Bacteriology of the Cervix in Human Sterility

Transcription

1 Bacteriology of the Cervix in Human Sterility Lee Buxton, M.D., Anna Southam, M.D., Walter Herrmann, M.D., Grace Girvin, Ph.D., and Hyman Nadel, Ph.D. THE OBSERVATION that certain bacteria cultured from the cervix had a deleterious effect on spenn in vitro, made in 1950 by Matthews and Buxton, prompted investigation of the possible etiologic role of these organisms in sterility. This paper is a summary and evaluation of further work done on this problem in the Sterility Clinic of the Sloane Hospital for Women. The ability of Escherichia coli to agglutinate spennatozoa as well as other cells was reported by Rosenthal in Barton and Wiesner in 1948 reported on a series of cases in which they recovered pathogens from cervical cultures. They found a high incidence of Esch. coli. Other pathogens recovered were chromogenic micrococci, Streptococcus viridans, Proteus vulgaris, Streptococcus faecalis, other streptococci, pneumococci, and Clostridium welchii. They found improved postcoital tests in many patients after appropriate antibiotics, estrogens, and judicious weight reduction. From the Departments of Obstetrics and Gynecology, Columbia University College of Physicians and Surgeons and the Sloane Hospital for Women, New York, N. Y. Present addresses: Yale University School of Medicine, New Haven, Conn. (L. B. and W. H.); Department of Bacteriology and Immunology, McGill University, Montreal, P. Q. (G. G.). Since this manuscript was completed. and submitted for publication, a papers has been published reporting essentially the same findings. The authors would like to express their gratitude to Dr. Harry Rose, Professor of Microbiology, Columbia College of Physicians and Surgeons, for his advice and suggestions throughout this study. This work was supported by grants from Lederle Laboratories, Pearl River, N. Y., and the National Institutes of Health, United States Public Health Service. 493

2 494 BUXTON ET At. Fertility & Sterility Shotton, in 1951, studied 32 cases with abnormal mucus. Esch. coli was found in only one of these. She pointed out that the diagnosis of abnormal cervical mucus can be made only if the patient is examined on repeated occasions. Postcoital tests and mucus may be good in many patients from Day 9 to Day 19 of the cycle, but in many sterility patients the length of time that the cervical mucus is clear and acellular may be much shorter. She found that the response to antibiotic therapy was disappointing and that pregnancy occurred at a fairly constant rate of 40 per cent if major causes of sterility were excluded. Laughton reported, in 1951, her observations on a group of 67 women and found a low incidence of pathogens. She concluded that bacterial infection was not an important factor in cervical dysfunction. In 1952 Sandler reported that pathogens, chiefly Esch. coli and streptococci, were found in about 15 per cent of sterile women with clinically abnormal cervices. Antibiotic therapy improved the conception rate in these patients. Cultures taken from the cervices of 43 women seeking contraceptive advice, who had had at least one child, yielded no pathogenic organisms. Barton, in 1952, reported that 85 per cent of cases with poor cervical mucus would be improved by chloramphenicol but that this antibiotic might need to be given on several successive cycles. Moloy, in 1950, found an increased conception rate after treatment with vaginal tampons saturated with streptomycin-sulfamylon solution. Horne and Rock (1952) administered oral terramycin to 43 infertile women with a diagnosis of clinical endocervicitis. Twenty-eight per cent of the treated group became pregnant. Gepfert and Davis, in 1953, reported a pregnancy rate of 28 per cent after treatment with aureomycin vaginal suppositories containing an antimonilial agent. This treatment did not Significantly decrease the incidence of spermicidal organisms. As a continuation of the study made by Matthews and Buxton, an investigation of the clinical effects of cervical spermicidal organisms on female sterility was begun in this laboratory in The first report 4 concerned the -t first 209 sterility patients investigated. Of the 176 patients who harbored what were then thought to be spermicidal" organisms, 95 patients were treated, 22 per cent becoming pregnant after antibiotic therapy. There were a number of variables to be considered, however, and as mentioned in the above report, the study presented so many undecided problems that it was thought advisable to pursue it further. The in vitro spermicidal effect of

3 Vol. 5, No.6, 1954 CERVICAL BACTERIOLOGY 495 certain organisms needed further investigation; the type of treatment was not considered satisfactory; there was an insufficient control series of cultures on normally fertile women; the quantitative aspects of both the bacterial and sperm populations of the postcoital cervix had not been studied; and several other problems presented themselves as the study progressed. The present report, therefore, is a continuation of the above investigation and deals with a total of 485 sterility patients on whom cervical cultures were taken, including the 209 aheady reported. It also concerns numerous further in vitro bacteriologic studies which partially confirm the findings of the previous work, but which have been subjected to different interpretation. One hundred and eighty-three of the 485 patients received various types of antibiotic therapy. In this group of patients we have studied (1) the incidence of organisms found in postcoital cultures; (2) the Similarity between vaginal flora and that obtained by cervical culture; (3) the effect of coitus on cervical flora; (4) the incidence of spermicidal organisms on repeat cultures in the same patient without intervening treatment; (5) the incidence of organisms at various times in the menstrual cycle; (6) the effect of treatment upon the cervical flora; (7) organisms associated with good postcoital tests; (8) organisms present in cervical cultures taken at the time of conception; and (9) the effects of antibiotic therapy upon the conception rate. It was hoped that a study of the above conditions, plus further bacteriologic investigation of the effects of bacteria on spermatozoa, would provide significant information concerning the validity of the suggestive clinical findings of the previous report. BACTERIOLOGIC MATERIALS AND METHODS Culture material taken from the cervix or vagina was inoculated onto two blood agar plates and in Brewer's meat broth. The plates were incubated aerobically and anaerobically at 37 C. and examined after 24 to 48 hours of incubation. Semen cultures were obtained by inoculation onto blood agar and MacConkey's plates. The isolates were identified according to Bergey's Manual of Determinative Bacteriology. The quantity of bacterial growth on the primary blood plates was estimated by recording a heavy growth as 4 +, a moderately heavy growth as 3 +, a moderately light growth as 2 +, and a light growth as 1 +.

4 496 BUXTON ET AL. Fertility & Sterility SPERMICIDAL EFFECT OF BACTERIA Determination of Spermicidal Activity All bacteria to be tested for spennicidal activity were first grown in heart infusion broth for 24 to 48 hours. The bacterial cells were separated from the culture medium by centrifugation and resuspended in normal saline solution. The spenn suspension was prepared by centrifuging the semen and then resuspending the spennatozoa either in seminal fluid or in nonnal saline so that the final volume would contain 100 million spermatozoa per cc. Only specimens of excellent quality were used. Equal quantities of each suspension were mixed in a small test tube and a drop of this mixture transferred to a glass slide. A wet-mount preparation was made by placing a coverslip over the drop and covering the edges with petroleum jelly to prevent drying. The preparation was kept at room temperature and periodically examined under the microscope for evidence of sperm agglutination and decreased motility. The following organisms isolated from the vagina, cervix, and semen were capable of clumping spennatozoa: Escherichia: coli, freundii, intermedium Aerobacter: aerogenes, cloacae Paracolobactrum: intermedium, aerogerwides, coliforme Alcaligenes: faecalis, bookeri, metalcaligenes Yeast All the organisms belonging to the genera Escherichia, Aerobacter, Paracolobactrum, and Alcaligenes were highly spermicidal and clumping began immediately after mixing the spennatozoa and the organisms. One hundred isolates of micrococci from semen were tested; only about 8 showed the ability to clump spennatozoa. None of the micrococci isolated from the vagina had the ability to clump spermatozoa. It was thought that the other criterion for spermicidal activity as described by Matthews and Buxton-evidence of longevity in certain bacterial suspensions-was probably not significant for two reasons. First, there were a number of extraneous factors involved, and second, the length of time before immobility occurred should not, from a practical point of view, prevent passage of spennatozoa through the cervix...

5 Vol. 5, No.6, 1954 CERVICAL BACTERIOLOGY 497 All organisms listed as being spermicidal were found at one time or another in the semen when it was presented to the laboratory. We have not determined whether all these organisms were due to contamination or if they were actually present in the male genital tract. Quantitative Studies of Spermicidal Activity Varying concentrations of bacteria. A stock preparation of Esch. coli bacterial cells was prepared as described previously. The concentrated mass of bacterial cells was suspended in normal saline. The number of bacteria present in the solution was estimated by comparison of turbidity to standard tubes of a McFarland nephelometer. All dilutions were made from this cell suspension. The sperm suspension was prepared by suspending the spermatozoa, separated from the seminal fluid by centrifugation, in normal saline so that the suspension contained 100 million spermatozoa per cc. An equal volume of this suspension was added to a series of small test tubes containing equal volumes, but varying concentrations of bacteria. Each tube then contained a constant of 50 million spermatozoa per cc. with concentrations of Esch. coli cells ranging from 300 million to 1 million per cc. End-point determinations were made by use of a macroscopic tube agglutination test which was found to be as reliable as microscopic examination of a wet-mount preparation. This was performed by placing the mixtures of spermatozoa and bacteria in Dunham fermentation tubes (50 X 6 mm.) and observing flocculation macroscopically. The end-point was defined as the last tube showing flocculation in one hour at room temperature. The control for this test consisted of the same sperm suspension without added bacteria. It remained uniformly turbid during the period of observation. Varying concentrations of sperm. In this study, the concentration of the test organism, Esch. coli, was kept constant at 10 million per cc. The spermatozoa were once again centrifuged out and resuspended in normal saline to yield concentrations varying from 6.2 million to 200 million per cc. Results. As Table 1 shows, the most effective spermicidal organisms found were Esch. coli and Paracolobactrum aerogenoides and the least effective were the micrococci. Most of our work dealt with Esch. coli because it was so effective in small numbers and because it was the most common of the enteric organisms found in the vaginal tract. Figure 1 illustrates how the end-point determinations of Table 1 were

6 498 BUXTON ET AL. Fertility & Sterility obtained. The spermatozoa have nearly completely settled out in the first three tubes (left to right). There are large aggregates of spermatozoa suspended in the fourth, fifth, and sixth tubes; smaller flocculi of spermatozoa in the seventh and eighth tubes, and no sperm agglutination in the last tube. TABLE 1. Macroscopic Tube Agglutination Test: Lowest Concentration of Organisms Exhibiting Spermicidal Activity Against 50 Million Spermatozoa Per ml. in 1 Hour Millions perml. Organism Esch. coli Esch. intermedium Esch. freundii Aerobacter aerogenes Aerobacter cloacae Alcaligenes faecalis Paracolobactrum aerogenoides Paracolobactrum intermedium Paracolobactrum coliforme Yeast Micrococci Control Fig. 1. Macroscopic tube agglutination test after one hour. The tubes contained a constant concentration of 50 million spermatozoa per ml. and varying concentrations of Esch. coli. The control tube remained uniformly turbid throughout the length of the test. The numbers under each tube indicate Esch. coli concentration in millions/cc. Our end-point, therefore, was the eighth tube containing 2.3 million Esch. coli per cc. Figure 2 illustrates the effect on motility of varying concentrations of Esch. coli in semen of good quality. The spermatozoa were affected by the organisms in a concentration as low as 1.1 million per cc. and were markedly affected in a bacterial concentration of 2.3 million per cc.

7 Percentage of Motility of Nona991utenated Spermatozoa Fig Spermatozoa and Saline \.~ Spermatozoa and 30 '" Esch. Coli 20.~ lS 7S ISO 300 Millions of Esdi. Co/i per ml. Effect on spenn motility of varying concentrations of Each. coli. Fig Percentage of Motility of 80 Nona991utenated 70 Spermatozoa /0_0_0 Spermatozoa and Saline / spe:~ozoa Esch.Coli / O+=--~--~~--~---.--,---, S ISO 200 Millions of Spermatozoa per cc. Effect on spenn motility of Each. coli in varying concentrations of spermatozoa.

8 500 BUXTON ET AL. Fertility & 5teriiity Figure 3 illustrates the effectiveness of a constant concentration of Esch. coli in the presence of low concentrations of spermatozoa and how its relative effectiveness was decreased as the sperm concentrations increased. At sperm levels of up to 12.5 million per cc., about 95 per cent were either clumped or nonmotile; at a sperm concentration of 50 million per cc., about 50 per cent were affected; and at a concentration of 100 million per cc. and over, about 10 per cent of the spermatozoa were clumped, while the remaining 90 per cent were actively motile and exhibited no adverse effects. STUDIES ON THE SPERM AGGLUTINATING FACTOR OF ESCH. COLI Rosenthal, in 1931 and 1943, presented evidence to indicate that the sperm agglutinating factor of Esch. coli is within the cell itself. The reaction occurs with fresh or formalized bacterial cells, in acid or alkaline medium, and with cells repeatedly washed in saline. He found that the Berkefeld filtrate of Esch. coli cultures was inactive, as well as saline washings. If these cells were killed by heating at 60 C. for 24 hours or more, they still possessed the ability to clump spermatozoa. Cells killed by boiling for one minute were inactive. He believed that the active principle may be due to a special protein substance or to an enzyme in the bacteria. These studies have been repeated in our laboratory with similar results. Bacterial extracts were prepared by growing Esch. coli on agar surfaces to obtain a large cell mass. The bacteria were washed with normal saline and centrifuged from the suspension. This cell paste was then ground in a mortar and pestle with powdered glass (30 mesh) for five minutes. 1S An extract was obtained which, when tested against spermatozoa, proved to be ineffective. The sediment, however, containing disrupted bacterial cells as well as whole cells, maintained its spermicidal activity. When Esch. coli were killed with formalin or with heat at 60 C. for one and a half hours, they were found to be as effective as living cells. However, when Esch. coli suspension was placed in a boiling water bath for 30 to 45 seconds, its sperm agglutinating ability was destroyed. The storage ofa saline suspension of Esch. coli in the refrigerator for 7 months. did not diminish its activity.

9 Vol. 5, No.6, 1954 CERVICAL BACTERIOLOGY 501 ADAPTATION FROM NONSPERMICIDAL TO SPERMICIDAL ACTIVITY An experiment was set up to determine whether an organism, at first innocuous to spermatozoa, might be adapted to cause sperm clumping. Proteus mirabilis #4, isolated from semen, was selected as the test organism because it is a gram-negative proteolytic bacillus and because, when originally isolated, it had no effect on spermatozoa. Three cc. of semen was inoculated with a heavy inoculum of this organism and incubated at 37 C. for 2 days. At 48-hour intervals, 0.5 cc. of the culture was transferred to a fresh substrate of semen for a total of 10 transfers. The organism was reisolated and identified before testing it against spermatozoa. This adapted strain proved to be highly spermicidal, whereas the parent stock culture remained ineffective. Several months later the experiment was repeated with the original parent culture and similar results were obtained. It is a well-established principle that biologic variation may be effected by altering environmental conditions, and generally the variants are more suited to the changed environment than the parental organisms. Thus it would appear possible that an organism at first innocuous to spermatozoa may become spermicidal, with or without benefit to itseu. Such an adaptation might conceivably occur in the male genital tract. BACTERIAL FLORA The purpose of this study was to determine the incidence of various organisms in the entire group of 485 patients. These cultures were taken at the time of the postcoital test before any treatment was given and were used as a control throughout this study. In addition to the organisms listed in Table 2, the following organisms were also isolated with an incidence of less than one per cent: Flavobacterium, Sarcina, GaDkya, bacterioids, and Actinomyces. Organisms which were probably lactobacilli were frequently isolated but are not tabulated. Escherichia coli was the only organism listed as to species, because it was highly spermicidal and most of the work on this problem was done with this organism. All other organisms were listed by genus only because in a number of cases it was only possible to identify the genera. Yeasts were not identified beyond establishing the fact that they were yeasts. Most of these were

10 502 BUXTONET AL. Fertility & Sterility isolated from the vagina and with few exceptions caused clumping of spermatozoa within one half hour. Beta hemolytic streptococci, some alpha hemolytic streptococci and nonhemolytic streptococci, as well as Clostridium perfringens, and Proteus vulgaris (not mirabilis) cause a progressive decrease in sperm motility. The TABLE 2. Incidence of Bacteria Found in the First Postcoital Cervical Cultures of 485 Patients Organism Esch. coli Aerobacter Pariwolo bactrum Alcaligenes Micrococcus Enterococcus Streptococci Beta hemolytic Alpha hemolytic Nonhemolytic Diphtheroides Bacillus Clostridium Achromobacter Gram-negative bacilli unidentified Gram-positive bacilli unidentified Nocardia Yeast No growth Incidence (%) time needed for this effect to become apparent is variable, but spennatozoa are often not significantly affected within the first one to two hours of observation. It is thought, therefore, that the clinical significance of this finding is questionable. Comparison of Cervical to Vaginal Flora It is probably impossible to obtain a culture from the cervical canal uncontaminated by vaginal organisms. The organisms isolated from our socalled cervical cultures may have been present only at the external cervical os, and, therefore, reflect only vaginal flora. Hecht-Lugari, in a study of 26 cases, in 1951, cultured the endocervical canal from above at the time of

11 Vol. 5, No.6, 1954 CERVICAL BACTERIOLOGY 503 supravaginal hysterectomy. These cultures were all negative except for the isolation of a micrococcus in one patient. Various technics of obtaining cervical cultures have been tried. Cultures taken by aspirating cervical mucus with a sterile pipette, either with or without first using an antiseptic, or by simply carefully inserting a sterile swab into the endocervical canal, show essentially the same type of growth. To further evaluate this problem, cultures were taken from the vagina at the same time the cervical cultures were taken. One hundred and forty-four comparative cultures have been studied. The amount of growth on the primary plates was graded from 1 + to 4 +. TABLE 3. Comparison of Cervical to Vaginal Flora (144 Cultures) Frequency (%) Average amount of growth Organism Cervix Vagina Cervix Vagina Each. coli Aerobacter Alcaligenes Micrococcus Enterococcus Paracolobactrum Streptococci Beta hemolytic Alpha hemolytic N onhemolytic Diphtheroides Bacillus Proteus Yeast No growth 5 0 Table 3 shows the comparative frequency of organisms isolated from the cervix and from the vagina, and the average amount of growth for each organism. It will be noted that all organisms are found with either equal or increased frequency in the vagina, and with either equal or increased amount of growth. In no instance was Esch. coli recovered from a cervical culture when it was not also present in the vaginal culture. Alpha hemolytic streptococci were recovered six times from the cervix when they were not identified on vaginal cultures. In all of these cases, there was a heavy growth of other organisms on the vaginal cultures which may have overgrown or obscured the streptococci. We must assume, then, either that organisms grow with equal facility in the vagina and cervix, or that our

12 504 BUXTON ET AL. Fertility & Sterility cervical cultures are so grossly contaminated when they are obtained that they do not represent the actual bacteriologic content of the cervical mucus. The medium of either the cervix or vagina is not deleterious to growth of the spermicidal organisms mentioned, even though there are differences in chemical constituents, ph, and other physical characteristics; and the proximity of the cervix and vagina makes the assumption of a spread of these organisms between them seem reasonable. On the other hand, if the spermicidal organisms are introduced into the vagina at the time of coitus, they presumably would not multiply and reach the cervix in time to provide a bacterial barrier for the spermatozoa. The only effectively spermicidal bacteria in this instance would presumably be ones which were introduced at the time of previous coitus. Therefore, it seemed advisable to investigate what changes in cervical bacteria population were affected by coitus. Effect of Coitus on Number and Type of Cervical Bacteria Cultures were taken from the cervix of 98 patients who had not had intercourse within at least 48 hours. Many of these patients were scheduled for insemination using the husband's semen in a cervical cap. Therefore, precoital and postcoital cultures on the same patient are not compared, but the precoital cultures are compared to the postcoital cultures of the entire group of 485 patients previously presented. Eighty-two semen specimens were cultured. These were not obtained under sterile conditions, but were used to show that the changes found in precoital and postcoital cultures cannot be attributed to the semen. The incidence of some organisms was lower in the cultures. taken at least 48 hours after intercourse, as shown by Table 4. This difference is most marked in the coliform group of organisms, but is also noticeable in the streptococcus and micrococcus groups. Even though the semen was not collected under sterile conditions, we cannot attribute this difference to organisms present in it. Mechanical transfer of skin contaminants at the ~ time of intercourse seems the most likely explanation. It is probable, therefore, that many of the coliform organisms isolated from postcoital cultures are not usual inhabitants of the female genital tract, but are introduced at the time of intercourse. Furthermore, the cervical and vaginal environment is apparently inimical to continued growth of these organisms, because they decrease greatly in numbers within 48 hours.

13 Vol. 5, No.6, 1954 CERVICAL BACTERIOLOGY 505 Cyclic Cultures It was desired to study possible cyclic variations in the bacterial content of cervical cultures. Cultures were, therefore, taken in the postmenstrual phase, at the time of ovulation, and in the premenstrual phase. No consistent pattern of occurrence was found in a group of 16 sterility patients other than changes that would be expected following coitus. ~ I TABLE 4. Effect of Coitus on Organisms Isolated from the Cervix Incidence in cultures (%) Organism Postcoitala Precoitalb Semenc Esch. coli Aerobacter Paracolobactrum Alcaligenes Micrococcus Enterococcus Streptococcus Beta hemolytic Alpha hemolytic N onhemolytic Diphtheroides Bacillus Proteus Yeast No growth a Cultures taken within 18 hours after coitus (485 patients). b Coitus 48 hours or more before culture was taken (98 patients). c Eighty-two patients. Six women not having had coitus were studied. Vaginal cultures were taken to avoid introducing a speculum through a virginal introitus. Each had a postmenstrual, ovulatory, and premenstrual culture. In eighteen cultures on these 6 women, Esch. coli were found once. Beta hemolytic streptococci were found once and alpha hemolytic streptococci three times. There was no indication in either the group of sterility patients or the group of women not having had coitus that these organisms were more frequent at one time of the cycle than at another. This study would seem to indicate that these organisms are cervical and vaginal inhabitants occasionally but not necessarily persistently, and, therefore, that one isolated finding is not necessarily indication for treatment.

14 506 BUXTON ET AL. Fertility & Sterility EFFECT OF TREATMENT ON CERVICAL FLORA Incidence of Organisms After Antibiotic Therapy Postcoital tests and cervical cultures were taken at the time of ovulation. When spermicidal organisms, as determined by in vitro tests, were found in the cervical cultures, specific antibiotics were given in the preovulatory phase of the following cycle. The intracervical treatment consisted of four injections of one of the following drugs: penicillin, 1 million units; streptomycin, 0.5 Gm.; or bacitracin, 2000 units per injection. These antibiotics each were dissolved in 2 cc. of normal saline or distilled water and injected into the submucosa of the cervical canal at several points. Other treatment consisted of terramycin or aureomycin applied locally in the form of vaginal suppositories or in a plastic cervical cap. Systemic therapy consisted of penicillin, 500,000 units; streptomycin, 1 Gm.; or aureomycin or terramycin, 2 Gm. daily, for four days immediately following the menstrual period. Gantrisin vaginal cream and ACi-Jel were applied on other occasions by the patient on four successive days following the cessation of menses. After treatment, cervical cultures and postcoital tests were repeated as near the time of the next ovulation as possible. Patients using contraceptive creams and diaphragms are included in this group for purposes of comparison. Results. The results of the posttreatment cultures are recorded in Table 5. It will be noted that the incidence of the coliform group of organisms which cause rapid clumping of spermatozoa in vitro are increased after penicillin therapy given either systemically or locally, and after intracervical bacitracin. The incidence of these organisms was not reduced by intracervical strept~mycin. This increase occurred for the most part in the patients injected intracervically and may possibly be due to the.repeated manipulations and cervical trauma involved. Only 9 patients were treated with systemic streptomycin. The coliform organisms were not found in the postcoital cultures of these patients, but the number of patients is too small to evaluate. The local use of terramycin and aureomycin, Gantrisin, and Aci Jel all reduced the incidence of these organisms in the small series in which they were used. In the case of the latter two medications, the series was again too small to be more than suggestive. Patients using contraceptive creams and douches had a similarly low incidence of coliform bacteria.

15 TABLE 5. Effect of Treatment on Cervical Flora Percentage of incidence Terramycin or Contra- Untreated Penicillin Streptomycin Bacitracin aureomycin Gantrisin Aci-lel ceptives I. C.a Syst. b I.C. Syst. I. C. Vaginal Systemic Vaginal Vaginal Vaginal Number of patients Esch. coli Aerobacter Paracolobactrum Alcaligenes Micrococcus Enterococcus Streptococcus Beta hemolytic Alpha hemolytic Nonhemolytic Diphtheroides Bacillus Proteus Yeast No growth a I. C. = intracervical. b Syst. = systemic.

16 508 BUXTON ET AL. Fertility & Sterility Yeasts were present in increased incidence after aureomycin or terramycin treatment. The occurrence of the alpha hemolytic group of streptococci is lower in the group of patients receiving local applications of terramycin or aureomycin. Since Table 4 shows that the incidence of coliform bacteria is higher in postcoital than precoital cultures and that in many instances these organisms were probably introduced mechanically at the time of intercourse, we would not expect therapy discontinued 24 to 48 hours before the postcoital culture to markedly affect these cultures. In the instances where antibiotic therapy markedly increased the incidence, we may suppose that this was due to alteration of the vaginal environment to one favorable to the growth of the coliform organisms.. It is interesting to note in this connection that Buxton and Wong also found a frequent occurrence of other spermicidal organisms following treatment. Actually, a postcoital cervical culture may not accurately represent the continued bacterial status of the cervical canal. CLINICAL SIGNIFICANCE OF SPERMICIDAL ORGANISMS FOUND IN THE FEMALE GENITAL TRACT Since a good postcoital test is dependent upon accurate timing in many cases, the bacterial flora in those patients having good results from postcoital tests have been studied. These results are shown in Table 6 without regard to therapy during the cycle except for those instances where Esch. coli probably occurred following treatment. Except for the treated patients, when it is higher, the incidence of Esch. coli is similar to that of the group of untreated patients. Cultures at Conception Time Forty-one patients from whom cultures were taken at conception time have been studied (Table 6). The incidence of various organisms in these cultures is similar to that found in the postcoital cultures of 485 patients, except in those who had antibiotic therapy, which raised the incidence of the coliform organisms. The coincidence of good results from postcoital sperm tests and average postcoital incidence of Esch. coli is hard to explain in the light of the fact that Esch. coli are highly spermicidal. Here again the postcoital culture may not be truly indicative of the real bacterial status of the cervix r

17 Vol. 5, No.6, 1954 CERVICAL BACTERIOLOGY 509 and the unmeasurable quantitative factor of bacteria and spermatozoa may be of great significance. A more detailed analysis of these cases is recorded in Table 7 where the results of the postcoital test, the day of the patients' menstrual cycle, and the semen counts are recorded. Four out of 34 recorded postcoital tests at conception time showed no spermatozoa, indicating either that a good postcoital test is not always present at conception time or that conception occurred on some other date. TABLE 6. Clinical Significance of Organisms Isolated from Cervical Cultures Incidence (%) Conception During Postcoital Good postcoital (postcoital) pregnancy Number of cases Each. coli Untreated All cases Aerobacter Paracolobactrum Alcaligenes Micrococcus Enterococcus Streptococcus Beta hemolytic Alpha hemolytic Nonhemolytic Diphtheroides Bacillus Proteus Yeast No growth Cultures During Pregnancy The organisms isolated from the cervix of women during pregnancy are shown in Table 6. These are not thought to serve as a control in any way. The incidence of Esch. coli is similar to that of sterility patients. EFFECT OF ANTIBIOTIC THERAPY UPON PREGNANCY RATE One hundred and eighty-three patients received one or more courses of antibiotic therapy. Sixty-two of these patients either had a major reason for sterility other than a possible cervical factor or were unavailable for

18 510 BUXTON ET At. Fertility & Sterility TABLE 7. Postcoital test Patients Studied During Conception Cycle Semen Case Time of Volume Count Spermicidal Mucus" Spermb cyclea (ml.) (millions/ml. ) organisms / Esch. coli / Esch. coli / Esch. coli / Esch. coli /28 Esch. coli / Esch. coli / Esch. coli / Esch. coli /28 Esch. coli / Esch. coli, a strep / a strep / a strep / a strep. 14 9/ a strep / a strep /28 a strep / a strep /? a strep /irreg a strep / f3 strep / f3 strep / Cl. welchii 23 15/ Yeast /28 None / None /29 None 27 14/27 ' None / None / None / None / None / None /30 None / None / None / None /? None / None /31 None / None /? None a I = poor; 2 = fair; 3 = good. b I = less than 5/H.P.F.; 2 = 5-I5/H.P.F.; 3 = more than I5/H.P.F. a Day of postcoital test/usual length of cycle.

19 Vol. 5, No.6, 1954 CERVICAL BACTERIOLOGY 511 follow-up. One hundred and twenty-one patients had no apparent cause for infertility. The untreated group consisted of 297 patients. Of these, 140 had no major cause for infertility. One hundred and fifty-seven either had a major cause found for their infertility or could not be followed. Results. Nine patients, or 7.4 per cent, of 121 treated patients without a major cause for infertility became pregnant during the same cycle that treatment was given (Table 8). One of these patients was not cultured after treatment. Of the remaining 8 patients, 3 had Esch. coli and one had alpha hemolytic streptococci in the post-treatment culture. TABLE 8. Pregnancies Occurring After Antibiotic Therapy (121 Patients with No Major Cause for Sterility) Time since treatment Number of pregnancies Same cycle 9 Six months or less 29 Over six months 10 TOTAL 48 Cumulative (%) TABLE 9. Pregnancies Occurring Without Antibiotic Therapy (140 Patients with No Major Cause for Sterility) Time since culture Same cycle Six months or less Over six months TOTAL Number of pregnancies Cumulative (%) Thirty-four treated patients became pregnant within three months, 38 within six months, and 10 after six months, making a total of 48 treated patients who became pregnant, or 40 per cent of a group with no other major cause of sterility. A comparison between these figures and those of the untreated group, however, is of considerable interest. Twenty-four patients (17 per cent of the untreated group without major cause for sterility) became pregnant in the same cycle in which a culture was taken and did not have antibiotic therapy (Table 9). Five of these or 20 per cent had cervical cultures containing Esch. coli. Beta hemolytic streptococci were found in two cultures. Only 2 patients with a culture at the

20 512 BUXTON ET Al. Fertility & Sterility time of conception had yeasts present. Nine patients who had cultures taken during the conception cycle had had treatment in previous cycles. Of these 9 patients, 2 had cultures containing Esch. coli, 3 had alpha hemolytic streptococci, and 1 had Cl. welchii. Forty per cent of the group receiving antibiotic therapy and 46 per cent of the group not receiving antibiotic therapy eventually became pregnant if patients with major causes of sterility were excluded. The pregnancy rate for both groups including all patients is 23 per cent, which is equal to the over-all pregnancy rate in the entire clinic. Table 10 indicates the over-all pregnancy rate for the entire series and shows the similarity of results in the treated and untreated patients. TABLE 10. Pregnancies in Treated and Untreated Groups Antibiotic therapy, all cases No major cause of sterility No antibiotic therapy, all cases No major cause for sterility Both groups, all cases No major cause for sterility Number of Pregnancy rate cases (%) DISCUSSION Several significant factors evolve from the above findings. It is clear that, as previously reported by us and others, the coliform group of organisms, especially Esch. coli, is highly spermicidal in vitro. Whether the spermicidal characteristics of various streptococci as previously reported by this laboratory have a rapid enough action to be of clinical significance is somewhat questionable. Although the over-all results in treated patients are generally similar to those of the preliminary report from this laboratory published by Buxton and Wong in 1950, an accumulation of adequate control material indicates that results in untreated patients are so similar that treatment has had no effect whatever on the pregnancy rate. This means either that the presence of spermicidal bacteria in the vagina and cervix has no effect on pregnancy, that the various types of treatment as carried out at the present time do not get at the basis of the problem, or that our present culture technics do not indicate the actual bacterial content of the cervical mucus.

21 Vol. 5, No.6, 1954 CERVICAL BACTERIOLOGY 513 It must be remembered that the patients reported here as having no other major cause for infertility were ones who, for the most part, had no obvious evidence of cervical infection so that a cervical barrier, if it existed, was not necessarily mechanical or chemical. Therefore, when poor results from postcoital tests occurred, suspicion was cast upon the possible spermicidal effect of bacteria which might be present. This, however, has not been proved. As a result of the present findings, it would appear reasonable to further investigate two problems. The first is to search for a more effective way of obtaining cultures from the cervical canal and, if this proves satisfactory, a more effective technic for treatment. The second is a search for other areas where spermicidal bacteria might be affecting spermigration which would result in poor postcoital tests and pregnancy failures. This suggests the possibility that the presence of spermicidal organisms in the male genital tract is a factor in changing the characteristics of spermatozoa. Plans for an investigation of this aspect of the problem are now under way. SUMMARY 1. Many of the coliform organisms, especially Esch. coli, and some yeasts are highly spermicidal in vitro. The concentration of organisms and the concentration of spermatozoa necessary to produce this effect have been determined. 2. Factors influencing spermicidal activity of these organisms are presented. 3. The spermicidal effect of various streptococci has such a long latent period that its significance is questionable. 4. Spermicidal organisms have been isolated from the cervix more frequently within the first 18 hours after intercourse than in cases where coitus has not occurred within two or more days before cultures were taken. 5. These organisms are not consistently found in anyone patient, as shown by repeat cultures without intervening treatment. Different phases of the menstrual cycle do not affect the occurrence of these organisms. 6. Adequate control studies indicate a similarity in incidence of spermicidal organisms in good postcoital tests and in cultures taken at the time of conception to the incidence found in routine cultures in sterility patients.

22 514 BUXTON ET AL. Fertility & Sterility 7. Adequate control studies indicate no diherence in pregnancy rate be~ tween those who have or who have not received antibiotic therapy. 8. Further technics for the investigation of the ehects of spennicidal or~ ganisms on fertility are suggested. REFERENCES 1. BARTON, M. The role of modern antibiotics in the treatment of infertility. Proc. Soc. Ferta., No.4: 55, BARTON, M., and WmsNER, B. P. The role of special diets in the treatment of female infecundity. Brit. M.J. 2:847, BICKERS, W. Patterns of uterine motility in relation to spermigration. Fertil. & SterlZ. 2:342, BUXTON, C. L., and WONG, A. S. H. Spermicidal bacteria in the cervix as a cause of sterility. Am. J. Ohst. & Gynec. 64:628, GEPFERT, R., and DAVIS,!. F. Endocervicitis: Its role in infertility; treatment with aureomycin. Ferta. & Stem. 4:318, HEClIT-LUGARI, C. Ricerche batteriologiche sui contenuto del canale cervicale dell'utero di dona. Glin. Ostet. 53: , HORNE, H. W., JR., and ROCK, J. Oral terramycin therapy of chronic endocervicitis in infertile women. Ferta. & Steril. 3:321, KAYE, B. M., COHEN, M. R., and MACLEAN, H. Significance of cervical bacteria in infertility. Ohst. & Gynec. 3:644, LAMAR, J. K., SHE'ITLES, L. B., and DELFS, E. Cyclic penetrability of human cervical mucus to spermatozoa in vitro. Am. J. PhysioZ. 129:234, LAUGlITON, N. Observations on the bacterial Bora of the cervical canal in infertile women with cervical dysfunction. Proc. Soc. Fertil., No.3: 18, MATrHEWS, C. S., and BUXTON, C. L. Bacteriology of the cervix in cases of infertility. Fertil. & SteriZ. 2:45, MOLOY, H. C. Sulfamylon in the treatment of chronic vaginal discharge associated with sterility. New York State J. Med. 50:992, ROSENTHAL, L. Spermagglutination by bacteria. Proc. Soc. Exper. BioI. & Med. 28:827, ROSENTHAL, L. Agglutinating properties of Escherichia coli. J. Bact. 45:545, RUBENSTEIN, B. B., STRAUSS, H., LAZARUS, M. L., and HANKIN, H. Sperm survival in women. FertiZ. & Steril. 2: 15, SANDLER, B. The investigation and treatment of sterility. Med. Press 227:56, SHOTTON, D. M. Some observations on the cervical factor in human infertility. Proc. Soc. FertiZ., No. 3:9, UMBREIT, W. W., BURRIS, R. H., and STAUFFER, J. F. Manometric Techniques and Tissue Metabolism (ed. 2). Minneapolis, Burgess, 1949.