Age of pregnant women and fertility rate in Rwanda
A previous epidemiological survey targeting 7525 pregnant women in 30 districts in Rwanda found that 17.0% of pregnant women were over 35 years old , and in a survey of 2,150 pregnant women in four districts, 18.7% were aged 35 years and over . On the other hand, in a survey targeting pregnant women in the capital Kigali, only 12.5% of the participants were over the age of 35 years . Older pregnant women, including those in their 40 s, were reported particularly in the Mibilizi Hospital zone, which was surveyed in this study. These findings suggest that the average age of pregnant women is higher in rural areas than in urban areas.
In Africa, the total fertility rate of women aged 15–49 years is known to be higher in rural areas than in urban areas . The total fertility rate in Rwanda peaked at 8.46 in 1979 and declined to 3.99 in 2019 , and a national survey in 2015 reported that the total fertility rate was 3.6 in the capital Kigali but 4.6 in the western province where the hospital is located . In rural areas, such as the zone where Mibilizi Hospital is located, many women become pregnant even after the age of 35 years, which is considered to be a factor in the high fertility rate in rural areas.
Relationship between carrier status and birth outcomes
The prevalence of periodontitis increases with age . In this study, the copy number of T. forsythia, which is the causative agent of severe periodontal disease, tended to increase as the age of the pregnant women increased, which is consistent with previous reports. Although the other bacterial species detected in this study did not correlate with the age of the pregnant women, our findings suggest that the bacterial species, whose mass is affected by the aging of the host, may differ depending on the lifestyle, eating habits or tooth brushing habits of the women.
It has been reported that periodontal disease among pregnant women promotes an inflammatory reaction and increases the risk of low birth weight . In this study, we further analyzed the relationship between the amount of bacteria and birth outcomes and tried to identify which bacterial species particularly affected birth outcomes. Regarding the development of the fetus, the copy numbers of total bacteria and T. forsythia and T. denticola were correlated with birth weight in this study. Previous studies have also reported that a large copy number of T. denticola in the oral cavity increases the risk of preterm low birth weight in China , so it is reasonable to assume that T. denticola generally influences fetal development regardless of the country.
Our findings also suggested that there may be weak correlation between the copy number of T. denticola and gestational age even though p value was not under 0.05. It’s possible that the high copy number of T. denticola affect to the reduction in both gestational age and birth weight in this area.
Previous studies have suggested that infections in pregnant women, especially oral, intrauterine, amniotic fluid, urinary tract, vaginal infections, and pneumonia, are associated with preterm birth [21, 22]. In particular, it has been suggested that periodontal bacteria induce preterm birth through increased levels of inflammatory cytokines and PGE2 . However, in reality, many studies have investigated the relationship between the progression of periodontal disease and pregnancy outcomes. In these studies, periodontal status was assessed by measuring the bleeding tendency from the gums, the depth of the periodontal pocket and so on. In addition, some reports stated that there was no correlation between the prevalence of preterm low birth weight and the severity of periodontal disease . In this study, we focused on periodontal bacteria, which are considered to be the cause of periodontal disease and induce pregnancy abnormalities, and tried to clarify the relationship between the amount of bacteria in the oral cavity of pregnant women and pregnancy outcomes in Rwanda. It was reported that the detection rate of T. denticola from the placenta was higher in pregnant women with threatened preterm birth than in normal pregnant women . Although only a weak correlation was found in this study, an increase in the number of T. denticola in the oral cavity may be a risk factor for preterm birth.
The sex ratio at birth was significantly higher in the pregnant women with high copy numbers of P. gingivalis and T. denticola. Previous studies have reported that the sex of the fetus may affect the maternal immune response. For example, when the serum of women who were pregnant with a female fetus was stimulated by lipopolysaccharide, they produced higher interleukin-6 (IL-6) and interleukin-1β (IL-1β) levels than women who were pregnant with a male fetus . There is also a report that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody titers are lower in pregnant women with coronavirus disease 2019 (COVID-19) when the fetus was a male . Thus, if the fetus is a male, the immune response of the mother may be suppressed and may lead to increased cytokine levels and antibody titers. Therefore, the immune response in pregnant women with male fetuses might be suppressed, and it might be reasonable to hypothesize that the growth of periodontal bacteria is promoted in the oral cavity of these women.
Transglutaminase 2 (TG2), a protein cross-linking enzyme, is thought to be associated with the activation of NF-κB, which is a regulator of inflammation. In fact, human periodontal ligament cells (HPDL cells) collected from patients with chronic periodontitis had higher levels of inflammatory cytokines, including IL-6, TNF-α, and HMGB-1, than HDPL cells collected from healthy individuals. In addition, the TG2 mRNA levels were also higher. In other words, TG2 may be involved in the early stages of the inflammatory response that occurs in periodontal disease. Therefore, measuring TG2 levels in the blood is also useful for monitoring inflammation during pregnancy, and in the future, simultaneous observation of the carrying status of periodontal bacteria and blood TG2 levels during pregnancy may clarify the relationship of TG2 with all pregnancy outcomes . It has also been reported that celecoxib is more effective than ibuprofen and placebos in terms of the incidence and severity of postoperative pain caused by the surgical removal of teeth. Pain intensity was assessed by a visual analog scale (VAS) in this study, and the anti-inflammatory effects of celecoxib may also be effective in preventing periodontitis . Further verification is required to see if celecoxib also has a preventive effect against excessive inflammatory reactions in pregnant women.
Oral cleaning habits among pregnant women
Tooth brushing at least twice a day is recommended to maintain oral hygiene . A survey of pregnant women aged 15–43 years in rural Zambia found that 38.5% of the participants brushed their teeth more than once a day . As a result of the current study, we speculate that pregnant women, especially in the Mibilizi Hospital zone, may not brush their teeth sufficiently to maintain oral hygiene. Furthermore, in a survey of adults living in the capital of Kigali, the percentage of participants who brushed their teeth more than once a day was only 27.2% . This suggests that tooth brushing guidance and education may be required in both urban and rural areas of Rwanda. A study has shown that the frequency of tooth brushing decreases during pregnancy ; therefore, oral cleaning guidance for pregnant women is even more important.
The frequency of toothbrush replacement also affects oral hygiene. Worn toothbrushes lose their ability to remove plaque; thus, it is recommended that toothbrushes be replaced when the bristles are worn or every 3 to 4 months of use at least . More than half of the pregnant women in the Mibilizi Hospital zone did not replace their toothbrush for more than a year. Considering that there are few opportunities to replace toothbrushes in rural than in urban areas, brushing teeth at least twice a day is considered to be particularly important in rural areas.
In addition to oral cleaning habits, restricted access to dental clinics is also a cause of deterioration of the oral environment. In a study conducted in Nigeria, only 12.5% of pregnant women had a history of access to a dental clinic , similar to the participants of the current study. There is a report that the presence of untreated dental caries and tartar is higher in rural areas than in urban areas , and the morbidity and severity associated with periodontal disease may also be related to accessibility to dental clinics.
Tooth brushing habits generally tend to deteriorate in the elderly generation [37, 38]. In the subjects in this study, the frequency of toothbrush replacement decreased and the proportion of those who brushed their teeth using inappropriate appliances increased with age in pregnant women (Additional file 1: Table S3); interventions need to be developed with regard to age for pregnant woman.
Effect of oral cleaning habits on carrier status
Among the participants in this study, the copy number of P. gingivalis was significantly higher in the pregnant women who brushed their teeth less frequently. Less frequent brushing has been reported to increase the prevalence of severe periodontal disease  and increase the number of positive bacterial species in the periodontal pocket . These results suggest that poor tooth brushing habits of the pregnant women in the Mibilizi Hospital zone may increase the risk of periodontal disease. In addition, since the copy numbers of P. gingivalis and T. forsythia were high in the pregnant women who infrequently changed their toothbrushes, it is necessary to provide health guidance and material support so that toothbrushes are changed every 3 months of use.
Insufficient tooth brushing habits increase periodontal bacteria and promote oral inflammation. The nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) is one of the constituents of the inflammasome, a protein complex involved in inflammatory response mechanisms. When NLRP3 knockout mice and wild-type mice were exposed to P. gingivalis, alveolar bone resorption was not enhanced in the knockout mice, suggesting that NLRP3 is strongly involved in P. gingivalis-induced bone metabolism and bone resorption, that is, alveolar bone dissolution . Metformin, an oral antidiabetic drug, exhibits anti-inflammatory effects by reducing the activity of inflammasomes containing NLRP3, suggesting that anti-inflammatory drugs may be useful in the prevention and treatment of periodontal disease . In addition, it has been reported that many drugs, such as allopurinol and nicotinamide riboside, inhibit intracellular signaling, and pralnacasan and emricasan inhibit caspase-1, and that many drugs act on various stages of the inflammatory reaction to exhibit anti-inflammatory responses . However, it is not easy to introduce these anti-inflammatory drugs in rural Africa and distribute them in a short period. On the other hand, chlorhexidine is a skin antiseptic that is used in a wide range of applications because it binds strongly to proteins in the skin and mucous membranes, which makes it easier to maintain its antibacterial effect. It is used not only for the skin but also as an ingredient in toothpaste and mouthwash . Various studies have reported the efficacy of chlorhexidine in perinatal care, including the efficacy of vaginal swabs and neonatal skin cleansing with chlorhexidine in Malawi and the efficacy of umbilical cord cleansing in Nepal . In dentistry, it has been shown that the combination of scaling and root planning (SRP), which removes plaque and polishes the tooth surface, with chlorhexidine irrigation improves periodontitis more than SRP alone . In addition, a study suggested that routine chlorhexidine mouthwashes may reduce the risk of premature birth . This study revealed that inadequate tooth brushing habits were associated with an increase in periodontal bacteria in the target population and that high amounts of periodontal bacteria correlated with lower birth weight. Therefore, chlorhexidine is effective in preventing preterm low birth weight, especially in rural Africa, where dental care resources are limited.
Our findings also indicate that it is important to visit a dental clinic to prevent periodontal disease and preterm low birth weight because the copy numbers of all four bacterial species tended to be low in the group of pregnant women who had visited a dental clinic. As shown in Additional file 1: Supplementary figure 1, the number of dentists per population in Rwanda is small compared with other less developed countries or low- and middle-income countries, such as Peru and Nepal. The number of dentists per 100,000 population is less than 10 in many countries, and Rwanda, which has 1.9 dentists per 100,000 population, can be said to have a serious shortage of dentists . If both access to dental clinics in rural areas and the oral cleaning habits of pregnant women are improved, birth outcomes may gradually improve. Furthermore, it is necessary not only to improve the antenatal care visit rate but also to incorporate dental examination as part of antenatal care.
In this study, for the first time, we simultaneously analyzed the relationship between the amount of periodontal bacteria in the oral cavity, tooth brushing habits, and pregnancy outcomes in pregnant women in Africa. Other preterm birth risk factors were also investigated. It was previously revealed that the risk of preterm low birth weight was increased in those with low lipid and vitamin E intake during pregnancy , malaria infection inhibiting angiogenesis and nutrient transport in the placenta , urinary schistosomiasis  and luxury goods, such as tobacco, consumption . Therefore, it is necessary to clarify in more detail the effects of periodontal bacteria on birth outcomes, taking into consideration the lifestyle and socioeconomic factors of local pregnant women and infectious diseases other than periodontal disease.