Cervical Mucus Analysis:
A Major Component in
Evaluation of Infertility
Tech Com:
Special Topics by Brooks A. Keel, B.S.
Male and female factors both con-
tribute to infertility. Cervical mucus-
sperm incompatibility can be a major
factor in infertility, and the analysis of
cervical mucus can be an important
tool in the evaluation of the infertile
couple, especially when examination
reveals that the male partner is ap-
parently normal. Several diagnostic
techniques used in the evaluation of
cervical mucus and of sperm-mucus
interactions are described. These
techniques, which can be performed
in any clinical laboratory, can greatly
assist the referring physician in mak-
ing a proper diagnosis and prognosis
when treating a couple for infertility.
(Key words: Cervical mucus; Infertil-
ity; Sperm-mucus interactions.)
Introduction
Before e n t e r i n g the h u m a n
uterus, sperm must first pass
through the uterine cervix. The
cervical mucus plays an active role
in facilitating this passage. Nor-
mally, both the cervix and the cer-
vical mucus undergo m o n t h l y
changes that reflect the different
phases of the hormonal cycle. The
hormonal changes responsible for
o v u l a t i o n normally cause the
mucus to become optionally re-
ceptive to sperm penetration.
Abnormal changes in mucus can
adversely influence the passage of
sperm into the uterus. Cervical
m u c u s - s p e r m incompatibility has
From the Tissue Bank and Clinical Laboratory,
XYTEX Corporation, Augusta, Georgia.
0007-5027/81/0300/161 $00.70 American Society of Clinical Pathologists
been suggested as a causative fac-
tor in 15% t o 50% of infertile
couples. 1 Therefore, analysis of
cervical mucus has become an im-
portant diagnostic tool in the eval-
uation of infertile couples, espe-
cially when examination of the
male partner shows no abnormality.
This article describes the meth-
ods that we use for routine cervical
mucus analysis. Along with a com-
plete semen analysis, this evalua-
tion can provide the physician with
valuable information concerning
the possible cause and treatment
of the infertile couple.
Monthly Changes in Cervical
Mucus
Cervical mucus undergoes dra-
matic changes during the men-
strual cycle. Hormones secreted
by the ovaries affect the structure
and secretory activity of the cervix,
resulting in both quantitative and
qualitative changes in cervical m u -
cus. 2 D u r i n g the preovulatory
(follicular) phase of the normal
menstrual cycle, increasing levels
of estrogen influence the cervix to
produce copious amounts of t h i n ,
watery mucus. By midcycle (around
the time of ovulation), the quality
and quantity of cervical mucus is
optimal for sperm penetration.
During the postovulatory (luteal)
phase of the menstrual cycle, the
levels of estrogen decrease and
progesterone begins to increase,
causing the cervix to produce
scanty amounts of thick, viscous
mucus. The penetrability of sper-
matozoa through the cervical m u -
cus is greatly inhibited during the
luteal phase. 2
Specimen Collection
Cervical mucus normally should
be examined at the time of ovula-
t i o n , when one w o u l d ordinarily
expect intercourse to result in
pregnancy. Ovulation may be de-
tected in a variety of simple ways,
one of which is a shift in basal
body temperature. Most experts
agree that cervical mucus should
be collected 24 t o 48 hours before
the temperature shift. 1 3
After the patient is prepared for
pelvic examination, cervical mucus
can be aspirated directly from the
endocervix using a tuberculin
syringe (without the needle). If the
cervix cannot be reached with the
syringe alone, a plastic tip may be
attached to the syringe. 3 Since the
characteristic ferning pattern of
the cervical mucus seen during
ovulation (described below) is due
in part to an increase in salt ions,
any apparatus used to collect the
mucus should be free of saline to
prevent false results. 4 After the mu-
cus has been collected, it may be
sent to the laboratory immediately
for analysis or it may be stored in
an ordinary refrigerator freezer
(-10C to -20C) for later analy-
sis. We have so stored mucus
specimens in the collection syringe
161
 cm case, the cervical mucus). There-
fore, a complete cervical mucus
7
analysis must include analysis of
the interactions of sperm and
6
mucus. For the following proce-
I l l
5 dures, sperm should be collected
from the male partner after a three-
A
4 day sexual abstinence. The ejacu-
D late should then be evaluated as
3
described previously. 7 If the part-
ner's semen is unavailable, semen
2
from a donor of proven fertility
1 may be used. The diagnostic value
of donor semen is substantially
0 less than the value of partner
semen.

3
Fig. 1. Measurement of cervical mucus spinnbarkeit:
(B) microscope slide; (C) cervical mucus extruded
syringe.
for several weeks w i t h o u t any
noticeable detrimental effects.
Gross Examination
Spinnbarkeit
Spinnbarkeit, an index to the
viscoelasticity or "threading capac-
ity" of cervical mucus, can be eval-
uated easily by extruding a small
portion of the mucus from the
syringe onto a clean, dry glass slide
(Fig. 1). The extruding portion of
the mucus is held on the slide w i t h
a coverslip, and the syringe is raised
to measure the maximum thread-
ing capacity of the mucus. This
height is measured with a centi-
meter ruler which is held vertically
behind the slide. 1 A spinnbarkeit
of 5 - 6 cm or more has been ac-
cepted as normal for ovulatory
mucus. 1 ' 5
Because variability in the ex-
truded volume of cervical mucus
can affect accurate measurement
of spinnbarkeit, it is important to
standardize the volumes by ex-
truding the same volume of mucus
from the syringe each time spinn-
barkeit is measured.
(A) centimeter ruler;
from (D) tuberculin
Ferning
When a drop of cervical mucus
obtained at midcycle from a nor-
mal patient is spread on a clean,
dry glass slide and allowed to dry,
a characteristic palm-leaf or fern-
ing effect (Fig. 2) can be seen un-
d e r t h e microscope. 6 Although the
criteria for quantifying the degree
of ferning are subjective, 3 ferning
is usually graded on a scale of 1 +
to 4 + ; the number rating repre-
sents the number of right angles
in the f e r n , with 4 + representing
maximum branching with four
right angles (Fig. 3).
Normal midcycle cervical mucus
should display good ferning with
3+ to 4 + branching. A low degree
of ferning or an irregular or " p u f f y "
appearance may indicate abnor-
mal cervical mucus and should be
reported.
Analysis of Sperm-Mucus
Interactions
Because infertility can be caused
by either sexual partner, an ac-
curate evaluation of the problem
can be accomplished only by eval-
uating both the male factor (se-
men) and female factors (in this
Slide Test
The slide test 1 8 is a simple quali-
tative evaluation of the ability of
sperm to penetrate cervical mucus.
A drop of semen and a drop of
cervical mucus are placed on a
clean, dry glass slide and then care-
fully brought together by placing
a coverslip on t o p , thus f o r m i n g
an interface between the sperm
and mucus. In a normal interface,
one is able to observe under the
microscope sperm migration
across the interface from the
seminal plasma into the mucus.
Results should be reported as pen-
etrable or nonpenetrable mucus.
CapiJJary-Tube Test
The quantitative capillary-tube
method of measuring the ability of
sperm to penetrate cervical mucus,
first described by Kremer, 9 utilizes
an easily constructed sperm pene-
tration meter (Fig. 4). A sperm
reservoir is made from a glass or
plastic test tube 7 cm long and 7
mm wide by longitudinally bisect-
ing the test tube using a diamond-
point pencil and retaining a 1-cm
" h e m i c u p " from the bottom of the
bisected tube. This hemicup is
glued to a clean glass slide to make
the reservoir. A glass capillary tube
2 cm long is then glued onto the
slide 1 cm from the aperture of
the reservoir and parallel to the
short side of the slide. The slide

162 LABORATORY MEDICINE VOL 12, NO. 3, MARCH 1981

can then be calibrated with a centi-
meter scale using ink.

To perform the capillary-tube

test, mucus is drawn into a capil-
lary tube. A drop of mucus is al-
lowed to f o r m at one end of the
capillary tube and the other end is
sealed with clay. The capillary tube
is then placed onto the penetra-
tion meter (Fig. 4) so that the mucus
drop comes into contact with the
semen that has been placed in the
reservoir. The capillary is then se-
cured to the meter with clay. The
meter is placed horizontally in an
incubator at 37C for three hours.
After the incubation p e r i o d , the
meter is placed under a microscope
and examined using low power. ' ! 3^>v
The distance that the sperm travels
Fig. 2. Photomicrograph demonstrating a typical 3-4+ cervical mucus
up the tube of mucus in three
ferning pattern.
hours is then recorded. It has been
suggested that the lower limit of
normal for this test is 20 mm/3 h,
and the lower limit for fertility is
6 mm/3 h. 5 1 0

Sperm-Mucus Immobilization
Test
The s p e r m - m u c u s immobiliza-
tion test is performed to determine
if any factors (e.g., chemical or
immunologic) are present in the
cervical mucus that may cause im-
mobilization of the sperm. To per-
form this test, a drop of semen
and a drop of mucus are mixed on
one end of a glass slide and cov-
ered with a coverslip. Another
drop of semen is placed at the op-
posite end of the slide and also
covered with a coverslip to serve
as a test control. Both drops of
semen are then evaluated micro-
scopically for percent motility by
examining ten high-power fields
(hpf) and estimating the percent-
age of motile sperm. The slide is
then placed in an incubator at 37C
for 30 minutes, and reexamined
after incubation for percent mo-
tility. The motility of the control
drop of semen is compared with the
s p e r m - m u c u s mixture, and any Fig. 3. Simplified diagram demonstrating the rating system used to grade
decrease in motility is recorded. cervical mucus ferning. The grade number (1 + to4 +) represents the num-
Chemical factors in the mucus or ber of right angles on the fern.

LABORATORY MEDICINE VOL. 12, NO. 3, MARCH 1981 1 6 3

 in the seminal plasma may cause a
simple decrease in motility. How-
ever, if a decrease in motility is
accompanied by sperm aggrega-
t i o n , one must consider i m m u n o -
logic factors.
Sims-Huhner Postcoital TestfPCTJ
The Sims-Huhner postcoital test
(PCT), a classical procedure in the
testing of the infertile couple, is
currently subject to widespread
debate. The method of performing
and interpreting this test has not
yet been standardized. 1 The re-
sults of the test are affected by
timing, coital technique and semen
quality as well as cervical mucus
physiology. 3 However, this simple
test can be performed in the physi-
cian's office during a routine pelvic
examination.
Basically, the test involves sam-
pling the cervical mucus after inter-
course and examining the mucus
for the presence of motile sperm.
A great deal of controversy exists
concerning the timing of the test.
Most experts agree that the PCT
should be performed during mid-
cycle or ovulation. However, the
recommended time interval from
coitus to analysis ranges f r o m 90
seconds 11 to eight days. 12 We rec-
o m m e n d that the physician use
the PCT as an office technique
to determine if further laboratory
studies are warranted.
After the cervical mucus speci-
men has been obtained, a d r o p is
placed on a slide, covered with a
coverslip and examined micro-
scopically under high power. Five
hpf are examined, and the num-
ber and percent motility of sperm
in each field are recorded. The
mean number of sperm per hpf
and the mean percent motility are
then calculated.
Although the interpretation of
the results requires a great deal of
clinical j u d g m e n t , the normal
range for a PCT may be considered
to be the presence of 10-20 motile
sperm per hpf within 8-10 hours
164 LABORATORY M E D I C I N E VOL. 12, NO. 3, MARCH 1981
mality is indicated. However, if
Fig. 4. Kremer sperm penetration
meter: left, front view; right, side
view (see text).
after coitus. 1 A normal PCT will
demonstrate more than ten sperm
per hpf w i t h 50% motility, and an
abnormal PCT will show less than
five sperm per hpf with less than
50% motility.
Sperm-Mucus Crossmatch
If cervical mucus is found to be
nonpenetrable to spermatozoa, or
if the results of a PCT are below
the normal range, a s p e r m - m u c u s
crossmatch is indicated. This test
compares the s p e r m - m u c u s in-
teractions of the infertile couple
with that of fertile donors. The re-
sults of this test can yield valuable
information in determining if the
nonpenetrability is due to a factor
in the husband's semen, the wife's
mucus, or both.
The s p e r m - m u c u s crossmatch
can be performed by using either
the slide test or the Kremer capil-
lary test m e t h o d . Cervical mucus
and semen are obtained both f r o m
the infertile couple being evalu-
ated and from suitable donors.
Donor semen and donor mucus
should be of good quality and
preferably obtained from donors
of proven fertility.
After an initial analysis of the
semen and the mucus has been
evaluated, crossmatch testing is
performed as follows: 1) the hus-
band's sperm is tested w i t h the
wife's mucus; 2) the husband's
sperm is tested w i t h the donor's
mucus; 3) the donor's sperm is
tested with the wife's mucus; and
4) the donor's sperm is tested with
the donor's mucus. If the hus-
band's sperm will penetrate the
donor's mucus but not the wife's
mucus, a cervical mucus abnor-
the husband's sperm will not pen-
etrate either the wife's or the
donor's mucus, a semen abnor-
mality is indicated. The interpre-
tation of the crossmatch results
will be reflected in the outcome
of interaction of the d o n o r ' s
sperm and the wife's mucus.
References
1. Davajan, V., and Nakamura, R.M., 1975. The
cervical factor. In Progress of Infertility. 2nd
edition. Edited by Behrman, S.J., and Kistner,
R.W. Boston, LittleBrown and Company, p. 17.
2. Hafez, E.S.E., 1976. Sperm transport in the
human and mammalian cervix. In The Cervix.
Edited by Jordan, J.A., and Singer, A. Phila-
delphia, W.B. Saunders Company, p. 164.
3. Blasco, L, 1977. Clinical approach to the
evaluation of sperm-cervical mucus inter-
actions. Fertil. Steril. 28:1133.
4. Grant, A., 1958. Cervical hostility. Fertil.
Steril. 9:321.
5. Ulstein, M., 1972. Evaluation of a capillary
tube sperm penetration method for fertility in-
vestigations. Acta Obstet. Gynecol. Scand.
51:287.
6. Campos, duPaz, A., 1953. Crystallization
phenomenon of cervical mucus in the human
being and in animals. Proc. Internat. Fertil.
Assoc. 1:595.
7. Keel, B.A., 1979. The semen analysis: An
important diagnostic evaluation. Lab. Med.
10:686.
8. Perloff, W.H., and Steinberger, E., 1963. In
vivo penetration of cervical mucus by
spermatozoa. Fertil. Steril. 14:231.
9. Kremer, J., 1965. A simple sperm penetration
test. Int. J. Fertil. 10:201.
10. Ulstein, M., 1972. Sperm penetration of
cervical mucus as a criterion of male fertility.
Acta Obstet. Gynecol. Scand. 51:335.
11. Noyes, R.W., Adams, C.E., and Walton, A.,
1958. Transport of spermatozoa into the
uterus of the rabbit. Fertil. Steril. 9:288.
12. Fraenkel, L, 1930. Sterilisierung and Kozep-
tionsverhutung. Arch. Gynakol. 144:86.