In situ variation of cervical mucus pH during exposure to atmospheric air

The objective of the present study was to determine if exposure of cervical mucus to air during specular examination could modify mucus pH. Detection of changes is justified because of their possible interference with sperm-mucus interaction, since an acidic pH is unfavorable to sperm penetration and is associated with infertility due to the cervical factor. Twenty women with good quality mucus were evaluated. pH measurements of ectoand endocervical mucus were made in situ using a glass electrode after 0-, 5and 10-min exposure to air. There was a progressive alkalinization of mucus pH. Mean values of ectocervical mucus pH were 6.91, 7.16 and 7.27, while mean values of endocervical mucus pH were 7.09, 7.34 and 7.46 at 0, 5 and 10 min, respectively. Significant differences were found between the mean values obtained at 0 and 5 min, and at 0 and 10 min (P<0.05), whereas the differences in mean values at 5 and 10 min were not significant at either site. We conclude that 5 to 10 min of exposure to atmospheric air affects cervical mucus pH in a significant way. Since tests used to evaluate sperm-mucus interaction generally have not considered this possibility, we suggest that they should be performed immediately after mucus collection in order to avoid misinterpretation of the results. Correspondence

Abnormalities of the cervix and its secretion are reported to be responsible for infertility in approximately 5 to 10% of women (1).Evaluation of sperm-cervical mucus interaction is important in infertility investigation.The traditional method for the evaluation of sperm-cervical mucus interaction in vivo is the postcoital test (PCT), but in vitro cervical mucus-sperm penetration tests are also used (2,3).
Sometimes, negative or abnormal PCT are attributed to immunological problems in particular.Anyway, recent studies have shown that the incidence of immunological factors in these cases is low, and that 80% of them can be reversed by means of a bicarbonate douche (4).The improvement observed with mucus alkalinization suggests that the low mucus pH (5) causes the poor spermmucus interaction.In addition, it was reported that some cases of negative PCT (absence of progressive sperm) or absent PCT (absence of sperm) involving ovulatory mucus and normal sperm became normal during the performance of in vitro cervical mucussperm penetration tests (6,7).It was suspected that this alteration occurred due to the variation of mucus pH after exposure to air.
Previous studies have reported pH variation of mucus exposed to air (8,9), but those studies were done in vitro, i.e., after mucus had been aspirated.The aim of this study was to determine if the exposure of cervical mucus to atmospheric air during specular examination could modify mucus pH in situ.
Twenty women attending this service during one year and who voluntarily agreed to take part in the study were evaluated.In all cases the women were of reproductive age, had regular cycles and presented good quality periovulatory cervical mucus.No pathological microorganisms were found on vaginal Gram-stained smears.Patients having organic or anatomic cervical changes, as well as patients using drugs that could change cervical mucus were excluded.
In order to collect the mucus for evaluation, the patient was examined in the gynecological position, the uterine cervix was exposed with a sterile, non-lubricated speculum and the ectocervix was cleaned of excess debris with a sterile cotton swab.Cervical mucus was obtained from the endocervix by gentle aspiration with a sterile tuberculin syringe (without a needle) daily from the 8th or 9th day of the cycle until the ideal score, described below, was reached.
Each item, except overheating, was scored from 0 to 3, with 3 being the optimal condition for each item.A score ³6 indicated preovulatory mucus receptive to sperm penetration, a score <6 was associated with unfavorable cervical secretion, and a score <3 represented hostile mucus (2).Overheated mucus showed color variations from totally light brown to totally clear during the periovulatory phase.
The pH of cervical mucus was determined when the cervical score was ³6/9 and the percentage of clear color was 50% or more upon overheating.Patients were examined daily and when the cervical mucus score was close to the predetermined values, pH measurement was programmed for the following day.On this occasion, pH was first measured and mucus quality was then evaluated.If mucus had not reached the predetermined score, the result was discarded and a new measurement was programmed for the following day or following cycle.For all measurements pH was determined with a glass microelectrode (Beckman ® No. 39535), 24 cm long and with a diameter of 5 mm, connected to a pHmeter (Digimed ® ), regularly calibrated against standard buffer solutions (pH 4.0 and 7.0).After being used for a patient, the microelectrode was wiped with tissue paper, washed with deionized water and disinfected with a solution of benzalkonium chloride.Prior to reuse it was washed with deionized water, recalibrated and washed again.
The first pH measurement (time zero) was made immediately after placement of the vaginal speculum and removal of vaginal secretion.The microelectrode was placed in contact with ectocervical mucus exactly in front of the external cervical os, where the measurement was made.The microelectrode was then gently inserted into the cervical canal until it reached the internal os, where endocervical pH was measured.These measurements were recorded as time zero.The electrode was removed from the cervix, cleaned with tissue paper and washed with deionized water.

The vaginal speculum was kept open and
In situ mucus pH variations the same procedure was repeated after 5 and 10 min.After the measurements, the mucus was aspirated with a tuberculin syringe and evaluated according to the criteria previously described.At the time of pH measurement, the following scores were recorded: volume -score 2 for four patients (20%) and score 3 for sixteen patients (80%); spinnbarkeit -score 2 for eight patients (40%) and 3 for twelve patients (60%); ferning -tertiary or quaternary (score 3) in all samples.Total cervical score was 7/9 for two patients (10%), 8/9 for eight patients (40%), and 9/9 for ten patients (50%).
Color at overheating -90% of the patients (N = 18) showed a percentage of clear color equal to or higher than 50%, while 10% (N = 2) showed a lower percentage of clear color.
The present study demonstrated that the lowest ectocervical pH value at time zero was 6.33 and the highest 7.35, at 5 min the lowest value was 6.78 and the highest 7.62, at 10 min the lowest value was 6.85 and the highest 7.73.Only one subject showed a reduction in ectocervical pH between 0 and 5 min.Mean ectocervical pH was 6.91 ± 0.235 at time zero, 7.16 ± 0.215 at 5 min and 7.27 ± 0.220 at 10 min.The values recorded at 0 and 5 min were significantly different, as also were the values recorded at 0 and 10 min (P<0.05,ANOVA), but there was no significant difference between the mean values recorded at 5 and 10 min.
The lowest endocervical pH value at time zero was 6.77 and the highest 7.40, at 5 min the lowest value was 6.89 and the highest 7.54, at 10 min the lowest value was 7.03 and the highest 7.69.Only one subject showed a reduction in endocervical pH between 0 and 5 min.Mean endocervical pH was 7.09 ± 0.161 at time zero, 7.34 ± 0.159 at 5 min and 7.46 ± 0.164 at 10 min.The values recorded at 0 and 5 min were significantly different, as also were the values recorded at 0 and 10 min (P<0.05,ANOVA), but there was no significant difference between the values obtained at 5 and 10 min.
The mean ectocervical and endocervical pH values obtained at different times are shown in Table 1.
The clinical relevance of pH changes after mucus exposure to the air is affected in practically all tests currently used to evaluate sperm-mucus interaction because in all of them aspirated mucus already exposed to the air is used and does not express physiological conditions.If lack of sperm penetration is due to a low mucus pH (9,(11)(12)(13), the penetration ability could not be preserved at the time of examination.In fact, some investigators have observed that, even with persistently negative PCT, in vivo penetration tests can be normal (6,7).A possible explanation could be the pH increase due to exposure to air when in vitro tests are performed.The contradictory results obtained in some PCT could also be explained by mucus pH changes at different times of interpretation.In addition, since pH values are different for each patient, there is no pattern for mucus alkalinization and subsequent correction at the time of test interpretation.Thus, PCT should be interpreted cautiously, since its results are obtained after cervical mucus has been aspirated and its pH is higher.This could mask the etiological factor, leading to a false diagnosis.
Ragni et al. (14) compared in vivo endocervical pH measurements first performed with paper and then with a microelectrode and observed that, although similar, the measurements made with an electrode were slightly superior.According to the findings in the present study, a possible explanation for these differences would be the period of time between the two measurements, rather than the different procedures.
In most cases of infertility due to cervical factor, the treatment is homologous artificial insemination (HAI).Intrauterine insemination is most frequently used (15).Although this is a low risk procedure, it is expensive, requiring sperm preparation in the laboratory and usually controlled ovarian stimulation and ultrasonography, generally restricting the treatment to specialized centers (16).Cervical factor cases due to low cervical mucus pH may be treated by means of a vaginal douche with sodium bicarbonate.This procedure is extremely simple and inexpensive and shows satisfactory results.
Several studies have shown a significant increase in PCT and in conception rates with the use of a vaginal douche with bicarbonate in previously selected couples (4,(17)(18)(19).
The authors suggest that greater attention should be paid to pH in the investigation of infertility and emphasize the need to perform pH measurements immediately after speculum placement in order to avoid mucus pH changes.Future research is needed to evaluate the exact period of time when cervical mucus pH begins to change and, consequently, the correct moment to perform sperm penetration tests in order to reproduce physiological conditions.We also recommend to check cervical pH before proceeding to HAI or evaluating other potential causes of poor sperm-mucus interaction, and to consider bicarbonate douche treatment if the pH is low.

Table 1 .
Mean ecto-and endocervical pH values in 20 subjects at each evaluation time.