Background and Objective: Female infertility may be attributed to several causes that are fundamentally related to the health status of women. The main objective of this project was to correlate the abundance of the microbiota in cervicovaginal fluid to infertility. Materials & Methods: A total of 36 married women who voluntarily came to the hospital in Riyadh for a routine visit participated in the study. To collect the cervicovaginal liquid, a Softcup TM menstrual device was used by the participant; the cup was then transported in a liquid nitrogen box to the laboratory for analysis. Results: The mean vaginal pH in normal women and infertile women was 3.96 and 5.06, respectively, and the difference between the two cohorts was significant (p < 0.0001). The results showed a clear correlation between the pH value and the floral species detected in the cervicovaginal liquid. In fact, normal flora is primarily composed of a heterogeneous group of Lactobacillus species that have been detected at pH range 3.0 - 4.5, whereas a second group of bacteria mainly composed of Gard-nerella vaginalis, Peptostreptococcus anaerobius and L. iners has been detected in a pH range between 4.5 and 5.5. The third group, with a pH range >5.5, is primarily composed of G. vaginalis, P. anaerobius, Mycoplasma hominis, Mobiluncus species and Atopobium vaginae. The protein content and viscosity of the cervicovaginal liquid were significantly lower in infertile women compared to normal women (p < 0.05).
The World Health Organization estimates that approximately 8% of couples, or 50 - 80 million people worldwide, have some forms of infertility [
The human microbiota is found various organs including the skin, mouth, gut and vagina. This microbiota plays a crucial role in the health/disease status in both man and women. While the gut is populated with more than 800 microbial species, the vaginal tract hosts approximately only 50 [
It is important to study the microbial communities in the reproductive organs of women within fertility risk and compare them to those in normal women. Such study will be able to determine whether or not the presence of some genera is significantly different between the two cohorts. While most of the bacteria found in the female reproductive organs are considered beneficial, some are considered as pathogens. Thus, the variation in bacterial communities between fertile and infertile women could be associated with level of infertility.
The study was approved by the Research Ethics Committee in King Saud University. The study has been conducted on married women who voluntarily came to the hospital for a routine visit on a random day of their cycle. The subjects were assigned into two groups according to the level of their fertility. The first group includes married non-pregnant women who have been pregnant at least one time since mirage but without clinical intervention (control, n = 22). The second group (Infertile group, n = 14) includes women who were married to normal men but were unable to get pregnant after at least one year of marriage. These women are defined as infertile according to WHO. The sampling criteria for the infertile group were: at least one year of marriage without successful fertilization, more than 10 times intercourses per month, have no antibiotics and have not used any vaginal medication in the last two months. Twenty five women with risk of fertility were recorded, however only 14 have matched the inclusion/exclusion criteria.
At admission, clinical measurements and health history were obtained. These include age, weight, height, date of marriage and number intercourse. Subjects were asked to provide information about any medications they used in the last two months. Women from both groups were asked to obtain a self-sample of the cervicovaginal liquid at admission using Softcup™ menstrual device (Evofem Inc., San Diego, CA, USA). A leaflet of clear instructions was provided to all subjects. Briefly, the Softcup was inserted by the participant into the vagina up to the cervix and removed after 20 minutes. This was repeated in the second admission. The Softcup was quickly placed inside an open 50 ml centrifuge tube and immediately placed into a box containing nitrogen. All cervicovaginal liquid samples were then transported to the laboratory for analysis.
The pH was measured using a digital pH meter (Φ11 pH meter, Beckman Instruments Inc., Fullerton, CA, USA) and a glass microelectrode designed to measure pH in small volumes. The pH level was calibrated each time using standard buffer solutions (Sigma-AldtichpH 4.0, 7.0 and 10.0). The microelectrode was washed with deionized water and disinfected with a solution of benzalkonium chloride each time.
The viscosity of each sample was measured using the Cambridge MicroSample Viscometer (Cambridge Viscosity, Medford, MA, USA). The cervicovaginal liquid of all samples was centrifuged at 2000g for 10 minutes at room temperature (25˚C), and the protein phase was removed for quantification. Finally, the samples were stored at −70˚C for future study.
Self-collected vaginal swabs from all subjects were treated according to the method described by Verstraelen et al. [
We used GraphPad prism version 5 for the statistical analysis. Statistical comparisons were made using a two-tailed independent sample t-test. All values are presented as the mean ± SD. Significance was assumed when p < 0.05.
The ages of the infertile women group (mean 25.27 years) were not significantly different (p = 0.42) compared to control women (mean 27.95 years). The mean weight of the normal women was higher (mean 67.86 kg) than that of the infertile women (mean 66.93 kg), but the difference was not significant (p = 0.73),
The pH levels of 36 Cervicovaginal samples were measured (22 normal women and 14 infertile women). The present study demonstrated that the mean vaginal pH levels in normal women and infertile women were 3.96 and 5.06, respectively, and the difference between the two cohorts was statistically significant (p < 0.0001). For normal women, the lowest pH value was 3.2, and the highest was 5.1 whereas for the infertile women, the lowest pH value was 3.9, and the highest was 5.8 (
The pH results show a clear correlation between the pH value and the floral species detected in the cervicovaginal liquid (
The protein content of the cervicovaginal liquid was significantly higher in the normal women compared to the infertile women (p < 0.0001) (
Normal women were found to have higher cervicovaginal viscosities (mean value 1.56) compared to women who were determined to be infertile (mean value 1.10). This difference was significant (p < 0.0001) (
Female infertility may be caused by many factors, including polycystic ovary syndrome, endometriosis, and fallopian tube problems. Many other factors could cause infertility in women. The physical properties of the cervicovaginal liquid must have an adequate consistency and a pH value for suitable proliferation of lactobacillus bacteria.
What attracted our attention in this study is the fact that in the second subgroup, L. iners (normal flora beneficial for vaginal health) coexisted with a
second type of harmful bacteria represented by Gardnerella vaginalis and Peptostreptococcus anaerobius. Our findings align with those of Jakobsson and Forsum [
explain their presence in the pH range of 4.5 to 5. Even G. vaginalis is not currently considered to be harmful because this species has been reported to be present in women without any symptoms [
However, during ovulation and under the effect of ovarian hormones (primarily LH), an increase in cervical mucus causes pH to move more alkaline (7 - 14), which allows sperm to travel through the vagina and cervix into the fallopian tubes for fertilization. Many other factors may also cause the vaginal alkalization, such as long menstrual periods, inadequate douches and the use of broad-spectrum antibiotics [
The differences in the protein quantity in the cervicovaginal liquid between the two groups suggest that the vaginal environments of the patients in the two groups are different and may be the cause of infertility. Chappell et al. [
Cervical mucus is considered to be a hydrogel that is composed of a high- molecular-weight component (gel phase) and a low-molecular-weight component (cervical plasma) [
The authors gratefully acknowledge the financial support provided by King Abdulaziz City for Science and Technology (S.F.306-35).
Al-Rezaki, A., Aldahmash, W., Alwassel, S. and Abotalib, Z. (2017) Assessment of the Microbiome Collected from the Reproductive Tracts of Women from Saudi Arabia and Its Potential Influence on Infertility. Advances in Reproductive Sciences, 5, 23-32. https://doi.org/10.4236/arsci.2017.52003