Molecular examination for Coxiella burnetii and Brucella spp. infections in Iranian women experiencing spontaneous miscarriage

Miscarriage is defined as the spontaneous termination of pregnancy and is estimated to occur in approximately 15–20% of pregnancies. While the exact causes of miscarriage remain unclear, several common factors contribute to this occurrence, including genetic abnormalities, uterine abnormalities, maternal age, maternal overweight, hypertension during pregnancy, hormonal imbalances, immunological factors, history of miscarriage, smoking, alcohol consumption, psychological stress, and infections [1‐3]. 15% of early abortions and 66% of late abortions are caused by infections [1]. Among the infections, some pathogens have been implicated in increasing the risk of spontaneous miscarriages. For instance, infections caused by bacteria such as Listeria monocytogenes, Ureaplasma urealyticum, and Chlamydia trachomatis have been associated with adverse pregnancy outcomes, including miscarriages [4]. Additionally, certain viral infections like cytomegalovirus (CMV) and herpes simplex virus (HSV) have also been linked to an increased risk of miscarriages [5]. These infections can lead to inflammation, placental damage, and ultimately fetal loss [6, 7]. However, the mechanisms responsible for infectious miscarriages are diverse and can involve direct fetal infection, placental injury, and severe maternal illness. In many cases, despite the presence of elevated levels of maternal inflammatory markers or histological evidence of chorioamnionitis, no specific pathogenic agents have been identified. Intracellular bacteria, such as Coxiella burnetii and Brucella, which cannot be easily detected using conventional culture media routinely used for pathogen detection in clinical specimens, may play a significant role as etiological agents of miscarriages [1, 4]. However, their contribution is likely underestimated [1]. Advancements in modern diagnostic techniques, particularly molecular methods, have led to increased identification of infectious agents that can cause pregnancy complications, including spontaneous miscarriages [4].

Coxiella burnetii is a Gram-negative bacterium and the etiological agent of the zoonotic disease known as Q fever. The main way of contracting Q fever is through the inhalation of aerosols and particles contaminated with C. burnetii. Direct contact and the consumption of unpasteurized dairy products are less common methods of transmitting the infection to humans [8, 9]. Q fever can cause various clinical manifestations in humans, ranging from asymptomatic to acute Q fever (resembling flu-like symptoms, pneumonia, and hepatitis) and chronic Q fever (endocarditis) [10]. During pregnancy, both acute and chronic Q fever can lead to complications such as premature delivery, growth retardation, fetal abnormalities, low birth weight, stillbirth, and abortion [11]. C. burnetii not only colonizes but also multiplies in various parts of the body, including the uterus, placenta, monocytes, macrophages, and human trophoblastic cells [12]. The prevalence of C. burnetii among pregnant women in Iran has been reported to be approximately 30% [13]. Q fever is endemic in Iran, and acute Q fever and Q fever endocarditis cases have been reported in various studies [14, 15]. The incidence of Q fever during pregnancy is likely underestimated, and there is limited information available regarding the role and prevalence of this bacterium in pregnancy complications such as miscarriages. Hence, it is of utmost importance to investigate the presence of C. burnetii in miscarriages, especially considering the high seroprevalence among livestock and milk in Iran [16, 17].

Brucella is a Gram-negative bacterium that causes the disease known as brucellosis. Humans typically contract the disease through direct contact with infected animals, consuming contaminated animal products, or inhaling airborne agents [18]. There are various Brucella species, such as B. abortus, B. melitensis, B. suis, and B. canis [19], all of which can cause brucellosis in humans. This disease can lead to complications during pregnancy, including miscarriage, premature birth, contagious or neonatal brucellosis, intrauterine infection, or intrauterine fetal death [20]. Brucellosis is an endemic disease in Iran and has been reported in different regions of the country; the prevalence rate varies from 98 to 130 per 100,000 population [21, 22]. In endemic areas, the prevalence rates of brucellosis in pregnant women range from 1.3 to 12.2% [23]. A study in Iran found that 53% of pregnant women with brucellosis experienced spontaneous miscarriages [24]. In Iran, there is a lack of research on the genetic proof of Brucella in women who have had abortions, making it imperative to examine the presence of Brucella in these women.

While the role of C. burnetii and Brucella spp. in spontaneous miscarriages is well-documented, our study focuses specifically on Iran, a region with a high prevalence of these pathogens, providing unique insights into their impact on spontaneous miscarriages. Therefore, this study aimed to investigate the presence of Brucella and C. burnetii in abortion samples from women with a history of abortion in Iran, as these infectious and miscarriage abortions are prevalent health issues in the country due to the endemic nature of both Brucella and C. burnetii.

In this study, the prevalence of C. burnetii and Brucella was investigated in samples from miscarriages. Miscarriage is a common complication of pregnancy that can occur frequently or sporadically [28]. Some intracellular bacteria, including L. monocytogenes, C. trachomatis, C. burnetii, and Brucella, have been found to affect embryonic units and cause serious diseases in both the mother and fetus [4]. For instance, a study conducted in Switzerland from 2006 to 2009 revealed that the prevalence of IgG against C. trachomatis was more common in the group experiencing miscarriages (15.2%) compared to the control group (7.3%; p = 0.018). Additionally, the DNA of C. trachomatis was more frequently detected from the products of conception or placenta in women who had miscarriages (4%) compared to those in the control group (0.7%; p = 0.026) [29]. However, few studies have been conducted on Q fever infections in pregnant women and the associated abnormal and complex complications. Adverse pregnancy outcomes resulting from Q fever infections in pregnant women are typically caused by vascular thrombosis, which leads to placental insufficiency and abortion. Studies have also reported direct infection of the fetus [30]. Serological studies of C. burnetii among pregnant women living in rural areas with direct contact with livestock were found to be 29.3% in southwestern and northern Iran and 48.4% in western Iran [13, 31]. However, our study did not detect any C. burnetii infections using the molecular method.

Acute Q fever infection during the first trimester of pregnancy significantly increases the risk of both the mother and fetus developing chronic Q fever infection. Furthermore, an asymptomatic infection during pregnancy can progress to a chronic state, increasing the chances of reinfection in future pregnancies [32, 33].. In Denmark, a study found a significantly higher seroprevalence rate (47%) of Q fever among pregnant women who were exposed to livestock than among nonexposed women (4.8%) [34].

Several studies have investigated the prevalence of C. burnetii in abortion samples from pregnant women using molecular methods. One study conducted in Turkey on 51 placental samples found no detection of C. burnetii [35]. Similarly, a study in France on 246 placenta samples also reported no infection with this pathogen [36], which is consistent with our findings. However, a study in Algeria reported a positive rate of 0.55% in placental samples [37]. In Iraq, a molecular study reported that 17.02% of blood samples from aborted women were positive for C. burnetii [38]. Many studies have focused on the prevalence of Q fever in pregnant women using serological methods. In two separate studies conducted in Turkey, the seroprevalence of Q fever among pregnant women was found to be 20.7% and 14%, respectively [35, 39]. In Iran, the seroprevalence of C. burnetii infection in pregnant women was found to be 22% in the southwest (Ahvaz City) and 36.5% in the north (Parsabad City). Furthermore, the overall prevalence of C. burnetii infection in women with a history of abnormal pregnancy (39.8%) was significantly higher than that in women with normal pregnancies (23.8%) [13]. Additionally, a study conducted among rural pregnant women in Khorramabad (western Iran), who were in contact with livestock, reported a seroprevalence of 48.4% for C. burnetii [31]. However, a possible explanation for the non-detection of C. burnetii in abortion samples during this study could be due to the exclusion of specific infection foci in the examined tissue samples. In addition, it is plausible that the infection was latent or had already resolved by the time of examination, rendering the molecular test insufficient for diagnosis.

Although the presence of erythritol in the placenta of livestock acts as a growth factor for Brucella, cases of abortion have also been reported in humans [40‐42]. The first case of brucellosis in a pregnant woman was reported in 1908 in a rural area [43]. In Egypt, the seroprevalence of this disease in pregnant women in endemic areas was found to be 12% [44].. In our study, a positive case of Brucella was only reported in the abortion sample of a pregnant woman living in a rural area. This positive sample was classified as B. melitensis, which causes brucellosis in humans and is known to primarily cause abortion and stillbirth [19]. In Saudi Arabia, a study reported a 3.5% prevalence of brucellosis in women living in rural areas. Furthermore, the incidence of abortion in pregnant women with Brucella titers greater than 1:160 was found to be 17.6%, while the incidence of abortion in women with a lower titer was significantly lower at 7.7% [45]. In 1954 in Spain, among 200 pregnant women infected with B. melitensis, 10% of these women experienced abortion in the first trimester (34). In Iran, the first study on human spontaneous abortion conducted in 1974 included 51 women from endemic regions in Isfahan Province who had abortions during their second trimester of pregnancy. Among these patients, six showed clinical and laboratory evidence of brucellosis infection, with five positive cultures of fetal remains and placenta samples [46].

In a study in Turkey, 24.1% of infected pregnant women suffered a miscarriage, whereas only 7.6% of the control group tested positive for brucellosis [47]. In Pakistan in 2016, the seroprevalence of Brucella in pregnant women was found to be 5.8%, with a significant rate of positive cases in rural areas. In addition, seroprevalence was reported to be 14.6% in women who experienced their first abortion, 12.5% in women in contact with infected animals, and 15.8% in women with a history of multiple abortions [20]. In a study in London in 2009, the seroprevalence of C. burnetii and Brucella among 438 pregnant women was found to be 4.6% and 0.5%, respectively [48]. Finally, in another study in Pakistan in 2021, it was discovered that 25.5% of women with a history of abortion were positive for Brucella using molecular methods. The predominant species identified in this study was B. abortus [49].

Since spontaneous miscarriages can be influenced by a multitude of factors beyond infections, such as B. melitensis, it should be noted that in this study and most studies, no adjustment was made for several risk factors that could be considered potential confounders. For instance, hypertensive disorders of pregnancy (HDP), which affect around 5–10% of pregnancies worldwide, can lead to complications such as fetal loss [50]. A study conducted in the Southern United States from 2000 to 2012 found that women with at least one fibroid were more likely to experience a miscarriage compared to those without fibroids. The odds ratio was 4.33 for women aged 35 years and above [51]. In addition, one study found that Polycystic ovary syndrome (PCOS), a common endocrine disorder, was present in 59.8% of women with recurrent miscarriages [52]. Our study was also limited by restricted access to patients’ medical records, a crucial limitation that impacted the comprehensive gathering and analysis of data. Consequently, multiple potential risk factors were not considered during the adjustment process.Therefore, it should be noted that other potential causes of spontaneous miscarriages, make it challenging to contextualize the significance of infection among other known risk factors.