Abortion is of great importance in public health, as it is among the main causes of maternal morbidity and mortality. In addition to sociodemographic- and lifestyle-related factors, studies have associated infections of the genital tract with higher rates of abortion. Therefore, the exacerbated presence and rise of Mollicutes in the genitourinary tract may be related to higher rates of abortion.

To perform an epidemiological analysis of women who had spontaneous abor-tions and placental colonization by Mollicutes in a maternity hospital.

This cross-sectional study involved the collection of data and biological material from women hospitalized due to spontaneous abortion or term delivery. The sample consisted of 89 women who miscarried and 20 women who had full term pregnancy. Data collection was carried out in three stages: (1) Conducting research on the information and clinical data in medical records of patients hospitalized due to abortion; (2) Application of a semi-structured questionnaire to identify the patient's epidemiological profile and (3) Collection of placental tissue. Placental samples were collected after the curettage procedure (abortion) and after placental expulsion (delivery), both performed by an obstetrician. Microbial identification in the fragments was performed by real-time polymerase chain reaction. In this study, the following explanatory variables were considered: (1) Sociodemographic variables; (2) Variables related to access to health services; (3) Variables related to lifestyle; and (4) Variables related to sexual and reproductive health, all of which were used to perform descriptive, univariate and multivariate analyses.

In the final model, placental colonization by Mollicutes was independently associated with the variables age [odds ratio (OR) = 7.55; CI: 2.37-24.03] and menarche (OR = 3.43; CI: 1.03-11.44). In this investigation, the prevalence of Mollicutes colonization by at least one of the following three species: Mycoplasma hominis, Ureaplasma urealyticum, Ureaplasma parvum in women who had spontaneous abortion was 73.0%. When comparing colonization between the two groups of participating women, there was an 8.12-fold risk of placental colonization by at least one Mollicutes species in the women who had an abortion, compared to those who completed pregnancy. The final multivariate analysis model revealed a statistically significant association between placental colonization by Mollicutes in abortion with the following variables: age, as women up to 29 years old had a 7.55-fold risk of spontaneous abortion, compared to those who were older than 29 years; menarche, where women who had menarche up to 13 years old had a 3.43-fold risk of miscarriage compared to those who had menarche over 13 years old; and a change in eating habits, after the discovery of pregnancy, was a protective factor (OR = 0.16).

These findings revealed a positive association between spontaneous abortion and placental colonization by Mollicutes. This indicates the need for further investigation of this issue, to guide decision-making for the prevention of abortion.

Abortion is considered the early termination of pregnancy at less than 22 wk and a fetus weighing less than 500 g. Approximately 10% to 15% of abortions occur spontaneously and mostly with an undetermined cause[1,2]. Abortion is of great importance in public health, as it is among the main causes of maternal morbidity and mortality, especially in developing countries such as Brazil[3].

According to the World Health Organization, each year, approximately 500 thousand women die from causes related to pregnancy, with 98% of cases occurring in developing countries. Of these deaths, 15% is associated with abortion compli-cations[4]. A global study revealed that in the period from 2010 to 2014, 25% of pregnancies worldwide resulted in abortion[5].

Studies have associated sociodemographic factors (such as age, marital status, social context, socioeconomic conditions, and education) and factors related to lifestyle (such as alcohol consumption, smoking, pre-gestational nutritional status, reproductive history, environmental and occupational exposure of woman) with spontaneous abortion[6-9].

Another important clinical aspect in pregnant women, is infections of the genital tract due to placental colonization with microorganisms related to bacterial vaginosis such as Gardnerella vaginalis, Mollicutes [Mycoplasma hominis (M. hominis), Ureaplasma urealyticum (U. urealyticum), Ureaplasma parvum (U. parvum)], Mobiluncus spp, Chlamydia trachomatis, among others[2,10] as a possible cause of spontaneous abortion.

Mollicutes constitute a class of bacteria that differ from the other bacteria as they do not have a cell wall. The genera of interest in this study are Mycoplasma and Ureaplasma. The former are important opportunistic pathogens that colonize the male and female urogenital tract, and are responsible for triggering various pathologies including spontaneous abortion[11]. Ureaplasma species are one of the main pathogens associated with genital infection, for example non-gonococcal urethritis in men and, in women, gestational complications such as premature birth and abortion, among others[12].

The presence of Mollicutes in the genitourinary tract and its pathogenic potential is related to age, hormones, sexual activity, pregnancies, low socioeconomic status, smoking, use of vaginal showers and the use of oral contraceptives[13]. The diagnosis of Mollicutes infection is quite difficult as they have low biosynthetic activity and require complex culture media, making the diagnosis using techniques such as Gram staining, complicated. They can also be diagnosed by immunoenzymatic reactions and by molecular biology, the latter being considered the gold standard for diagnosis[14].

In pregnant women with modification of the vaginal microbiota, a reduction in Lactobacillus species is frequently found, triggering an increase in vaginal pH[15]. As a result, changes occur in the human vaginal microbiome, which contains a varied number of bacterial species, including members of the Mollicutes class, which may find conditions which allow entry into the uterine cavity[16]. In the uterus, they may be able to induce an immune response that can lead to excessive inflammation and consequently damage and death to the cellular components of the placental and fetal unit[17].

Thus, in the present study we analyzed the association between spontaneous abortion and placental colonization by Mollicutes, in order to provide evidence capable of contributing to reduced health costs. In addition, the study of spontaneous abortion in women with placental colonization by Mollicutes has been little explored and may encourage further studies on this issue, thus increasing our understanding and management of spontaneous abortion. Therefore, we performed an epidemiological analysis of women treated for spontaneous abortion with placental colonization by Mollicutes in a maternity hospital in southwest Bahia, Brazil.

This is a cross-sectional study, derived from a larger project, entitled “Evaluation of the participation of Mollicutes and other microorganisms of genital interest in the etiopathogenesis of spontaneous abortions”. The present study aimed to detect Mollicutes and other microorganisms of genital interest in the placental biopsy of women who underwent spontaneous abortion, to assess their probable relationship in etiopathogenesis. It involved the collection of data and biological material from two groups: patients hospitalized due to spontaneous abortion and patients who had term delivery and was carried out by researchers from the Universidade Federal da Bahia and the Universidade Estadual de Santa Cruz.

The main objective of the present investigation was to determine the association between patients hospitalized due to spontaneous abortion and placental colonization by Mollicutes. For the purpose of comparison and verification of this association, we assessed the prevalence of placental colonization by Mollicutes from the group of women who had term pregnancy.

The study was carried out in a public hospital, in a maternal and child health care unit for the municipality of Vitória da Conquista, Bahia, Brazil and the southwestern macro-region of the state of Bahia, Brazil. Vitória da Conquista it is the third largest city in Bahia, with a total population of 348718 inhabitants[18].

The participants were women aged 18 years or older, hospitalized due to spontaneous abortion, with a gestational age greater than or equal to 8 wk and less than 22 wk and a fetus weighing less than 500 g. In addition, a group of women who underwent vaginal delivery at term (gestational age between 38 and 42 wk), who voluntarily agreed to participate in the study after being approached by the researchers was also included.

The study excluded women who used antibiotics less than two weeks after the abortion occurred. Also excluded were those with confirmed immunodeficiency, and chronic diseases previously diagnosed (hypertension, diabetes, endocrine disorders) and a history of recurrent abortions due to anatomical abnormalities and/or congenital malformation.

In this study the sample consisted of 89 women who miscarried and were treated between July 2017 and August 2018 and 20 women who had full term pregnancy at the hospital.

Data collection took place in three stages, which are shown in Figure 1. The first stage involved the collection of information and clinical data from the medical records of the patients hospitalized due to abortion. The second stage was the application of a semi-structured questionnaire in order to identify the patient's epidemiological profile, divided into modules, as a result of the adaptation of the National Health Survey, and the instrument used in the study by Campos et al[19]. The third stage consisted of collecting placental tissue[20]. Placental samples were collected after a curettage procedure using a sterile speculum performed by an obstetrician. The fragment was stored in a transport medium for Mycolpasma and transported in thermal boxes to the Microbiology and Immunology laboratory of the Instituto Multidisciplinar em Saúde, at the Universidade Federal da Bahia. For microbial identification of placental tissue, genomic deoxyribonucleic acid was initially extracted using the Invitrogen PurelinkTM Genomic deoxyribonucleic acid Kit (Invitrogen, Waltham, MA, United States). Subsequently, real-time polymerase chain reaction was performed using StepOne Plus (Life Technologies) with a final volume of 25 µL and the use of Master Mix (Thermo Fisher Scientific, Waltham, MA, United States). To identify the Mollicutes species, TaqMan probes were used, following the basic amplification protocol for each species. The studied species were: M. hominis, U. urealyticum and U. parvum.

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Figure 1 Flowchart of the data collection procedure. ICF: Informed consent form.

The identification of placental Mollicutes in patients with abortion constituted the outcome variable of this study. The following explanatory variables were considered: (1) Sociodemographic variables: Age group, income, marital status, education, and skin color; (2) Variables related to access to health services: Prenatal consultations; (3) Variables related to lifestyle: Type of food consumed, physical activity, alcohol and tobacco use; and (4) Variables related to sexual and reproductive health: Historical existence of sexually transmitted infection, presence of genital alterations (ulcers, blisters, lumps, discharge), presence of urinary tract infection, changes in the reproductive system such as: presence of polycystic fibroids and ovaries, number of previous pregnancies, number of abortions, menarche and first sexual intercourse.

A descriptive analysis of the variables was performed by comparing frequencies and using Pearson's Chi-square test with P < 0.05 and a 95% confidence interval, using a Microsoft Office Excel database and analyzed using the statistical package EPI-INFO (version 7.2). To obtain measures of association of the variables, the odds ratio calculation was then used. All variables with P < 0.20 in the univariate analysis were included in multivariate analysis using logistic regression, performed in SPSS version 21. In the final multivariate model, a significance index of P < 0.05 was considered and the final model which obtained the best fit was evaluated by the Hosmer-Lemeshow test.

This study took place after approval by the teaching and research nucleus of the Health Foundation of Vitória da Conquista and approval by the Human Research Ethics Committee of the Instituto Multidisciplinar em Saúde (Universidade Federal da Bahia), opinion 1.764 332.

After analyzing the placental tissue, the prevalence of colonization by Mollicutes with at least one of the three species: M. hominis, U. urealyticum and/or U. parvum among women who had a spontaneous abortion was 73.0%. In the larger study, in addition to studying women who had abortions, the presence of Mollicutes in the placenta of women who completed their term pregnancy was also investigated. In this group, the presence of at least one species of those listed above was detected in 25% (n = 5) of women. When comparing colonization between the two groups of women, there was an 8.12-fold risk (CI: 2.66-24.78, P < 0.001), for placental colonization by at least one Mollicutes species in women who had an abortion, compared to those who completed their pregnancy.

Table 1 shows the descriptive analysis of women undergoing abortion who were included in the study (n = 89). The average age of the participants was 28 years. Most women reported having a partner, were from an urban area, were non-white and had more than 8 years of education. 65.2% (n = 58) of the study participants reported having a family income of up to one minimum wage, which at the time of the interview was R$ 95400.

With regard to life habits, most of them reported not having changed their eating habits after the discovery of pregnancy, not smoking, not drinking alcohol and not taking part in physical activity. Regarding sexual and reproductive health, the majority declared that they did not have any gynecological disorders, such as fibroids, polycystic ovaries and never had a sexually transmitted infection. Of the interviewees, 65.1% (n = 56) started their sexual life aged more than 15 years, and reported having had more than one sexual partner in her life and not using condoms during sexual intercourse. Just over half of the participants reported having had up to 2 pregnancies in their lifetime.

The average gestational age at which the abortion occurred was 12 wk, ranging from 8 to 22 wk. 58.4% (n = 52) of the interviewees had at least one prenatal consultation and among all the women studied an average of 1.7 consultations occurred before the abortion. Most of the participants reported not having planned their pregnancy and having had some type of vaginal discharge in the last three months. A small number of women reported having had pelvic pain and dysuria.

Table 2 shows the results of the univariate analysis between the variables selected for the study and placental colonization by at least one of the studied Mollicutes species. It was found that women up to 29 years old had a 7.26-fold risk (CI: 2.49-21.12) of placental colonization by at least one of the studied Mollicutes species, compared with those older than 29 years. Women who reported menarche age of up to 13 years had a 3.15-fold risk (CI: 1.13-8.79) of placental colonization by at least one of the studied Mollicutes species compared with those who had menarche over 13 years. Women who had up to 2 pregnancies had a 2.73-fold risk (CI: 1.04 -7.05) of spontaneous abortion and placental colonization by at least one of the studied Mollicutes species, when compared to those who had more than 2 pregnancies. For those with up to one live child, there was a 3.33-fold (CI: 1.25-8.84) risk compared to those who had more than one live child.

The final multivariate analysis (Table 3) revealed a statistically significant association between placental microbial colonization by Mollicutes in abortion and the variables: Age (women up to 29 years of age had a 7.55-fold risk of having a miscarriage, compared to those older than 29 years); menarche (women who had menarche up to 13 years of age had a 3.43-fold risk of having a miscarriage than those who had menarche over 13 years of age). A change in eating habits after the discovery of pregnancy appeared to be a protective factor.

Microbial colonization by Mollicutes in the urogenital tract may predispose to spontaneous abortion[11,21]. In this study, the prevalence of microbial colonization by at least one of three Mollicutes species (U. urealyticum, U. parvum, M. hominis) in the placenta of women with spontaneous abortion was 73.0%. The prevalence of U. urealyticum in endocervical material from patients who had an abortion was 16.5% in another study[22]. Previous studies were also carried out in other countries such as Germany, Switzerland and Australia, and showed evidence that the presence of Mollicutes and other microorganisms in the vaginal microbiota may be related to adverse pregnancy outcomes, including spontaneous abortion[21,23,24].

In the present study, women who had up to one live child had a 3.33-fold risk (CI: 1.25-8.84) of abortion compared with those who had more than one live child. In addition, women who had 2 pregnancies had a 2.73-fold risk of abortion when colonized by Mollicutes, compared to those with more than 2 pregnancies. The literature demonstrates conflicting data on this issue, some studies have shown a protective effect against spontaneous abortion in women with more than one live child[7], and some have indicated an increased risk in women with more than one live child[8,21].

Results also vary with parity: the difficulty of becoming pregnant or the existence of complications in previous pregnancies may be the reason for past spontaneous losses[6,7,20]. In other studies, women with more live children indicated a higher number of pregnancies, and had the greatest risk of abortion[8,25].

Age up to 29 years was independently associated (OR = 7.55; CI: 2.37-24.03) with the presence of Mollicutes in the placenta of women with abortion. Age between 20 and 30 years was also associated with abortion and the presence of infectious diseases such as STORCH syndrome (syphilis, toxoplasmosis, rubella, cytomegalovirus and herpes virus)[1] or the presence of Ureaplasma spp in the female genital tract[26]. These findings are in line with other studies, where it was shown that young age (women under 29 years old) is a risk factor for the occurrence of spontaneous abortion, and younger pregnant women may have less access to health services. This implies fewer possibilities for early detection of infections associated with abortion[1,6,22].

Findings from studies that evaluated prenatal programs and cervical screening using Pap smear, which has often shown a higher prevalence of the use of these services by older women, corroborates these findings[27,28]. However, other studies diverge from these findings where the occurrence of spontaneous abortion occurs in a greater proportion of women over 35 years of age[8,25], these studies are usually population-based and self-reported, and only point out that the older the age, the longer the time of exposure to pregnancy and abortion.

After final adjustment, women who had their first menstruation up to 13 years of age had a 3.43-fold risk (CI: 1.03-11.44) of developing spontaneous abortion with placental colonization by Mollicutes when compared to women who menstruated after 13 years. Early menarche was associated with spontaneous abortion[6], and in Brazilian girls this age has decreased further, implying precocious reproduction, which may be related to a greater number of pregnancies among young women. This, together with other factors, can lead to an increase in the occurrence of spontaneous abortion[29,30]. Thus, it can be inferred that sex education, and the provision of family planning by health services, should be started early, in order to avoid early pregnancies.

Prenatal care was also one of the variables examined in this study. Even though there was no statistical significance, it still deserves to be mentioned, as most of the participants stated that they had prenatal care before the abortion occurred. However the average number of consultations was 1.7 per woman, a very low rate, considering that the average gestational age at abortion was 12 wk. According to technical norms of the Ministry of Health, prenatal care must occur as early as possible. Right at the first consultation it is necessary to ​​request laboratory tests and to perform a gynecological examination (specular and cytopathological examination). Also, at this time, the existence of urinary tract infections and the presence of bacterial vaginosis should be investigated. The latter is defined as infection of the female genital tract, where among several pathogens, Mollicutes can be the causative agents. This can provide an opportunity for the early diagnosis and treatment of these infections, preventing gestational adverse effects such as the occurrence of spontaneous abortion[28,31,32].

Socioeconomic factors have been identified as important elements related to spontaneous abortion. Some studies have shown a significant association between low income levels, low education level and spontaneous abortion[3,7,8]. In this study, income was not significantly associated with spontaneous abortion, but due to the homogeneity in this variable, most participants 65.2% (n = 58) reported income of up to one minimum wage, resulting in low power for comparison purposes.

Schooling has been reported in the literature to be an important factor related to the occurrence of spontaneous abortion. In countries such as Brazil, women who had up to 8 years of education had a 1.66-fold greater risk (CI: 1.11-2.49) of having a miscarriage, than those with more than 8 years of education[6], and similar results have been found in other studies[7,8]. These findings were not observed in this study, most women reported having more than 7 years of education; thus, education level was not significant. Among the women studied, the problem was access to health services than the lack of information.

In summary, it should be noted that this is a pioneering study in the southwestern region of Bahia, Brazil, which revealed a positive association between spontaneous abortion and placental colonization by Mollicutes. The study also showed an association between abortion and women up to 29 years of age, who had their first menstruation up to 13 years old. These findings require more detailed studies within this area, in order to provide a better epidemiological profile of women undergoing abortion and to support decision-making by health managers. Decision-making in abortion can have an important impact on reducing health costs, as managing the complications of abortion and its occurrence is much more costly than preventing it.

This is a pioneering study in the southwestern region of Bahia, which revealed a positive association between spontaneous abortion and placental colonization by Mollicutes, and abortion was also associated with women up to 29 years of age, who had their first menstruation up to 13 years of age. These findings are important, but point to the need for more detailed studies within this area, in order to provide a better epidemiological profile of women undergoing abortion to support decision-making by health managers, with a view to decreasing health costs, since the prevention of abortion is much less costly than managing its occurrence and complications.