Changes of Vaginal Microecology of Women with Intrauterine Adhesions

Introduction

Intrauterine adhesion (IUA) refers to endometrial basal layer damage caused by various reasons such as miscarriage, postpartum curettage, uterine artery embolism, postpartum uterine hemorrhage, manual placenta retrieval, and infection.¹ During the repair process, the endometrium is prone to form fibrous scar tissue, causing adhesion and occlusion in the uterine cavity and (or) the cervix.² As an endometrial injury disease, IUA could cause symptoms such as decreased menstrual flow, amenorrhea, infertility, and repeated miscarriages, which seriously affects women’s health.³ With the change of sexual concept, the increase of uterine cavity operation and the improvement of diagnostic technology, the incidence of IUA in China has been increasing.⁴ Mechanical or infectious endometrial injury caused by intrauterine operation is the main cause of IUA, but a small number of women without a history of intrauterine operation may also develop IUA, suggesting that there may be a synergistic effect of other factors in the occurrence of IUA.⁵

The vaginal microbiome of women is an ecosystem composed of vaginal microflora, endocrine regulation system, vaginal anatomy and local mucosal immunity.⁶ The balance of vaginal microflora plays an important role in maintaining the stability of vaginal microecology.⁷ The vaginal microflora is diverse, with Lactobacillus as the dominant bacteria, and the cytotoxins produced by it can inhibit cervical cancer cells by promoting apoptosis.⁸ The reduction of vaginal lactobacilli, the imbalance of flora and the increase of pH value favor the growth of other pathogens and increase the risk of cervical HPV infection.⁹

The role of endometritis in the development of IUA is gradually being paid attention to.¹⁰ When the balance of vaginal microbiota is disrupted, conditional pathogens in turn inhibit the growth of lactobacilli, leading to a decrease in vaginal defense ability. In addition, some pathogenic bacteria can also ascend to infect the uterine cavity, causing endometritis.¹¹ A study has found that there is a certain similarity between the microbiota in the official cavity and the microbiota parasitized in the lower reproductive tract, which further confirms that the bacterial colonies in the official cavity may originate from the lower reproductive tract.¹² Previous studies have found that the incidence of vaginal microbiota imbalance in patients with IUA is significantly higher than that of healthy women, with an increase in the diversity and density of vaginal microbiota.¹⁰ In this study, we speculate that the imbalance of vaginal microecology will lead to the decline of vaginal immunity, which may cause pathogenic bacteria to ascend along the reproductive tract or with the operation of the uterine cavity, resulting in endometrial infection and IUA. Therefore, this work aims to study the changes of vaginal microecology in IUA patients compared with healthy controls.

Methods

Study Subjects

Patients with clinical symptoms such as decreased menstrual flow, amenorrhea or infertility and were diagnosed with IUA by hysteroscopy were selected as the research subjects (n=150). Patients with normal uterine cavity who were admitted to hospital for diagnosis and treatment with clinical symptoms such as decreased menstrual flow, amenorrhea or infertility as the main complaints and excluded intrauterine adhesions by hysteroscopy were selected as the control group (n=150). The research method was reviewed by the ethics committee of Daqing Oilfield General Hospital, and all subjects signed the informed consent. This study was performed in strict accordance with the Declaration of Helsinki, Ethical Principles for Medical Research Involving Human Subjects.

Both menstrual regular and irregular menstruation were performed hysteroscopy (Nissan Olympus) 3–7 days after menstruation was clean. The uterine cavity is expanded with a dilatation medium, and a cold light source is introduced into the uterine cavity through a fiber guide beam, a lens, and a hysteroscope. Under the direct vision of hysteroscopy, the cervical canal, internal cervical orifice, endometrium, fallopian tube opening, etc. are examined, and the lesion tissue is sampled and sent for pathological examination. Mild adhesion refers to the presence of slender or thin adhesions in less than 1/4 of the uterine cavity, the upper end of the uterine cavity and the orifice of the fallopian tubes are normal, and the shape of the uterine cavity is normal. Moderate adhesions refer to the presence of adhesions in 1/4–3/4 of the uterine cavity but not progressing to the uterine wall, with atresia at the upper end of the uterine cavity and the orifice of the fallopian tubes, and abnormal shape of the uterine cavity. Severe adhesion refers to the adhesion of more than 3/4 of the uterine cavity, which is relatively hypertrophic and serious, and has progressed to the uterine wall, the upper end of the uterine cavity and the orifice of the fallopian tubes are completely closed, and the shape of the uterine cavity is deformed.

Exclusion criteria include having received vaginal local medication within 1 month, using estrogen and progesterone drugs, suffering from immune diseases or malignant tumors, and suffering from reproductive system diseases such as endometrial polyps, uterine fibroids, and adenomyosis, genital tract malformations, perimenopausal women, previously diagnosed or treated IUA by hysteroscopy.

All research subjects (150 cases in the control group and IUA group) underwent vaginal microecological examination. Routine vaginal secretion smears are dried and fixed for Gram staining, and the vaginal microecology is examined under a microscope. The inspection indicators include the bacterial flora density, bacterial flora diversity, dominant bacterial species, fungi, and trichomonas in the vagina. The detection kits for vaginal pH, hydrogen peroxide (H₂O₂), leukocyte esterase (LE), sialidase (SNA), 3-glucuronidase (GUS) and acetylglucosaminidase (NAG) were purchased from Jiangsu Bioperfectus Technologies Co., Ltd.

Detection of Vaginal Flora Density, Diversity and Dominant Bacteria

The vaginal secretions were made into saline glass slides, and fungal hyphae, trichomonas, etc. were observed under a microscope; after Gram staining, the vaginal flora density, diversity, and dominant bacteria were observed and analyzed under an oil microscope.

Bacterial density index: on average, 1–9 bacteria can be seen in each field of view under the oil microscope, which is “level I”; 10–99 bacteria, which is “level II”; 100–999 bacteria, which is “level III”; ≥ 1000 bacteria, which is “level IV”.

Bacterial diversity index: 1 to 3 bacteria can be identified in each field of view under the oil microscope, which is “level I”; 4 to 6 bacteria, which is “level II”; 7 to 9 bacteria, which is “level III”; ≥10 species of bacteria, which is “level IV”.

Dominant bacteria: the most common microorganisms seen under the oil microscope.

Detection of Pathogenic Microorganisms

Vulvovaginal candidiasis (VVC) detection: vaginal secretions were collected using the hanging drop method, and spores and hyphae were observed under a microscope.

Trichomonas vaginitis (TV) detection: leukocytosis and trichomonas with wave-like movement can be seen under the microscope. Bacterial vaginosis (BV) test: the dominant bacteria are Vibrio or Gram-negative bacilli, and the sialidase is positive. A Nugent score ≥ 7 is diagnosed as BV.

Microecological Evaluation

“Normal microecology” means that the density and diversity of vaginal flora are both II to III, the dominant bacteria are Lactobacillus, white blood cells or pus cells can be seen under a 0–5 magnification microscope, and the pH value is less than or equal to 4.5 and the H₂O₂ level is greater than 2 μmol/L. “Microecological imbalance” refers to any abnormality in vaginal flora density, diversity, dominant bacteria, H₂O₂ level and pH value.

Statistical methods

SPSS 23.0 statistical software was used. The data presented are mean ± SD or n (percentage). The comparisons of data between the two group were done by Mann–Whitney test, Chi-square test or Fisher’s exact test. Binary logistic regression analysis was applied for multivariable logistic analysis. P<0.05 was considered statistically significant.

Results

Clinical Characteristics of Patients with IUA

A total of 150 patients with decreased menstrual flow, amenorrhea, or infertility who were diagnosed with IUA after hysteroscopy were included in the study. The clinical characteristics of 150 participants and 150 healthy control volunteers are shown in Table 1. There were no significant differences in BMI and age between IUA patients and healthy controls. The number of pregnancies and abortions and the number of uterine evacuations in IUA patients were all significantly higher than those in the control group. IUA patients were divided into mild (64, 42.7%), moderate (47, 31.3%) and severe (39, 26%) according to the severity of the disease.

Table 1 Clinical Characteristics of Women with Intrauterine Adhesions (IUA) and Control

Comparisons of Vaginal Microecology Between the Women with IUA and Controls

To assess the vaginal microenvironment of IUA patients, we took intravaginal samples from IUA patients and healthy controls and analyzed vaginal pH and multiple microbes in Table 2. The number of IUA patients with vaginal pH value less than 4.5 (89, 59.3%) was significantly lower than that of the control group (123, 82%), suggesting that the vaginal pH value of IUA patients was increased. Lactobacillus is a common vaginal dominant bacterium in ordinary people. Among 150 healthy volunteers, the predominant vaginal species in 119 patients was Lactobacillus. However, only 96 people had Lactobacillus as the predominant vaginal species, which was significantly lower than the control group. In addition, the bacterial species diversity and distribution density in the vagina of IUA patients were significantly lower than those of healthy individuals. The incidence of TV and BV were significantly higher in IUA patients than in controls. Overall, the vaginal microecology assessment indicated that the vaginal microecological imbalance accounted for 25.3% in the control group and 48.7% in the IUA group, with a statistically significant difference.

Table 2 Comparisons of Vaginal pH Value and Vaginal Microecology Between the Women with Intrauterine Adhesions (IUA) and Control

Comparisons of Vaginal Functions Between the Women with IUA and Controls

To further analyze the vaginal microenvironment of IUA patients, we compared the vaginal function of IUA patients with healthy controls and presented the results in Table 3. Test contents included H₂O₂, LE, SNA, GUS and NAG. The number of patients with intravaginal hydrogen peroxide levels greater than 2 was significantly lower in the IUA group relative to the healthy controls. However, both intravaginal IE and SNA levels were significantly higher in IUA patients than in healthy controls. In addition, the number of positive vaginal NAG in IUA patients was also significantly higher than that in healthy controls.

Table 3 Comparisons of Vaginal Functions Between the Women with Intrauterine Adhesions (IUA) and Control

Multivariable Logistic Analysis of IUA

To analyze the risk factors of IUA, we performed multivariable logistic regression analysis on IUA patients. We took the occurrence of IUA (0=no, 1=yes) as the dependent variable, and selected patient age, BMI, Gravidity ≥ 2, Abortion ≥ 2, Uterine curettage ≥ 2, PH value> 4.5, non-Lactobacillus dominant bacteria, vaginal flora diversity (grade I, IV), density (grade I, IV), VVC positive, TV positive, BV positive, H₂O₂ positive (< 2 μmol/L), LE ≥ 9 U/L, SNA ≥ 7 U/L, GUS≥ 0.5 g/L, and NAG ≥ 0.1 U/mL as independent variables. We used a stepwise method to exclude irrelevant items. Finally, statistically significant IUA related factors were presented in Table 4. Related factors for IUA include gravidity ≥ 2, abortion ≥ 2, uterine curettage ≥ 2, pH > 4.5, non-Lactobacillus dominant bacteria, vaginal flora diversity (grade I, IV), density (grade I, IV), BV positive, H₂O₂ positive and NAG positive.

Table 4 Multivariable Logistic Analysis of Intrauterine Adhesions (IUA)

Comparisons of Vaginal pH Value, Microecology and Functions Between the Women with Mild to Moderate IUA and Severe IUA

IUA patients were divided into mild (64, 42.7%), moderate (47, 31.3%) and severe (39, 26%) according to the severity of the disease. We further analyzed vaginal pH value, microecology and functions between the women with mild to moderate IUA and severe IUA in Table 5. The proportion of vaginal pH greater than 4.5 was significantly higher in patients with more severe IUA than in patients with mild to moderate IUA. Patients with more severe IUA had a higher proportion of vaginal primary flora that were not Lactobacilli compared with patients with mild to moderate IUA. The diversity and density of vaginal flora in patients with more severe IUA were significantly different from those with mild to moderate IUA. In addition, the severe IUA patient group had more TV and BV positive patients. However, the proportion of SNA and GUS positivity in the severe IUA group was not significantly different from that in the mild to moderate IUA group.

Table 5 Comparisons of Vaginal pH Value, Microecology and Functions Between the Women with Mild to Moderate Intrauterine Adhesions (IUA) and Severe IUA

Discussion

At present, it is believed that the occurrence of IUA is mainly related to the uterine cavity injury after intrauterine operation, but there are still many other factors that play an important role in the occurrence and development of IUA, such as infection, congenital developmental abnormalities, and genetic factors.¹³ Long-standing chronic endometritis is a high-risk factor for the formation of intrauterine adhesions.¹⁴ Some studies have reported that women of childbearing age who suffer from chronic genital tract inflammation before abortion have a relatively high probability of intrauterine adhesions, which may be related to the fact that bacteria and various other pathogens are easily brought into the uterine cavity during uterine cavity operations and lead to infection.¹⁵ Inflammation can inhibit endometrial hyperplasia, induce fibrosis between the basal layers of the endometrium, and lead to the occurrence of IUA.¹⁶

The vagina of women is a micro-ecosystem of the human body.¹⁷ There are a variety of microbial flora in the vagina, and the flora is coordinated with each other to maintain the balance of the vaginal flora.¹⁸ Due to the interaction of vaginal anatomical structure, vaginal microflora, local immunity of vaginal mucosa, and the endocrine state of the human body, the vaginal microecological system is maintained in a dynamic and stable state.¹⁹ Vaginal microecological system is closely related to reproductive tract infection and other diseases, which is a research hotspot in recent years.²⁰ Vaginal microecological disorders will cause changes in the cleanliness and pH of the vagina, which will easily induce vaginitis, and also create favorable conditions for the ascending infection of endogenous and exogenous pathogens, leading to the occurrence of pelvic inflammatory disease and further leading to IUA.²¹ Some studies have found that¹⁵ the microbes in the uterine cavity are similar to the flora parasitic in the lower genital tract, which further confirms that the colonies of the uterine cavity may originate from the lower genital tract.²² In order to explore the changes of vaginal microecology in IUA patients and improve the accuracy of the research conclusions, the subjects of this study were all women with IUA or normal uterine cavity confirmed by endoscopy. In this study, we found that the proportion of vaginal microecological imbalance in the control group was 25.3%, and the proportion of vaginal microecological imbalance in the IUA group was 48.7%, with a statistical difference. The incidence of abnormal vaginal microecological morphological and functional indicators in the IUA group was remarkably higher than that in the control group, mainly manifested as relatively high pH value, reduction of Lactobacillus, increased ratio of flora density I, IV and flora diversity I, IV, and higher detection rate of TV and BV.

Lactobacilli are the most important flora for maintaining the balance of the vaginal microbiome, producing lactic acid and bacteriocins that help prevent bacterial growth.²³ Lactobacilli adhere to the surface of vaginal or cervical epithelial cells and can prevent bacterial adhesion to infected epithelial cells or facilitate the clearance of cells infected by microorganisms such as bacteria and viruses.²⁴ The reduction or disappearance of the number of lactobacilli can lead to a series of microbial imbalances such as the alkaline pH of the vagina, the growth of miscellaneous bacteria, the increase of white blood cell count, and the increase of cleanliness level.²⁵ The anatomical position of the vagina and cervix is adjacent to the vagina, and the external orifice of the cervix also has normal vaginal flora growth.²⁶ Therefore, the state of the cervix has a certain correlation with the microecology in the vagina. In the present study, we found that Lactobacilli were significantly reduced in IUA patients. In addition, the increase in the positive rate of H₂O₂ further demonstrated the reduction of vaginal lactobacilli, and the increase in the positive rate of NAG and SNA revealed that the enhanced probability of anaerobic infection. Further analysis found that gravidity ≥ 2, abortion ≥ 2, uterine curettage ≥ 2, pH > 4.5, non-Lactobacillus dominant bacteria, vaginal flora diversity (grade I, IV), density (grade I, IV), BV positive, H₂O₂ positive and NAG positive were risk factors for IUA. In addition, this paper found that there were also differences in vaginal microecological flora among patients with different adhesions. Patients with severe adhesions had the most abnormal degree of vaginal microecological flora, and the dysbacteriosis was more obvious. Lactobacillus was the predominant bacterium in 72.1% of mild to moderate IUA patients, but only 41% in severe IUA patients, suggesting that Lactobacillus is crucial to the occurrence of IUA. In addition, recurrence after IUA treatment is also a difficult clinical problem. Therefore, we plan to elucidate in future studies whether IUA recurrence is related to the vaginal microbiota status before and after treatment.

Conclusion

In this study, we reported that vaginal microecological imbalance is a risk factor for synergistic formation of IUA. Gravidity ≥ 2, abortion ≥ 2, uterine curettage ≥ 2, pH value>4.5, dominant bacteria not Lactobacillus, diversity of vaginal microbiota (grade I, IV), density (grade I, IV), BV positive H2O2<2 μ mol/L and NAG ≥ 0.1 U/mL are risk factors for IUA.