*4.1. Obstetric Mode of Delivery: Vaginal Birth vs. Caesarean Section (C-Section)*

As infants delivered by vaginal birth move through the birth canal, they get colonized by their mother s vaginal microbiota, and thus, adopted a resembling gut microbiome. In contrast, infants born via c-section are colonized by the microbiota of their mother s skin. Therefore, the delivery mode affects the composition of the gut microbiota in infants [121].

Results of various studies showed that in comparison to vaginally born infants, babies delivered by c-section had a decreased gut microbiota diversity including lower levels of *Bifidobacterium* spp. and Bacteroidetes, but increased levels *Clostridium di*ffi*cile* [122] up until the age of two years [123].

Several research groups studied the correlation between c-section delivery and ADHD (see Table 2). An animal study showed a correlation between offspring born via c-section and altered dopamine metabolism throughout development [124]. It is important to note that these results might have been confounded by indication, which means that the altered dopamine response might be due to triggers that lead to a c-section [125]. In contrast to the above findings, two previous case-control studies found no significant correlation between c-sections and ADHD [126,127] in humans. A systematic review by Curran et al. initially showed a slight increase in the prevalence of ADHD in children born via c-section [128]. This correlation was challenged in their later study due to confounders, such as not differentiating between elective and emergency c-sections [129]. The only correlation that still seemed to be consistent was an increased prevalence of ADHD in children born via emergency c-sections. Confirmative data were obtained in a prospective cohort study using 671,592 Danish children. They found a significantly increased chance of children developing ADHD (Hazard Ratio 1.21) for intrapartum c-sections, but no effect when born by an elective c-section [130]. In contrast, the Millenium UK cohort study testing 13,141 children found no correlation between ADHD and mode of delivery despite differentiating between emergency, planned and induced c-sections [131].

**Table 2.** List of seven studies that tested the effects of c-section delivery on the development of ADHD. The table describes if the studies differentiated between the types of c-sections and their effects, and finally shows the sample size and statistical significance level of the individual studies. The symbol represents that for these studies, this information could not be found as the studies were systematic reviews. The data shows that elective vs. emergency c-sections seem to have different effects on ADHD. *p* levels less than 0.05 were considered statistically different.


The reasons for finding a positive correlation between intrapartum c-sections and ADHD development cannot unequivocally be explained as multiple confounders, such as unobserved familial factors, birth weight or gestational age also directly influence the mode of delivery and ADHD. However, there is a strong indication that the microbiota plays a subordinate role in this correlation as Axelsson et al. discovered that exposure of the newborn to ruptured vs. non-ruptured membranes prior to c-section did not influence the correlation between c-section and ADHD development [130].

To conclude, the accumulative data show that the mode of delivery affects the composition of the gut microbiota. However, a clear correlation between c-section delivery and a higher chance of developing ADHD cannot be found as results depend on various confounders and the type of c-section, whereby intrapartum c-sections show a positive correlation with the development of ADHD in comparison to elective c-sections.

#### *4.2. Stress of the Mother*

A prospective follow up study, and a Dutch population-based cohort study concluded a correlation between prenatal maternal stress exposure and an increase in ADHD in their offspring [132,133]. This data was confirmed by a Canadian study enrolling 203 pregnant women exposed to stress. Sixty-two of them were exposed to severe prenatal stress (experienced physical or sexual abuse, or death of a close relative) and delivered children with more severe ADHD symptoms in comparison to the 48 mothers who experienced moderate stress (financial or marital troubles) [134].

An animal study using quantitative PCR determined that maternal stress significantly decreased one of the most abundant taxa in the maternal vaginal flora, *Lactobacillus* spp. [135]. Consequently, *Lactobacillus* spp. was also significantly decreased in the distal colon of the offspring of stress exposed mothers. Additionally, a review focusing on the immunomodulatory effects of *Lactobacillus* spp. shows that stress reduces the abundance of this species independent of the host being pregnant or not [136]. *Lactobacillus* spp. is important for the synthesis of acetylcholine, while together with *Bifidobacteria* spp. it is contributing to the production of the main inhibitory neurotransmitter GABA [137]. Alterations in the GABAergic system have been associated with neurodevelopmental diseases, such as autism spectrum disorder and ADHD. This system is especially susceptible to alterations during development as GABAergic neurons originate from a different part of the neural tube than GABA s most important counterpart, the glutamatergic system. ADHD symptoms may be explained by the hypothesis that inhibitory functions of the cerebral cortex are reduced, leading to a reduction of filtering sensory influences, and finally having difficulties choosing the right behavioral reaction [138].

As described above, several studies have associated low levels of cerebral GABA concentrations with symptoms of ADHD [139–141]. Furthermore, a randomized controlled study showed that *Lactobacillus rhamnosus* also has a preventive effect as the administration of this bacterium in the first six months of life reduced the risk of ADHD and Asperger Syndrome (AS) [142]. The positive effects of this species may be due to the fact that *Lactobacillus rhamnosus* is, on the one hand, implicated in the development of tight junctions responsible for a strong gut barrier, and on the other hand, important for the immunoglobulin A and mucin production [143].

Various factors influence the development of ADHD, among which the neuroinhibitory neurotransmitter GABA seems to play a crucial role. However, to what extent low levels of *Lactobacillus* spp. and decreased concentrations of GABA are associated and how they precisely affect the development of ADHD remains unclear and has to be thoroughly investigated.
