THE CERVIX AND ITS SECRETIONS have been the subject of numerous histologic

Transcription

1 Fractional In-Vivo and In-Vitro Examination of Postcoital Cervical Mucus in the fiuman VAL DAVAJAN, M.D., and GEORGE M. KUNITAKE, PH.D., M.D. THE CERVIX AND ITS SECRETIONS have been the subject of numerous histologic and biochemical studies Much information concerning human semen has a1so been published However, there is a significant lack of data regarding the interaction of spermatozoa and cervical mucus in both fertile and infertile couples. Malfunction of cervical secretions is thought to be the major etiologic factor in one-third to one-half of all instances of infertility These conclusions usually are based on the premise that an unidentified physical or chemical component in the cervical mucus immobilizes or kills the sperm. A cursory microscopic examination of postcoital cervical mucus generally is used to substantiate this theory. The examination of postcoital cervical mucus was first described by Sims in 1869; Huhner amplified and popularized the method in Since then little additional information has been presented in this area of research. The Sims-Huhner test is still useful, but the procedure is not standardized. Therefore, it is extremely difficult to interpret the variety of results reported in the literature. The purpose of this paper is to describe a method for performing a fractional in-vivo cervical mucous test. In addition, a fractional in-vitro test is a1so described. The latter method has been developed to allow a more thorough investigation of the interaction of cervical mucus and sperm in the infertile couple. From the Division of Reproductive Biology, and the Departments of Obstetrics and Gynecology, and of Pathology, Harbor General Hospital, Torrance, Calif., and the UCLA School of Medicine, Los Angeles, Calif. Supported by a Grant from the Ford Foundation and by Grant 5T1HD31 from the National Institute of Health, U. S. Public Health Service. This paper was the recipient of the Squibb Prize A ward at the American Fertility Society Meeting, San Francisco, Calif., Mar ,

2 DAvAJAN & KuNITAKE 198 FERTILITY & STERILITY MATERIALS AND METHODS Cervical Mucus Collection In order to evaluate the relation between cervical mucus and spermatozoa, a new method of mucous collection has been developed. The cervical mucus is aspirated gently with a 10-cc. disposable syringe attached to a No. 14 polyethylene suction catheter. The catheter is stabilized by grasping it 3 em. from the distal end with an atraumatic cervical tenaculum (Fig. 1). The grasping part of the clamp ( atragrip) should be adjusted carefully, with the clamp set at the first ratchet. The closed clamp must allow the atragrip to be loose enough to permit gentle aspiration of the mucus at the same time acting as a trap in order to preclude the loss of the mucus into the more proximal portion of the catheter. This procedure, if done properly, will ensure an even spread of the mucus in the distal 3 em. segment of the collecting catheter. Aspiration is initiated as the catheter is introduced into the external cervical os. A constant negative pressure should be maintained with the syringe while the catheter is advanced slowly to the level of the internal os ( 2.5 em.). Aspiration is terminated at this time and the atragrip is closed by locking the rachets completely. When aspiration of the mucus is performed in this manner, the mucous sample closest to the atragrip represents mucus collected from the level of the external os, while the mucus at the distal end of the catheter represents mucus collected from the level of the internal os. The position of the aspirated mucus contained Fig. 1. Suction catheter (polyethylene No. 14) grasped with atraumatic cervical tenaculum. Atragrip acts as a mucous trap. The cervical mucus sample is confined to the 3-cm. distal segment.

3 VoL. 20, No.2, 1969 PosTcOITAL CERVICAL Mucus 199 in the 3 em. segment catheter thus will be related directly to its original position in the endocervical canal. As the catheter is withdrawn from the cervical canal, it is necessary to cut away any trailing mucus with long curved scissors. If the aspirated mucus is not cut away from the mucus remaining in the cervical canal, the tenacity of the mucus may either pull the sample out of the catheter or draw off the remaining mucus from the cervical canal allowing the sample to become contaminated by the contents of the vagina. In-Vivo Fractional 2-Hr. Postcoital Test After two days of abstention, the couple is instructed to have sexual intercourse within 2 hr. prior to the time scheduled for mucous collection. The cervical mucus is then collected as described above between 1 and 2 hr. following coitus. The 3-cm. section of the catheter containing the sample is cut into four segments with scissors (Fig. 2). Each segment is then examined grossly and microscopically. In-Vitro Fractional Compatibility Test The couple is again instructed to abstain from intercourse for 2 days prior to the scheduled examination. On the day of the examination, instead of having intercourse as is done in the in-vivo test, the wife is directed to bring a fresh sample of her husband's semen to the clinic. A cervical mucous sample is then collected from the patient as described above, but unlike the in-vivo test, the 3-cm. cylinder containing the mucus is not cut. Instead, the catheter containing the cervical mucus is Fig. 2. Four cut segments of 3-cm. segment catheter containing cervical mucous sample. placed in a small beaker in which the husband's fresh semen sample has been placed. The mucus is exposed to the spermatozoa at 37 C. for 90 min. (Fig. 3). After exposure to the semen, the cylinder is removed, cut into four segments, and examined as in the in-vivo test.

4 200 DAvAJAN & KuNITAKE FERTILITY & STERILITY Gross and Microscopic Examination of Cervical Mucus Volume Determination. The approximate volume of each specimen is determined by comparing it to a similar length of catheter containing a known amount of oil medium. A B '------~0 CERVICAL MUCUS - SAMPLE l Fig. 3. In-vitro fractional postcoital test: 3-cm. segment of catheter (A) containing cervical mucus is exposed to fresh semen sample (B) at 37 C. for 90 min. Catheter is then cut into four segments (C) and examined as in the in-vivo test. -=/==~=~=~====::J/ ph Measurement. The approximate ph of each segment of catheter is determined by using phydrion ph paper. One end of each segment is placed gently on a strip of the phydrion paper and allowed to remain in contact for 10 sec. Color change is then used to determine approximate ph of the cervical mucus. Spinnbarkheit. Each segment of the catheter containing the cervical mucus is tested for the capacity of the mucus to be drawn into a thread (spinnbarkheit). With a small hemostat, the catheter segment is pressed against a microscope slide. The extruding portion of the mucous plug is held against the slide by means of a coverslip. The catheter segment is then elevated to the maximum capacity of mucus threadability. This maximum height is measured with a centimeter ruler positioned vertically behind the slide (Fig. 4). Microscopic Examination. The entire portion of mucus in each segment of the catheter is then spread in a thin layer on a glass microscope slide and covered with a coverslip. With a binocular microscope, five separate fields are examined at 40X magnification. The total number of sperm

5 VoL. 20, No.2, 1969 PosTCOITAL CERVICAL Mucus 201 present are counted and recorded. In order to provide consistency, all counts are best performed by the same examiner. Counting may be performed rapidly and accurately by counting one quadrant of the field at a time in a clockwise manner. Following the total count, the number of immobile sperm per field is determined by a second rapid count. Thus, the number of motile sperm can be calculated by subtracting the second count (immobile) from the first (total). This procedure is followed in each of the five high-power fields examined. The mean count of the five fields is recorded as the total count per high-power field. The percent motility for each segment of cervical mucus also is recorded. Fig. 4. Spinnbarkheit: cervical mucus is held against the glass microscope slide (A) by means of a coverslip (B). Catheter segment is elevated (C) to maximum capacity of mucous threadability. Preliminary Application of Described Methods The methods described were used in examination of the interaction of the cervical mucus and spermatozoa of 15 couples from the fol1owing selected groups: Group I-five fertile couples with previous proven fertility who were not using any type of contraception; Group II-Five infertile

6 202 DAVAJAN & KuNITAKE FERTILITY & STERILITY couples who had no demonstrable reason for their infertility and who had had a normal Sims-Huhner postcoital test performed by their referring gynecologist; and Group III-Five infertile couples who had had a poor Sims-Huhner postcoital test as the referring diagnosis. Each patient was examined initially at a time calculated to be 2 days prior to the expected rise in the basal body temperature as determined by the recording of this parameter in previous cycles. Volume of Cervical Mucus RESULTS The volume of cervical mucus obtained at the time of the initial examination varied from 0.10 mi. in a patient from Group III to 0.7 mi. in a patient from Group I (Table 1). There was no appreciable difference in the amount of cervical mucus collected between Group I and Group II patients. The mean volume from the patients in Group III was decreased as compared with Group I, but 4 of the 5 patients did have a volume consistent with the lower range of the normal group (Group I). ph Determination The ph of the cervical mucus from all patients was alkaline ( Fig. 5). The mucus collected from the external os level had a lower mean ph than the mucus aspirated from a higher level in the cervical canal in all the samples tested. The mean ph noted at the 2-cm. level of patients in Group III was appreciably lower than the other groups. Spinnbarkheit The cervical mucus obtained from each of the 15 patients was tested for its threadability ( spinnbarkheit) at the four cervical levels of the catheter (Fig. 6). When comparing the threadability at the external os level of Group I and Group II patients, no apparent difference was noted. The TABLE I. Volume of Cervical Mucus Obtained at Initial Examination Volume of cervical mucus (ml.) Patient Group I Group II Group Ill ,

7 VoL. 20, No.2, 1969 PosTCOITAL CERVICAL Mucus 203 Group III patients showed a marked difference at this level as well as at all other levels. The spinnbarkheit in this latter group was consistently poor. The fractional method revealed a significant difference in this physical property when comparing Group I and Group II samples. The cervical mucus of Group II patients had a definite decrease in its threadability -at Fig. 5 (top). Mean ph values of cervical mucus in each group of 5 patients for each level of endocervical canal examined. Solid line represents Group I; hatched line Group II; dotted line Group III, in this and subsequent figures. Fig. 6 (middle). Mean values of spinnbarkheit for each group at level of endocervical canal examined. Fig. 7 (bottom). Mean number of sperm for each group of patients at each level of endocervical canal examined (1-2 hr. after coitus) PH 7.0 ' 'l CM ~.----,.-.: ~-...,,, "' CM CERVICAL CANAL a No. of SPERM H.P.F CM CERVICAL CANAL...,... ~--..,. \... \ \. \... ~ CM CERVICAL CANAL

8 204 DAVAJAN & KuNITAKE FERTILITY & STEmLITY each of the three levels above the external os. The mucus from the fertile group collected from levels above the external os invariably had a spinnbarkheit of at least 12 em. Microscopic Examination In-Vivo Postcoital Test. The total number of sperm per high power field at 40X magnification in each cervical mucus segment was determined for all15 patients (Fig. 7). In Group I (normal) patients the mean number at the internal os level ( 2.5 em.) was 11. In all 3 groups the mean value at the external os level ( 0.5 em.) was higher than that found at the internal os ( 2.5 em.). This difference was most apparent in Group III samples where no sperm were found in the mucus collected from the 2.0- and 2.5- cm. levels. At the external os level there was no such apparent difference between the groups. The range of total number of sperm per high-power field (Table 2) was 7-38 in Group I, 3-36 in Group II, and 0-18 in Group III. In Group I patients, the range at the internal os (2.5 em.) was 7-15 compared to 3-10 in Group II patients. The number of actively motile sperm was determined for each highpower field examined (Fig. 8). These determinations were then calculated as the percent of sperm that were mobile per high-power field for each group of patients at the four levels of cervical canal. Group I and Group II patients showed very little difference in motility at the external os level. At this level, however, Group III patients contained very few motile sperm. A striking difference, not apparent in determining the total number of sperm per high-power field, was noted when comparing Group I and Group II patients as to percent of sperm motility at the internal os level. In Group I, the percent of active sperm at the internal os level was 82 as compared to 33 in Group II patients. In Group III patients, no active sperm were noted above the external os level. In-Vitro Simulated Postcoital Test. The total number of sperm per highpower field was determined for each segment as in the in-vivo test (Fig. 9). TABLE 2. Total Number of Spenns per High-Power Field Sperm per high-power field (No.) Cervical canal level (em.) Group I Group II Group Ill Q Q

9 VoL. 20, No.2, 1969 PosTCOITAL CERVICAL Mucus 205 These determinations were calculated as mean values for each of the four cervical mucus segments tested. In comparison to the in-vivo test, the mean value of the total number of sperm as determined by the in-vitro test was lower at the external os level in all three groups of patients tested. In 100 Fig. 8 (top). Mean percent values of motile sperm for each group at each level of endocervical canal examined (1-2 hr. after coitus). Fig. 9 (middle). Mean of the total number of sperm for each group at each level of catheter examined (in-vitro simulated endocervical postcoital test-90 min.). Fig. 10 (bottom). Mean percent values of motile sperm for each group at each level of catheter examined (in-vitro simulated endocervical postcoital test-90 min.). % MOTILE NO.OF 8 SPERM H.P.F CM CERVICAL CANAL ~ ~ CM (SIZE 14 FRENCH) RESIFLEX SUCTION CATHETER % MOTILE CM (SIZE 14 FRENCH) RESIFLEX SUCTION CATHETER

10 206 DAVAJAN & KuNITAKE FERTILITY & STERILITY Group I and Group II patients, the two test methods had similar total sperm counts at the internal os level ( 2.5 em.); but a marked difference between the in-vivo and in-vitro tests was observed in Group III patients at this level. In the in-vitro test, all Group III patients had sperm at the internal os level ( 2.5 em.) in contrast to the in-vivo test, where none of the patients had sperm above the 1-cm. level. The percent of actively motile sperm per high-power field was determined by the same method used in the in-vivo test (Fig. 10). The results of the in-vivo and in-vitro tests for motility were similar. As also noted in the in-vivo test, the Group I patients in the in-vitro test had a significantly higher percent of active sperm at the internal os level when compared to the Group II patients. Again, none of the Group III patients had any motile sperm at the internal os level. DISCUSSION An attempt to standardize and improve the postcoital test was performed successfully. The utilization of plastic catheters and the method developed provided the necessary tools for easier collection and systematic examination of the postcoital cervical mucus. A method of in-vitro testing was also developed. Although the results of the two methods of testing are not identical, the preliminary results are similar enough to allow the in-vitro method to be used for a more thorough examination of the postcoital cervical mucus. The major advantage of this fractional method of testing postcoital cervical mucus was the difference demonstrated between the fertile patients and the infertile patients with no previously determined etiologic factor. Therefore, patients who have no demonstrable causes for infertility following a routine Sims-Huhner postcoital test may show a defect in interaction between cervical mucus and spermatozoa when the examination is performed by this fractional technic. This point is well demonstrated in this study when the Group I and Group II patients are compared as to spinnbarkheit and percent of motile sperm at a given level in the endocervical canal. The Group II patients who were referred to this clinic with no demonstrable reason for their infertility, when compared to the normal group, were all found to have decreased spinnbarkheit at the higher levels of their cervical canals and a concomitant decrease in number of motile sperm at the internal os levels. These findings had not been demonstrated previously by the routine infertility work-up. Thus, patients who previously had been thought to be infertile for no known reason may now have the etiology of their infertility detected by this technic.

11 VoL. 20, No.2, 1969 PosTCOITAL CERVICAL Mucus 207 Volume of Cervical Mucus Since the comparison of the volume of cervical mucus collected from Group I and Group II patients revealed very little difference, volume determination is apparently of little value in evaluating patients with infertility due to undetermined etiology. There was a mean decrease in volume noted in Group III patients. This difference may be due to the greater tenacity of the cervical mucus in this group. The decrease in volume, therefore, may be attributable in part to the technical difficulty of aspirating this viscid mucus rather than to an actual reduction in volume. ph Determination Since no appreciable difference in the ph of cervical mucus was noted in the three groups of patients, this parameter appears to be of little aid in evaluating cervical mucus, and its determination is probably unnecessary. Spinnbarkheit A major significant difference in cervical mucus characteristics between Group I and Group II patients was noted in this test. The Group II patients had been referred to this clinic with supposedly normal spinnbarkheit. The results obtained show that if the cervical mucus being tested is obtained mainly from the external os level, there may be very little difference between the two groups. However, when the cervical mucus was examined fractionally, a marked difference in this property was noted at higher levels of the endocervical canal. The greatest difference was noted at the internal os level. The decreased threadability of cervical mucus obtained at this level from the Group II patients may be the result of chemical changes due to endometrial secretions. The latter would be expected to be at the highest concentration at the internal os level of the endocervical canal. The importance of this observation remains to be investigated. Microscopic Examination In-Vivo Postcoital Test. No difference was noted when comparing the mean number of sperm in Group I and Group II patients at all four levels examined. However, when the lower range of the total number was compared, the Group II patients had a noticeable decrease in the number of sperm at the internal os level. This difference in the range might account for the infertility in the second group, but this supposition cannot be verified at the present time. Again, this finding might prove to be due to

12 208 DAVAJAN & KuNITAKE FERTILITY & STERILITY the influence of endometrial secretions on the receptibility of the cervical mucus to spermatozoa. The mean percent of motile sperm found at all levels again revealed a marked difference between the normal Group I patients and the infertile Group II patients. In the first group, there were 82% motile sperm as compared to 33% in the second group. Because of the small number of patients used in this preliminary study, it is again difficu,lt to assess the significance of this difference. The percent motility corresponds well with the difference noted in spinnbarkheit findings. Whether there is a relation between these observations remains to be investigated. At this time, it should be emphasized again that there was a demonstrable difference between normal and infertile couples revealed by the use of this method of testing, especially in the parameter of spinnbarkheit and percent of motile sperm. In-Vitro Simulated Postcoital Test. The good correlation observed between the in-vivo and in-vitro methods of testing allows one to use the latter method for cross-testing of cervical mucus taken from infertile pa- PATIENT'S MUCUS DONOR'S MUCUS (;Ac ~ liusband's SEMEN Q: : I I I DONOR'S SEMEN I I I 0 I Fig. 11. In-vitro cross-testing-cervical mucus from infertile patient (A) is cross-tested with known fertile semen (B). Spermatozoa from patient's husband (C) are also cross-tested against cervical mucus from a known fertile woman (D). l==;=a==ff=~l I~- I tients against fertile spermatozoa, and to test sperm of males with unproven fertility against cervical mucus obtained from donors who have demonstrated their fertility (Fig. 11). This type of cross-testing by this in-vitro technic currently is being performed.

13 VoL. 20, No.2, 1969 PosTCOITAL CERVICAL Mucus 209 The results of the in-vitro testing method revealed that, in 90 min., more sperm penetrated the simulated endocervical canal in all three groups when compared to sperm penetration in the in-vivo test. This was noticeable especially in Group III patients where no sperm were found at the 2.0- and 2.5-cm. levels in any of the samples tested by the in-vivo method. In contrast, as many as 12 sperm were noted at these same levels in the in-vitro testing method. Therefore, the question of whether the in-vitro testing method represents a true picture of spermatozoa penetration or superimposes other effects such as chemotoxicity or capillary action has not yet been determined. In all three groups the mean percent of motile sperm at all four levels of simulated endocervical canal correlated much more closely to the results obtained in the in-vivo method. This correlation, therefore, makes it feasible to use the in-vitro testing method as an adjunct to the in-vivo method in investigation of possible causes of infertility. The cross-testing of the spermatozoa and cervical mucus from infertile couples with known fertile spermatozoa and normal cervical mucus by the in-vitro technic currently is being performed. With these technics a thorough investigation of the interaction of cervical mucus and spermatozoa can now be carried out. SUMMARY Fractional in-vivo and in-vitro technics for examination of the interaction of cervical mucus and spermatozoa has been developed. A difference in spinnbarkheit and percent of motile sperm was noted when comparing normal patients with infertile patients who, up to this time, had no demonstrable cause of their infertility. An in-vitro cross-testing system has been developed successfully and will be utilized in future work in order to better categorize the factors that may be causing incompatibility of cervical mucus and spermatozoa. Department of Obstetrics and Gynecology Harbor General Hospital 1000 West Carson St. Torrance, Calif REFERENCES ATXINSON, W. B., SHETTLES, L. B., and ENGLES, E. T. Histochemical studies on secretion of mucus by the human endocervix. Amer ] Obstet Gynec 56:112, AusTIN, C. R. Number of sperms required for fertilization. Nature (London) 162:534, BRECKENRIDGE, M.A., PETERSON, A. B., and PoMMERENCE, W. T. A. ph study of human cervical secretions. Fertil Steril1:421, 1950.

14 210 DAvAJAN & KuNITAKE FERTILITY & STERILITY 4. BuNcE, R. G., and SHERMAN, J. K. Liquefaction of human semen by alphaamylase. Fertil Steril 5:.'353, CLAYERO, N. A. Statistical study of the cervix as a factor in sterility in 16:31 cases: Results of treatment. Rev Esp Obstet Ginec 13:266, DAVAJAN, V., and KuNITAKE, G. M. Disc electrophoretic and disc-gel ouchterlony analyses of human genital fluids. Fertil Steril19:623, HuHNER, M. Sterility in the Male and Female and 1ts Treatment. Rohman, New York, HUHNER, M. The Hubner test as a diagnosis of sterility due to necrospermia. I Obstet Gynec 44:334, KELLY, J. V. Myometrial participation in human sperm transport: A dilemma. Fertil Steril13:84, MANN, E. C., McLARN, W. D., and HAYT, D. B. The physiology and clinical significance of uterine isthmus. Amer I Obstet Gynec 81:209, MAcLEOD, J. Metabolism and motility of human spermatozoa. I Endocr 29:583, MAcLEOD, J., and GoLD, R. The male factor in fertility and infertility. Fertil Steril 4:10, MAcLEoD, J., and HoTCHKISS, R. S. Distribution of spermatozoa and of certain chemical constituents in human ejaculate. I Ural 48:225, MAcLEOD, J., MASTEN, F., SILBERMAN, C., and SoBRERO, A. J. The postcoital and postinsemination cervical mucus and semen quality. Stud Fertil 10:41, MARcus, S. L., and MARcus, C. C. Review: Cervical mucus and its relation to infertility. Obstet Gynec Survey 18: , MAZER, C., and IsRAEL, S. L. Diagnosis and Treatment of Menstrual Disorders and Sterility. Hueber, New York, BEBLAD, E. The physics of cervical mucus. Acta Obstet Gynec Scand 38 (Suppl. 1) :44, PoMMERENKE, W. T., and VIERGIVER, E. The relationship between cervical mucus and basal body temperature cycles. Amer I Obstet Gynec 54:616, RAI-'OTT, A. E. The physiology of the endocervix. Clin Obstet Gynec 6:305, SHETTLES, L. B., and GUTTMACHER, A. F. Normal and abnormal variations in human cervical mucus. Amer I Physiol129:462, SIMMONS, F. A., and TAYMOR, M. L. Failure of conception in 100 completely studied couples. Fertil Steril 6:320, SIMS, J. M. On the microscope, as an aid in the diagnosis and treatment of sterility. NY Med I 8:393, STEINBERG, W. The role of the cervical factor in sterility. Fertil Steril 6:169, STEINBERG, W. The role of the cervical factor in sterility. Fertil Steril 9:401, VIERGIVER, E., and PoMMERENKE, W. T. Measurement of the cycle variations in quality of cervical mucus and its correlation with basal temperature. Amer J Obstet Gynec 48:321, 1944.