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I
FERTILITY AND STERILITY Vol. 62, No. 6, December 1994
Copyright © 1994 The American Fertility Society Printed on acid-free paper in U S. A.

Sperm recovery and survival: two tests that predict in vitro

fertilization outcome

Dale W. Stovall, M.D.t Joel S. Krasnow, M.D.

David S. Guzick, M.D., Ph.D. Anthony J. Zeleznik, Ph.D.
Sarah L. Berga, M.D.

Division of Reproductive Endocrinology, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh
School of Medicine, Magee- Womens Hospital, Pittsburgh, Pennsylvania

Objectives: To determine if human sperm recovery during swim-up and sperm survival after 24
hours, as obtained from a screening semen specimen, are predictive of subsequent IVF and clinical
pregnancy rates (PRs) and to determine if these techniques can identify men with normal semen
analysis parameters and poor IVF success.
Design: Historical prospective study.
Setting: All semen evaluations and IVF cycles were performed at the University of Pittsburgh,
Magee-W omens Hospital, Pittsburgh, Pennsylvania.
Patients, Participants: Couples undergoing IVF at Magee-W omens Hospital from August
1988 through June 1993.
Interventions: A screening semen analysis and swim-up procedure were performed on all cou-
ples undergoing IVF. The number of spermatozoa recovered after swim-up and the percentage of
motile spermatozoa present after a 24-hour incubation were recorded.
Main Outcome Measures: Fertilization and PRs were compared according to the parameters
obtained from routine semen analysis, the number of spermatozoa obtained with swim-up, and the
percentage of motile spermatozoa at 24 hours.
Results: Using x 2 or Fisher's exact test, fertilization rates were significantly different according
to the number of spermatozoa recovered after swim-up (:s;2.0 and >2.0 X 106 spermatozoa recov-
ered, 48.3% versus 71.4%) as were PRs (16.9% versus 29.8%). Similarly, the percentage of motile
spermatozoa present at 24 hours (:s;20% and >20%) discriminated between fertilization rates
(45.9% versus 65.8%) and PRs (16.4% versus 36.5% ). Among a subset of men with normal semen
analyses and total motile sperm counts~ 40 X 106 , the results from swim-up and survival discrimi-
nated between men with high and low fertilization and PRs. Receiver operating characteristic
analysis revealed that swim-up results better discriminated between pregnant and nonpregnant
IVF patients than sperm motility, but that the percentage of motile spermatozoa present at 24
hours was no better in this regard than sperm motility.
Conclusions: The number of spermatozoa recovered after swim-up and the percentage of sper-
matozoa that maintain their motility after 24 hours were both helpful in assessing IVF and PRs and
may be helpful in altering physicians to a subset of men having normal semen analysis parameters
yet poor IVF success. Fertil Steril1994;62:1244-1249

Key Words: Spermatozoa, in vitro fertilization, clinical pregnancy rates, swim-up, sperm sur-
vival, receiver operating characteristic analysis

Received April 1, 1993; revised and accepted June 30, 1994.
* Presented at the 48th Annual Meeting of The American Fer-
tility Society, New Orleans, Louisiana, October 31 to November
5, 1992.
t Reprint requests: Dale W. Stovall, M.D., Department of Ob-
stetrics, Gynecology, and Reproductive Sciences, Magee-
Womens Hospital, 300 Halket Street, Pittsburgh, Pennsylvania
15213 (FAX: 412-641-1133).
Several methods of semen analysis have been
used to help predict the outcome of male infertility
treatment IVF. Included are some semen analysis
as defined by the World Health Organization
(WHO) (1), the sperm penetration assay (SPA) us-
ing zona-free hamster oocytes, evaluation of the
acrosome reaction, the human sperm hypo-osmotic

1244 Stovall et al. Sperm recovery and survival Fertility and Sterility
swelling test, the hemizona assay (HZA), and com-
puter-aided sperm analysis (CASA) (2, 3). Exclud-
ing the human sperm hypo-osmotic swelling test
and WHO semen analysis, which may not reflect
the fertilizing ability of human spermatozoa (4),
each of these tests requires expensive laboratory
equipment, significant time expenditure, and/or
highly trained laboratory personnel.
A swim-up procedure is often used in IVF to seg-
regate motile and immotile spermatozoa. This tech-
nique requires minimal technician time and can eas-
ily be performed by andrology personnel. The
ability of a spermatozoa to "swim" through a col-
umn of culture media within a given time may be
indicative of its cell velocity and trajectory. Be-
cause sperm motility is important not only for
sperm transport but in sperm -egg interaction, one
might anticipate that this information would be
predictive of IVF success.
Moreover, to fertilize an oocyte in vivo, sperma-
tozoa must sustain their motility until they have
become capacitated and have reached the portion of
the fallopian tube containing the egg. Under culture
condition, spermatozoa obtained by swim-up can
maintain their motility for many hours. In human
spermatozoa incubated for 48 hours, a correlation
can be demonstrated between the length of sperm
survival and fertilization rates (5). Like the swim-
up test, assessment of sperm motility after incuba-
tion is a simple and easily repeated procedure. One
might suspect that the ability of spermatozoa to
maintain their motility for an extended time would
be predictive of IVF. The purpose of this study was
twofold: [1] to determine iftwo routine assessments
obtained at a screening semen evaluation, recovery
of spermatozoa after swim-up, and 24-hour motility
are predictive of subsequent IVF and clinical preg-
nancy rates (PRs) and [2] to determine ifthese tech-
niques can identify men with normal semen analy-
sis parameters but poor IVF success.
MATERIALS AND METHODS
Patient Population
Patients undergoing IVF at the University of
Pittsburgh, Magee-W omens Hospital from August
1988 through June 1993 were considered for inclu-
sion in the analysis. Couples with male factor infer-
tility (11.6%), endometriosis (14.5%), tubal disease
(67.4%), and unexplained infertility (6.5%) were
Vol. 62, No.6, December 1994
included. Men with severe oligospermia (sperm
density < 1 X 106 /mL), azoospermia, previous va-
sectomy, prostatitis, retrograde ejaculation, and se-
men volume < 1.0 mL were excluded (57 of 674,
8.5%). Patients with residual oocytes available for
fertilization after gamete intrafallopian transfer
were also excluded from the analysis.
Semen Collection and Analysis
Semen samples were obtained for a screening
analysis within 6 weeks of oocyte retrieval. The
men were instructed to abstain from ejaculation for
3 days before collecting a semen sample. All sam-
ples were obtained by masturbation and collected in
a sterile specimen container (Fisher Scientific,
Norcross, GA). After liquefication, a routine semen
analysis including semen volume, sperm count, per-
centage of motile spermatozoa, and sperm morphol-
ogy was performed as outlined in the WHO labora-
tory manual (1).
A swim-up was accomplished as follows. Two
milliliters ofT6 culture media (made in-house) sup-
plemented with Albuminar (10% by volume; Alpha
Therapeutic Corporation, Los Angeles, CA) were
placed in a 12 x 75-mL round-bottom tube (Falcon,
Lincoln Park, NJ). Then, to avoid mixing semen
and culture media, 1 mL of liqueified semen was
gently placed in the bottom of the column of media.
Semen samples that were not completely liqueified
and homogeneous within 60 minutes of collection
were gently pushed through a 21-gauge injection
needle before preparing the swim-up. To increase
surface area, the specimen tubes were placed at a
45° angle and incubated for 90 minutes at 37°C in
5% C0 2 • A sperm count and assessment of sperm
motility were performed on the upper 0.5 mL of
media. The mean percentage of motile spermatozoa
recovered after swim-up was virtually always
~90%. To evaluate the preservation of sperm motil-
ity, the top 0.5-mL aliquot of media was incubated
for an additional 24 hours at 37°C in 5% C0 2 • The
percentage of motile spermatozoa was re-evaluated
after 24 hours.
In Vitro Fertilization Cycle and Evaluation
of Outcome
Ovarian stimulation was obtained by administra-
tion of leuprolide acetate (Lupron; Tap Pharma-
ceutical, Inc., Chicago, IL) in a midluteal phase, fol-
lowed by hMG and FSH (Pergonal or Metrodin;
Stovall et al. Sperm recovery and survival 1245

Serono Laboratories, Inc., Randolph, MA) once
menstrual bleeding occurred and ovarian suppres-
sion was confirmed. Oocytes were recovered using
transvaginal ultrasound guidance approximately 36
hours after the administration of hCG, 10,000 IU.
Oocytes were graded on a 1 to 5 scale as previously
described (6). Only mature eggs were used to assess
fertilization success. Each mature egg was insemi-
nated with 150,000 motile spermatozoa. A clinical
pregnancy was defined as an intrauterine gesta-
tional sac as identified by transvaginal ultrasonog-
raphy or the presence of chorionic villi from uterine
currettings in the case of a spontaneous abortion.
Data Analysis
Fertilization of mature oocytes and clinical preg-
nancy were the outcome variables under study. The
x 2 test was used to assess differences in clinical
pregnancy and fertilization rates according to cate-
gories of semen analysis parameters, the number of
spermatozoa recovered after swim-up, and the per-
centage of motile spermatozoa present after 24-
hour incubation. Fisher's exact test was used when
expected values were <5.
Because cutoff values for any semen analysis pa-
rameter (e.g., :s; or >50% sperm motility) are essen-
tially arbitrary, receiver operating characteristic
(ROC) curves were generated for each semen analy-
sis parameter in regards to PRs. Using this method-
ology, the true positive fraction (i.e., sensitivity) is
plotted against the false-positive fraction (i.e., 1 ·
specificity). Each ROC curve indicates the trade-
offs between sensitivity and specificity for a given
test and is a measure of the performance of the test
over the entire range of values. Here, the ROC anal-
ysis was used to determine the ability of a given test
to discriminate between those who did and did not
conceive from IVF. Areas under the curve (AUC)
for two ROC curves were compared using a statisti-
cal test described by Metz et al. (7) and pro-
grammed for a personal computer.
RESULTS
Six hundred seventeen couples were included in
the analysis. The women in the study ranged in age
from 26 to 44 with a mean (±SE) of 34.6 ± 3.8 years.
An average of 3.2 embryos were transferred per cy-
cle. The mean pregnancy and fertilization rates
were 27% and 68%, respectively.
Analysis of PRs and fertilization rates of mature
oocytes in relation to screening semen analysis pa-
rameters revealed significant differences with re-
1246 Stovall et al. Sperm recovery and survival
1
spect to motility but not count nor morphology.
Cross-tabulation of fertilization rates and PRs by
deciles of percent motility suggested a threshold
value of 30% motility: FR for :::;30% and >30% mo-
tility were 49.0% and 66.7%, respectively (x 2 = 48.0,
P < 0.0001). Pregnancy rates for :s;30% and >30%
motility were 18.1% and 27.2%, respectively (x 2
= 3.7, p = 0.053).
Pregnancy rates and fertilization rates of mature
oocytes based on the number of spermatozoa recov-
ered after swim-up and the percentage of motile
spermatozoa at 24 hours are shown in Table 1.
Cross-tabulation of fertilization rates and PRs by
0.5 X 106 motile sperm increments suggested thresh-
old values of 0.5 and 2.0 X 106 motile spermatozoa
recovered after swim-up. Pregnancy rates and fertil-
ization rates showed a significant, stepwise incre-
ment as the number of spermatozoa recovered after
swim-up increased (x 2 = 14.2, P < 0.001 and x 2
= 145.0, P < 0.0001, respectively). Pregnancy and
fertilization rates also showed a significant, step-
wise increment as the percentage of motile sperma-
tozoa at 24 hours increased, using threshold values
of 20% and 80% as determined by cross-tabulation
of fertilization rates and PRs by deciles of 24-hour
motility (x 2 = 6.4, P < 0.04 and x2 = 53.4, P
< 0.0001, respectively).
To determine if the information obtained from
swim-up and 24-hour sperm survival was helpful in
assessing couples with normal semen analysis pa-
rameters, a subset of couples whose husbands had
normal semen analyses and total motile sperm
counts ;:;:: 40 X 106 were isolated. Table 2 lists the
PRs and fertilization rates for men with total mo-
tile sperm counts ;:;:: 40 X 106 based on the results
found during swim-up and 24-hour incubation. The
fertilization rates were significantly different ac-
cording to both the swim-up and 24-hour incuba-
tion data (x 2 = 9.48, P < 0.009 and x 2 = 11.389, P
< 0.003, respectively). Pregnancy rates in the sam-
ple of men with total motile spermatozoa ;:;:: 40 X 106
were significantly different based on the number of
spermatozoa recovered during swim-up (P < 0.002,
Fisher's exact test) but not the percentage of motile
spermatozoa present at 24 hours (x 2 = 3.025, P
= 0.220).
Receiver operating characteristic curves for
pregnancy were estimated for sperm motility, the
number of spermatozoa recovered after swim-up,
and the percentage of motile spermatozoa at 24
hours. There was a significant difference between
the curve generated for the number of spermatozoa
recovered after swim-up (AUC index= 0.5711) and
Fertility and Sterility
Table 1 Clinical Pregnancy and Fertilization Rates Based on Swim-up and 24-Hour Sperm Motility for Couples Undergoing IVF

No. of motile sperm

in upper 0.5 mL (X106 ) 24-h motility (%)

0.1 to 0.4 0.5 to 2.0 >2.0 :o;20 21 to 80 z80

Clinical pregnancy/retrieval 9/81 25/120 124/416 11/67 55/239 92/311

(PR) (11.1)* (20.8)* (29.8)* (16.4)t (23.0)t (29.6)t
Fertilization of mature oocytes 134/341 265/485 1,142/1,600 128/279 531/862 882/1285
(fertilization rate) (39.3)* (54.6)* (71.4)* (45.9)* (61.6)* (68.6)*

* P < 0.001. Values in parentheses are percents. t p < 0.04.

the curve for sperm motility (AUC index= 0.5238,
P < 0.028). There was no difference in the area
indexes between the percentage of motile sperma-
tozoa at 24 hours (AUC index= 0.5596) and motil-
ity (AUC index = 0.5238, P = 0.148).
DISCUSSION
Our data indicate that the number of spermato-
zoa obtained from a screening swim-up and the per-
centage of motile sperm at 24 hours are predictive
of subsequent fertilization rates and PRs in IVF.
Even in patients with normal screening semen anal-
ysis and ~40 X 106 motile spermatozoa, these two
tests could delineate between patients with high
and low fertilization rates and PRs. In the subset of
patients with normal semen analysis parameters,
the PR for those with 24-hour motility ~ 80% was
double that of those with 24-hour motility :::; 20%,
but this difference did not reach statistical signifi-
cance because of low sample size.
Comparisons between different semen analysis
parameters using either a x2 or Fisher's exact test
require the derivation of cut-points. These values
can be selected in many ways but in the end are
usually value judgments. If a given cut-point is
strict, then there will be many false negatives (with
male factor infertility cases missed), and if the cut-
point is relaxed, the false-positive fraction will rise.
We used ROC analysis, which assesses the perfor-
mance of a test over the entire range of values. This
analysis showed the number of spermatozoa recov-
ered at swim-up to discriminate better between
those who did and did not conceive than motility by
WHO criteria. However, the ROC curves generated
for the percentage of motile spermatozoa at 24
hours and the percentage of motile spermatozoa by
WHO criteria were not significantly different.
Therefore, each of these three measures (the per-
centage of motile spermatozoa by WHO criteria,
the percentage of motile spermatozoa at 24 hours,
and the number of spermatozoa recovered at swim-
up) discriminated between subjects who did and did
not conceive, but the number of spermatozoa recov-
ered at 24 hours was best according to ROC anal-
ysis.
Although several tests are available to screen
male infertility patients undergoing IVF, no single
test or parameter has consistently proven to be
most useful. In some studies, the percentage of mo-
tile spermatozoa obtained from a routine semen
analysis appears to correlate best with fertilization
success or failure (8, 9). Others have found that
sperm morphology is the more reliable index of IVF
success (10, 11). Using CASA, Holt et al. (12) was
able to correlate sperm velocity measurements with
successful human oocyte penetration. Conversely,
Grunert et al. (13) reported that sperm motility and

Table 2 Clinical Pregnancy and Fertilization Rates of Couples Based on Results of Swim-up and Percentage of Motile
Spermatozoa at 24 hours in a Subset of Men Whose Routine Semen Analysis Revealed a Total Motile Sperm Count z 40 X 106

No. of motile sperm

in upper 0.5 mL (X106 ) 24-h motility(%)

0.1 to 0.49 0.5 to 2.0 >2.0 :o;20 21 to 79 z80

Clinical pregnancy /retrieval

(PR) 0/3 (0.0)* 9/48 (18.7)* 75/179 (29.5)* 3/22 (13.6) 30/97 (30.9) 51/190 (26.8)
Fertilization of mature oocytes
(fertilization rate) 6/12 (50.0)* 101/152 (86.5)* 644/850 (75.8)* 47/75 (62.7)* 245/308 (79.5)* 465/649 (71.6)*

* P < 0.01. Values in parentheses are percents.

Vol. 62, No.6, December 1994 Stovall et al. Sperm recovery and survival 1247
morphology obtained from routine semen analysis
were more predictive of IVF success than sperm ve-
locity measurements calculated by CASA. Bioas-
says such as the SPA, HZA, and human sperm
hypo-osmotic swelling test, have been developed to
evaluate IVF success. Again, these tests may (14) or
may not (15) be useful in assessing IVF.
We evaluated two sperm tests that are easy to
perform and more universally available than
CASA, SPA, or the HZA. Although the swim-up
and survival tests are technically simple, they are
more dynamic than a routine semen analysis.
These tests measure two important physiological
events: the quality of sperm mbtion and sperm lon-
gevity.
Of the tests previously mentioned, only CASA
directly evaluates sperm motion characteristics.
Using CASA motion parameters such as straight
line velocity, a significant difference between a fer-
tile and sub fertile population of men can be as small
as 10.0 f.lm/s with speeds from 24.2 to 34.2 f.lm/s
(12). The swim-up procedure evaluates sperm mo-
tion characteristics by determining the number of
cells that progressed to the top of the swim-up me-
dia within a given time interval. This system is less
precise. Both gravity and seminal viscosity may im-
pede sperm progression, and some spermatozoa
may travel to the top of the swim-up media several
times. These two techniques may evaluate very dif-
ferent sperm movement characteristics. Whether
the number of spermatozoa recovered from a swim-
up procedure correlates with sperm motion charac-
teristics such as cell velocity and trajectory remains
to be investigated.
Sperm longevity or survival is not directly mea-
sured by any of the semen analysis methods previ-
ously mentioned. However, an analysis similar to
the one reported here was evaluated by Fuse (5).
Spermatozoa from oligospermic men were incu-
bated for varying time intervals from 6 to 48 hours.
The survival test was interpreted as positive if only
motile sperm were present after a given incubation
period and negative if only immotile sperm re-
mained. A significant difference in fertilization
rates was found between patients with positive and
negative 36-hour sperm survival tests. Although
the fertilizatin rate reported by Fuse for a positive
sperm survival test at 24 hours (68.1%) compares
well with the fertilization rate we found for ~80%
motility at 24 hours (68.6%), we believe our system
is more user friendly. In an active IVF program, a
single assessment of motility at 24 hours is much
less time-consuming.
1248 Stovall et al. Sperm recovery and survival
Several aspects of our swim-up procedure deserve
discussion. In general, to be considered a fair test,
spermatozoa from each sample should have an
equal opportunity to reach the top of the swim-up
media. Factors that influence this system include
packing or crowding of cells, semen consistency,
and mixing of semen and culture media. We did not
perform a sperm wash or centrifugation before pre-
paring the swim-up. This avoids packing cells into a
small pellet, which may interfere with sperm motil-
ity. Some semen samples did not display a normal
consistency. These samples were gently pushed
through a 21-gauge injection syringe to help break
up the sample and reduce the impedance to motility
caused by a viscous plasma. Placing liquefied semen
at the bottom of a column of media helps to avoid
mixing of the two solutions but offers little advan-
tage over gently overlaying semen with culture me-
dia. Finally, by avoiding centrifugation, one can
avert the inconsistency presented by the need to
discard a supernate and disturbing the underlying
pellet.
The data in this study suggest that evaluation of
semen within 6 weeks of a subsequent IVF cycle for
recovery of spermatozoa after swim-up and for the
percentage of motile spermatozoa at 24 hours can
serve as a useful adjunct to the screening semen
analysis. In a population of patients with normal
semen analysis parameters, the results obtained
from swim-up and 24-hour incubation could delin-
eate between patients with high and low fertiliza-
tion rates. Further study is needed to compare these
methods with the more technically difficult and ex-
pensive analyses such as CASA and the HZA.
Acknowledgment. This program (CLABROC) was graciously
provided by Charles E. Metz, Ph.D., Department of Radiology,
The University of Chicago, Chicago, Illinois.
REFERENCES
1. World Health Organization. WHO Laboratory manual for
the examination of human semen and semen-mucus inter-
action. 2nd ed. Cambridge: The Press Syndicate of the Uni-
versity of Cambridge, 1987:27.
2. Smith RG, Johnson A, Lamb D, Lipshultz Ll. Functional
tests of spermatozoa. Urol Clin North Am 1987;14:451-8.
3. Collins JA. Diagnostic assessment of the infertile male
partner. Curr Probl Obstet Gynecol Fertil1987;10:173-224.
4. Aitkin RJ, Best FSM, Warner P, Templeton A. A prospec-
tive study of the relationship between semen quality and
fertility in cases of unexplained infertility. J Androl
1984;11:369-78.
5. Fuse M. Sperm survival test assessing the change of sperm
Fertility and Sterility
 motility after long-term incubation. Nippon Sanka Fujioka
Gakkai Zasshi 1990;42:1678-84.
6. Ben-Rafael Z, Kopf GS, Blasco L, Flickinger GL, Tureck
RW, Strauss JW, et al. Follicular maturation parameters
associated with the failure of oocyte retrieval, fertilization
and cleavage in vitro. Fertil Steril1986;45:51-7.
7. Metz CE, Kronman HB. Statistical significance tests for
binormal ROC curves. J Math Psycho! 1980;22:218-43.
8. Talbert LM, Hammond MG, Halme J, O'Rand M, Fryer
JG, Ekstrom RD. Semen parameters and fertilization of hu-
man oocytes in vitro: a multivariable analysis. Fertil Steril
1987;48:270-7.
9. Mahadevan MM, Trounson AO. The influence of seminal
characteristics on the success rate of human in vitro fertil-
ization. Fertil Steril 1984;42:400-5.
10. Kruger TF, Acosta AA, Simmonds KF, Swanson RJ, Matta
JF, Oehninger S. Predictive value of abnormal sperm mor-
phology in in vitro fertilization. Fertil Steril 1988;49: 112-7.
11. Comhaire FH, Vermeulen L, Hinting A, Schoonjans F. Accu-
Vol. 62, No.6, December 1994
racy of sperm characteristics in predicting the in vitro fertil-
izing capacity of semen. J In Vitro Fert Embryo Transf
1988;5:326-31.
12. Holt WV, Moore HDM, Hillier SG. Computer-assisted mea-
surement of sperm swimming speed in human semen: corre-
lation of results with in vitro fertilization assays. Fertil
Steril1985;44;112-9.
13. Grunert JH, DeGeyter C, Bordt J, Schneider HPG, Niesch-
lag E. Does computerized image analysis of sperm move-
ment enhance the predictive value of semen analysis for in
vitro fertilization results? Int J Androl1989;12:329-38.
14. Oehninger S, Coddington CC, Scott R, Franken DA, Burk-
man LJ, Acosta AA, et al. Hemizona assay: assessment of
sperm function and prediction of in vitro fertilization out-
come. Fertil Steril1989;51:665-70.
15. Chan SYW, Wang C, Chan STH, Ho PC. Differential evalu-
ation of human sperm hypoosmotic swelling test and its re-
lationship with the outcome of in-vitro fertilization of hu-
man oocytes. Hum Reprod 1990;5:84-8.
Stovall et al. Sperm recovery and survival 1249