor of subfertility in three of the four articles, with concentration and motility/ progressive motility also showing
good predictive power27–30. Calculated thresholds ranged from 4 to 10% for morphology, 13.6×106/ml and 34×106/ml for concentration, and from 31.8 to 52% for
motility. A second set of much lower thresholds was calculated in three of the articles. The adjusted lower thresholds were between 3 and 5% for morphology,
9×106/ml for concentration, and between 20 and 30% for motility. Because these lower thresholds have a much higher positive predictive value, it was suggested that
they should be used to identify the subfertile male. The lower thresholds for morphology also fit IVF and IUI data calculated previously. It was concluded that using the
parameters in combination increases the clinical value of a semen analysis.
Study of a fertile population: three continents, four centers
In recent publications, thresholds of 8–10% for normal sperm morphology have been suggested to distinguish between fertile and infertile men in in vivo situations27–30.
It was also shown that sperm morphology is the semen parameter with the best prognostic value in these couples. The aim of this study on the fertile population was to
compare the basic semen parameters of proven fertile patients from four different international fertility centers (three continents).
Men who had impregnated their wives within 1 year of unprotected sexual intercourse were recruited to give a semen sample. The wives of these men were all
pregnant at the time of evaluation. Semen parameters (concentration, motility) were evaluated according to WHO 1999 guidelines, while the sperm morphology was
evaluated according to the strict criteria.
In this study, there were 57 patients in the Argentinian center, of which 18 were from one center (Argenl) and 39 from a second center (Argen2), 36 in the South
African center (Tygerberg), and 68 in the Turkish center. Crosstabulations of the morphology groups by pattern were done. There was a significant difference in the
morphologic profile as expressed by P, G, and N categories (p<0.0001). The Tygerberg center had 19% in the Ppattern group and 6% in the Npattern group. The
reason for this finding is still uncertain, and further studies in different population groups are under way.
When the four centers were added together, there were 9/161 (5.6%) P patterns, 91/161 (56.5%) G patterns, and 61/161 (37.9%) N patterns. The percentage
motility in the Tygerberg center was the lowest as well as in the percentage normal morphology. The Argenl center had the highest concentration per/ml compared with
the other three centers (Table 1.2).
Table 1.1 Pregnancy rate per cycle: risk difference for pregnancy rate (strict criteria, 4% threshold). Value not included (whole population) in final meta
analysis
Study ≤4% >4% Weight Risk difference (95% Cl)
Idiopathic
MontanaroGauci et al.20 1/36 35/274 77.9 –0.10 (–0.17 to –0.04)
Matorras et al.21 13/120 3/23 22.1 –0.02 (–0.17 to 0.13)
Subtotal λ2
=1.22 (df=1)
100 –0.08 (–0.1 6 to –0.01)
Whole population
Toner et al.22 6/86 35/309 20.9 –0.04 (–0/11 to 0.02)
Ombelet et al.23 40/335 76/469 24.7 –0.05 (–0.09 to 0.00)
Karabinus et al.24 3/53 44/485 20.3 –0.03 (–0.10 to 0.03)
Lindheim et al.25 1/99 15/77 15.5 –0. 19 (–0.28 to –0.09)
Matorras et al.21 18/172 10/99 18.6 –0.00 (–0.07 to 0.08)
Subtotal λ2
=10.74 (df=4)
100 –0.06 (–0.11 to –0.01)
Total λ2
=11.79 (df=5)
18. Page 17
The concentration per/ml and percentage motility were in the same range for all the centers, except for Argenl regarding concentration per/ml. The difference in
percentage morphology in the Ppattern group of Tygerberg needs further evaluation. The majority of patients were, however, in the Gpattern sperm morphology
group or higher. Patients in the Gpattern group can therefore be considered as fertile, while those below 5% should be regarded with a higher potential for subfertility.
Conclusion
From the reviews mentioned, the main problem was one of obtaining sufficient data from the old WHO criteria (1992 and earlier) in order to look at the morphology
impact on IVF and IUI results. Not only did the threshold differ in different papers, but also insufficient data meant that a metaanalysis could not be performed. On the
other hand, thresholds on strict criteria and sufficient papers with data resulted in a consensus opinion on the thresholds. This is the reason why the World Health
Organization accepted the strict criteria as an international guideline31.
It seems as if the Ppattern group (normal morphology 0–4%) is the group with the lowest pregnancy success rate in IUI, IVF, and in vivo fertilization. It is,
however, important to view these thresholds in perspective. We do not claim that a pregnancy is not possible within the Ppattern group, but only that there is a
significantly lower chance of a pregnancy. It is, however, important to note that all the other semen parameters are also contributing, and that if the other parameters are
below suggested thresholds, the chance of success will be even lower.
On the other hand, when there is an excellent concentration/ml and percentage motility >50%, these parameters will compensate for patients with Ppattern
morphology, with a significantly better chance of pregnancy than IUI20.
It is also suggested that these morphological thresholds can be used to direct a patient towards controlled IVF or IVFintracytoplasmic sperminjection (ICSI)
treatment if sufficient oocytes are available during stimulation.
References
1. Van Zyl JA, Kotze TJ van W, Menkveld R. Predictive value of spermatozoa morphology in natural fertilization. In Acosta AA et al., eds. Human Spermatozoa in
Assisted Reproduction, Chap. 11. Baltimore: Williams & Wilkins, 1990
2. Kruger TF, et al. Sperm morphologic features as a prognostic factor in in vitro fertilization outcome. Fertil Steril 1986; 46:1118
3. Kruger TF, et al. Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril 1988;49:112
4. Coetzee K, Kruger TF, Lombard CJ. Predictive value of normal sperm morphology: a structured literature review. Hum Reprod 1998;4:73
5. Kruger TF, et al. Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril 1988;49:111
6. Oehninger SC, et al. Corrective measures and pregnancy outcome in IVF in patients with severe morphology abnormalities. Fertil Steril 1988;50:283
7. Yue Z, et al. Sperm morphology using strict criteria after Percoll density separation: influence on cleavage and pregnancy rates after in vitro fertilization. Hum Reprod
1995; 10:1781
8. Figueiredo H, et al. Isolated teratozoospermia and in vitro fertilization. J Asst Reprod Genet 1996;13:64
9. Enginsu ME, et al. Male factor as determinant of in vitro fertilization outcome. Hum Reprod 1992; 7:1136
10. Enginsu ME, et al. Predictive value of morphologically normal sperm concentration in the medium for in vitro fertilization. Int J Androl 1993;16:113
11. Ombelet W, et al. Teratozoospermia and in vitro fertilization: a randomized prospective study. Hum Reprod 1994;9:147
12. Grow DR, et al. Sperm morphology as diagnosed by strict criteria: probing the impact of teratozoospermia on fertilization rate and pregnancy outcome in a large in vitro
fertilization population. Fertil Steril 1994;62:559
13. Vawda AJ, Gunby J, Younglai V. Semen parameters as predictors of in vitro fertilization: the importance of strictcriteria sperm morphology. Hum Reprod 1996;
11:1445
Table 1.2 Descriptive statistics of semen parameters
Center Concentration (ml) Motility (%) Morphology Volume
Argen1 138.3 (74.4) 64.5 (12.9) 12.2 (3.7) 2.7 (1.5)
Argen2 65.4 (56.1) 58.1 (17.1) 16.1 (6.8) –
Tygerberg 66.7 (54.1) 47.6 (16.3) 6.8 (3.8) 2.6 (1.8)
Turkey 50.4 (30.9) 59.8 (14.6) 14.9 (6.1) 3.9 (1.9)
19. Page 18
14. Robinson JN, et al. Does isolated teratozoospermia affect performance in in vitro fertilization and embryo transfer? Hum Reprod 1994;9:870
15. Sevenster CBvO, et al. In vitro fertilization when normal sperm morphology is less than fifteen percent. S Afr Med J 1990;78:203
16. Hernandez M, et al. Prognostic value of strict criteria: an Argentinian experience. Arch Androl 1996;37:85
17. Kobayashi T, et al. Sperm morphology assessment based on strict criteria and in vitro fertilization outcome. Hum Reprod 1991;6:903
18. AlHasani S, et al. The combination of two semen preparation techniques (glass wool filtration and swim up) and their effect on the morphology of recovered
spermatozoa and outcome of IVFET. Int J Androl 1996;19:55
19. Van Waart J, et al. Predictive value of normal sperm morphology in intrauterine insemination (IUI): a structured literature review. Hum Reprod Update 2001;7:495
20. MontanaroGauci M, et al. Stepwise regression analysis to study male and female factors impacting on pregnancy rate in an intrauterine insemination programme.
Andrologia 2001;33:135
21. Matorras R, et al. Sperm morphology analysis (strict criteria) in male infertility is not a prognostic factor in intrauterine insemination with husband’s sperm. Fertil Steril
1995;63:608
22. Toner JP, et al. Value of sperm morphology assessed by strict criteria for prediction of the outcome of artificial (intrauterine) insemination. Andrologia 1995;27:43
23. Ombelet W, et al. Intrauterine insemination after ovarian stimulation with clomiphene citrate: predic tive potential of inseminating motile count and sperm morphology.
Hum Reprod 1997;12:1458
24. Karabinus DS, Gelety T. The impact of sperm morphology evaluated by strict criteria on intrauterine insemination success. Fertil Steril 1997;67:536
25. Lindheim SR, et al. Abnormal sperm morphology is highly predictive of pregnancy outcome during controlled ovarian hyperstimulation and intrauterine insemination. J
Assist Reprod Genet 1996;13:569
26. Van der Merwe FH, et al. What is a normal semen analysis: a structured literature review (submitted)
27. Günalp S, et al. A study of semen parameters with emphasis on sperm morphology in a fertile population: an attempt to develop clinical thresholds. Hum Reprod
2001;16:110
28. Ombelet W, et al. Semen parameters in a fertile versus subfertile population: a need for change in the interpretation of semen testing. Hum Reprod 1997;12:987
29. Menkveld R, et al. Semen parameters, including WHO and strict criteria morphology, in a fertile and subfertile population: an effort towards standardization of invivo
thresholds. Hum Reprod 2001; 16:1165
30. Guzick DS, et al. Sperm morphology, motility, and concentration in fertile and infertile men. N Engl J Med 2001;345:1388
31. World Health Organization. WHO Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interaction. 4th edn. Cambridge, UK:
Cambridge University Press, 1999
20. Page 19
2
The role of sperm cell morphology in intracytoplasmic sperm injection
(ICSI)
M.L.Windt and T.F.Kruger
Sperm cell morphology and classical IVF
In classical in vitro fertilization (IVF), a decrease in sperm concentration, progressive motility and particularly sperm cell morphology (Tygerberg strict criteria) has
been shown to be of significant value in predicting fertilization and pregnancy rates1–5.
In a structured literature review of the IVF situation, Coetzee et al.5 reported on the impact of sperm morphology on fertilization and pregnancy rates. When looking
at all the studies available at the time, 92% of the articles evaluated showed a positive association between sperm morphology and IVF success. Parinaud et al.6 found
that poor sperm morphology (particularly sperm head abnormalities) resulted in poor embryo quality with subsequent implications for pregnancy outcome. Grow et al.7
identified strict sperm cell morphology as an excellent biomarker for sperm fertilizing capacity. Miller et al.8 showed that sperm cell morphology could be significantly
correlated with blastocyst development, and Salumets et al.9 concluded that blastomere cleavage rate was also determined by sperm cell morphology. Høst et al.10,11,
however, have shown that neither Tygerberg strict criteria nor the WHO criteria correlated with fertilization rate, embryo development, and score or pregnancies in
couples undergoing IVF.
Sperm cell morphology and ICSI
The introduction of intracytoplasmic sperm injection (ICSI), currently the most efficient technique to treat malefactor infertility, made fertilization with very impaired
semen parameters possible. In the case of ICSI fertilization, the selective barrier of the zona pellucida is eliminated and replaced with the judgment of the embryologist
performing the ICSI procedure. If possible, and also if available, morphologicallynormal spermatozoa will be selected for injection. The role of sperm cell morphology
in ICSI fertilization is somewhat difficult to investigate. Evaluation of the sperm cell morphology of the raw semen sample is probably not of real importance, since the
sperm cells selected for injection are not necessarily representative of the sperm population in the initial sample.
ICSI and sperm cell morphology: showing no effect
However, numerous studies have shown that sperm cell morphology cannot be correlated with outcomes in ICSI success. Studies by Nagy et al.12,13 and others
concluded that none of the three semen parameters analyzed (sperm concentration, motility, and morphology) influenced the outcome of ICSI12–16.
21. Page 20
Mansour et al.17 showed that patients with >95% and <95% teratozoospermia had similar fertilization and pregnancy outcomes. Svalander et al.18, using the
Tygerberg strict criteria to identify and compare three sperm morphology groups, also concluded that sperm morphology is not related to the outcome of ICSI. In a
study carried out by Lundin et al.19, strictcriteria sperm morphology showed a significant correlation with fertilization in classical IVF, but not with ICSI fertilization
and pregnancy outcome. Similar results were found by Høst 11, showing no predictive value of sperm cell morphology (strict criteria and WHO) and teratozoospermia
index (TZI) for ICSI outcome. They also found, however, no predictive value for classical IVF either.
Results from our own clinic also showed that strict morphology evaluation did not correlate with fertilization and pregnancy rates after ICSI with ejaculated
spermatozoa. Injection of spermatozoa from the goodprognosis (Gpattern, 5–14% normal) morphology group resulted in a fertilization rate of 66.1% and a
pregnancy rate of 25.3%. The Ppattern group (0–4% normal morphology) showed similar results (64.3% and 25.5%, respectively). ICSI results with testicular
spermatozoa also showed similar results (65.0% and 30.2%, respectively).
The outcome of a study using the Integrated Visual Optical System (IVOS) system to evaluate sperm cell morphology also showed no relation between sperm cell
morphology and ICSI fertilization rate20.
In a study by Yavetz et al.21, the sperm cell morphology of testicular spermatozoa was determined (head dimensions, acrosome, and midpiece irregularities) and
was shown not to affect the fertilization rate in ICSI.
From the studies discussed above, it seems therefore that sperm cell morphology (as determined by strict criteria and WHO criteria on raw semen) has no relation to
ICSI outcomes. This result can possibly be explained by the fact that, during the ICSI procedure, the embryologist tries to select the spermatozoon with apparent
normal morphology and also with motility. Sperm cell morphology may therefore not be a critical factor for ICSI fertilization, since many of the natural processes such
as the binding to, and penetration of, the zona pellucida are bypassed.
ICSI and sperm cell morphology: showing an effect
However, some studies have shown a correlation between sperm cell morphology and ICSI outcome. Tasdemir et al.4 reported that in cases with 100%
teratozoospermia, fertilization and cleavage rates were relatively high (50.8% and 93.2%, respectively), but pregnancy rates were lower than in cycles where normal
spermatozoa were injected (17% vs. 27%). Ongoing pregnancies in the 100% teratozoospermia group were very low (5.88%), and the abortion rate increased. The
authors suggest that abnormal head morphology may reflect abnormalities in spermatogenesis that are manifested by embryos with a low implantation potential.
Moomjy et al.22 found total teratozoospermia to be the only apparent reason for total fertilization failure in four of 20 couples presenting with this outcome.
The advantage of using ICSI in cases with poorprognosis sperm morphology (i.e. <5% morphologically normal spermatozoa), compared with IVF, was highlighted
in studies by O’Neil et al.23 and Moon et al.24. Although the fertilization rate increased in the ICSI group, however, spontaneous abortion was very high and ongoing
pregnancy after ICSI with poormorphology spermatozoa decreased.
A number of more recent studies also indicate that sperm morphology (strict criteria) does in fact influence ICSI fertilization and subsequent pregnancy outcome.
Levran et al.25 recorded the sperm morphology of each injected sperm cell, and found that fertilization declined with the following morphological appearance: short
acrosome region, round head, and amorphoushead defect. No pregnancy occurred in the amorphoushead defect group. The authors concluded that the injection of
spermatozoa with head defects results in decreased fertilization rates and poor embryo quality.
De Vos et al.26 also evaluated the influence of individual spermatozoa on ICSI fertilization and pregnancy outcome. Their results showed a lower fertilization,
pregnancy, and implantation rate after injection of morphologicallyabnormal spermatozoa. Embryo transfers were unmixed, and sperm morphology was assessed on
live sperm using Tygerberg strict criteria (‘within the limits of the inverted microscope’ and ‘given the limited magnification’). A sperm cell was considered normal if it
had a head of normal shape and size, and an acrosome,
22. Page 21
and was lacking midpiece and tail defects. The main defects recorded were elongated and tapered heads, amorphous heads, broken necks, and cytoplasmic droplets.
Fertilization with amorphous heads resulted in the lowest fertilization rate. Once fertilization occurred, embryo quality was good for all types of abnormal spermatozoa
except for spermatozoa with broken necks. The effect of a neck defect on fertilization and embryo quality (and subsequently pregnancy outcome) in ICSI was also
addressed in a case study by Rawe et al.27. They suggested that alteration in headtail junction and attachment in this case was caused by a centriolar dysfunction. This
caused insufficient sperm aster formation, lack of syngamy and cleavage, or even defective embryos leading to early abortions in successive treatment cycles.
The majority of sperm cells showed an abnormal development of the neck region. Heads were attached either to the tip or to the sides of the midpiece without linear
alignment with the sperm axis. Very strict selection of normallooking spermatozoa resulted in goodquality embryos, but after transfer ended in a preclinical abortion.
The sperm centrosome provides the active division center for the embryo, and plays a very important role in the first division of the embryo at syngamy28. The
functional, proximal sperm centriole is carried into the oocyte at fertilization, persists during sperm decondensation, and organizes the sperm aster and the first mitotic
spindle28,29. It has been shown that immotile spermatozoa have more centriolar defects than motile spermatozoa. Such a centriole dysfunction can therefore cause
lower fertilization rates and can also compromise embryo development in IVF and ICSI28–31.
Kahraman et al.32,33 found decreased fertilization and pregnancy rates with the injection of megalohead (macrocephalic) and multipletail spermatozoa. They suggest
that a high incidence of chromosomal abnormalities may be associated with these morphological forms and can therefore be the reason for low fertilization and
pregnancy. This suggestion was confirmed in separate studies by Viville et al.34 and Devillard et al.35. In both studies, macrocephalic sperm showed highly elevated
aneuploidy/diploidy rates. The majority of sperm cells showed both X and Y chromosomes35, and in study by Viville et al.34 a sexratio distortion in the favor of Y
bearing spermatozoa was found. Devillard et al.35 concluded that;
in the case of macrocephalic sperm, both meiosis I and II are affected, finally resulting in failure of
nuclear cleavage. Both authors34,35 concluded that ICSI should not be recommended as a method of treatment for these patients.
In a study by Osawa et al.36, it was reported that only in the case of severely tapered sperm heads was a decrease in ICSI fertilization (13%) observed. They
commented that such spermatozoa have a prolonged and incomplete decondensation pattern that could possibly explain the decrease.
Finally, Bartoov et al.37 developed a method by which sperm was assessed in real time—the motile sperm organelle morphology examination (MSOME) method—
and found that the normality of the entire sperm cell was associated with ICSI fertilization rate, but not with pregnancy outcome. A normal sperm nucleus (as defined by
MSOME) was, however, significantly associated with both ICSI fertilization rate and pregnancy outcome. ICSI pregnancy rate was therefore affected by
malformations of the sperm nucleus. These malformations can remain undetected by the embryologist during routine selection of spermatozoa.
Globozoospermia
One of the rare cases of abnormal sperm cell morphology is roundheaded spermatozoa (globozoospermia). The identification of a male patient suffering from
globozoospermia is a relatively rare occurrence, with an incidence in the infertile population of less than 0.05%. Owing to the absence of the acrosomal structures and
contents, roundheaded spermatozoa have been reported to have severely reduced capacity to bind to the zona pellucida and penetrate an oocyte normally These
spermatozoa have also been shown to be deficient in oocyteactivation factor and may have chromosomal defects38–40.
ICSI allows fertilization even in cases of extreme oligoasthenoteratozoospermia. Roundheaded spermatozoa lacking acrosomes are, however, an exception, and
fertilization rates with such spermatozoa are reduced in ICSI, although fertilization and some pregnancies have been reported38,41,42. The injection of roundheaded
spermatozoa has resulted in varying levels of success (fertilization and cleavage), ranging from 0 to 90%, but the general consensus is that it is lower than that of
standard intracytoplasmic spermatozoa injection cases39,41,42. In our own clinic, a
23. Page 22
globozoospermia ICSI fertilization cycle resulted in three embryos (3/7; 43% fertilization rate). The transfer of these three embryos resulted in a live birth of healthy twin
boys43.
Results are therefore somewhat contradictory but in many cases patients have a heterogeneous sperm population, and the injected sperm cell is not always
representative of the initial diagnosis38. The underlying pathology of roundheaded spermatozoa and the intracytoplasmic sperminjection technique itself may be the
major contributors to this variation. The examination of the semen sample must confirm whether the spermatozoa from the sample are type I (complete lack of
acrosomal structures and contents) or type II (limited acrosomal structures)42. The results in a study by Rybouchin et al.38 also revealed that roundheaded
spermatozoa are deficient in oocyteactivation capacity due to the absence of spermassociated oocyte activating factor (SAOAF)38. Battaglia et al.44 also concluded
that roundheaded spermatozoa used in ICSI were unable to induce oocyte activation. Treatment of unfertilized oocytes with calcium ionophore showed that activation
was in fact possible. This study also speculates that roundheaded spermatozoa are deficient in SAOAF.
ICSI and genetic status/DNA status and sperm cell morphology
Although morphologically abnormal spermatozoa do not necessarily reflect a genetic abnormality of the male gamete12,45, several studies have reported on the
increased risk of chromosome abnormalities in spermatozoa used in ICSI fertilization. In a study by Ryu et al.46, another aspect of strict sperm morphology (Tygerberg
criteria) and chromosome abnormalities was highlighted. Individual spermatozoa were evaluated for normal sperm morphology and also for chromosomes X,Y, and 18,
using FISH. A fertile and infertile group was investigated. Chromosomal aneuploidies were also present in normal spermatozoa, and a significantly higher rate of
chromosomal aneuploidy was detected in morphologically normal sperm from the infertile group compared with the fertile group. The authors concluded that normal
sperm morphology is not an absolute indicator for the selection of genetically normal sperm.
Kunathikom et al.47 and Rubio et al.48 showed that aneuploidy rates in spermatozoa of ICSI patients were increased compared with patients with normal semen
parameters. This was also the finding of Calogero et al.49, where ICSI patients (mainly OAT patients) had significantly higher aneuploidy and diploidy rates in
spermatozoa compared with fertile donor spermatozoa. This study also showed a direct correlation between the frequency of aneuploidy and the number of abnormal
spermatozoa (Tygerberg strict criteria). Similar data were published by Lee et al.50, showing an association of some sperm morphological abnormalities (amorphous,
round and elongated heads) with structural chromosome aberrations. When Bernardini et al.51 compared control, unexplained infertility and severe malefactor
oligoastenoteratozoospermia (OAT) male patients, the severe malefactor group had a significantly higher abnormal chromosome constitution.
VirantKlun et al.52 evaluated the effect of sperm singlestranded DNA and classical semen parameters on ICSI fertilization rate and embryo development. An
increase in sperm singlestranded DNA with decreased ICSI fertilization was found, as well as heavy embryo fragmentation. Classical semen parameters did not affect
the embryo quality of ICSI embryos.
Several recent studies have shown correlations between chromosomal abnormalities and abnormal sperm morphology. The studies of Viville et al.34 and Devillard et
al.35 have already been mentioned. In both studies, macrocephalic sperm showed highly elevated aneuploidy/diploidy rates. It is interesting to note that the results of the
study by Lee et al.50 did not show increased chromosome aberrations in spermatozoa with large (and also small) heads. The study by Morris et al.53, using WHO
sperm morphology evaluation, showed with multivariate analysis that DNA damage increased as a function of patient age, sperm motility, and abnormal forms. When
comparing infertility patients and normospermic donors, a significant increase in DNA damage was found as the proportion of morphologically normal forms decreased.
Roundheaded spermatozoa (globozoospermia) have also been associated with an elevation in abnormal chromosome structure and DNA strandbreaks40.
ICSI and testicular spermatozoa
The use of testicular spermatozoa in ICSI has been met with some concerns regarding the increased risk of malformations in offspring. A large study by
24. Page 23
Ludwig et al.54 showed, however, that the course of pregnancy, as well as the outcome after ICSI, was not affected by the origin of spermatozoa (testicular or
ejaculated).
In a study by Yavetz et al.21, a high proportion of testicular spermatozoa were shown to be morphologically normal when head dimensions, acrosome, and mid
piece irregularities were determined. The morphological characteristics were also not associated with ICSI fertilization rates.
ICSI with testicular spermatozoa has been shown to be very successful, although some studies have reported lower fertilization rates (particularly from non
obstructive azoospermia) than with ejaculated spermatozoa22,30,55–58.
Results from other studies showed that ICSI results using ejaculated, testicular, or epididymal spermatozoa did not differ significantly12,59–62. There was however, a
significant decrease in the fertilization rates in cases of nonobstructive azoospermia when compared with obstructive azoospermia in a study by Mansour60, but not in a
study by Bukulmez et al.63.
Conclusive remarks
Based on the current literature, we conclude that low sperm morphology, as determined on the raw semen sample according to strict criteria, serves as a warning that
decreased fertilization rates in vitro can be expected. In these cases, ICSI is the treatment of choice. To improve ongoing pregnancy rates it is, however, of utmost
importance (based on the literature) to select the individual spermatozoon used in ICSI, based on normal strictcriteria morphology principles2,64.
References
1. Kruger TF, et al. Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril 1986;46:1118
2. Kruger TF, et al. Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril 1988;49:112
3. Ombelet W, et al. Teratozoospermia and invitro fertilization: a randomised prospective study Hum Reprod 1994;9:1479
4. Tasdemir I, et al. Effect of abnormal sperm head morphology on the outcome of intracytoplasmic sperm injection in humans. Hum Reprod 1997; 12:1214
5. Coetzee K, Kruger TF, Lombard CJ. Predictive value of normal sperm morphology: a structured literature review. Hum Reprod Update 1998;4:73
6. Parinaud J, et al. Influence of sperm parameters on embryo quality. Fertil Steril 1993;60:888
7. Grow DR, et al. Sperm morphology as diagnosed by strict criteria: probing the impact of teratozoospermia on fertilization rate and pregnancy outcome in a large in vitro
fertilization population. Fertil Steril 1994;62:559
8. Miller JE, Smith TT. The effect of intracytoplasmic sperm injection and semen parameters on blastocyst development in vitro. Hum Reprod 2001;16:918
9. Salumets A, et al. Influence of oocytes and spermatozoa on early embryonic development. Fertil Steril 2002;78:1082
10. Høst E, et al. Sperm morphology and IVF: embryo quality in relation to sperm morphology following the WHO and Kruger’s strict criteria. Acta Obstet Gynecol
Scand 1999;78:526
11. Høst E, et al. Morphology of spermatozoa used in IVF and ICSI from oligozoospermic men. Reprod Biomed Online 2001;3:212
12. Nagy ZP, et al. The result of intracytoplasmic sperm injection is not related to any of the three basic sperm parameters. Hum Reprod 1995;10:1123
13. Nagy ZP, et al. Special applications of intracytoplasmic sperm injection: the influence of sperm count, motility, morphology, source and sperm antibody on the outcome
of ICSI. Hum Reprod 1998;13: (Suppl.1),143
14. Hammadeh ME, et al. The effect of chromatin condensation (Aniline blue staining) and morphology (strict criteria) of human spermatozoa on fertilization, cleavage and
pregnancy rates in an intracytoplasmic sperm injection programme. Hum Reprod 1996;11:2468
15. Kupker W, Schukze W, Diedrich K. Ultrastruture of gametes and intracytoplasmic sperm injection: the significance of sperm morphology. Hum Reprod 1998;13:
(Suppl.1),99
16. Sallam HN, Sallam AN, Agamia AF. The correlation between eight sperm parameters assessed objectively and the fertilization rate in IVF and ICSI. Fertil Steril
1998;70:(Suppl.1),371
17. Mansour RT, et al. The effect of sperm parameters on the outcome of intracytoplasmic sperm injection. Fertil Steril 1995;64:982
18. Svalander P, et al. The outcome of intracytoplasmic sperm injection is unrelated to ‘strict criteria’ sperm morphology. Hum Reprod 1996;11:1019
19. Lundin K, Söderlund B, Hamberger L. The relationship between sperm morphology and rates of fertilization, pregnancy and spontaneous abortion in an
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invitro fertilization/intracytoplasmic sperm injection programme. Hum Reprod 1997;12:2676
20. Sukcharoen N, et al. Sperm morphology evaluated by computer (IVOS) cannot predict the fertilization rate in vitro after intracytoplasmic sperm injection. Fertil Steril
1998;69:564
21. Yavetz H, et al. Morphology of testicular spermatozoa obtained by testicular sperm extraction in obstructive and nonobstructive azoospermic men and its relation to
fertilization success in the in vitro fertilizationintracytoplasmic sperm injection system. J Androl 2001;22:376
22. Moomjy M, et al. Implications of complete fertilization failure after intracytoplasmic sperm injection for subsequent fertilization and reproductive outcomes. Hum
Reprod 1998;13:2212
23. O’Neil J, et al. Should strict morphology be used to indicate the need for intracytoplasmic sperm injection? Fertil Steril 1998;70:(Suppl.1),442
24. Moon SY, et al. Assessment of oocyte morphology, oolemma breakage, and suction types as a predictor of normal fertilization after ICSI. Fertil Steril 1998;70:
(Suppl.1),323–27
25. Levran D, et al. The impact of injected sperm morphology on fertilization rate, embryo quality and pregnancy rate in IVFICSI cycles. Fertil Steril 1998;70:
(Suppl.1),972
26. De Vos A, et al. Influence of individual sperm morphology on fertilization, embryo morphology, and pregnancy outcome of intracytoplasmic sperm injection. Fertil
Steril 2003;79:42
27. Rawe VY, et al. A pathology of the sperm centriole responsible for defective sperm aster formation, syngamy and cleavage. Hum Reprod 2002;17:2344
28. Sathananthan AH, et al. The sperm centriole: its inheritance, replication and perpetuation in early human embryos. Hum Reprod 1996;11:345
29. Ng SC, et al. Review: Microinjection of human sperm directly into human oocytes. J Assist Reprod Genet 1993;10:337
30. Palermo G, Cohen J, Rosenwaks Z. Intracytoplasmic sperm injection: a powerful tool to overcome fertilization failure. Fertil Steril 1996;65:899
31. Wall MB, et al. Cytogenetic and fluorescent insitu hybridization chromosomal studies on invitro fertilized and intracytoplasmic sperminjected ‘failedfertilized’ human
oocytes. Hum Reprod 1996; 11:2230
32. Kahraman S, et al. Fertility of pinhead and multipletail megalohead spermatozoa only in the ejaculate with ICSI. Fertil Steril 1998;70:(Suppl.1),812
33. Kahraman S,. et al. Fertility of ejaculated and testicular megalohead spermatozoa with intracytoplasmic sperm injection. Hum Reprod 1999;14:726
34. Viville S, et al. Do morphological anomalies reflect chromosomal aneuploidies? Hum Reprod 2000; 15:2563
35. Devillard F, et al. Polyploidy in largeheaded sperm: FISH study of three cases. Hum Reprod 2002;17:1292
36. Osawa Y, et al. Assessment of the dominant abnormal form is useful for predicting the outcome of intracytoplasmic sperm injection in the case of severe
teratozoospermia. J Assist Reprod Genet 1999;16:436–38
37. Bartoov B, et al. Realtime fine morphology of motile human sperm cells is associated with IVFICSI outcome. J Androl 2002;23:1
38. Rybouchkin A, et al. Analysis of the oocyte activating capacity and chromosomal complement of roundheaded human spermatozoa by their injection into mouse
oocytes. Hum Reprod 1996;11:2170
39. Edirisinghe WR, et al. Cytogenetic analysis of unfertilized oocytes following intracytoplasmic sperm injection using spermatozoa from a globozoospermia man Hum
Reprod 1998;13:3094
40. Vicari E, et al. Globozoospermia is associated with chromatin structure abnormalities. Hum Reprod 2002;17:2128
41. Kilani ZM, et al. Triplet pregnancy and delivery after intracytoplasmic injection of roundheaded spermatozoa. Hum Reprod 1998;13:2177
42. Stone S, et al. A normal livebirth after intracytoplasmic sperm injection for globozoospermia without assisted oocyte activation. Hum Reprod 2000;15:139
43. Coetzee K, et al. An intracytoplasmic sperm injection pregnancy with a globozoospermia male. J Assist Reprod Genet 2000;18:311
44. Battaglia, DE, et al. Failure of oocyte activation after intracytoplasmic sperm injection using roundheaded sperm. Fertil Steril 1997;68:118
45. Martin RH, Rademaker A. The relationship between sperm chromosomal abnormalities and sperm morphology in humans. Mutat Res 1988;207:159
46. Ryu HM. et al. Increased chromosome X, Y, and 18 nondisjunction in sperm from infertile patients that were identified as normal by strict morphology: implication for
intracytoplasmic sperm injection. Fertil Steril 2001;76:879
47. Kunathikom S, Rattanachaiyanont M, Makemaharn O. Analysis of aneuploidy in minipercoll gradient centrifuged human sperm for intracytoplasmic sperm injection
(ICSI) using fluorescence in situ hybridization. J Obstet Gynaecol Res 2002;28:224
48. Rubio C, et al. Incidence of sperm chromosomal abnormalities in a risk population: relationship with sperm quality and ICSI outcome. Hum Reprod 2001;16:2084
49. Calogero AE, et al. High sperm aneuploidy rate in unselected infertile patients and its relationship with
26. Page 25
intracytoplasmic sperm injection outcome. Hum Reprod 2001;16:1433
50. Lee JD, Kamiguchi Y, Yanagimachi R. Analysis of chromosome constitution of human spermatozoa with normal and aberrant head morphologies after injection into
mouse oocytes, Hum Reprod 1996;11:1942
51. Bernardini L, et al. Comparison of gonosomal aneuploidy in spermatozoa of normal fertile men and those with severe male factor detected by insitu hybridization. Mol
Hum Reprod 1997;3:431
52. VirantKlun I, Tomazevic T, MedenVrtovec H. Sperm singlestranded DNA, detected by acridine orange staining, reduces fertilization and quality of ICSIderived
embryos. J Assist Reprod Genet 2002;19:319
53. Morris ID, et al. The spectrum DNA damage in human sperm assessed by singlecell gel electrophoresis (Comet assay) and its relationship to fertilization and embryo
development. Hum Reprod 2002;17:990
54. Ludwig M, Katalinic A. Pregnancy course and health of children born after ICSI depending on parameters of malefactor infertility. Hum Reprod 2003;18:351
55. Aboulghar MA, et al. Fertilization and pregnancy rates after intracytoplasmic sperm injection using ejaculate semen and surgically retrieved sperm. Fertil Steril
1997;68:108
56. Calderon I, et al. Is the fertilization rate of testicular sperm and its capacity to achieve pregnancy as good as or comparable to that of donor sperm? Fertil Steril
1998;70:(Suppl.1),462
57. Shulman A, et al. Invitro fertilization treatment for severe male factor: the fertilization potential of immotile spermatozoa obtained by testicular biopsy, Hum Reprod
1999;14:749
58. Goker EN, et al. Comparison of ICSI outcome of ejaculated sperm with normal, abnormal parameters and testicular sperm. Eur J Obstet Gynecol Reprod Biol
2002;104:129
59. Ghazzawi IM, et al. Comparison of the fertilizing capability of spermatozoa from ejaculates, epididymal aspirates and testicular biopsies using intracytoplasmic sperm
injection. Hum Reprod 1998;13:348
60. Mansour R, Intracytoplasmic sperm injection: a state of the art technique. Hum Reprod Update 1998;4:43
61. Schoysman R, et al. Pregnancy obtained with human testicular spermatozoa in an in vitro fertilization program. J Androl 1994;15:10S13S
62. Silber SJ, et al. High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa obtained from testicle biopsy. Hum Reprod 1995;10:148
63. Bukulmez O, et al. The origin of spermatozoa does not affect intracytoplasmic sperm injection outcome. Eur J Obstet Gynecol Reprod Biol 2001;94:250
64. Menkveld R, Stander, FSH, et al. The evaluation of morphological characteristics of human spermatozoa according to stricter criteria. Hum Reprod 1990;5:586
28. Page 27
3
Relationship between sperm morphology and binding capacity to the zona
pellucida: a critical step leading to fertilization
S.C.Oehninger
Introduction
Conventionally measured features of semen using the World Health Organization (WHO) criteria1, including sperm concentration, motility, and morphology, all affect
rates of fertilization in vitro and the outcome of assisted reproductive techniques, as demonstrated in both IVF (in vitro fertilization) and GIFT (gamete intrafallopian
transfer) therapies2–4 The evaluation of sperm morphology by a more stringent technique (strict criteria) has been shown to enhance significantly the prediction of IVF
outcome3,5–7.
Although positively correlated with fertilization rates in IVF, the assessment of spermmotion characteristics by computerassisted semen analysis still cannot be
reliably used to predict fertilization outcome8–10. Therefore, attention has also focused on bioassays of spermoocyte interaction11–14.
The hemizona assay (HZA) has been introduced as a new diagnostic test for the binding of human spermatozoa to its homologous zona pellucida to predict
fertilization potential12. In the HZA, the two matched zona hemispheres created by microbisection of the human oocyte provide three main advantages:
(1) The two halves (hemizonae) are functionally equal surfaces, allowing controlled comparison of binding and reproducible measurements of sperm binding from a
single egg.
(2) The limited number of available human oocytes is amplified because an internally controlled test can be performed on a single oocyte.
(3) As the oocyte is split microsurgically, even fresh oocytes cannot lead to inadvertent fertilization and preembryo formation12,15.
Spermzona pellucida binding tests evaluate the first, crucial step of spermoocyte interaction that leads to fertilization, that is, tight binding of spermatozoa to the zona
pellucida16. Overstreet and Hembree17 were the first to propose an assay for the evaluation of zona pellucida penetration by human spermatozoa using human oocytes
recovered from the ovaries of cadavers. Initially developed as a zonapenetration assay, sperm binding to the zona was not defined as an endpoint for the assay. The
two most common zonabinding tests currently used are the hemizona assay12 and a zona pellucidabinding test14. Both bioassays have the advantage of providing a
functional homologous test for sperm binding to the zona, comparing populations of fertile and infertile spermatozoa in the same assay. The internal control offered by
the HZA represents an advantage by decreasing the number of oocytes needed during the assay and diminishing the intraassay variation9,10,12,15,18–22.
The objective of this chapter is to present studies that were performed in order to assess the relationship between the basic sperm parameters and the spermzona
pellucida binding capacity using the
29. Page 28
HZA model. These studies have provided further evidence to validate the significance of sperm morphology assessment using strict criteria, and the benefits of the HZA
as a spermfunction test in the screening process and clinical management of malefactor patients.
Methodology
Different sources of human oocytes were used in the HZA: oocytes recovered from surgically removed ovaries or postmortem ovarian tissue, and surplus oocytes
from IVF treatments (following patients’ consent in approved protocols). As fresh oocytes are not always available for the test, different alternatives have been
implemented for storage. Others have described the storage of human oocytes in dimethylsulfoxide (DMSO) at ultralow temperatures17. Additionally, Yanagimachi
and colleagues showed that highly concentrated salt solutions provided effective storage of human oocytes, such that the spermbinding characteristics of the zona
pellucida were preserved23. In developing the HZA, we examined the binding ability of fresh, DMSO, and saltstored (under controlled pI I conditions) human
oocytes, we concluded that the spermbinding ability of the zona remains intact under all these conditions24–27. Subsequently, we have used saltstored oocytes and
assessed the kinetics of sperm binding to the zona; we showed maximum binding at 4–5h of gamete coincubation, with similar binding curves both for fertile and
infertile semen samples12,25,26,28–31.
A detailed description of oocyte collection, handling, and micromanipulation, as well as semen processing and sperm suspension preparations for the HZA, has been
published12,32. The assay has been standardized to 4h gamete coincubation, exposing each hemizona to a sperm droplet (100μl of a motile sperm dilution of
500000/ml, prepared after swimup). Human tubal fluid supplemented with human serum albumin is usually the medium utilized for sperm preparation and gamete co
incubation. After coincubation, the hemizonae are subjected to pipetting through a glass pipette in order to dislodge loosely attached sperm. The number of tightly
bound spermatozoa on the outer surface of the zona is finally counted using phasecontrast microscopy (×200). Results are expressed as hemizona index (HZI),
calculated as the number of sperm tightly bound for the patient sample/control sample×10012. The assay has been validated by a clearcut definition of the factors
affecting data interpretation; i.e., kinetics of binding, egg variability and maturation status, intraassay variation and influence of spermconcentration morphology,
motility, and acrosome reaction status25,28–31,33–36.
During the course of 90 days of evaluation, spermatozoa from fertile men do not exhibit a timedependent change in zonabinding potential, ensuring their suitability as
controls in the bioassay29. Within each donor pool utilized in the assay, a cutoff value or minimal threshold of binding has to be established in order to validate each
assay for the purpose of identification of a poor control semen specimen and/or a poor zona. In our control population, this cutoff value is approximately 20 sperm
tightly bound to the control hemizona (fertile donor)29; each laboratory should statistically assess its own control data in order to establish a reasonable lowerlimit
assay acceptance.
If the control hemizona (matching hemizona exposed to fertile semen) has a good binding capability, that is, tightly binds at least 20 spermatozoa after the 4 h
incubation period (information derived from a statistical evaluation of a fertiledonor pool), then a single oocyte will give reliable information about the fertilizing ability of
test spermatozoa (sperm from infertile patients) under IVF conditions. Because of the definition of the assay’s limitations and its small intraassay variation (<10%), the
power of discrimination of the HZA has been maximized. Conversely, for other spermzona binding tests, several oocytes have to be used because of the high interegg
variation, and, in fact, an intraassay coefficient of variation of 30% has been reported14.
The interegg variability is high, not only for oocytes at different stages of maturation (immature vs. mature eggs), but also within a certain population of eggs at the
same maturation stage. However, this factor is internally controlled in the assay by the utilization of matching hemizona; this allows a comparison of binding of a fertile
vs. an infertile semen sample in the same assay under the same oocytequality conditions. Incubating matching hemizona from eggs at the same maturational stage with
homologous spermatozoa from the same fertile ejaculate, we have been able to determine the intraegg (intraassay) viability for human oocytes38. Overall, the mean
value of the difference between the two matching halves shows an intraegg variability of approximately 10% for all categories of egg nuclear maturation. Additionally,
we have shown
30. Page 29
that full meiotic competence of human oocytes is associated with an increased spermbinding potential. It would appear that zona maturation is associated with the
nuclear developmental progression of the oocyte, perhaps in parallel with cytoplasmic and membrane maturation, leading to a fully fertilizable status.
The specificity of the interaction between human spermatozoa and the human zona pellucida under HZA conditions is strengthened by the fact that the sperm tightly
bound to the zona are acrosomereacted20,31. Results of interspecies experiments performed with human, cynomolgus monkey, and hamster gametes have
demonstrated a high speciesspecificity of human spermzona pellucida functions under HZA conditions, providing further support for the use of this bioassay in
infertility and contraception testing.
Results—assisted reproduction
In prospective blinded studies, we investigated the relationship between sperm binding to the hemizona and IVF outcome21,22,26,35–37,39. Results have shown that the
HZA can successfully distinguish the population of malefactor patients at risk for failed or poor fertilization. Using either a cutoff value of fertilization rate of 65%
(mean –2 SD of the overall fertilization rate in the Norfolk Program for nonmalefactor patients), or distinguishing between failed vs. successful fertilization (0% vs. 1–
100%), the hemizonaassay results, expressed as HZI, provide a valuable means of separating these categories of patients10,18,19,20. This HZI cutoff value is
approximately 35%.
Overall, for failed vs. successful and poor vs. good fertilization rate, the correct predictive ability (discriminative power) of the HZA was 80 and 85%, respectively.
The information gained may be extremely valuable for counseling patients in the IVF setting (i.e., considering an HZI of below 35%, the chances of poor fertilization are
90–100%, whereas for an HZI of over 35%, the chances of good fertilization are 80–85%)18,19,35,36 (Figure 3.1).
The assay has an excellent sensitivity and specificity with a low incidence of falsepositive results. For an HZI of 35%, the positive predictive value of the HZA is
79%, and its negative predictive value is 100% (considering good vs. poor fertilization rates).
Figure 3.1 Clinical use of HZA results using an HZI threshold of 35 (cluster analysis of fertilization outcome versus HZI and its
regression line)
31. Page 30
In the HZA, falsepositive results can be expected, since other functional steps follow the tight binding of sperm to the zona pellucida and are essential for fertilization
and preembryo development. Thus, although binding is not crucial to achieve fertilization, it does not guarantee that fertilization will ensue. However, tight binding to the
zona is an absolute prerequisite for fertilization to follow naturally.
Powerful statistical results allow us to use the HZA in the prediction of fertilization rate18,19,21,22. Logistic regression analysis provides a robust HZI rangepredictive
of the oocyte’s potential to be fertilized (Table 3.1). As a consequence, patients may be directed to intracytoplasmic sperm injection (ICSI) therapy based upon
defined abnormalities diagnosed by the screening tests and not empirically40–42.
Of the classical sperm parameters, sperm morphology is the best predictor of the ability of spermatozoa to bind to the zona pellucida (Table 3.2). Patients with
severe teratozoospermia (‘poorprognosis’ pattern or <4% normal sperm scores as judged by strict criteria) have an impaired capacity to bind to the zona under HZA
conditions (possibly owing to membrane/receptor deficiencies?)18,19. We agree with Liu et al. that the ability of sperm to achieve tight binding to the zona pellucida
might reflect multiple functions of human spermatozoa14.
Under HZA conditions, patients with severe teratozoospermia demonstrate a higher binding capacity following an increase in the sperm concentration during sperm
and hemizona coincubation29. This may explain the improved fertilization results following high insemination concentration during IVF in some malefactor patients43,44.
We have also investigated the morphological characteristics of those spermatozoa that are tightly
bound to the zona pellucida under HZA conditions, and we compared the morphological normality of zonabound spermatozoa to that observed in the original semen
samples and in the swimup fractions45. A significantly higher number of normal forms were found among the zonabound sperm, both for normospermic as well as
teratozoospermia patients45. These results indicated that spermatozoa classified as normal or slightly abnormal have the potential for selectively achieving binding to the
zona in preference to abnormal (bizarre) spermatozoa. In particular, those sperm cells with acrosome abnormalities and other severe head defects are either actively
excluded or simply cannot bind to the zona, or do it with a low efficiency owing to inherent defects.
This newly identified human zona property of sperm selectivity points to another potential use of HZA, i.e. selection of sperm to be used in ICSI for assisted
fertilization. For such a purpose, using micromanipulators, morphologically normal sperm can be identified more efficiently and removed from the hemizona, after which
they can be used for surgical fertilization45. Additionally this would ensure that the physiological acrosome reaction has occurred.
Results—semen analysis
In support of this development, Huszar and coworkers have utilized creatine kinaseimmunocytochemistry to evaluate human hemizonasperm complexes to examine
whether the distribution of creatine kinasestained cells bound to the hemizona would follow the incidence of these sperm cells in semen samples, or if there is
preferential binding by the normal sperm46. Huszar et al.47,48 have previously shown a relationship between sperm creatine
Table 3.1 Logistic regressionanalysis of the impact of the basic sperm parameters and HZA results (expressed as HZI) on fertilization outcome
Fertilization rate
Sperm parameter (r) pValue
Concentration 0.35 <0.05
Motility 0.60 0.0001
Velocity 0.50 0.01
Linearity 0.13 0.4
Morphology 0.57 0.0001
HZI 0.62 0.0001
Table 3.2 Logistic regressionanalysis of the impact of the basic sperm parameter on spermzona pellucida binding capacity under HZA conditions
Spermzona binding capacity
Sperm parameter (r) pValue
Concentration 0.30 <0.04
Motility 0.29 0.06
Velocity 0.23 0.2
Linearity 0.20 0.3
Morphology 0.52 0.0001
32. Page 31
kinase concentrations and fertilizing potential in men. The binding to the hemizona was selective for normal sperm, as the incidence of intermediate and dark sperm in
the semen samples was significantly higher than in those bound to the hemizona (normal=clear heads; intermediate and immature= light and dark staining, respectively.
Huszar and coworkers have suggested that this high degree of selectivity is not related to the properties of the zona, but rather to the fact that immature and abnormal
spermatozoa are defective in the site(s) of oocyte recognition and binding. Therefore, creatine kinasestaining patterns in the hemizona complexes support the use of
strict criteria for identification of the malefactor population and the use of the HZA in assisted reproduction and in assisted fertilization.
Discussion
Under HZA conditions, the minimum concentration of motile spermatozoa from fertile donors necessary to achieve valid results is approximately 250000/ml. The
‘effective number of sperm’ (morphologically normal sperm with high motility) can be determined under such HZA conditions and may be an indication of the actual
number of normal spermatozoa necessary to achieve binding and thereby successful fertilization37. Others have proposed that the HZA may be particularly useful for
identifying those patients that may benefit from insemination with increased concentration (patients with severe oligozoospermia, in which microinsemination methods
are utilized to try to enhance fertilization rates during IVF)49. Our data extend such observations and demonstrate that morphology assessment using strict criteria and
HZA results can be used to recommend IVF or ICSI as the treatment of choice in the individual clinical situation.
Failure of fertilization owing to a defective spermzona pellucida interaction is a relatively common problem, thereby underscoring the potential of the HZA as a
diagnostic/predictive test. However, it must be realized that postzona binding defects may occur, thereby stressing the necessity of evaluating patients with fertilization
disorders utilizing other bioassays that assess complementary sperm functions. The order of progression of the predictive bioassays may be important, because those
sperm defects that prevent tight binding to the zona pellucida, or its penetration, will negate subsequent func tional tests. Accordingly, the HZA should be applied first.
Men whose specimens show adequate tight binding or penetration under HZA conditions should be examined next for acrosome reaction, oolemma fusion,
decondensation, and pronuclear formation in sequence.
Our results demonstrate that the interaction between spermatozoa and the zona pellucida is a critical event leading to fertilization and reflects multiple sperm functions
(i.e. completion of capacitation as manifested by the ability to bind to the zona pellucida and to undergo ligandinduced acrosome reaction)18,19,40.
The induced acrosomereaction assays appear to be equally predictive of fertilization outcome and appear to be simpler in their methodologies. At the present time,
the use of a calcium ionophore to induce acrosome reaction is the most widely used methodology50,51. Nevertheless, the implementation of assays using small volumes
of human solubilized zonae pellucidae52,56, biologically active recombinant human ZP353–55, or active, synthetic ZP3 peptides57 will probably allow for the design of
improved, physiologically oriented acrosomereaction assays.
Franken et al.56 devised a new microassay that is easy and rapid to perform, and facilitates the use of minimal volumes of solubilized zona pellucida (even a single
zona) for assessment of human acrosome reaction. The microassay has been validated as compared with standard macroassays and, consequently, offers a unique
arena to test for the physiological induction of acrosomal exocytosis by the homologous zona pellucida. Moreover, preliminary clinical studies using the microassay
have demonstrated that the zonainduced acrosome reaction (ZIAR) can predict fertilization failure in the IVF setting. The microassay ZIAR can therefore refine the
therapeutic approach for male infertility prior to the onset of therapy58,59.
Recently, Bastiaan et al.60 prospectively evaluated the relationship between sperm morphology, acrosome responsiveness to solubilized zona pellucida using the
microassay, spermzona binding potential (HZA), and IVF outcome. Receiver operator characteristics (ROC) curve analyses indicated ZIAR to be a robust indicator
for fertilization failure during IVF therapy, with a sensitivity of 81% and specificity of 75%. In addition, a positive and significant correlation existed between ZIAR
results and sperm morphology (r=0.65) and spermzona binding (r= 0.57).
33. Page 32
Stateoftheart management of the infertile man
If sperm abnormalities are observed in the ‘basic’ semen analysis or if the couple is diagnosed as having ‘unexplained’ infertility the workup should proceed to the
analysis of sperm function/biochemical tests. The diagnosis of subfertility or infertility based upon the firsttier (initial ‘basic’ evaluation) and the
‘expanded’ (functional/biochemical) screenings, will direct management towards a variety of therapeutic options21,22,35,36,40,41. We have previously proposed that
laboratory evaluation of sperm quality/quantity for assisted reproduction should be approached using such a sequential, multistep diagnostic scheme18,19,21,35,36 (Table
3.1). This concept has also been proposed by others61.
Male infertility problems may be successfully treated with (a) urological interventions (surgical or nonsurgical treatments, such as conventional, microsurgical, or
laparoscopic surgery, including correction of varicocele, epididymo and vasovasostomy and modern approaches for ejaculatory disorders); (b) medical therapies
(such as specific treatment of hypogonadism, hyperprolactinemia, and infection); while (c) a significant proportion will proceed to low and highcomplexity ARTs
(assistedreproduction techniques).
It is at this time that sperm function/biochemical tests may be of highest value in order to direct the couple to ART. Currently recommended ART options include:
‘lowcomplexity’ intrauterine insemination (IUI) therapy ‘standard’ IVF and embryo transfer, and IVF augmented with ICSI. Assisted reproduction can be indicated as
a result of (a) the failure of urological/medical treatments, (b) the diagnosis of ‘unexplained’ infertility in the couple; (c) the presence of ‘basic’ sperm abnormalities of
moderatetohigh degree; or (d) abnormalities of sperm function as diagnosed by predictive bioassays (such as the HZA or induced acrosome reaction test).
In our program, patients are selected for IVF augmented with ICSI according to the following indications: (1) poor sperm parameters (i.e. <1.5× 106 total
spermatozoa with adequate progressive motility after separation, severe teratozoospermia with <4% normal forms in the presence of a borderlinetolow total motile
fraction, and/or poor spermzona pellucida binding capacity with a hemizona assay index <30%)21; (2) failure of IUI therapy in cases presenting with abnormal sperm
parameters including presence of antisperm antibodies); (3) previous failed fertilization in IVF, and (4) presence of obstructive or nonobstructive azoospermia, where
ICSI is combined with sperm extraction from the testes or the epididymis40,41. In the presence of severe oligoasthenoteratozoospermia, or if the outcome of sperm
function testing indicates a significant impairment of fertilizing capacity, couples should be immediately directed to ICSI. This approach is probably more costeffective
and will avoid loss of valuable time (particularly in women >35 years). Excellent pregnancy results can also be achieved with ICSI using ejaculated, epididymal (using
MESA or microsurgical epididymal sperm aspiration), and testicular sperm (using TESA or testicular sperm extraction) in cases of obstructive and nonobstructive
azoospermia62.
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