Fecal Microbiota Transplantation Role in the Treatment of Alzheimer's Disease: A Systematic Review

Alzheimer's, a neurodegenerative disease that starts slowly and worsens progressively, is the leading cause of dementia worldwide. Recent studies have linked the brain with the gut and its microbiota through the microbiota-gut-brain axis, opening the door for gut-modifying agents (e.g., prebiotics and probiotics) to influence our brain's cognitive function. This review aims to identify and summarize the effects of fecal microbiota transplantation (FMT) as a gut-microbiota-modifying agent on the progressive symptoms of Alzheimer's disease (AD). This systematic review is based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines. A systematic search was done using Google Scholar, PubMed, PubMed Central, and ScienceDirect databases in June 2022. The predefined criteria upon which the studies were selected are English language, past 10 years of narrative reviews, observational studies, case reports, and animal studies involving Alzheimer's subjects as no previous meta-analysis or systematic reviews were done on this subject. Later, a quality assessment was done using the available assessment tool based on each study type. The initial search generated 4,302 studies, yielding 13 studies to be included in the final selection: 1 cohort, 2 case reports, 2 animal studies, and 8 narrative reviews. Our results showed that FMT positively affected AD subjects (whether mice or humans). In humans, the FMT effect was measured by the Mini-Mental State Examination (MMSE), showing improvement in Alzheimer's symptoms of mood, memory, and cognition. However, randomized and nonrandomized clinical trials are essential for more conclusive results.


Introduction And Background Alzheimer's disease
Alzheimer's disease (AD) is the most prevalent dementia worldwide, affecting more than 45 million patients without a known cure yet [1]. The pathogenesis of AD starts with the brain accumulating damage over 15-20 years, such as synaptic and mitochondrial alterations, vessel injury, chronic neuroinflammation, cognitive dysfunction, and neuronal cell death affecting multiple systems, after which the clinical symptoms become overt [1]. AD's dominant histological lesions identified in the brain are amyloid-beta (Aβ) extracellular plaques and intracellular neurofibrillary tangles enriched with hyperphosphorylated Tau protein [1,2]. The Aβ-soluble oligomeric form is the most neurotoxic species and the key correlate of the disease gravity [2]. However, multiple anti-Aβ-targeting trials have failed as a route of treating AD; thus, perhaps, the complexity of its pathogenesis requires alternative and multitarget therapies [3].

The microbiota-gut-brain axis
The microbiota-gut-brain axis is an association between the microbiota, the gut, and the brain that synchronizes the gut with the CNS to modify behavior and the brain's immune homeostasis [4]. Microbial metabolites, including short-chain fatty acids (SCFAs), protein, and tryptophan metabolites; the immune system; the vagus nerve; and the enteric nervous system run the bidirectional communication between the gut and the brain (Figure 1) [4].  This association explains the recent studies showing how the gut microbiota plays a vital role in neuropsychiatric [5,6] and neurodegenerative [7,8] disorders by metabolizing the soluble fibers, peptides, and proteins, and their main products are SCFAs. The gut microbiota also metabolizes the dietary proteins, bringing out tryptophan and other metabolites. The SCFAs and tryptophan metabolites are ligands of the aryl hydrocarbon receptor (AHR), and by activating this receptor, a decrease in gut inflammation, in addition to a reduction in the gut and blood-brain barrier (BBB) permeabilities and blockage of the microglia and astrocyte activation, is noticed to trigger the gut and brain homeostasis ( Figure 2) [4]. AHR, aryl hydrocarbon receptor; SCFA, short-chain fatty acid; Trp, tryptophan; BBB, blood-brain barrier Understandably, considerable shifts in human gut microbiota have been spotted in the CNS disorders such as anxiety, autism, and depression [5,6] and neurodegenerative diseases such as Alzheimer's and Parkinson's [7,8]. It has then been thought that the microbiome-modifying agents, e.g., probiotics and prebiotics, will improve some AD symptoms [9].
In this review, another microbiome-modifying method and its effect on AD symptoms and progression will be studied.

Fecal microbiota transplantation
Fecal microbiota transplantation (FMT) is the process of transferring fecal material from a fit donor to a receiver to calibrate the recipient's intestinal composition and, thus, the function of intestinal microbiota [10]. FMT has successfully treated many gastrointestinal disorders [11], mainly used to treat recurrent Clostridium difficile infections [12].
FMT has also been described as lessening intestinal dysbiosis, alleviating physical impairment in Parkinson's disease mice [13], and reducing alcohol-induced anxiety and depression in animal models [14,15].
Therefore, this review aims to focus on FMT's role in AD symptoms and progression, for it is about time we start looking for the potential involvement of the gastrointestinal microbiota in AD pathophysiology. This new linkage may provide worthy targets for new treatment approaches and modalities to delay the onset, slow the progression, or reverse the disease process, eventually reducing the ever-increasing prevalence of AD.

Review Method
This study is based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines [16].

Eligibility Criteria
This review question was formulated based on the participants, intervention, and outcome (PIO) elements: participants, Alzheimer's patients, or AD mice models; intervention, fecal microbiota transplantation; and outcome, any change, whether for the better or the worse, of AD symptoms. Besides, additional inclusion and exclusion criteria were added. Inclusion criteria: English language, free full-text articles published within the past 10 years, human and animal studies, literature reviews, gray literature, randomized controlled trials (RCTs), observational studies, case reports, systematic reviews, and meta-analyses. Exclusion criteria: editorials and papers that were published in languages other than English.

Databases and Search Strategy
The search was conducted systematically using Google Scholar, PubMed, PubMed Central (PMC), and ScienceDirect databases. The last search of all the databases was conducted on June 2022. The key terms used in the search engines were AD, fecal microbiota transplantation, dementia, bacteriotherapy, and the Medical Subject Heading (MeSH) strategy used in PubMed. Details of the databases and search strategies can be found in Table 1.

PMC, PubMed Central
All references were grouped and alphabetized using EndNote, and duplicate removal was done both by EndNote and manually. Then, the records were screened based on the titles and abstracts, where the exclusion of irrelevant studies was implemented. Retrieval of the full-text articles was done. The articles successfully retrieved were assessed according to the appropriate tool for quality appraisal to minimize the risk of bias in this study.

Risk of Bias in Individual Studies
The full articles retrieved were assessed for quality assessment and risk of bias using tools depending on the study type: cohort studies, Newcastle Ottawa Scale (NOS); case reports, Joanna Briggs Institute (JBI) Critical Appraisal Checklist; animal studies, Systematic Review Centre for Laboratory animal Experimentation's (SYRCLE) risk of bias tool; and narrative reviews, Scale for the Assessment of Narrative Review Articles 2 (SANRA 2) [17][18][19][20]. The assessment tools differed in their criteria and passing scores, and a 70% score was required for each assessment tool to be accepted ( Eight items: (1) representativeness of the exposed cohort, (2) selection of the nonexposed cohort, (3) ascertainment of exposure, (4) demonstration that outcome of interest was not present at the start of the study, (5) comparability of cohorts based on the design or analysis,* (6) assessment of outcome, Animal studies Ten items: (1) Was the allocation sequence adequately generated and applied? (2) Were the groups similar at baseline, or were they adjusted for confounders in the analysis? (3) Was the allocation adequately concealed? (4) Were the animals randomly housed during the experiment?

Data Collection, Items, and Analysis
As the topic of this study is still recent, this study is the first systematic review acknowledging this topic as per our knowledge. Furthermore, as we lack previous meta-analyses, systematic reviews, and human clinical trials, the first author has elected to include animal studies and the two published case studies regarding the FMT effect on AD.

Study Selection and Quality Assessment
At the start of the database search, there were 4,302 potentially relevant titles. After duplicates were removed, 3,723 records were retained. In the next step, 3,692 articles were removed while screening the records' titles and abstracts based on this review's PIO elements and eligibility criteria, leaving 31 articles for retrieval. Finally, a quality assessment for reports retrieved was conducted by the first author and reviewed and agreed upon by the second and third authors, yielding the 13 studies that scored more than 70% and were accepted in this study. These were eight narrative reviews, one cohort, two case studies, and two animal studies. A flow diagram showing the screening process and study selection is presented in Figure  3.

FIGURE 3: Flowchart of the study search selection.
The PRISMA 2020 statement [16] PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-Analyses; SANRA 2, Scale for the Assessment of Narrative Review Articles 2; SYRCLE, Systematic Review Centre for Laboratory animal Experimentation Tables 3-6 show how each study was evaluated according to the corresponding study type and the final result of the evaluation process. Table 3 demonstrates the scoring of narrative reviews using the SANRA 2 checklist based on six items.  The JBI tool was used in assessing the two case reports in the review. Both of the studies have scored more than 70% (Table 4).     Finally, Table 6 demonstrates the assessment process of animal studies using the SYRCLE assessment tool.

Human Gut Microbiota and Dysbiosis
The microbes of the human gut exhibit a sizeable interindividual variability that is regularly explained by intrinsic, e.g., genetics, and extrinsic factors, e.g., diet, antibiotics, lifestyle, and illness [21]. As we get older, this microbiota undergoes dynamic changes, as proved by the fact that the diversity and number of gut microbiota decrease noticeably with aging [21,27]. In the study by Ling et al. in a Chinese population, 100 AD patients' samples were collected in addition to 71 age-and gender-matched, cognitively normal controls to investigate the functional and structural alterations of the fecal microbiota by targeting the V3-V4 region of the 16S rRNA gene by MiSeq sequencing and to correlate their results with the clinical features. The data demonstrated a notable reduction in bacterial diversity along with changes in the taxonomic composition of the fecal microbiota of AD patients [27].
Dysbiosis, a phenomenon defined as the atypical changes in the content of gut flora, appears to be directly related to the pathophysiology of AD as it affects several distant organs [21]. For instance, two phyla seem more abundant in healthy individuals than others, Bacteroidetes and Firmicutes, which composite about 90% of the gut microbiota; the rest is composed of Actinobacteria, Proteobacteria, Fusobacteria, and Verrucomicrobia [23].
In patients with AD, studies have shown an increase in the number of Escherichia/Shigella along with a decrease in Eubacterium rectale and Bacteroides fragilis when comparing fecal samples of cognitively impaired patients with healthy controls using real-time quantitative polymerase chain reaction (qPCR) [24]. B. fragilis is an anti-inflammatory bacterium that can enhance the intestinal barrier and repair gut leakiness [24]. This may stand as one of the reasons why intestinal leakage is caused by a change in the gut microbiota along with bacterial components entering the brain. On the other hand, Escherichia, which promotes the body to maintain an inflammatory environment by inducing pro-inflammatory cytokines through an NLRP3-dependent mechanism, is generally upregulated in AD patients [24]. In addition, many bacteria have been reported to be increased in AD patients, such as Prevotellaceae, Enterococcaceae, Ruminococcaceae, Lactobacillaceae, Dorea, Lactobacillus, and Streptococcus.
In contrast, other types of bacteria have shown a decreased abundance such as Negativicutes, Fusobacteriaceae, Eubacterium rectale, Veillonellaceae, Lanchnospiraceae, and Bacteroidaceae [24]. This represents how different types of bacteria have varying effects on the brain and its cognitive function and explains the notion of gut dysbiosis as a potential target as a noninvasive biomarker to distinguish AD patients from the healthy population [27]. With that being said, studies breaking down the good vs. bad microbes are also essential in solidifying our understanding of the development and progression of AD.

Short-Chain Fatty Acids Part in AD and the Role of FMT in Restoring a Healthy Gut Microbiome
Bioactive products produced by bacteria explain how the gut microbiota modulates central physiological and pathological processes. Short-chain fatty acids (SCFAs), which are one of the metabolites coproduced by the host and its gut microbiota, influence host cells through diverse mechanisms and, therefore, are considered essential for host health with a pertinent role in AD prevention employing altering histone acetylation and cell proliferation, and activation of G-protein-coupled receptors [25,26]. SCFAs comprise butyrate, propionate, and acetate manufactured by Clostridium, Eubacterium, and Butyrivibrio bacteria [25]. Studies have shown butyrate to have neuroprotective qualities positively influencing the brain's functioning. It is also considered a significant energy substrate as it increases the mitochondrial production of adenosine triphosphate (ATP). In addition, it enforces BBB integrity while minimizing intestinal permeability and exhibiting an anti-inflammatory effect by inhibiting the immune system from secreting pro-inflammatory cytokines [25]. Sodium butyrate has positively affected AD mouse models, enhancing their learning and memory abilities, even in progressive and advanced stages of the disease [25]. Propionate, on the other hand, protects the BBB by decreasing the impact of pro-inflammatory and oxidative factors. Following their lead, acetate, another common SCFA, reduces the permeability of the BBB. This compound effect decreases the exposure of the CNS to active compounds originating from outside the CNS. In addition, acetate can cross the BBB, inducing a satiety feeling and causing a change in the neurotransmitters' levels ( Figure 4) [25].

FIGURE 4: Types of short-chain fatty acids.
BBB, blood-brain barrier; CNS, central nervous system Thus, as is reasonable and expected, restoration of optimal levels of SCFAs and a healthy gut microbiome have been shown by multiple studies to reduce neurodegenerative pathogenesis. In particular, SCFAs have disrupted the assembly of Aβ oligomers in AD into neurotoxic aggregates by meddling with their proteinprotein interactions with Aβ peptides [28].
Furthermore, a recent study using APPswe/PS1dE9 transgenic (Tg) mouse model revealed that FMT could lessen the brain deposition of Aβ and the phosphorylation of Tau protein along with the levels of Aβ40 and Aβ42 [28]. Besides, cognitive function refinement and synaptic plasticity rise have also been noted in Tg mice [23,28]. Accordingly, the FMT effect on restoring such active mediators may serve as a future therapeutic target for neurodegenerative disorders, particularly AD.

FMT in Animal and Human Studies
FMT has already been used successfully to treat C. difficile infections in humans [12]. However, due to technical and ethical concerns, FMT has not been implemented and used routinely in clinical practice in neurological diseases; it is still restricted in animal models' scientific studies [24]. Nevertheless, these initial studies have shown full of promise, though not irrefutable, results [22]. Table 7 shows two of these animal studies in detail.   [30]. The second case study involved a 90-year-old woman (the recipient) with AD and severe C. difficile infection who received FMT twice from a 27-year-old healthy male. In this case, an improvement in cognitive function, microbiota diversity, and SCFAs production were also observed [31].

Limitations
This review only included studies published in the English language, using four databases from 2012 to 2022. Most of the evidence in this review referred to animal studies due to the lack of clinical trials on human objects, further limiting our findings. In addition, only full free-text articles were acquired, and thus, this may have precluded some of the eligible studies.

Conclusions
In conclusion, FMT can potentially become one of the modalities in treating AD, exerting its effect through the microbiota-gut-brain axis. The diversity of the gut microbiota in AD patients is widely changed compared with the healthy population as the types of bacteria abundant in healthy people differ from those in AD patients. This is speculated to participate in AD pathophysiology because the original gut bacteria metabolize peptides, soluble fibers, and dietary proteins, bringing out SCFAs and tryptophan along with other metabolites. These products work to lessen gut inflammation along with BBB permeability. Animal studies have demonstrated the FMT effect to restore the SCFAs and a healthy microbiome to disrupt the Aβ oligomers, decreasing AD's pathogenesis. However, the lack of clinical trials due to ethical concerns has hindered our findings in humans.
Nevertheless, two case reports were identified in which 82-and 90-year-old patients had received FMT from fit donors and the recipients exhibited cognition improvements. Future recommendations from this review would be to conduct further studies, especially randomized controlled trials, where FMT is implemented in patients of AD at various stages of the disease, following them up for longer durations. Thus, a further detailed assessment of its effect as a possible AD intervention could be evaluated.

Conflicts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.