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Mycoplasma in the uterine cervix
Mycoplasma in the uterine cervix JAMES
E.
FRANCIS Ann
Arbor,
GREGORY, E.
PAYNE,
Dr.P.H. M.D.
Michigan
A study of Mycoplasma in the uterine cervix was made on 2 groups of 150 women each. One group consisted of women attending a venereal disease clinic; the ether of participants of a family planning clinic. Ages ranged from 16 to 61 years in the rlenereal di.rease group and 15 to 45 years in the family planning women. Both groups were from lower income family units and of similar ethnic background. The prevalence of Mycoplasma in the cervix was 92 per cent in the venereal disease group, as compared to 38 per cent in the family planning group. Mycoplasma hominis, type 1 was the predominant species of mycoplasma isolated from both groups. Within either the venereal disease or family planning group, age, phase of the menstrual cycle, type of contraception, or non-Mycoplasma genitourinary tract infections did not significantly relate to the prevalence of cervical Mycoplasma. Mycoplasma isolations did not relate to cytologic evidence of premalignant or malignant alteration of cervical cells in either group of women. Coccoid bodies associated :uith the cytoplasm of squamous cells were present in cervical smears from about 50 per cent of women yielding M. hominis as opposed to about 10 per cent of women not yielding Mycoplasma.
produce R E P o R T s T H .k T Mycoplasma chromosomall and other alterations*+ in cells cultured in vitro led to a consideration of the possible effects of Mycoplasma infection on cells of the uterine cervix. The feasibility of investigating this possibility depended on the prevalence of the infection in the populations to be studied. Among previous reports, there was wide variation in the frequency with which Mycoplasma were isolated from the female genitourinary tract.5-12 A possible explanation for this variation was sought in the present study by comparing the prevalence of cervical Mycoplasma to the age of the women, the phase of the menstrual cycle at the time of sampling, the contraceptive tech-
nique used, and the presence of non-Mycoplasma genitourinary tract infections. Cultures for Mycoplasma and smears for cytologic studies were prepared from the same cervical sampling of each woman. All Mycoplasma isolated were identified serologically. Materials and methods Population studied. The FP group consisted of 150 women examined at the Family Planning Clinic, Inkster, Michigan. Their ages ranged from 15 to 45 years. In the venereal disease group, I7 women were esamined at the Wayne County Health Department Venereal Disease Clinic, Eloise, Michigan, and 133 were examined at the Detroit Health Department Social Hygiene Clinic, Detroit, Michigan. The ages of these women ranged from 16 to 61 years. Subjects in both groups were of similar ethnic background and were from low income family units. Women who visited clinics and who had not had a cytologic examination of the cervix during the previous year were so examined. A specimen for Mycoplasma culture was
From the Department of Epidemiology, School of Public Health, University of Michigan. This study was supported by United States Public Health Service Grant 5TI AI 60-09. In part from. a doctoral dissertation submitted by the senior author in partial fulfillment of requirements for the degree of Doctor of Public Health, School of Public Health, University of Michigan.
220
Volume Number
Mycoplasma
107 2
taken at the same time. Each study was examined just once.
woman
Diagnosis of non-Mycoplasma infections. All clinical specimens for testing were obtained at the time of sampling the subject for Mycoplasma. The cardiolipin microflocculation test for syphilis was used. Gonorrhea was diagnosed from smears and cultures; trichomoniasis and yeast infection were diagnosed from cervical smears.
Mycoplasma cultures and identification. The :media used were suggested by Dr. Denys K. Ford, Department of Medicine, University of British Columbia, Vancouver, British Columbia. PPLO broth (Difco Laboratories, Detroit, Michigan) was supplemented to contain 10 per cent horse serum (Baltimore Biological Labora tory, Baltimore, Maryland) , 1 per cent yeast extract (Difco) , 5 mg. per cent urea, and 0.02 mg. per cent phenol red.
isolations
from
Results Since the prevalence of Mycoplasma in women attending the County Venereal Disease Clinic was found to be the same as in women attending the Social Hygiene Clinic, the women attending either of these venereal disease clinics were considered together in the results presented.
the cervices of two populations No.
Group
Venereal Farnily
/
disease clinic planning
clinic
N”k~!n?~en
150 150
221
PPLO agar (Difco) was supplemented to contain 0.25 per cent PPLO broth base, 10 per cent horse serum, and 1 per cent yeast extract. Both the broth and agar contained 1,000 units of penicillin per milliliter and both were adjpusted to pH 6.0. These media supported the growth of strains of M. hominis, type 1, M. fermentans, M. orale, type 1, M. salivarium, and M. pneumoniae (generously furnished by Dr. Lee E. Bartholomew of the University of Michigan Medical Center) and of a T-strain Mycoplasma (kindly furnished by Dr. Denys K. Ford). All cultures were incubated until positive or for 14 days at 34’ C. in a humidified atmosphere at 5 per cent CO, and 95 per cent N,. When colonies were seen on the agar culture, the corresponding broth culture was passaged and used for antigenic identification. The growth inhibition test described by ClydeI” was employed using specific antisera prepared in rabbits against the various species of Mycoplasma (Robbin Laboratories, Chapel Hill, North Carolina).
in the
Collection of specimens. In the family planning group a cervical scraping was secured by means of a wooden spatula. A sterile cotton swab was rubbed over one side of the spatula, gently rolled over the surface of a PPLO-agar plate, and then placed in a screw-capped tube containing 5 ml. of PPLO broth. The second side of the spatula was used to prepare a slide for cytology. Of the specimens from the venereal disease group, 17 were collected as described for the family planning group and were obtained from women attending the County Venereal Disease Clinic. The remaining 133 specimens from the venereal disease group were collected in a somewhat different manner and were from women attending the Social Hygiene Clinic. From the latter women, a wooden spatula was used to prepare the smear for cytology. A cotton swab, used to sample the cervical OS was placed in a screwcapped tube containing 5 ml. of PPLO broth and stored at 4” C. overnight. The broth was then used IO inoculate a PPLO agar plate. Cytology. Each smear was immediately fixed in absolute methanol. The smears were stained by the method of Lillie,13 modified by the use of azure II and eosin Y.
Tabk I. Mycoplasma
in cervix
1 Total 138 57
of women
1 M~~~~?ir 137 56
yielding
Mycoplasma
1 ?yiy?
1 M. 1 0
fermentans 0 1
222
Gregory
and
Payne
Amer.
Table II. Mycoplasma
“~~~~~p
(-
isolations
from
Venereal
di.rease
No.
clinic
tested
Totals --___
occurrence
to subject’s Family
positive
No.
of Mycoplasma
age planning
clinic
women
) No.
tested
11 60 24 15 9 1 3 5 4
150
The
in relation
women No.
14 65 26 15 9 1 3 5 4 2 3 1 2
15..17 18-20 21-23 24-26 27-29 30-32 33-35 36-38 39-41 42-44 45-47 48-50 50-+
Table III.
the cervix
May 15, 1970 J, Obstet. Cynec.
positiue
3 1 1
4 22 33 25 18 10 17 7 7 6 1 0 0
4 11 11 10 7 3 2 2 4 2 1 0 0
138
150
57
in relation
to phase of the menstrual
cycle* Days of mensirual cycle
Venereal No.
I-5t 6-10 11-15 16-20 21-25 26-:30 3o+s
disease
women No.
Totals
ora! hormones groups.
Table IV. Mycoplasma
isolations
Positive (total
STS’ cases)
Positive
23
Negative
1
No.
7 32 23 22 17 15 15
140
Qome women employing the extended periods in both
Family
positive
7 33 27 22 18 15 18
from
the venereal
failed
to
experience
from
cervices
Gonorrhea (total cases)
“STS := serologic test for syphilis; gonorrhea for Mycoplasma. Two suk-groups, “trichomoniasis
41 1
planning
clinic
women No.
tested 7 33 20 36 24 10 16
131
*Not included in this tabulation are 10 women were found to be pregnant at the time of sampling. tDays I to 5: Menstrual bleeding.
Mycopla.rma isolation
clinic
tested
1 14 9 10 13 ; 4
146 disease
group
withdrawal
and
flow
of subjects
for
with
Trichomoniasis (total cases)
4 from
one
other
57 the
or
two
family
planning
consecutive
genital
tract
Positive STS* only
103 5
was diagnosed by means of cervical and yeast” and “trichomoniasis and
positive
smears and yeast and
group
cycles,
who
hence
infections
or a
Gonorrhea only
6
10
1
0
culture. Trichomoniasis and gonorrhea,” each containing
yeast one
Volume Number
Mycoplasma
107 2
Mycoplasma were recovered from 92 per cent of the women attending the venereal diease clinics, as compared to 38 per cent of those visiting the family planning clinic (Table I). This difference between the venereal disease and family planning groups probably jis not attributable to the difference in technique of specimen collection used for these two groups: since the two techniques gave the same isolation rates within the venereal disease group. The predominance of M. hominis, type 1 in the cervix uteri, when Mycoplasma were isolated, was conspicuous. One isolate each of M. fermentans and M. orale type 1 was obtained. The proportion of women yielding Mycoplasma did not vary significantly (x20,95), with the age of subjects attending either the venereal disease clinics or the family planning clinic (Table II). The data presented also indicate the age distribution in the two groups. The very high prevalence of Mycoplasma in the venereal disease group was apparent in all phases of the menstrual cycle (Table III). Although the organisms were least prevalent during the period of menstrual bleeding in the family planning group no significant difference (x20.95) in the prevalence of Mycoplasma was discernible among the various phases of the menstrual cycle. The prevalence of Mycoplasma infection among the venereal disease women did not relate to concurrent venereal disease (Table IV), suggesting that Mycoplasma infection was iassociated with some factor common to, essentially, all -the women attending the venereal disease clinic. In the family plan-
positive
serologic ~~
Trichomoniasis only
test for syphilis Trichomoniasis and positive STS
60 4 infection subject
were diagnosed from from whom Mycoplasma
12
disease clinic)
Trichomoniasis gonorrhea
and
Trichomoniasis Positive STS gonorrhea
All diagnoses are not shown
and and I
Yeast only
5
1 smears. isolated
223
ning group, as in the venereal disease group, trichomoniasis did not significantly ( x’~.~;) influence the prevalence of Mycoplasma isolations from the cervix (Table V) . The family planning group provided a population for comparing birth control techniques and the prevalence of Mycoplasma in the cervix. Among groups that used different forms of contraception, the proportion of women yielding Mycoplasma varied (Table VI). However, the difference in prevalence between any two of these groups was not significant at the 5 per cent level. In neither the venereal disease or family planning group did Mycoplasma significantly (x?~,~~) influence the occurrence of cervical cellular dyscrasias as classified by Papanicolaou (Table VII). With the exception of 1 isolate of M. fermentans, from a woman with a Class III smear, the species of Mycoplasma recovered from those women with abnormal smears was M. homini.r, type 1. Examination of azure-eosin-stained cervical smears revealed, in addition to the cervical dyscrasias noted above, the presence of cytoplasmic coccoid bodies in squamous cells (Fig. 1) from approximately one third of the women examined. In both the venereal disease and family planning subjects, there were cytoplasmic bodies in smears from approximately 50 per cent of those women from whom Mycoplasma was isolated (Table VIII). Most noteworthy was the absence of these bodies in smears from all but 10 of 93 family planning women from whom Mycoplasma were not isolated. A similar relationship was present in the relatively few venereal disease subjects who did not yield Mycoplasma.
24
0 cervical was
(veneral
in cervix
clinical
I
0
0 were made from in the table.
Negative STS and no other infection 19
1 materials
secured
5 at
the
time
of
sampling
224
Gregory
and
Payne
Amer.
Comment
In considering what factors might have been responsible for the very high prevalence of cervical Mycoplasma in the venereal disease group as compared to the family planning group, it should be noted that the two groups of women were not intentionally matched according to age, phase of menstrual cycle, or type of contraception employed. However, data were presented indicating that none of these variables significantly influenced the prevalence of cervical Mycoplasma. The two groups of women were considered to be of simiIar socioeconomic and ethnic background. It would seem reasonable to assume that sexual promiscuity was more
Mycoplasma was isolated from the cervix of 92 per cent of the women in the venereal disease group in contrast to 38 per cent of those in the famiiy planning group. Previous investigators’5-17l IS’ *O have found that a high proportion of women with syphilis, gonorrhea, or trichomoniasis had Mycoplasma in the vagina. In the present study, the prevalence of Mycoplasma in the cervix did not significantly relate to any of these diseases, per se. A similar lack of association between vaginal Mycoplasrna and classical venereal diseases has been reported by KlienebergerNobel.!’
Table V. Mycopla.sma (family ---
planning
isolations
from
May 15, 1970 J Obstet. Gynec.
subjects
with
other
genital
tract
infections
clinic) Other
organisms*
uaginalis
Mycoplasma isolations
T. vaginalis (totai cases)
Yeast (total cases)
Positive
16
t
15
0
1
4t
4
16
4
0
73
Negative *Detected
Fig.
16 in stained
cwvical
1. Cytoplasmic
T.
only
Yeast Only
T. uaginalis and yeast
None
smears.
coccoid
bodies
in
squamous
cells
of cervical
smear.
(Azure-eosin
stain.)
Volume Number
107 2
Mycoplasma
prevalent in the venereal disease group than in the family planning group. Shepard18 found,, in a group of men, that the prevalence of Mycoplasma in the genitourinary tract was directly related to sexual promiscuity. A similar relationship would appear to be a Mycoplasma in the cervices of subjects using various contraceptive measures (family planning clinic) --___
for
Mycoplesma Contraceptive technique
No. tested
No. positive
53
25
47
75 5
27 1
36 20
2 I 1 1 12
0 0 0 1 3
0 0 0 100 25
150
57
Intrauterine device (Lippes loop) Oral hormone (ethynodiol diacetate with mestranol) Pareni.eraI hormone (med roxyprogesterone acetate) Foams Creams Condoms Douch.es None Totals
Table VII.
Mycoplasma
Per
cent
positive
and cellular
dyscrasias -.__~ Cytologic
Group Venereal Clinic:
I
) II
class”
( III
1 zv
I v
Disease
Mycoplasma+ Mycoplasma-
135 I?
2 0
0 0
1 0
(1 0
53 86
0 6
3 0
1 1
0 0
Fami1.v Planning Clinic:
Mycoplasma+ Mycoplasma-*Papanicolaou
classilication.
Table VIII.
Mycoplasma
and cytoplasmic
coccoid
bodies
~~___ Venereal Mvcoblasmat Coccoid Coccoid Totals
bodies+ bodies-
disease 1
225
likely explanation for the high prevalence of cervical mycoplasma in the venereal disease group women of the present study. Two previous reportssllg have suggested that the age of women might relate to the prevalence of Mycoplasma in the genitourinary tract. In both of these studies, the age groups also separated the women into groups that might be expected to have different degrees of sexual activity. In contrast to the present report, Mclen and OdebladlO suggested that the presence of Mycoplasma in the vagina or cervix might relate to the menstrual cycle. The latter study, reported in 1952, may reflect the rclative insensitivity of the isolation technique empIoyed at that time and the varied frcquency of isolation of Mycoplasma may have resulted from variations in the numbers of Mycoplasma present in the vagina during the menstrual cycle. That almost all of the large colony Mycoplasma isolated from the cervix in both the venereal disease and family planning groups were M. hominis, type 1 is in agreement with previous reports.l’j, lo, ‘O M. fermentan. and M. orale were each isolated from one woman in the present study. Some of the strains isolated from the female genitourinary tract in Ford’s studyzO may have been M. fermentam. Isolation of M. orale from the female genitourinary tract has not been preThe absence of T-strain viously reported. Mycoplasma among the isolates in the present study is conspicuous since other?‘, “O have frequently found this species in the frmale genitourinary tract. Although the prototype T strain grew well on the media employed, it is possible that these media were not adcquate for growing freshly isolated T strains. It is also possible that heavy growth of hl.
Table VI.
Women cultured
in cervix
Family planning
clinic Mvcoblasma-
Mvcoblasmat
67
3
71
9
138
12
27 30 57
1
clinic Mvcoblasma10
83 93
226
Gregory
and
May
Payne
hominis
interfered with the detection of T strains in some cultures. Although Mycoplasma isolations did not relate to premalignant or malignant changes in cervical cells, cytoplasmic coccoid bodies were observed in cervical smears from about 50 per cent of women yielding M. hominis as opposed to about 10 per cent of women not yieiding Mycoplasma. Immunofluorescent staining has thus far failed to demonstrate M. hominis antigen in these bodies. It is of interest that Jones and Davson”l observed minute coccoid bodies in cervical smear,s and found that presence of these bodies related to the density of growth of M. horn&is in cultures of vaginal specimens. In the present study no attempt was made to quantitate the amount of M. hominis in the cervical specimens; however, in our series and in that of Jones and Davson approximately half the women from whom Mycoplasma was isolated had cervical smears showing coccoid bodies.
15, 1970
Amer. J. Obslet. Gynec.
The high prevalence of Mycoplasma in the female genitourinary tract in some populations, such as those in the present study, along with recent reports which associate Mycoplasma with spontaneous abortior?“” and neonatal eye infectionsz5 serves to emphasize the need for continued awareness of these organisms in considering factors which might affect the health of the patient or her fetus or newborn. In obtaining clinical material, the generous cooperation of Enrique Cabrera, M.D., and Miss Ann Suttles of the Family Planning Clinic, Inkster, Michigan, George Pickett, M.D., and Mrs. Juanita Reese of the Wayne County Health Department, and Benjamin Schwimmer, M.D., and Mrs. Virginia Emmanuel of the Detroit Health Department is gratefully acknowledged. We also thank Lee E. Bartholomew, M.D., and Miss Frances Nelson of the Kresge Research Foundation for assisting us to initiate this research.
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
13.
Paton, G. R., Jacobs, J. P., and Perkins, F. T.: Nature 207: 43, 1965. Robinson, L. B., Wichelhausen, R. H., and Roizman, B.: Science 124: 1147, 1956. Kraemer, P. M.: Proc. Sot. Exp. Biol. Med. 115: 206, 1964. Butler, M., and Leach, R. H.: J. Gen. Microbial. 34: 285, 1964. Klieneberger-Nobel, E.: Lancet 2: 46, 1945. Beveridge, W. 1’. B., Campbell, A. D., and Lind, P. E.: Med. J. Austr. 1: 179, 1946. Salaman, M. H: J. Path. Bact. 58: 31, 1946. Randall, J. H., Stein, R. J., and Ayres, J. C.: AMER. J. OBSTE.T. GYNEC. 59: 404, 1950. Mel&n, B., and Odeblad, E.: Stand. J. Clin. Lab. Invest. 3: 47, 1951. MelCn, B., and Odeblad, E.: Acta Dermat. 32: 74, 1952. Stokes, E. J.: Brit. Med. J. 1: 510, 1959. Bercovici, B., Persky, S., Rozansky, R., and Razin, S.: AMER. J. OBSTET. GYNEC. 84: 687, 1962. Lillie, R. D.: Histopathologic Technic and Practical Histochemistry, New York, 1965, The Blakiston Company, pp. 116-l 19.
14. 15. 16. 17. 18. 19.
20. 21. 22.
23. 24. 25.
Clyde, W. A., Jr.: J. Immunol. 92: 958, 1964. Harkness, A. H., and Henderson-Begg, A.: Brit. J. Vener. Dis. 24: 50, 1948. Nicol, C. S., and Edward, D. G.: Brit. J. Vener. Dis. 29: 141, 1953. Horoszewicz, J.: Brit. J. Vener. Dis. 37: 183, 1961. Shepard, M. C.: Amer. J. Syph. 38: 113, 1954. Csonka, G. W., Williams, R. E. O., and Corse, J.: Ann. N. Y. Acad. Sci. 143: 794, 1967. Ford, D. K.: Ann. N. Y. Acad. Sci. 143: 501, 1967. Jones, D. M., and Davson, J.: Nature 213: 828, 1967. Harwick, H. J., Iuppa, J. B., Purcell, R. H., and Fekety, F. R.: AMER. J. OBSTET. GYNEC. 99: 725, 1967. Jones, D. M.: Brit. Med. J. 1: 338, 1967. Kundsin, R. B., Driscoll, S. G., and Ming, P. L.: Science 157: 1573, 1967. Jones, D. M., and Tobin, B.: Brit. Med. J. 3: 467, 1968.
E.
FRANCIS Ann
Arbor,
GREGORY, E.
PAYNE,
Dr.P.H. M.D.
Michigan
A study of Mycoplasma in the uterine cervix was made on 2 groups of 150 women each. One group consisted of women attending a venereal disease clinic; the ether of participants of a family planning clinic. Ages ranged from 16 to 61 years in the rlenereal di.rease group and 15 to 45 years in the family planning women. Both groups were from lower income family units and of similar ethnic background. The prevalence of Mycoplasma in the cervix was 92 per cent in the venereal disease group, as compared to 38 per cent in the family planning group. Mycoplasma hominis, type 1 was the predominant species of mycoplasma isolated from both groups. Within either the venereal disease or family planning group, age, phase of the menstrual cycle, type of contraception, or non-Mycoplasma genitourinary tract infections did not significantly relate to the prevalence of cervical Mycoplasma. Mycoplasma isolations did not relate to cytologic evidence of premalignant or malignant alteration of cervical cells in either group of women. Coccoid bodies associated :uith the cytoplasm of squamous cells were present in cervical smears from about 50 per cent of women yielding M. hominis as opposed to about 10 per cent of women not yielding Mycoplasma.
produce R E P o R T s T H .k T Mycoplasma chromosomall and other alterations*+ in cells cultured in vitro led to a consideration of the possible effects of Mycoplasma infection on cells of the uterine cervix. The feasibility of investigating this possibility depended on the prevalence of the infection in the populations to be studied. Among previous reports, there was wide variation in the frequency with which Mycoplasma were isolated from the female genitourinary tract.5-12 A possible explanation for this variation was sought in the present study by comparing the prevalence of cervical Mycoplasma to the age of the women, the phase of the menstrual cycle at the time of sampling, the contraceptive tech-
nique used, and the presence of non-Mycoplasma genitourinary tract infections. Cultures for Mycoplasma and smears for cytologic studies were prepared from the same cervical sampling of each woman. All Mycoplasma isolated were identified serologically. Materials and methods Population studied. The FP group consisted of 150 women examined at the Family Planning Clinic, Inkster, Michigan. Their ages ranged from 15 to 45 years. In the venereal disease group, I7 women were esamined at the Wayne County Health Department Venereal Disease Clinic, Eloise, Michigan, and 133 were examined at the Detroit Health Department Social Hygiene Clinic, Detroit, Michigan. The ages of these women ranged from 16 to 61 years. Subjects in both groups were of similar ethnic background and were from low income family units. Women who visited clinics and who had not had a cytologic examination of the cervix during the previous year were so examined. A specimen for Mycoplasma culture was
From the Department of Epidemiology, School of Public Health, University of Michigan. This study was supported by United States Public Health Service Grant 5TI AI 60-09. In part from. a doctoral dissertation submitted by the senior author in partial fulfillment of requirements for the degree of Doctor of Public Health, School of Public Health, University of Michigan.
220
Volume Number
Mycoplasma
107 2
taken at the same time. Each study was examined just once.
woman
Diagnosis of non-Mycoplasma infections. All clinical specimens for testing were obtained at the time of sampling the subject for Mycoplasma. The cardiolipin microflocculation test for syphilis was used. Gonorrhea was diagnosed from smears and cultures; trichomoniasis and yeast infection were diagnosed from cervical smears.
Mycoplasma cultures and identification. The :media used were suggested by Dr. Denys K. Ford, Department of Medicine, University of British Columbia, Vancouver, British Columbia. PPLO broth (Difco Laboratories, Detroit, Michigan) was supplemented to contain 10 per cent horse serum (Baltimore Biological Labora tory, Baltimore, Maryland) , 1 per cent yeast extract (Difco) , 5 mg. per cent urea, and 0.02 mg. per cent phenol red.
isolations
from
Results Since the prevalence of Mycoplasma in women attending the County Venereal Disease Clinic was found to be the same as in women attending the Social Hygiene Clinic, the women attending either of these venereal disease clinics were considered together in the results presented.
the cervices of two populations No.
Group
Venereal Farnily
/
disease clinic planning
clinic
N”k~!n?~en
150 150
221
PPLO agar (Difco) was supplemented to contain 0.25 per cent PPLO broth base, 10 per cent horse serum, and 1 per cent yeast extract. Both the broth and agar contained 1,000 units of penicillin per milliliter and both were adjpusted to pH 6.0. These media supported the growth of strains of M. hominis, type 1, M. fermentans, M. orale, type 1, M. salivarium, and M. pneumoniae (generously furnished by Dr. Lee E. Bartholomew of the University of Michigan Medical Center) and of a T-strain Mycoplasma (kindly furnished by Dr. Denys K. Ford). All cultures were incubated until positive or for 14 days at 34’ C. in a humidified atmosphere at 5 per cent CO, and 95 per cent N,. When colonies were seen on the agar culture, the corresponding broth culture was passaged and used for antigenic identification. The growth inhibition test described by ClydeI” was employed using specific antisera prepared in rabbits against the various species of Mycoplasma (Robbin Laboratories, Chapel Hill, North Carolina).
in the
Collection of specimens. In the family planning group a cervical scraping was secured by means of a wooden spatula. A sterile cotton swab was rubbed over one side of the spatula, gently rolled over the surface of a PPLO-agar plate, and then placed in a screw-capped tube containing 5 ml. of PPLO broth. The second side of the spatula was used to prepare a slide for cytology. Of the specimens from the venereal disease group, 17 were collected as described for the family planning group and were obtained from women attending the County Venereal Disease Clinic. The remaining 133 specimens from the venereal disease group were collected in a somewhat different manner and were from women attending the Social Hygiene Clinic. From the latter women, a wooden spatula was used to prepare the smear for cytology. A cotton swab, used to sample the cervical OS was placed in a screwcapped tube containing 5 ml. of PPLO broth and stored at 4” C. overnight. The broth was then used IO inoculate a PPLO agar plate. Cytology. Each smear was immediately fixed in absolute methanol. The smears were stained by the method of Lillie,13 modified by the use of azure II and eosin Y.
Tabk I. Mycoplasma
in cervix
1 Total 138 57
of women
1 M~~~~?ir 137 56
yielding
Mycoplasma
1 ?yiy?
1 M. 1 0
fermentans 0 1
222
Gregory
and
Payne
Amer.
Table II. Mycoplasma
“~~~~~p
(-
isolations
from
Venereal
di.rease
No.
clinic
tested
Totals --___
occurrence
to subject’s Family
positive
No.
of Mycoplasma
age planning
clinic
women
) No.
tested
11 60 24 15 9 1 3 5 4
150
The
in relation
women No.
14 65 26 15 9 1 3 5 4 2 3 1 2
15..17 18-20 21-23 24-26 27-29 30-32 33-35 36-38 39-41 42-44 45-47 48-50 50-+
Table III.
the cervix
May 15, 1970 J, Obstet. Cynec.
positiue
3 1 1
4 22 33 25 18 10 17 7 7 6 1 0 0
4 11 11 10 7 3 2 2 4 2 1 0 0
138
150
57
in relation
to phase of the menstrual
cycle* Days of mensirual cycle
Venereal No.
I-5t 6-10 11-15 16-20 21-25 26-:30 3o+s
disease
women No.
Totals
ora! hormones groups.
Table IV. Mycoplasma
isolations
Positive (total
STS’ cases)
Positive
23
Negative
1
No.
7 32 23 22 17 15 15
140
Qome women employing the extended periods in both
Family
positive
7 33 27 22 18 15 18
from
the venereal
failed
to
experience
from
cervices
Gonorrhea (total cases)
“STS := serologic test for syphilis; gonorrhea for Mycoplasma. Two suk-groups, “trichomoniasis
41 1
planning
clinic
women No.
tested 7 33 20 36 24 10 16
131
*Not included in this tabulation are 10 women were found to be pregnant at the time of sampling. tDays I to 5: Menstrual bleeding.
Mycopla.rma isolation
clinic
tested
1 14 9 10 13 ; 4
146 disease
group
withdrawal
and
flow
of subjects
for
with
Trichomoniasis (total cases)
4 from
one
other
57 the
or
two
family
planning
consecutive
genital
tract
Positive STS* only
103 5
was diagnosed by means of cervical and yeast” and “trichomoniasis and
positive
smears and yeast and
group
cycles,
who
hence
infections
or a
Gonorrhea only
6
10
1
0
culture. Trichomoniasis and gonorrhea,” each containing
yeast one
Volume Number
Mycoplasma
107 2
Mycoplasma were recovered from 92 per cent of the women attending the venereal diease clinics, as compared to 38 per cent of those visiting the family planning clinic (Table I). This difference between the venereal disease and family planning groups probably jis not attributable to the difference in technique of specimen collection used for these two groups: since the two techniques gave the same isolation rates within the venereal disease group. The predominance of M. hominis, type 1 in the cervix uteri, when Mycoplasma were isolated, was conspicuous. One isolate each of M. fermentans and M. orale type 1 was obtained. The proportion of women yielding Mycoplasma did not vary significantly (x20,95), with the age of subjects attending either the venereal disease clinics or the family planning clinic (Table II). The data presented also indicate the age distribution in the two groups. The very high prevalence of Mycoplasma in the venereal disease group was apparent in all phases of the menstrual cycle (Table III). Although the organisms were least prevalent during the period of menstrual bleeding in the family planning group no significant difference (x20.95) in the prevalence of Mycoplasma was discernible among the various phases of the menstrual cycle. The prevalence of Mycoplasma infection among the venereal disease women did not relate to concurrent venereal disease (Table IV), suggesting that Mycoplasma infection was iassociated with some factor common to, essentially, all -the women attending the venereal disease clinic. In the family plan-
positive
serologic ~~
Trichomoniasis only
test for syphilis Trichomoniasis and positive STS
60 4 infection subject
were diagnosed from from whom Mycoplasma
12
disease clinic)
Trichomoniasis gonorrhea
and
Trichomoniasis Positive STS gonorrhea
All diagnoses are not shown
and and I
Yeast only
5
1 smears. isolated
223
ning group, as in the venereal disease group, trichomoniasis did not significantly ( x’~.~;) influence the prevalence of Mycoplasma isolations from the cervix (Table V) . The family planning group provided a population for comparing birth control techniques and the prevalence of Mycoplasma in the cervix. Among groups that used different forms of contraception, the proportion of women yielding Mycoplasma varied (Table VI). However, the difference in prevalence between any two of these groups was not significant at the 5 per cent level. In neither the venereal disease or family planning group did Mycoplasma significantly (x?~,~~) influence the occurrence of cervical cellular dyscrasias as classified by Papanicolaou (Table VII). With the exception of 1 isolate of M. fermentans, from a woman with a Class III smear, the species of Mycoplasma recovered from those women with abnormal smears was M. homini.r, type 1. Examination of azure-eosin-stained cervical smears revealed, in addition to the cervical dyscrasias noted above, the presence of cytoplasmic coccoid bodies in squamous cells (Fig. 1) from approximately one third of the women examined. In both the venereal disease and family planning subjects, there were cytoplasmic bodies in smears from approximately 50 per cent of those women from whom Mycoplasma was isolated (Table VIII). Most noteworthy was the absence of these bodies in smears from all but 10 of 93 family planning women from whom Mycoplasma were not isolated. A similar relationship was present in the relatively few venereal disease subjects who did not yield Mycoplasma.
24
0 cervical was
(veneral
in cervix
clinical
I
0
0 were made from in the table.
Negative STS and no other infection 19
1 materials
secured
5 at
the
time
of
sampling
224
Gregory
and
Payne
Amer.
Comment
In considering what factors might have been responsible for the very high prevalence of cervical Mycoplasma in the venereal disease group as compared to the family planning group, it should be noted that the two groups of women were not intentionally matched according to age, phase of menstrual cycle, or type of contraception employed. However, data were presented indicating that none of these variables significantly influenced the prevalence of cervical Mycoplasma. The two groups of women were considered to be of simiIar socioeconomic and ethnic background. It would seem reasonable to assume that sexual promiscuity was more
Mycoplasma was isolated from the cervix of 92 per cent of the women in the venereal disease group in contrast to 38 per cent of those in the famiiy planning group. Previous investigators’5-17l IS’ *O have found that a high proportion of women with syphilis, gonorrhea, or trichomoniasis had Mycoplasma in the vagina. In the present study, the prevalence of Mycoplasma in the cervix did not significantly relate to any of these diseases, per se. A similar lack of association between vaginal Mycoplasrna and classical venereal diseases has been reported by KlienebergerNobel.!’
Table V. Mycopla.sma (family ---
planning
isolations
from
May 15, 1970 J Obstet. Gynec.
subjects
with
other
genital
tract
infections
clinic) Other
organisms*
uaginalis
Mycoplasma isolations
T. vaginalis (totai cases)
Yeast (total cases)
Positive
16
t
15
0
1
4t
4
16
4
0
73
Negative *Detected
Fig.
16 in stained
cwvical
1. Cytoplasmic
T.
only
Yeast Only
T. uaginalis and yeast
None
smears.
coccoid
bodies
in
squamous
cells
of cervical
smear.
(Azure-eosin
stain.)
Volume Number
107 2
Mycoplasma
prevalent in the venereal disease group than in the family planning group. Shepard18 found,, in a group of men, that the prevalence of Mycoplasma in the genitourinary tract was directly related to sexual promiscuity. A similar relationship would appear to be a Mycoplasma in the cervices of subjects using various contraceptive measures (family planning clinic) --___
for
Mycoplesma Contraceptive technique
No. tested
No. positive
53
25
47
75 5
27 1
36 20
2 I 1 1 12
0 0 0 1 3
0 0 0 100 25
150
57
Intrauterine device (Lippes loop) Oral hormone (ethynodiol diacetate with mestranol) Pareni.eraI hormone (med roxyprogesterone acetate) Foams Creams Condoms Douch.es None Totals
Table VII.
Mycoplasma
Per
cent
positive
and cellular
dyscrasias -.__~ Cytologic
Group Venereal Clinic:
I
) II
class”
( III
1 zv
I v
Disease
Mycoplasma+ Mycoplasma-
135 I?
2 0
0 0
1 0
(1 0
53 86
0 6
3 0
1 1
0 0
Fami1.v Planning Clinic:
Mycoplasma+ Mycoplasma-*Papanicolaou
classilication.
Table VIII.
Mycoplasma
and cytoplasmic
coccoid
bodies
~~___ Venereal Mvcoblasmat Coccoid Coccoid Totals
bodies+ bodies-
disease 1
225
likely explanation for the high prevalence of cervical mycoplasma in the venereal disease group women of the present study. Two previous reportssllg have suggested that the age of women might relate to the prevalence of Mycoplasma in the genitourinary tract. In both of these studies, the age groups also separated the women into groups that might be expected to have different degrees of sexual activity. In contrast to the present report, Mclen and OdebladlO suggested that the presence of Mycoplasma in the vagina or cervix might relate to the menstrual cycle. The latter study, reported in 1952, may reflect the rclative insensitivity of the isolation technique empIoyed at that time and the varied frcquency of isolation of Mycoplasma may have resulted from variations in the numbers of Mycoplasma present in the vagina during the menstrual cycle. That almost all of the large colony Mycoplasma isolated from the cervix in both the venereal disease and family planning groups were M. hominis, type 1 is in agreement with previous reports.l’j, lo, ‘O M. fermentan. and M. orale were each isolated from one woman in the present study. Some of the strains isolated from the female genitourinary tract in Ford’s studyzO may have been M. fermentam. Isolation of M. orale from the female genitourinary tract has not been preThe absence of T-strain viously reported. Mycoplasma among the isolates in the present study is conspicuous since other?‘, “O have frequently found this species in the frmale genitourinary tract. Although the prototype T strain grew well on the media employed, it is possible that these media were not adcquate for growing freshly isolated T strains. It is also possible that heavy growth of hl.
Table VI.
Women cultured
in cervix
Family planning
clinic Mvcoblasma-
Mvcoblasmat
67
3
71
9
138
12
27 30 57
1
clinic Mvcoblasma10
83 93
226
Gregory
and
May
Payne
hominis
interfered with the detection of T strains in some cultures. Although Mycoplasma isolations did not relate to premalignant or malignant changes in cervical cells, cytoplasmic coccoid bodies were observed in cervical smears from about 50 per cent of women yielding M. hominis as opposed to about 10 per cent of women not yieiding Mycoplasma. Immunofluorescent staining has thus far failed to demonstrate M. hominis antigen in these bodies. It is of interest that Jones and Davson”l observed minute coccoid bodies in cervical smear,s and found that presence of these bodies related to the density of growth of M. horn&is in cultures of vaginal specimens. In the present study no attempt was made to quantitate the amount of M. hominis in the cervical specimens; however, in our series and in that of Jones and Davson approximately half the women from whom Mycoplasma was isolated had cervical smears showing coccoid bodies.
15, 1970
Amer. J. Obslet. Gynec.
The high prevalence of Mycoplasma in the female genitourinary tract in some populations, such as those in the present study, along with recent reports which associate Mycoplasma with spontaneous abortior?“” and neonatal eye infectionsz5 serves to emphasize the need for continued awareness of these organisms in considering factors which might affect the health of the patient or her fetus or newborn. In obtaining clinical material, the generous cooperation of Enrique Cabrera, M.D., and Miss Ann Suttles of the Family Planning Clinic, Inkster, Michigan, George Pickett, M.D., and Mrs. Juanita Reese of the Wayne County Health Department, and Benjamin Schwimmer, M.D., and Mrs. Virginia Emmanuel of the Detroit Health Department is gratefully acknowledged. We also thank Lee E. Bartholomew, M.D., and Miss Frances Nelson of the Kresge Research Foundation for assisting us to initiate this research.
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13.
Paton, G. R., Jacobs, J. P., and Perkins, F. T.: Nature 207: 43, 1965. Robinson, L. B., Wichelhausen, R. H., and Roizman, B.: Science 124: 1147, 1956. Kraemer, P. M.: Proc. Sot. Exp. Biol. Med. 115: 206, 1964. Butler, M., and Leach, R. H.: J. Gen. Microbial. 34: 285, 1964. Klieneberger-Nobel, E.: Lancet 2: 46, 1945. Beveridge, W. 1’. B., Campbell, A. D., and Lind, P. E.: Med. J. Austr. 1: 179, 1946. Salaman, M. H: J. Path. Bact. 58: 31, 1946. Randall, J. H., Stein, R. J., and Ayres, J. C.: AMER. J. OBSTE.T. GYNEC. 59: 404, 1950. Mel&n, B., and Odeblad, E.: Stand. J. Clin. Lab. Invest. 3: 47, 1951. MelCn, B., and Odeblad, E.: Acta Dermat. 32: 74, 1952. Stokes, E. J.: Brit. Med. J. 1: 510, 1959. Bercovici, B., Persky, S., Rozansky, R., and Razin, S.: AMER. J. OBSTET. GYNEC. 84: 687, 1962. Lillie, R. D.: Histopathologic Technic and Practical Histochemistry, New York, 1965, The Blakiston Company, pp. 116-l 19.
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Clyde, W. A., Jr.: J. Immunol. 92: 958, 1964. Harkness, A. H., and Henderson-Begg, A.: Brit. J. Vener. Dis. 24: 50, 1948. Nicol, C. S., and Edward, D. G.: Brit. J. Vener. Dis. 29: 141, 1953. Horoszewicz, J.: Brit. J. Vener. Dis. 37: 183, 1961. Shepard, M. C.: Amer. J. Syph. 38: 113, 1954. Csonka, G. W., Williams, R. E. O., and Corse, J.: Ann. N. Y. Acad. Sci. 143: 794, 1967. Ford, D. K.: Ann. N. Y. Acad. Sci. 143: 501, 1967. Jones, D. M., and Davson, J.: Nature 213: 828, 1967. Harwick, H. J., Iuppa, J. B., Purcell, R. H., and Fekety, F. R.: AMER. J. OBSTET. GYNEC. 99: 725, 1967. Jones, D. M.: Brit. Med. J. 1: 338, 1967. Kundsin, R. B., Driscoll, S. G., and Ming, P. L.: Science 157: 1573, 1967. Jones, D. M., and Tobin, B.: Brit. Med. J. 3: 467, 1968.