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The Crystallization Test as a Guide to the

Treatment of Cervical Hostility

A. Campos do Paz, M.D.

IT HAS BEEN pointed out by many researchers that cervical mucus suffers
physicochemical modifications during the ovulation period which makes
it more receptive to spermatozoa. The studies of Seguy and Simmonet
( 1933) indicated that these modifications were associated with an increase
in estrogen excretion, an observation later confirmed by Moricard and Wat-
son. These investigators verified the increase of cervical mucus secretion,
its greater fluidity and receptivity to spermatozoa, following the use of
estrogen. Guttmacher and Shettles and Bennett in administering estrogen
to women after the menopause also observed the reappearance of an
abundant, translucent cervical secretion, of low viscosity. Abarbanel, in
studying women previously submitted to supracervical hysterectomy and
bilateral oophorectomy, also verified this action of the estrogens on the ( re-
maining) cervix. Palmer's studies of castrated women led him to conclude
that whereas estrogen stimulated cervical secretion, progesterone seemed to
have an inhibitory action.
In 1950 I described a new test for the diagnosis of cervical sterility based
on the presence or absence of fern-like crystallization of the cervical mucus,
which has been used since then without failure. I have also made studies
of the fern-like crystallization of cervical mucus at different phases of the
menstrual cycle, during pregnancy, and after the menopause and verified
the fact that the use of estrogen in women after the menopause, besides
stimulating the cervical secretion, promotes the appearance of fern-like
crystallization.
These studies have led me to the following conclusions:
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138 CAMPOS DA PAZ [Fertility & Sterility

1. The fern-like pattern of the cervical mucus is present in the first half
of the menstrual cycle and during the ovulation period. It is absent after
ovulation, during pregnancy, and after the menopause, at which times the
mucus exhibits only cellular content.

Fig. 1. The author's technic for the fernlike crystallization test of cervical mucus.
The ectocervix is initially cleaned with cotton or gauze. The mucus is then aspired
from the interior of the cervical channel spread on a glass slide, dried by heat, and
examined under the microscope at a magnification of 100 X. When we started to
investigate the fernlike phenomen in 1948, our technic was to wait for the mucus to
dry spontaneously at room temperature and examine it 24 hours after the material was
taken, as done by Rydberg. The observations reported in our first work (1950) resulted
from this technic. Later we used the heat of a flame or put the slide on the hot lid of
a sterilizer; the mucus dried more quickly and could be examined immediately under
the microscope. Three types of microscopic image were invariably obtained with this
technic: typical, atypical, and negative. Cervical mucus dried at room temperature did
not always give satisfactory results. The fernlike phenomenon is influenced by varia-
tions in humidity. The fernlike phenomenon, with its typical features, does not occur
when the atmosphere is saturated with water vapor.

2. Only during anovulatory menstrual cycles with normal estrogen level


can we find fern-like crystallization of the cervical mucus at any time of the
cycle.
Vol. 4, No. 2, 1953] FERN TEST OF CERVICAL MUCUS 139

3. Fern-like crystallization is closely related to the estrogen function.


Progesterone does not cause its appearance; on the contrary, it seems to
inhibit it.
4. Fern-like crystallization is closely related to the receptivity of the
cervical mucus to spermatozoa. As the crystallization loses its characteristic
features, the receptivity of the mucus diminishes. When the crystallization
is absent, the mucus, as a rule, is hostile to spermatozoa.

Fig. 2. Typical fernlike crystallization phenomenon of the cervical mucus


(100 per cent).

Maxwell Roland has recently published a paper suggesting this test for
the determination of ovulation, estrogen activity, and early pregnancy. All
of my findings were confirmed in his study.
Basing my work on the observation of other authors that the administra-
tion of estrogen increases the receptivity of cervical mucus to spermatozoa
and on my own observations of the appearance of fern-like crystallization
with the use of estrogen, I have tried to study the effects of estrogen in
140 CAMPOS DA PAZ [Fertility & Sterility

cases of cervical sterility due to hostility of the cervical mucus. Our first
results were presented to the Brazilian College of Surgeons. 6
The results obtained with this treatment in patients coming to our private
clinic are reported in this paper. Actually our experience is much wider
than the number of cases presented here indicates. We have treated approx-
imately 40 cases of cervical hostility at the Sterility Clinic of the Hospital

3 4
Fig. 3. The cervical mucus pattern before estrogen therapy of a case from Group I;
there is no fernlike crystallization. Fig. 4. The same case after 1 mg. of estrogen
has been administered on the fifth, sixth, and seventh days of the cycle. No effect is
visible; crystallization, negative.

dos Servidores do Estado, observing nearly 200 menstrual cycles. These


cases were not, for obvious reasons, included in the present paper.
The present study is based upon the use of estrogens in the first phase
of 215 menstrual cycles in 53 sterile women. All patients treated presented
a negative Sims-Huhner test during the ovulation period and absence of
the fern-like crystallization of the cervical mucus. Muscle injections of
Stradiol-benzoate were used in all cases.
The following treatment procedure was tried:
Group I: 1 mg. daily for 3 days, starting on the fifth day of the menstrual
cycle. Result: No effect in 10 cycles ( 0 per cent).
Vol. 4, No. 2, 1953] FERN TEST OF CERVICAL MUCUS 141

Group II: 1 mg. daily for 6 days, starting on the fifth day of the cycle.
Result: No effect in 10 cycles (0 per cent).
Group III: 2 mg. daily for 3 days, starting on the fifth day of the cycle.
Result: Atypical fern-like crystallization in 12 cycles ( 100 per cent atypical).
Group IV: 2 mg. during 5 days, starting on the fifth day of the cycle.
Result: Typical fern-like crystallization in 9 cycles ( 42.80 per cent) and
atypical in 12 ( 57.10 per cent); total, 21 cycles.

5 6
Fig. 5. Cervical mucus before estrogen therapy of a case from Group III. There is
no fernlike crystallization. Fig. 6. The same case after 2 mg. of estrogen have been
administered on the fifth, sixth, seventh, eighth, and ninth days of the cycle: Atypical
fernlike crystallization, low receptivity of the cervical mucus. The fern palms are not
typical. There are zones without the fern feature.

Group V: 3 mg. on the tenth day of the cycle. Result: Typical fern-like
crystallization in 4 cycles ( 30.70 per cent) and atypical in 9 ( 69.20 per
cent); total, 13 cycles.
Group VI: 5 mg. on the fifth day of the cycle. Result: Typical fern-like
crystallization in 20 cycles ( 48.70 per cent) and atypical in 21 ( 51.11 per
cent); total, 41 cycles.
Group VII: 5 mg. on the tenth day of the cycle. Result: Typical fern-like
142, CAMPOS DA PAZ [Fertility & Sterility

crystallization in 47 cycles ( 88.60 per cent) and atypical in 6 ( 11.30 per


cent); total, 53 cycles.
Group VIII: 5 mg. on the fifth and tenth days of the cycles. Result: Typi-
cal fern-like crystallization in 55 cycles ( 100 per cent).

7 8
Fig. 7. The cervical pattern before estrogen therapy of a case from Group V:
no fern crystallization. When expelled from the pipette, the mucus was very viscous
and was deposited as great air bubbles. Fig. 8. The same case after 3 mg. of
estrogen, have been administered on the tenth day of the menstrual cycle. Great air
bubbles show that the mucus is still very viscous. Fern crystallization appei1rs in some
zones only.

COMMENTS
We can conclude from this data that the use of 5 mg. of estrogen on the
tenth day of the cycle in certain cases and on the fifth and tenth day in
other cases were the schemes of treatment which gave the best results.
However, treatment should be individualized with the prime objective of
securing a normal cervical receptivity to spermatozoa with the smallest pos-
sible dose of estrogen.
The plan of treatment given below is being followed at present:
Initially, we try 5 mg. of estrogen on the fifth or tenth days of the cycle.
10
Fig. 9. The cervical mucus of a case from Group VII before treatment: no fern
crystallization. Fig. 10. The same case after 5 mg. of estrogen was administered
on the tenth day of the cycle: total and typical fernlike crystallization.

12
Fig. II. The cervical mucus before treatment: no fern phenomenon. Fig. 12.
The same case after 5 mg. of estrogen were administered on the fifth and tenth days
of the menstrual cycle: total and typical fernlike crystallization of the cervical mucus.
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Fig. 13. Menstrual calendar and basal body temperature (author's original diagram)
of a case from Group VII. March 3 (twelfth day of the menstrual cycle): Fern test
negative. March 5 (fourteenth day of the menstrual cycle): Sims-Huhner test negative;
Vol. 4, No. 2, 1953] FERN TEST OF CERVICAL MUCUS 145

If results are not satisfactory, we then give 5 mg. on both the fifth and tenth
days. With these doses, the receptivity of the mucus reappears and persists
during the ovulation period.
Basal body temperature curves were taken for all patients. None of these
have showed any changes which might suggest inhibition of ovulation or of
the formation of a corpus luteum.
We would also point out that estrogen gives results only in the menstrual
cycle in which it is used. In our experience, cervical hostility reappears in
the following cycle if estrogen is not used on the proper day. For this reason,
it is necessary to repeat treatment during every cycle until pregnancy occurs.
To date, ~ll tests designed to verify the receptivity of the cervical mucus
were based on the behavior of the spermatozoa in vivo or in vitro in the
presence of the cervical mucus. These tests cannot, by their nature, be
repeated too often. In certain cases, they cannot be made at all because of
the husband's refusal to co-operate.
With the fern-like crystallization test, we can verify at will the receptivity
of the cervical mucus to spermatozoa and control the exact dose of estrogen
to be used in the treatment of certain cases of cervical hostility. Its simplicity

Fern test negative. March 7 (sixteenth day of the menstrual cycle): Fern test negative.
April 3 (thirteenth day of the menstrual cycle): Fern test negative. April 5 (fifteenth
day of the menstrual cycle): Fern test negative. April 7 (seventeenth day of the menstrual
cycle): Fern test negative. April 27 (tenth day of the menstrual cycle): 5 mg. of estrogen
administered. May 3 (fifteenth day of the menstrual cycle): Sims-Huhner test positive;
Fern test positive, typical. May 5 (seventeenth day of the menstrual cycle): Fern test
positive, typical. May 26 (tenth day of the menstrual cycle): 5 mg. of estrogen adminis-
tered. June I (15th day of the menstrual cycle): Fern test positive, typical. June 26th (tenth
day of the menstrual cycle): 5 mg. of estrogen administered. June SO (fourteenth day of
the menstrual cycle): Fern test positive, typical. July 24 (tenth day of the menstrual
cycle): 5 mg. of estrogen. July 29 (fourteenth day of the menstrual cycle): Fern test
positive, typical. August 20 (tenth day of the menstrual cycle): 5 mg. of estrogen. August
25 (fifteenth day of the menstrual cycle): Fern test positive, typical. September 17
(tenth day of the menstrual cycle): 5 mg. of estrogen. September 22 (fifteenth day of
the menstrual cycle): Fern test positive, typical. October 12 (32 days after the begin-
ning of the last menstrual flow): Fern test negative (pregnancy?) October 28 (51 days
after the last menstrual period): Gailli-Mainini urinary test positive. The basal body
temp.erature during October, November, and December are typical of pregnancy. This
is a perfect illustrative case. Normal receptivity of the cervical mucus was obtained in
each cycle with 5 mg. of estrogen given every tenth day of the menstrual cycle without
any alteration of the basal body temperature curve or of the menstrual rhythm.
Pregnancy occurred in the sixth cycle of treatment.
146 CAMPOS DA PAZ [Fertility & Sterility

constitutes, in our opinion, a strong reason to include this test in the diag-
nostic routine of female sterility.
Of the 53 cases treated by this method, pregnancy has occurred in only 9.
These apparently poor results, however, are explained by the following
facts: 17 patients submitted to this treatment for 1 to 3 cycles only, aban-
doning it afterwards; 9 patients suffer from other causes of sterility, not
yet removed; 18 are still under treatment.
Where pregnancy was accomplished, fecundation occurred after the
menstrual cycle in which treatment by estrogen was. tried: 2 cases in the
fourth cycle of treatment; 3 cases in the fifth cycle of treatment; 2 cases in

AUGUST

Fig. 14. The basal body temperature curve of a case from Group VIII with 5 mg. of
estrogen used on the fifth and tenth days of a menstrual cycle. No effect is apparent.

the sixth cycle of treatment; 1 case in the eighth cycle of treatment; and
1 case in the tenth cycle of treatment.
An analysis of these results leads us to the conclusion that treatment must
be repeated during as many cycles as may be necessary until pregnancy
occurs. If the treatment does not give satisfactory results after at least 6 to 8
cycles, the couple must be studied further. Later study frequently reveals
other causes of sterility not previously diagnosed, which account for the
failure of this treatment. This has occurred in several of our cases, demon-
strating the need for close observation in all cases of conjugal sterility.

CONCLUSIONS
1. Estrogen constitutes another therapeutic weapon in many cases of
cervical hostility.
Vol. 4, No. 2, 1953] FERN TEST OF CERVICAL MUCUS 147

2. The usual dose to be used varies with the case; generally, 5 mg. of
estrogen are administered intramuscularly on the fifth or tenth day of the
menstrual cycles, or on both days.
3. The fern-like crystallization test of cervical mucus is the easiest and
simplest test capable of determining the exact dose of estrogen to be used
in each case.
4. Estrogen therapy, although not definitely removing cervical hostility,
is capable of producing the cervical receptivity necessary to the progression
of spermatozoa into the cervix in every cycle.
5. No effect in the basal body temperature curve or in the regularity of
the menstrual cycle was observed with the dose used.
Rua Sao Jose 50-4° andar, Rio de Janeiro, Brazil.

REFERENCES
1. ABARBANEL, A. R. Tr. Am. Soc. Study Steril. p. 46, 1946.
2. BENNETT, H. J. Am. ]. Obst. & Gynec. 44:296, 1942.
3. CAMPos DA PAz, A. 1 o Convenio Soc. Bras. Esteril., 1951.
4. CAMPOS DA PAz, A. Am.]. Obst. & Gynec. 61A:790, 1951.
5. CAMPOS DA PAz, A. III Congresso Bras. Ginec. e Obst., 1951.
6. CAMPOS DA PAz, A. Meeting of Dec. 17, 1951, Colegio Brasileiro de Cirurgioes.
7. GUTTMACHER, A. F., and SHETTLES, L. B. Human Fertil. 5:4, 1940.
8. MoRICARD, R. Bull. Soc. d'obst. et gynec. 25:426, 1936.
9. PALMER, R. lOth Congress Frant;ais de Gynec. Lyon, 1946.
10. RoLAND, M. Am. ]. Obst. & Gynec. 63:81, 1952.
11. RYDBERG, E. Acta obst. et gynec. Scandinav. 28:172, 1948.
12. SEGUY, J., and SIMMONET, H. Gynec. et obst. 28:657, 1933.
13. WATSON, M. C. Canad. M.A.]. 40:542, 1939.

DISCUSSION
DR. J. P. GREENHILL, Chicago, Ill.: The role of the cervix in infertility and
sterility has been variously estimated. In some statistical reports the cervix is
given a very minor role of responsibility; in other reports it is blamed for 40 to
70 per cent of the sterility cases. Unfortunately I did not have an opportunity
to read Dr. da Paz Filho's paper before the meeting, so I could not analyze it
and prepare a discussion. Nevertheless, I would like to take up some important
points.
As I recall, Papanicolaou was the first to discover these fern-like crystals in
the cervical mucus. Rydberg in Copenhagen also demonstrated them. Roland
called attention to these crystals as a test for pregnancy. As shown by Dr. da Paz,
the appearance of these fern-like crystals indicates the presence of estrogen
activity.
There have been important studies on cervical secretion by two French authors,
Seguy and Simmonet; Dr. Pommerenke has also made important contributions
about the cervical mucus. You know about the Sims-Huhner test, and that some-
148 CAMPOS DA PAZ [ Ferti 1ity & Steri 1ity

times cervical secretion prevents the penetration of sperm into the uterine cavity.
You know the effects of hormones and antibiotics on hostile cervical secretions.
Dr. da Paz Filho has shown that the hypodermic injection of 5 mg. of estrogen
on the fifth and tenth days of the cycle will overcome infertility in most cases
of hostility of the cervix, as shown by the Sims-Huhner test or by the much more
simple demonstration of fern-like crystals. This is a simple and inexpensive
treatment and we should all try it. I want to thank Dr. da Paz Filho for coming
here and telling us about this very simple remedy.
DR. W. T. PoMMERENKE, Rochester, N.Y.: I wish to thank Dr. Campos da Paz
Filho for the opportunity of reviewing his presentation. His method, illustrated
by excellent pictures of the fern patterns of crystallized cervical mucus, would
appear to offer a simple means of appraising the quality of this secretion in
relationship to its amicability or hostility to spermatozoa. The ease with which
the test can be performed, the small amount of apparatus and time required, and
the sharpness of the end points are features that should commend the test. It is
a familiar observation that estrogen administration in the castrate enhances the
quality of the cervical secretion, such as occurs physiologically at the time of
ovulation in the intact woman. One may, however, offer the suggestion that
efforts at improving the character of the cervical mucus by administration of
estrogens in the menstrual or early postmenstrual phase may, in fact, not work
a converse net result by inhibiting ovulation. The cervix, constituting as it does
the initial barrier to the progress of spermatozoa ejected into the female genital
tract, deserves serious study of the type suggested by Dr. Campos da Paz.