- Email: [email protected]
A New Method for Evaluating Cervical Penetrability Using Daily Aspirated and Stored Cervical Mucus
FERTILITY AND STERILITY Copyright © 1976 The American Fertility Society
Vol. 27, No.5, May 1976 Printed in U.SA.
A NEW METHOD FOR EVALUATING CERVICAL PENETRABILITY
USING DAILY ASPIRATED AND STORED CERVICAL MUCUS AMNON MAKLER, M.D.
Department of Obstetrics and Gynecology, Rambam Government Hospital, Technion, Aba Khoushy School of Medicine, Haifa, Israel
Cervical mucus samples from 33 women undergoing fertility work-up and married to normospermic husbands were aspirated daily with a tuberculin syringe during the periovulatory period. Samples were transferred to polyethylene tubing, sealed, and stored at 4° C for 7 to 14 days. In vitro sperm penetration tests were performed in each case, using only one sample of husband's semen with all of the mucus samples that were collected during one cycle. Preliminary studies with 50 samples of cervical mucus taken at random showed that no difference in physicochemical and penetrability properties existed between fresh mucus and mucus stored for 10 to 14 days. Very often the optimal penetrability of the cervical mucus occurred on unpredictable days. In contrast, many tests performed on days which would have been predetermined for in vivo postcoital tests were poor; consequently, those in vivo tests would have been falsely interpreted as negative. This new test using daily aspirated and stored mucus, which is simple to perform, is valuable mainly because it may eliminate the false negative results of other penetration tests. In this way the diagnosis of a truly hostile cervix may be more accurate.
The postcoital test (peT) is one of the most important and prevalent tests used in the investigation of the infertile couple. Nevertheless, there are still many disadvantages caused by lack of definite standards regarding performance by the couple, the technique of the test itself, and assessment of the results by the investigator. 1 It is generally agreed that a positive correlation between a "good" peT and fertility does exist. 1 -3 However, very little information can be obtained from a "poor" peT because a complete lack of sperm or motility cannot be due to a cervical factor and may be attributable to many other causes. 4 Accepted December 24, 1975.
Two of the most important reasons for a poor peT are: 1. Time difference between sexual intercourse and perfOl:mance of the test. There is as yet no agreement as to the ideal time interval, and it fluctuates somewhere between 1 and 2 hours and 14 and 16 hours. 1, 4, 5 A negative result may sometimes be attributed to improper interval. 2. Failure to determine the proper day in the woman's cycle for performance of the test. Although it is agreed by many investigators that the ideal day for performance of the peT is 1 to 2 days before midcycle,1. 4 it is very difficult to predict exactly when this occurs. As it is necessary to fix the day for the test at the beginning ofthe cycle or a few days later,
533
534
MAKLER
May 1976
it often happens that the test is per- 1 to 16); (2) women with a good PCT formed either too early, before the mucus performed once in a previous cycle on has reached its optimal properties, or too one certain day (patients 17 to 25); and late, when the mucus is already under (3) women with a poor PCT performed the influence of the postovulatory proges- once in a previous cycle on one certain tative phase. Since drastic changes in the day (patients 26 to 33). cervical mucus may occur from one day to another, some authors6 suggest daily Mucus Samples PCTs in the treatment of anovulatory women with gonadotropins. However, it For the purpose of this study only, is not practical to ask the couple to have instructions were given to the couple to sexual intercourse daily, because sperm abstain from intercourse during the study quality may thus be reduced 7 or technical cycle. The wives were asked to come problems may arise when the couple has to the clinic daily, starting from the 1st "to perform on demand."8 In such cases, or 2nd day after the end of menstruathe couple is often asked to repeat the tion, and the cervix was inspected with test during the next cycles. a bivalve speculum. When cervical mucus In order to overcome the first problem, first appeared, it was aspirated with a several in vitro tests have been suggested, I-ml disposable tuberculin syringe. The such as the slide test by Miller and aspirated sample was given a score of Kurzrok9 or different kinds of capillary + 1 to + 10 according to the following tests,10-16 and the correlation between criteria: these tests and the in vivo test was found Volume. Less than 0.1 ml, + 1; 0.1 to be good. In some of these tests the to 0.2 ml, + 2; 0.2 to 0.5 ml, + 3; 0.5 ml results can be observed several times or more, +4. during one test. Transparency. Completely opaque, 0; In order to overcome the second prob- less than 50% of the volume transparent, lem-the proper day for the test in the + 1; more than 50% of the volume transfemale cycle-in this study we tried to parent, + 2; completely transparent, solve it by combining one of the in +3. vitro tests with the use of mucus samples Spinnbarkheit. Completely unelastic, obtained during several consecutive 0; thread of 1 to 2 cm, + 1; thread of proper days of the cycle and stored at 2 to 5 cm + 2; thread of more than 5 cm, low temperature. The test itself was per- +3. formed in one stage, using all of the stored The mucus was immediately transmucus with one sample of the husband's ferred to a transparent polyethylene semen. Thus, this in vitro test may tube with an inner diameter exactly supplement unsatisfactory or negative in fitting the end of the syringe and approxivivo tests. mately 3 to 4 cm long. The mucus was transferred in its entirety or only part of it, but not over half the volume MATERIALS AND METHODS of the tube (Fig. 1). The tube was imThirty-three female patients from our mediately sealed hermetically with sterility clinic, of reproductive age, paraffin film (Parafilm M, Neenah, Wis.) married to husbands with normal sperm, and kept in a vertical position for storage and undergoing routine investigation for at 4° C. Additional samples were obtained fertility, were divided into three groups: daily until the mucus showed all signs (1) women with normal cycles and normal of postovulatory disappearance (approxibasal body temperature charts (patients mately five to eight samples per cycle).
Vol. 27, No.5
EVALUATING CERVICAL PENETRABILITY
535
FIG. 1. Aspirated cervical mucus is transferred into a polyethylene tube to approximately one-half the length of the tube.
In cases of anovulation or in cases where mucus disappeared slowly, a greater number of samples was necessary. After all samples had been obtained from a wife, her husband was asked to deposit one specimen of semen, at his convenience, into a glass jar after an abstinence of 2 or 3 days. In specific cases (see ccPreliminary Investigations") donor's semen was obtained according to the same instructions.
Penetration Test The penetration test itself was carried out with all the wife's samples obtained
FIG. 2. Seminal fluid is injected into the upper part of the tube, just above the level of the stored · cervical mucus.
FIG. 3. All samples that were stored during the cycle are tested with one sample of husband's semen.
during one cycle, after rewarming to room temperature. The covering Parafilm was removed and, with the aid of a syringe, a certain amount of fresh semen was introduced into the empty upper part of each tube, taking care not to let air bubbles form in the area of contact between semen and mucus (Fig. 2). All of the tubes were left standing upright at a temperature of 31' C for 60 minutes, and precautions were taken to avoid vibrations (Fig. 3). Each tube was then cut somewhat below the demarcation line, taking care not to mix mucus with seminal fluid. The cutting was done quickly with scissors (Fig. 4) in order
FIG. 4. After sperm-mucus contact for 60 minutes the tube is cut somewhat below the demarcation line, within the region of the mucus.
536
May 1976
MAKLER
FIG. 5. The remaining mucus is tranferred to the surface of a slide with the aid of delicate forceps.
to prevent the mucus from sticking to the scissors or to the upper part of the tube, which was discarded. With the aid of delicate forceps the remaining mucus was extracted, put on a slide (Fig. 5), and covered with a cover glass. The smear was examined in a high power field (HPF) for presence of sperm, number of sperm, and motility and percentage of normal forms. The results were graded as follows: no motile sperm per HPF, 0; 1 to 5 motile sperm per HPF, + 1; 5 to 10 motile sperm per HPF, +2; 10 to 20 motile sperm per HPF, +3; more than 20 motile sperm per HPF, + 4. Two hundred sixty-four penetration tests were performed in which the earliest starting day in the cycle was the 7th day and latest was the 24th day. The lowest number of tests performed in any one wife was 5, the highest number was 13.
Preliminary Investigations In order to clarify whether the properties of the mucus changed as a result of the storage process, preliminary tests were performed on 50 samples of cervical mucus taken at random from various women on various days during the periovulatory period. Each sample wasdivided into two parts. One half was examined immediately and the other half was examined 10 to 14 days after storage at 4° C. The following parameters were
examined: (1) volume, (2) color and transparency, (3) spinnbarkheit, (4) arborization (ferning), (5) pH, (6) cellular and microbial content, (7) penetration of live sperm, and (8) penetration of sperm killed by heating. Changes in volume were determined' by marking the level of mucus in the tube and looking for swelling or shrinkage of mucus occurring as a result of the storing process. Changes in color or transparency were judged by description of the mucus before and after storage. Changes in spinnbarkheit were determined by measuring in centimeters the length of the mucus thread stretching from a slide. Changes in ferning were determined by comparing gradings of arborization from + 1 to + 4 before and after storage. pH was measured before and after storage with pH indicator paper (Spezial Indikator Papier; Merck AG, Darmstadt, Germany). Changes in cellular and microbial content were 'determined by description of these contents before and after storage. Penetration tests of live or killed sperm were performed either by the above method or by that of Miller and Kurzrock, 9 and rates of penetration, were compared before and after storage. All preliminary penetration tests were performed with high-quality donor semen. RESULTS
Preliminary Investigations. Table 1 lists some results of the preliminary tests made in order to compare properties of cervical mucus before and after storage. In summary, cervical mucus showed no change in volume either through shrinking or swelling as a result of the storing process. No changes were observed in color or transparency. No significant changes were found in the degree of spinnbarkheit (Fig. 6) and
Vol. 27, No.5
EVALUATING CERVICAL PENETRABILITY
537
TABLE 1. Comparison of Some Properties of Cervical Mucus before and after Storage (N = 50) Before storage
Property Range
Spinnbarkheit (em) Arborization pH Penetration score
2-17 1-4 6.7-8.6 1-4
After storage
Mean ± 2 BE
5.82 2.83 7.85 2.63
arborization. Properties present before storage were found to be the same after storage. No significant changes in pH were found before and after storage. Mucus containing epithelial cells, leukocytes, and microbes showed almost no changes in these contents. Live sperm penetrated almost at the same rate before and after lO-day storage, with some reduction toward the end of 2 weeks. This was found either by the above-described test or the Miller-Kurzrock test. By either technique, killed sperm did not penetrate either fresh or stored mucus. Penetration Tests Performed during the Periovulatory Period. Figure 7 shows the days on which samples were taken, number of samples taken from each patient, the approximate day of ovulation according to the basal body temperature chart (day of temperature nadir),
± ± ± ±
0.91 0.42 0.03 0.41
Mean ± 2 BE
2-15 1-4 6.6-8.5 1-4
5.76 2.91 7.83 2.59
± ± ± ±
0.78 0.53 0.02 0.62
duration of menstruation, length of cycle, grading of mucus quality on each day it was taken, and the results of the penetration tests, respectively.
10
12
,. 15 16
17
"
19
20 21 22 23
"
25
26 27
21
:19 30 JI 32
33
FIG. 6. Spinnbarkheit of 9 em is demonstrated in mucus stored for 14 days.
Range
,
~iH
~
~iB~nluJ,fu~Hf ....... YHHU~ooo U6787S1
FIG. 7. Description of 33 cycles in which penetration tests were performed. The length of each cycle is hown by dots indicating days of menstruation. Horizontal lines indicate days on which mucus samples were taken. Figure below this line indicate scoring of cervical mucus (see text) and figures above this line indicate the corresponding penetration score. Figures between two short vertical lines indicate the ovulation day according the basal body temperature nadir.
MAKLER
538
May 1976
TABLE 2. Frequency Distribution of Results of Penetration Tests for Each Score of Cervical Mucus (N = 264) Penetration grade Total
Mucus score
1 2 3 4 5 6 7 8 9 10
0
+1
51 16
5 34 27 7 1
1 1 1 2 1
1 1 1
In each patient a typical pattern could be seen in which the scoring of cervical mucus increased to maximum, then again decreased. It usually began 2 to 3 days before the highest score and diminished 1 to 2 days after it, as described by MacDonaldY The high scoring and positive penetration tests occurred between the 10th and 18th days of the cycle, while negative results could be found on any day of the cycle. Of these women who in previous tests showed negative in vivo peTs (patients 26 to 33), four showed positive in vitro penetration tests on days 9, 16, 17, and 18 when checked daily in the present test. One patient (patient 33) showed negative results in all of her penetration tests, although the scoring of her mucus was as high as + 9.
+2
2 2 8 18 9 6
+3
1 12 10 13 7 2
+4+
1 1 6 9 7
56 52 29 16 21 23 20 21 17 9
Table 2 shows the frequency distribution of the results of all the 264 penetration tests for each score of cervical mucus. Of 56 tests performed with cervical mucus of grade + 1, in 51 cases no penetration was found and in 5 cases penetration of + 1 was found. Of nine tests performed with cervical mucus of grade + 10, two cases showed + 3 penetration and seven cases showed + 4 penetration. All other figures fell between those boundaries. In Figure 8 mucus scoring is plotted against the mean ± 2 SE of the results of penetration tests in the same scoring, for all 33 patients. The correlation is positi ve and shows that the better the quality of the mucus the higher the degree of penetration test.
DISCUSSION
The correlation between postcoital tests and fertility, and the correlation between in vivo and in vitro tests, have already been reviewed extensively by others. 1. 4 , 6, 8, 13, 18·23 Even though some authors have criticized the value of either +2 in vivo or in vitro penetration tests,19 most agree that a fair correlation does +1 exist and some even point to the advantages of the in vitro test in special cases o + 1 +- 2 +-3 +-4 +- 5 + 6 + 7 +- 8 + 9 +10 such as repeatedly negative in vivo tests, FIG. 8. Correlation between cervical mucus score low seminal volume, technical coital prob(ordinate) and sperm penetration grade (abscissa) according to the 33 in vitro tests. The heavy line lems, and vaginal infection. The purpose indicates means; thin lines, ± 2 SE. of this work was mainly to determine
Vol. 27, No.5
EVALUATING CERVICAL PENETRABILITY
539
found in our study in four of eight patients whose previous peT, made once on a certain day, was negative. On the other hand, the fact that a high-scoring cervical mucus was almost never negative in the penetration test proves the test to From the preliminary results of this be reliable and excludes the possibility study it can be stated that storing of that stored mucus loses its penetrability. cervical mucus for approximately 2 weeks This method eliminates other possible does not change the physicochemical prop- difficulties, technical or psychologic, deerties of the mucus and its penetrability. riving from the need to perform in vitro This finding is in agreement with the work or in vivo penetration tests on a certain of Ulstein 15 and Reichman et al.,r6 even fixed day. In cases where the husband's though they later used estrogen-stimu- semen sample is accidentally poor, the lated cervical mucus. In both of these test can be deferred to another day when studies the tests with stored mucus were the sample presents its characteristic only a concomitant observation while they count. tested other properties of the mucusThe results obtained in our study during sperm penetration interrelationship, and ovulatory cycles are in accord with those not much attention was given to the stored made by in vivo and in vitro tests desmucus and no practical implication or con- cribed by others. 20, 22, 23 The correlation clusion was drawn from studying stored between the penetration rate and the days mucus. in the cycle in which the mucus reaches Ulstein 15 did not find any reduction its peak activity is identical to that found in penetrability after as much as 3 weeks in tests made with fresh mucus. In a great of storage, whereas Reichman et al. 16 number of cases positive results would not found some reduction after 1 week's stor- have been obtained from in vivo peTs age. The fact that mucus properties are if the tests had been performed only once not changed after almost 2 weeks of on a predetermined day of the cycle storage at low temperature has enormous (Fig. 7). The optimal day could be deterimportance, as it makes it easier for us to mined only after obtaining daily samples. follow daily changes in the cervical mucus. This fact explains why, in half of the cases These cyclic changes in the mucus can previously showing negative results, norbe observed readily when samples are ob- mal penetration was found when the test tained daily for 5 to 8 days and examined was made during several days. From prelater at one time. In this way there is liminary results of slide tests, we may no need to rely on memory and, if the conclude that, although the tube test is examination is performed by more than technically very simple to perform, identione person, the possibility of difference cal results would have been obtained b:t those preferring the slide test (Kurzrockin interpretation is eliminated. Miller test) if they are trained in perOf even greater importance is the fact forming this kind of test, We could not that with this method it is possible to confirm Davajan et al.'s! observation diagnose the presence or absence of hos- that in in vitro tests the seminal sample tile cervix, since it is unncessary to· fix must be below the cervical mucus because the optimal day for an in vitro peT and sperm have to climb up into the mucus. the possibility of false negative results In all of our tests, sperm penetrated is eliminated in cases where the test is readily "down" from the upper into not done on the proper day. This was the lower mucus compartment. whether it was possible to perform in vitro tests with stored cervical mucus instead of fresh mucus and to determine the practical advantages as well as the informative value of such a method.
MAKLER
540 REFERENCES
1. Davajan V, Nakamura RM, Kharim K: Spermatozoal transport in cervical mucus: review. Obstet Gynecol Survey 25:1, 1970 2. Buxton CL, Southam A: Human Infertility. New York, Hoeber-Harper, 1958 3. Jette NT, Glass RH: Prognostic value of the postcoital test. Fertil Steril 23:29, 1972 4. Marcus CC, Marcus SL: The cervical factor. In Progress in Infertility. Boston, Little, Brown & Co, 1968 5. London GD, Lawrence PB: Alteration of the postcoital examination by systematic medication. Fertil Steril 8:1, 1967 6. Friberg J, Gemzell C: Daily PCT in the conception cycle during treatment of anovulatory women with human gonadotropins. Int J Fertil 17:178, 1972 7. Freund M: Interrelationship among the characteristics of human semen and factors affecting semen specimen quality. J Reprod Fertil4:143, 1962 8. Speroff L, Glass RH, Kase NG: Investigation of Infertility. Baltimore, Williams & Wilkins Co, 1973 9. Miller EG Jr, Kurzrok R: Biochemical studies of human semen. Ill. Factors affecting migration of sperm through the cervix. Am J Obstet Gynecol 24:19, 1932 10. Perlof WH, Steinberger E: In vitro penetration of cervical mucus by spermatozoa. Fertil Steril 14:231, 1963 11. Kremer J: A simple sperm penetration test. Int J Fertil 10:201, 1965 12. Davajan V, Kunitake GM: Fractional in vivo and in vitro examination of postcoital cervical mucus in the human. Fertil Steril 20:197, 1969
May 1976
13. Kunitake GM, Davajan V: A new method of evaluating infertility due to cervical mucusspermatozoal incompatibility. Fertil Steril 21:706, 1970 14. Bottella-Llusia J: Quantitative tests for sperm motility. In Fertility Disturbances in Men and Women. Basel, Karger, 1971 15. Ulstein M: Evaluation of capillary tube sperm penetration for fertility investigation. Acta Obstet Gynecol Scand 51:287,1972 16. Reichman J, Insler V, Serr D: A modified in vitro spermatozoal penetration test. I. Method. Int J Fertil 18:232, 1973 17. MacDonald. RR: Cyclic changes in cervical mucus. J Obstet Gynaecol Br Commonw 76: 1090, 1969 18. Moghissi KS: Cyclic changes of cervical mucus in normal and progestin-treated women. Fertil Steril17:663, 1966 19. Gibor Y, Garcia CJ, Cotten MR: Cyclic changes in the physical properties of cervical mucus and the results of the postcoital test. Fertil Steril 21:20,1970 20. Moghissi KS: The function of the cervix in fertility. Fertil Steril 23:295, 1972 21. Ulstein M: Sperm penetration of cervical mucus as a criterion of male fertility. Acta Obstet Gynecol Scand 51:335, 1972 22. Reichman J, Insler V, Serr D: A modified in vitro spermatozoal penetration test. II. Application in fertility investigation. Int J Fertil 18:241, 1973 23. Moran J, Davajan V, Nakamura R: Comparison of fractional PCT with Sims-Huhner PCT. Int J Fertil19:93, 1974
Vol. 27, No.5, May 1976 Printed in U.SA.
A NEW METHOD FOR EVALUATING CERVICAL PENETRABILITY
USING DAILY ASPIRATED AND STORED CERVICAL MUCUS AMNON MAKLER, M.D.
Department of Obstetrics and Gynecology, Rambam Government Hospital, Technion, Aba Khoushy School of Medicine, Haifa, Israel
Cervical mucus samples from 33 women undergoing fertility work-up and married to normospermic husbands were aspirated daily with a tuberculin syringe during the periovulatory period. Samples were transferred to polyethylene tubing, sealed, and stored at 4° C for 7 to 14 days. In vitro sperm penetration tests were performed in each case, using only one sample of husband's semen with all of the mucus samples that were collected during one cycle. Preliminary studies with 50 samples of cervical mucus taken at random showed that no difference in physicochemical and penetrability properties existed between fresh mucus and mucus stored for 10 to 14 days. Very often the optimal penetrability of the cervical mucus occurred on unpredictable days. In contrast, many tests performed on days which would have been predetermined for in vivo postcoital tests were poor; consequently, those in vivo tests would have been falsely interpreted as negative. This new test using daily aspirated and stored mucus, which is simple to perform, is valuable mainly because it may eliminate the false negative results of other penetration tests. In this way the diagnosis of a truly hostile cervix may be more accurate.
The postcoital test (peT) is one of the most important and prevalent tests used in the investigation of the infertile couple. Nevertheless, there are still many disadvantages caused by lack of definite standards regarding performance by the couple, the technique of the test itself, and assessment of the results by the investigator. 1 It is generally agreed that a positive correlation between a "good" peT and fertility does exist. 1 -3 However, very little information can be obtained from a "poor" peT because a complete lack of sperm or motility cannot be due to a cervical factor and may be attributable to many other causes. 4 Accepted December 24, 1975.
Two of the most important reasons for a poor peT are: 1. Time difference between sexual intercourse and perfOl:mance of the test. There is as yet no agreement as to the ideal time interval, and it fluctuates somewhere between 1 and 2 hours and 14 and 16 hours. 1, 4, 5 A negative result may sometimes be attributed to improper interval. 2. Failure to determine the proper day in the woman's cycle for performance of the test. Although it is agreed by many investigators that the ideal day for performance of the peT is 1 to 2 days before midcycle,1. 4 it is very difficult to predict exactly when this occurs. As it is necessary to fix the day for the test at the beginning ofthe cycle or a few days later,
533
534
MAKLER
May 1976
it often happens that the test is per- 1 to 16); (2) women with a good PCT formed either too early, before the mucus performed once in a previous cycle on has reached its optimal properties, or too one certain day (patients 17 to 25); and late, when the mucus is already under (3) women with a poor PCT performed the influence of the postovulatory proges- once in a previous cycle on one certain tative phase. Since drastic changes in the day (patients 26 to 33). cervical mucus may occur from one day to another, some authors6 suggest daily Mucus Samples PCTs in the treatment of anovulatory women with gonadotropins. However, it For the purpose of this study only, is not practical to ask the couple to have instructions were given to the couple to sexual intercourse daily, because sperm abstain from intercourse during the study quality may thus be reduced 7 or technical cycle. The wives were asked to come problems may arise when the couple has to the clinic daily, starting from the 1st "to perform on demand."8 In such cases, or 2nd day after the end of menstruathe couple is often asked to repeat the tion, and the cervix was inspected with test during the next cycles. a bivalve speculum. When cervical mucus In order to overcome the first problem, first appeared, it was aspirated with a several in vitro tests have been suggested, I-ml disposable tuberculin syringe. The such as the slide test by Miller and aspirated sample was given a score of Kurzrok9 or different kinds of capillary + 1 to + 10 according to the following tests,10-16 and the correlation between criteria: these tests and the in vivo test was found Volume. Less than 0.1 ml, + 1; 0.1 to be good. In some of these tests the to 0.2 ml, + 2; 0.2 to 0.5 ml, + 3; 0.5 ml results can be observed several times or more, +4. during one test. Transparency. Completely opaque, 0; In order to overcome the second prob- less than 50% of the volume transparent, lem-the proper day for the test in the + 1; more than 50% of the volume transfemale cycle-in this study we tried to parent, + 2; completely transparent, solve it by combining one of the in +3. vitro tests with the use of mucus samples Spinnbarkheit. Completely unelastic, obtained during several consecutive 0; thread of 1 to 2 cm, + 1; thread of proper days of the cycle and stored at 2 to 5 cm + 2; thread of more than 5 cm, low temperature. The test itself was per- +3. formed in one stage, using all of the stored The mucus was immediately transmucus with one sample of the husband's ferred to a transparent polyethylene semen. Thus, this in vitro test may tube with an inner diameter exactly supplement unsatisfactory or negative in fitting the end of the syringe and approxivivo tests. mately 3 to 4 cm long. The mucus was transferred in its entirety or only part of it, but not over half the volume MATERIALS AND METHODS of the tube (Fig. 1). The tube was imThirty-three female patients from our mediately sealed hermetically with sterility clinic, of reproductive age, paraffin film (Parafilm M, Neenah, Wis.) married to husbands with normal sperm, and kept in a vertical position for storage and undergoing routine investigation for at 4° C. Additional samples were obtained fertility, were divided into three groups: daily until the mucus showed all signs (1) women with normal cycles and normal of postovulatory disappearance (approxibasal body temperature charts (patients mately five to eight samples per cycle).
Vol. 27, No.5
EVALUATING CERVICAL PENETRABILITY
535
FIG. 1. Aspirated cervical mucus is transferred into a polyethylene tube to approximately one-half the length of the tube.
In cases of anovulation or in cases where mucus disappeared slowly, a greater number of samples was necessary. After all samples had been obtained from a wife, her husband was asked to deposit one specimen of semen, at his convenience, into a glass jar after an abstinence of 2 or 3 days. In specific cases (see ccPreliminary Investigations") donor's semen was obtained according to the same instructions.
Penetration Test The penetration test itself was carried out with all the wife's samples obtained
FIG. 2. Seminal fluid is injected into the upper part of the tube, just above the level of the stored · cervical mucus.
FIG. 3. All samples that were stored during the cycle are tested with one sample of husband's semen.
during one cycle, after rewarming to room temperature. The covering Parafilm was removed and, with the aid of a syringe, a certain amount of fresh semen was introduced into the empty upper part of each tube, taking care not to let air bubbles form in the area of contact between semen and mucus (Fig. 2). All of the tubes were left standing upright at a temperature of 31' C for 60 minutes, and precautions were taken to avoid vibrations (Fig. 3). Each tube was then cut somewhat below the demarcation line, taking care not to mix mucus with seminal fluid. The cutting was done quickly with scissors (Fig. 4) in order
FIG. 4. After sperm-mucus contact for 60 minutes the tube is cut somewhat below the demarcation line, within the region of the mucus.
536
May 1976
MAKLER
FIG. 5. The remaining mucus is tranferred to the surface of a slide with the aid of delicate forceps.
to prevent the mucus from sticking to the scissors or to the upper part of the tube, which was discarded. With the aid of delicate forceps the remaining mucus was extracted, put on a slide (Fig. 5), and covered with a cover glass. The smear was examined in a high power field (HPF) for presence of sperm, number of sperm, and motility and percentage of normal forms. The results were graded as follows: no motile sperm per HPF, 0; 1 to 5 motile sperm per HPF, + 1; 5 to 10 motile sperm per HPF, +2; 10 to 20 motile sperm per HPF, +3; more than 20 motile sperm per HPF, + 4. Two hundred sixty-four penetration tests were performed in which the earliest starting day in the cycle was the 7th day and latest was the 24th day. The lowest number of tests performed in any one wife was 5, the highest number was 13.
Preliminary Investigations In order to clarify whether the properties of the mucus changed as a result of the storage process, preliminary tests were performed on 50 samples of cervical mucus taken at random from various women on various days during the periovulatory period. Each sample wasdivided into two parts. One half was examined immediately and the other half was examined 10 to 14 days after storage at 4° C. The following parameters were
examined: (1) volume, (2) color and transparency, (3) spinnbarkheit, (4) arborization (ferning), (5) pH, (6) cellular and microbial content, (7) penetration of live sperm, and (8) penetration of sperm killed by heating. Changes in volume were determined' by marking the level of mucus in the tube and looking for swelling or shrinkage of mucus occurring as a result of the storing process. Changes in color or transparency were judged by description of the mucus before and after storage. Changes in spinnbarkheit were determined by measuring in centimeters the length of the mucus thread stretching from a slide. Changes in ferning were determined by comparing gradings of arborization from + 1 to + 4 before and after storage. pH was measured before and after storage with pH indicator paper (Spezial Indikator Papier; Merck AG, Darmstadt, Germany). Changes in cellular and microbial content were 'determined by description of these contents before and after storage. Penetration tests of live or killed sperm were performed either by the above method or by that of Miller and Kurzrock, 9 and rates of penetration, were compared before and after storage. All preliminary penetration tests were performed with high-quality donor semen. RESULTS
Preliminary Investigations. Table 1 lists some results of the preliminary tests made in order to compare properties of cervical mucus before and after storage. In summary, cervical mucus showed no change in volume either through shrinking or swelling as a result of the storing process. No changes were observed in color or transparency. No significant changes were found in the degree of spinnbarkheit (Fig. 6) and
Vol. 27, No.5
EVALUATING CERVICAL PENETRABILITY
537
TABLE 1. Comparison of Some Properties of Cervical Mucus before and after Storage (N = 50) Before storage
Property Range
Spinnbarkheit (em) Arborization pH Penetration score
2-17 1-4 6.7-8.6 1-4
After storage
Mean ± 2 BE
5.82 2.83 7.85 2.63
arborization. Properties present before storage were found to be the same after storage. No significant changes in pH were found before and after storage. Mucus containing epithelial cells, leukocytes, and microbes showed almost no changes in these contents. Live sperm penetrated almost at the same rate before and after lO-day storage, with some reduction toward the end of 2 weeks. This was found either by the above-described test or the Miller-Kurzrock test. By either technique, killed sperm did not penetrate either fresh or stored mucus. Penetration Tests Performed during the Periovulatory Period. Figure 7 shows the days on which samples were taken, number of samples taken from each patient, the approximate day of ovulation according to the basal body temperature chart (day of temperature nadir),
± ± ± ±
0.91 0.42 0.03 0.41
Mean ± 2 BE
2-15 1-4 6.6-8.5 1-4
5.76 2.91 7.83 2.59
± ± ± ±
0.78 0.53 0.02 0.62
duration of menstruation, length of cycle, grading of mucus quality on each day it was taken, and the results of the penetration tests, respectively.
10
12
,. 15 16
17
"
19
20 21 22 23
"
25
26 27
21
:19 30 JI 32
33
FIG. 6. Spinnbarkheit of 9 em is demonstrated in mucus stored for 14 days.
Range
,
~iH
~
~iB~nluJ,fu~Hf ....... YHHU~ooo U6787S1
FIG. 7. Description of 33 cycles in which penetration tests were performed. The length of each cycle is hown by dots indicating days of menstruation. Horizontal lines indicate days on which mucus samples were taken. Figure below this line indicate scoring of cervical mucus (see text) and figures above this line indicate the corresponding penetration score. Figures between two short vertical lines indicate the ovulation day according the basal body temperature nadir.
MAKLER
538
May 1976
TABLE 2. Frequency Distribution of Results of Penetration Tests for Each Score of Cervical Mucus (N = 264) Penetration grade Total
Mucus score
1 2 3 4 5 6 7 8 9 10
0
+1
51 16
5 34 27 7 1
1 1 1 2 1
1 1 1
In each patient a typical pattern could be seen in which the scoring of cervical mucus increased to maximum, then again decreased. It usually began 2 to 3 days before the highest score and diminished 1 to 2 days after it, as described by MacDonaldY The high scoring and positive penetration tests occurred between the 10th and 18th days of the cycle, while negative results could be found on any day of the cycle. Of these women who in previous tests showed negative in vivo peTs (patients 26 to 33), four showed positive in vitro penetration tests on days 9, 16, 17, and 18 when checked daily in the present test. One patient (patient 33) showed negative results in all of her penetration tests, although the scoring of her mucus was as high as + 9.
+2
2 2 8 18 9 6
+3
1 12 10 13 7 2
+4+
1 1 6 9 7
56 52 29 16 21 23 20 21 17 9
Table 2 shows the frequency distribution of the results of all the 264 penetration tests for each score of cervical mucus. Of 56 tests performed with cervical mucus of grade + 1, in 51 cases no penetration was found and in 5 cases penetration of + 1 was found. Of nine tests performed with cervical mucus of grade + 10, two cases showed + 3 penetration and seven cases showed + 4 penetration. All other figures fell between those boundaries. In Figure 8 mucus scoring is plotted against the mean ± 2 SE of the results of penetration tests in the same scoring, for all 33 patients. The correlation is positi ve and shows that the better the quality of the mucus the higher the degree of penetration test.
DISCUSSION
The correlation between postcoital tests and fertility, and the correlation between in vivo and in vitro tests, have already been reviewed extensively by others. 1. 4 , 6, 8, 13, 18·23 Even though some authors have criticized the value of either +2 in vivo or in vitro penetration tests,19 most agree that a fair correlation does +1 exist and some even point to the advantages of the in vitro test in special cases o + 1 +- 2 +-3 +-4 +- 5 + 6 + 7 +- 8 + 9 +10 such as repeatedly negative in vivo tests, FIG. 8. Correlation between cervical mucus score low seminal volume, technical coital prob(ordinate) and sperm penetration grade (abscissa) according to the 33 in vitro tests. The heavy line lems, and vaginal infection. The purpose indicates means; thin lines, ± 2 SE. of this work was mainly to determine
Vol. 27, No.5
EVALUATING CERVICAL PENETRABILITY
539
found in our study in four of eight patients whose previous peT, made once on a certain day, was negative. On the other hand, the fact that a high-scoring cervical mucus was almost never negative in the penetration test proves the test to From the preliminary results of this be reliable and excludes the possibility study it can be stated that storing of that stored mucus loses its penetrability. cervical mucus for approximately 2 weeks This method eliminates other possible does not change the physicochemical prop- difficulties, technical or psychologic, deerties of the mucus and its penetrability. riving from the need to perform in vitro This finding is in agreement with the work or in vivo penetration tests on a certain of Ulstein 15 and Reichman et al.,r6 even fixed day. In cases where the husband's though they later used estrogen-stimu- semen sample is accidentally poor, the lated cervical mucus. In both of these test can be deferred to another day when studies the tests with stored mucus were the sample presents its characteristic only a concomitant observation while they count. tested other properties of the mucusThe results obtained in our study during sperm penetration interrelationship, and ovulatory cycles are in accord with those not much attention was given to the stored made by in vivo and in vitro tests desmucus and no practical implication or con- cribed by others. 20, 22, 23 The correlation clusion was drawn from studying stored between the penetration rate and the days mucus. in the cycle in which the mucus reaches Ulstein 15 did not find any reduction its peak activity is identical to that found in penetrability after as much as 3 weeks in tests made with fresh mucus. In a great of storage, whereas Reichman et al. 16 number of cases positive results would not found some reduction after 1 week's stor- have been obtained from in vivo peTs age. The fact that mucus properties are if the tests had been performed only once not changed after almost 2 weeks of on a predetermined day of the cycle storage at low temperature has enormous (Fig. 7). The optimal day could be deterimportance, as it makes it easier for us to mined only after obtaining daily samples. follow daily changes in the cervical mucus. This fact explains why, in half of the cases These cyclic changes in the mucus can previously showing negative results, norbe observed readily when samples are ob- mal penetration was found when the test tained daily for 5 to 8 days and examined was made during several days. From prelater at one time. In this way there is liminary results of slide tests, we may no need to rely on memory and, if the conclude that, although the tube test is examination is performed by more than technically very simple to perform, identione person, the possibility of difference cal results would have been obtained b:t those preferring the slide test (Kurzrockin interpretation is eliminated. Miller test) if they are trained in perOf even greater importance is the fact forming this kind of test, We could not that with this method it is possible to confirm Davajan et al.'s! observation diagnose the presence or absence of hos- that in in vitro tests the seminal sample tile cervix, since it is unncessary to· fix must be below the cervical mucus because the optimal day for an in vitro peT and sperm have to climb up into the mucus. the possibility of false negative results In all of our tests, sperm penetrated is eliminated in cases where the test is readily "down" from the upper into not done on the proper day. This was the lower mucus compartment. whether it was possible to perform in vitro tests with stored cervical mucus instead of fresh mucus and to determine the practical advantages as well as the informative value of such a method.
MAKLER
540 REFERENCES
1. Davajan V, Nakamura RM, Kharim K: Spermatozoal transport in cervical mucus: review. Obstet Gynecol Survey 25:1, 1970 2. Buxton CL, Southam A: Human Infertility. New York, Hoeber-Harper, 1958 3. Jette NT, Glass RH: Prognostic value of the postcoital test. Fertil Steril 23:29, 1972 4. Marcus CC, Marcus SL: The cervical factor. In Progress in Infertility. Boston, Little, Brown & Co, 1968 5. London GD, Lawrence PB: Alteration of the postcoital examination by systematic medication. Fertil Steril 8:1, 1967 6. Friberg J, Gemzell C: Daily PCT in the conception cycle during treatment of anovulatory women with human gonadotropins. Int J Fertil 17:178, 1972 7. Freund M: Interrelationship among the characteristics of human semen and factors affecting semen specimen quality. J Reprod Fertil4:143, 1962 8. Speroff L, Glass RH, Kase NG: Investigation of Infertility. Baltimore, Williams & Wilkins Co, 1973 9. Miller EG Jr, Kurzrok R: Biochemical studies of human semen. Ill. Factors affecting migration of sperm through the cervix. Am J Obstet Gynecol 24:19, 1932 10. Perlof WH, Steinberger E: In vitro penetration of cervical mucus by spermatozoa. Fertil Steril 14:231, 1963 11. Kremer J: A simple sperm penetration test. Int J Fertil 10:201, 1965 12. Davajan V, Kunitake GM: Fractional in vivo and in vitro examination of postcoital cervical mucus in the human. Fertil Steril 20:197, 1969
May 1976
13. Kunitake GM, Davajan V: A new method of evaluating infertility due to cervical mucusspermatozoal incompatibility. Fertil Steril 21:706, 1970 14. Bottella-Llusia J: Quantitative tests for sperm motility. In Fertility Disturbances in Men and Women. Basel, Karger, 1971 15. Ulstein M: Evaluation of capillary tube sperm penetration for fertility investigation. Acta Obstet Gynecol Scand 51:287,1972 16. Reichman J, Insler V, Serr D: A modified in vitro spermatozoal penetration test. I. Method. Int J Fertil 18:232, 1973 17. MacDonald. RR: Cyclic changes in cervical mucus. J Obstet Gynaecol Br Commonw 76: 1090, 1969 18. Moghissi KS: Cyclic changes of cervical mucus in normal and progestin-treated women. Fertil Steril17:663, 1966 19. Gibor Y, Garcia CJ, Cotten MR: Cyclic changes in the physical properties of cervical mucus and the results of the postcoital test. Fertil Steril 21:20,1970 20. Moghissi KS: The function of the cervix in fertility. Fertil Steril 23:295, 1972 21. Ulstein M: Sperm penetration of cervical mucus as a criterion of male fertility. Acta Obstet Gynecol Scand 51:335, 1972 22. Reichman J, Insler V, Serr D: A modified in vitro spermatozoal penetration test. II. Application in fertility investigation. Int J Fertil 18:241, 1973 23. Moran J, Davajan V, Nakamura R: Comparison of fractional PCT with Sims-Huhner PCT. Int J Fertil19:93, 1974