FERTILITY AND STERILITYt

VOL. 72, NO. 1, JULY 1999

Copyright ©1999 American Society for Reproductive Medicine
Published by Elsevier Science Inc.
Printed on acid-free paper in U.S.A.

Spermac stain analysis of human sperm

acrosomes
Philip J. Chan, Ph.D., H.C.L.D.,*† Johannah U. Corselli, Ph.D.,†
John D. Jacobson, M.D.,† William C. Patton, M.D.,† and Alan King, M.D.†
Loma Linda University School of Medicine, Loma Linda, California

Objective: To study the association between low percentages of intact sperm acrosomes and fertilization
failures in conventional IVF procedures.
Design: A retrospective study.
Setting: Clinical and academic research environment.
Patient(s): Patients undergoing treatment of infertility.
Intervention(s): Sperm cells were fixed and stained using the Spermac stain.
Main Outcome Measure(s): Percentages of intact acrosomes and fertilization.
Result(s): There was a significant association between specimens with ,40% intact acrosomes and failed
conventional IVF procedures. Among the 29 cases with ,40% intact acrosomes, 9 cases (31%) resulted in
zero penetration of the oocytes. The mean (6SEM) percentage of fertilization was lower in the abnormal
acrosome group (43.3% 6 6.5%) than in the normal acrosome group (64.1% 6 5.6%). The status of the sperm
acrosome was not correlated with the results of fertilization in intracytoplasmic sperm injection procedures.
Conclusion(s): Sperm with low percentages of intact acrosomes were associated with failed fertilization. The
Spermac stain was useful for assessing acrosomes and identifying possible male factor infertility problems.
The results suggested that a minimum percentage of sperm with intact acrosomes are needed for fertilization
to occur in vitro. (Fertil Sterilt 1999;72:124 – 8. ©1999 by American Society for Reproductive Medicine.)
Key Words: IVF-ET, acrosome reaction, spermatozoa, Spermac stain, ICSI

The acrosome reaction (1–3) of the ejacu- preferred approach when appropriate. Hence, it
lated sperm cell occurs either spontaneously or is important to determine the status of the
at the surface of the zona pellucida after bind- sperm acrosome to help in planning the course
ing to the ZP3 receptor protein (4, 5). The of treatment with assisted reproductive tech-
mechanism involves fusion of the sperm nology.
plasma membrane with the outer acrosomal Methods of assessing the sperm acrosome
Received July 24, 1998; membrane, calcium influx (4), and vesiculation include the Pisum sativum agglutinin-fluores-
revised and accepted and exocytosis of the inner acrosomal enzymes cein isothiocyanate assay (10), the triple stain
March 12, 1999. (6, 7). The sperm head enters the oocyte by procedure (11), the indirect immunofluores-
Reprint requests: Philip
J. Chan, Ph.D., Loma Linda endocytosis, decondenses, and forms the pro- cence assay (12), and the chlortetracycline as-
University Center for nucleus, which shortly thereafter participates in say (13). However, these methods are laborious
Fertility and In Vitro syngametic approximation with the oocyte pro-
Fertilization, 11370
to perform and difficult to incorporate into the
Anderson Street, Loma nucleus (8). routine semen analysis. The Spermac stain
Linda, California 92354 (14 –18), originally developed for domestic an-
(FAX: 909-558-2450; E- Failure to fertilize is thought to occur when
mail: www.llu.edu/llumc/ the sperm lacks acrosomal enzymes (i.e., the imal species, has been shown to be method-
fertility). round-headed syndrome [(9)]) or prematurely ologically simple to use and rapid in the as-
* Department of Physiology loses acrosomal enzymes because it undergoes sessment of acrosomal status in human sperm.
and Pharmacology. The accuracy of this stain for human sperm
† a spontaneous acrosome reaction. The intracy-
Department of analysis has been verified using electron mi-
Gynecology and toplasmic sperm injection (ICSI) procedure can
Obstetrics. bypass acrosomal dysfunction, but because of crographs (15).
recent concerns about injecting genetically de- The objective of this study was to determine
0015-0282/99/$20.00
PII S0015-0282(99)00201-0 fective sperm, conventional IVF remains the whether the status of sperm acrosomes ana-

124
lyzed with the use of the Spermac stain during routine semen
analysis would be predictive of fertilization failure in con-
ventional IVF procedures. The information obtained would
provide further supporting evidence for the usefulness of this
simple staining method as part of the routine semen analysis.
MATERIALS AND METHODS
Semen Samples
In this retrospective study, semen specimens were ob-
tained from the male partners of 63 couples undergoing
conventional IVF procedures (n 5 22), ICSI procedures
(n 5 24), or a combination of both procedures (n 5 17). The
study was approved by the institutional review board at our
institution. Signed informed consent forms were obtained
from all patients who participated in the study. The sperm
were assessed during routine semen analyses (19) performed
as part of the pretreatment infertility workup in a retrospec-
tive analysis of data design. Cases involving donor sperm or
canceled ovulation induction cycles were omitted from the
study.
Several stains, including the Diff-Quik, the Papanicolaou,
and the Spermac stains, were available for use in the mor-
phologic analysis of sperm. In our laboratory (17), the Sper-
mac stain already was being used for morphologic analysis.
Hence, nothing different was added to the routine semen
analysis procedure except that sperm morphology and acro-
somal status were evaluated together for each stained slide.
A study of the semen analysis parameters associated with the
Spermac stain data has been published previously (17).
Spermac Stain Procedure
A 100-mL aliquot of semen was mixed with an equal
volume of culture medium (modified human tubal fluid kept
at 23°C) and the diluted semen was smeared on a glass slide.
The staining procedure was carried out according to the
Spermac kit manufacturer’s guidelines (Stain Enterprises,
Onderstepoort, South Africa). The sperm smear was allowed
to air-dry before being fixed in the formalin solution (Fixa-
tive I) provided in the Spermac kit for 5 minutes at room
temperature (23°C).
Each slide with fixed sperm was washed in tapwater, and
stain solution A was pipetted out and used to flood the slide,
which was kept in a horizontal position. Stain solution A was
kept on the slide for 2 minutes and then rinsed off with
water. Stain solution B was used to flood the slide for 1
minute and then was rinsed off with water. Stain solution C
was used to flood the slide for 1 minute and then it was
rinsed off. The stained slides were air-dried for 10 minutes
and then analyzed in oil immersion (31,000) and by light
microscopy to assess the percentage of sperm with intact
acrosomes.
Regardless of the shape of the sperm head, sperm were
considered to have an intact acrosome when the anterior
acrosomal region stained green (14, 15) and the dark green,
FERTILITY & STERILITYt
FIGURE 1
Ejaculated sperm cells after staining with the Spermac acro-
somal stain. Sperm with intact acrosomes have a dark green,
“rubber-band,” semicircle cap line with red counterstained
postacrosomal regions (left). Acrosome-reacted (center) and
acrosome-defective (right) sperm do not have the continuous
dark green band of even thickness at the acrosomal cap.
thickened, “rubber-band” border forming a semicircle at the
tip of the head remained unbroken or continuous (Fig. 1).
The posterior postacrosomal region of each sperm head was
red-pink in coloration. Sperm that lacked the red-pink coun-
terstain in the posterior head region were inadequately
stained and were not counted. Sperm with nonintact acro-
somes (reacted or defective acrosomes) showed peeled ac-
rosomal membranes, spotting, irregular thickness in the
green band, or partial green coloration. Another type of
sperm with nonintact acrosomes (missing acrosomal en-
zymes) were stained either white or red at the acrosomal
region and had no green color at the head.
For each sperm smear, the percentage of sperm with
intact acrosomes was calculated by dividing the number of
sperm with intact green acrosomes by the total number of
sperm analyzed and multiplying by 100. At least 100 sperm
cells were assessed for each slide, and the same technician
performed all the Spermac stain analyses. The cutoff value
of 40%, with higher percentages interpreted as normal per-
centages of intact sperm acrosomes (Table 1), was based on
the lowest percentage that did not result in failed fertilization
with conventional IVF.
The same Spermac-stained slides also were used to de-
termine the percentage of sperm with strict normal morphol-
ogy according to the Tygerberg strict criteria developed by
Kruger et al. (20) on at least 100 sperm. A sperm was
classified as normal when the head was oval, the acrosome
occupied 40%–70% of the head, there were no midpiece or
tail defects, cytoplasmic droplets were absent or negligible,
and the dimensions of the head were appropriate.
Assisted Reproductive Technologies
The IVF procedures were performed according to estab-
lished protocols (21). Briefly, female patients and their part-
ners scheduled to undergo IVF procedures at the Center for
125
 Data Analyses
TABLE 1 Statistical analyses were performed using commercial
Relation between sperm specimens with abnormal or computerized statistical programs. Student’s t-test was used
normal percentages of intact acrosomes and the outcome to determine statistically significant differences between
of conventional IVF or intracytoplasmic sperm injection. means. Categorical data were analyzed using the x2 test
statistic. P,.05 was considered statistically significant.
,40% intact $40% intact
Parameter acrosomes group acrosomes group
RESULTS
Conventional IVF
Total no. of cases 29 12 The results showed a significant association between se-
No. of failed fertilizations/total 9/29 (31) 0/12 (0)* men specimens with ,40% intact acrosomes and failed
no. of cases (%) fertilization with conventional IVF procedures (Table 1).
Fertilization in vitro (%) 43.3 6 6.5 64.1 6 5.6*
Among the 29 cases in which the sperm of the male partner
Strict normal morphology (%) 8.5 6 1.7 14.5 6 2.1*
Sperm concentration (3106/ 63.8 6 7.7 83.9 6 11.7* had ,40% intact acrosomes, 9 cases (31%) resulted in no
mL) penetration of the oocytes. Two of the 9 cases were in
Total motility (%) 61.7 6 2.5 67.3 6 2.6 couples with male factor infertility. Altogether, there were
Intact acrosomes (%) 24.3 6 1.8 51.3 6 2.7* only 3 cases of male factor infertility in the 63 patients based
ICSI
on semen parameters (sperm count of ,20 3 106/mL,
Total no. of cases 34 4
No. of failed fertilizations/total 0/34 (0) 0/4 (0) ,50% total motility, and ,30% normal sperm forms by
no. of cases (%) World Health Organization criteria).
Fertilization by ICSI (%) 71.1 6 3.1 74.0 6 8.4
The mean percentage of fertilization in the abnormal
Strict normal morphology (%) 4.3 6 0.6 7.5 6 2.7
Sperm concentration (3106/ 45.5 6 6.2 86.3 6 18.8* intact acrosome group was significantly lower than in the
mL) group with .40% intact sperm acrosomes. Similarly, there
Total motility (%) 51.3 6 2.5 65.5 6 5.5* were differences in the percentage of strict normal morphol-
Intact acrosomes (%) 18.8 6 1.6 48.3 6 4.3* ogy and the sperm count between the abnormal and normal
Note: Data are means 6 SEM unless otherwise noted. ICSI 5 intracyto- acrosome groups. There were 16 clinical pregnancies and 2
plasmic sperm injection. miscarriages in the abnormal acrosome group and 5 clinical
* P,.05.
pregnancies and no miscarriages in the normal acrosome
group. However, these data were not applicable because
Fertility and In Vitro Fertilization were given routine exam- some cases involved the transfer of embryos derived from
inations. For the female patients, ovulation induction was both conventional IVF and ICSI. There were 2 clinical
achieved after several days of leuprolide acetate suppression pregnancies from ICSI-derived embryos among the 9 cases
and was initiated using recombinant FSH (an average of four of failed conventional IVF.
ampules given in split doses daily, 75 IU per ampule) for
All cases of ICSI yielded fertilized embryos (Table 1).
9 –12 days; 10,000 U of hCG generally was administered on
There was no difference in the percentage of fertilization.
the 10th day if the E2 level was .500 pg/mL and the largest
However, there were differences between the abnormal and
follicle was .18 mm in diameter. Patients who underwent
normal acrosome groups in the percentage of strict normal
leuprolide acetate cycles followed a similar induction proto-
morphology, the sperm count, and motility. The abnormal
col, except that 0.5 mg of leuprolide acetate was adminis-
acrosome group had 13 clinical pregnancies and 1 miscar-
tered beginning on day 21 of the previous cycle and ending
riage, whereas the normal acrosome group had 1 clinical
on the last day of stimulation (21).
pregnancy and no miscarriages.
The culture medium used for all protocols was human
tubal fluid (Irvine Scientific, Santa Ana, CA) supplemented DISCUSSION
with synthetic serum substitute (Irvine Scientific). At the
time of the IVF procedure, the sperm cells were washed The results demonstrated an association between ejacu-
according to standard wash procedures and used to insemi- lated sperm with low percentages of intact acrosomes
nate the oocytes at a concentration of 100,000 sperm per (,40%) and failed fertilization. This finding suggested that
milliliter for routine cases. Intracytoplasmic sperm injection the Spermac acrosome stain procedure may be used to help
was performed in cases of suspected male factor infertility. screen for possible cases of male factor infertility. It is
Day 3 embryos were returned transcervically to the uterine logical that sperm cells with low percentages of intact acro-
lumen of the originating patients. Each patient was moni- somes would not fertilize oocytes, and this is supported by
tored for evidence of clinical pregnancy. The criteria for the finding of significantly lower fertilization rates in the
clinical pregnancy were an elevated serum hCG concentra- abnormal acrosome group in the present study. These data
tion and detection of a uterine sac with a beating heart on are consistent with those in previous reports (10). Other
ultrasound examination. studies (22, 23) have shown that in cases of failed fertiliza-

126 Chan et al. Spermac acrosome stain Vol. 72, No. 1, July 1999
tion, the sperm cells do not have appropriate head-directed
mannose ligand activity and membrane cholesterol levels,
and that they have low percentages of spontaneous acrosome
loss. Such cases have been referred to as “nonresponders” by
Benoff et al. (22, 23).
In the present study, explanations for the observed low
percentages of intact acrosomes in sperm include abnormal
spermiogenesis with a disrupted Golgi transformation step,
premature discharge of acrosomal contents caused by lumi-
nal receptors and autoantibodies, or an artifact resulting from
genetic defects (8). Sperm with abnormally low percentages
of intact acrosomes also had low percentages of strict normal
morphology. As expected, the acrosomal status of the sperm
was not associated with the fertilization results at ICSI.
When comparing Spermac staining with advanced tests
such as the mannose binding assay, several advantages of
Spermac staining are apparent: extra preparatory steps are
not needed because Spermac staining already is being used
for sperm morphology assessment; the method is simple
and rapid, yielding results within 20 minutes; a fluorescent
microscope and darkroom are not needed; and repeated
low-cost analyses can be performed on specimens at dif-
ferent times (14, 17, 18). Further, assessment of the sperm
acrosomal status using Spermac staining has been shown
to correlate with the results of electron microscopy studies
(15, 16).
Sperm acrosomes stained by the Spermac stain (Fig. 1)
can be categorized as intact (the green acrosome band is
continuous), reacted (the band appears as spots or dashes, or
it is missing), or defective (the band appears uneven in
thickness). Colored photographs are available on the internet
website, www.llu.edu/llumc/fertility/research.
One concern with the staining procedure is disruption of
the acrosome during the air-drying process. The procedure
may be improved by diluting the semen in the Spermac
fixative solution (formalin) provided, making the smear, and
then air-drying. Other concerns, such as distinguishing be-
tween dead and viable sperm, have been addressed by com-
bining the Spermac stain with the hypo-osmotic swelling
solution (18) or multiplying the percentage of intact acro-
somes by the fraction of viable sperm determined with the
use of the supravital stain during the routine semen analysis
(19).
Although the traditional semen analysis parameters of
sperm count, motility, and morphology have been criticized
severely for their lack of correlation with sperm function and
fertilizing capacity, little has been done to improve this
problem (19). Adding the evaluation of acrosomal status to
the semen analysis provides some improvement with mini-
mal effort. The 31% incidence of failed fertilization in men
with low percentages of intact acrosomes suggests that the
Spermac stain cannot predict with 100% accuracy and is not
meant to be used alone. The collective interpretation of
FERTILITY & STERILITYt
several other sperm tests, such as strict criteria morphology,
is necessary. In addition, variations in sperm quality occur
with different ejaculates, so repeated testing may be required
(19).
The consistent identification of male infertility factors in
sperm specimens using the Spermac stain as part of routine
semen analyses would assist clinicians in deciding between
conventional infertility treatments and the need for ICSI.
Further, the detection of sperm with low percentages of
intact acrosomes would trigger a thorough evaluation for
possible round-headed sperm syndrome.
In summary, our findings suggest that the minimal extra
effort expended for sperm acrosome evaluation may help
improve the traditional semen analysis for the detection of
male factor infertility.
Acknowledgments: The authors thank members of the Loma Linda Univer-
sity Gynecology and Obstetrics Medical Group, Inc., and the Loma Linda
University Center for Fertility and In Vitro Fertilization, for their help and
support in the preparation of this manuscript.
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Vol. 72, No. 1, July 1999