The Genome and Microbiome Implications for Endometriosis’s Etiology
Endometriosis is a chronic illness that affects one in ten[1] women. Its medical definition is when endometrial tissue grows somewhere other than the uterus; endometrial tissue is the tissue that grows on the uterus that is shed during the monthly menstrual cycle. If it grows elsewhere, that person will experience a menstrual period inside their body, where the blood has nowhere to go, and instead creates inflammation. Although the disease impacts roughly 400 million people worldwide, our knowledge of the disease and its treatment is extremely limited beyond this definition.
It takes a median of 7.0 years[2] for someone to get diagnosed with endometriosis. This is because we can only identify the disease via a laparoscopic surgery (allowing the surgeon to see the abdominal region and note if endometrial tissue is present). Since endometriosis is difficult to identify, it is also difficult to study. Therefore, cheaper, quicker and more precise diagnostic tools could support thousands of people annually and help further the body of endometriosis science. To find new diagnostic tools, we must update our 100-year-old theories that still govern our hypotheses around the disease. We can do this through modern quantitative biology techniques in genetics and microbiome testing. Genetics is the study of our genes and the microbiome is the collection of microorganisms, like bacteria, in the body. Both of these topics have been traditionally omitted from our understanding of endometriosis, yet they likely affect the progression of the disease. Thus, this paper will explore how the genome and microbiome affect the formation of endometriosis and how this information can improve future diagnostic tests. To answer this question, I will first outline the definitions pertaining to endometriosis, theories on why it happens and how we diagnose it. Then, I will explore the genetic and microbiome research related to endometriosis. Finally, I will conclude the implications of genetics and metabolomics for informing future diagnostic methods.
The Basis of the Disease: Definition and Theories
A person with endometriosis has endometrial tissue growth outside of their uterus. “Endometrial tissue” is the blood-rich tissue that lines the uterus monthly and sheds during the menstrual period. The change in hormones (mainly progesterone and estrogen) during the menstrual cycle triggers the building and shedding of endometrial tissue. When endometrial tissue grows elsewhere, such as the ovaries, fallopian tubes and pelvic tissues, the tissue still responds to hormonal changes in the body. However, unlike the uterus, these areas do not have a place for the shed blood to exit. Therefore, people with endometriosis have internal menstrual periods, resulting in extreme inflammation within the body. Correspondingly, the top four symptoms of endometriosis are dysmenorrhea (severe, painful and frequent menstrual cramps), chronic pelvic pain, deep dyspareunia (pain with deep vaginal penetration) and infertility[3]. Thus, endometriosis penetrates the lives of individuals vastly: inflicting physical pain, affecting sexual activity and fertility.
The origins of endometriosis (also known as its “etiology”) in the body are unclear. There have, however, been some theories proposed. One of these theories is retrograde menstruation: the backwards flow of menstrual blood through the body. Rather than exiting the uterus as shed endometrial tissue normally does, the theory suggests that some blood may flow backwards, entering the fallopian tubes where some endometrial cells are trapped there and re-grow. However, this leading theory has logical inconsistencies. During a laparoscopic study 70–90%[4] of menstruating patients experienced retrograde menstruation while only 10%[1] of menstruating people have endometriosis. The discrepancies in these statistics suggest that endometriosis is far more complex than explained by retrograde menstruation. Although it is from 1927, Retrograde menstruation is the most widely accepted theory for endometriosis[5]. The fact that we are using a one-hundred-year-old theory to explain a chronic illness illustrates how little progress we have made on our understanding of endometriosis.
Nonetheless, there have been other less widespread speculations. An alternative theory suggests that endometriosis originates from extrauterine cells (cells outside the uterus) that abnormally transform into endometrial cells6. Moreover, because endometrial tissue growth happens in harmony with the rise and fall of estrogen and progesterone, hormones are clearly a fundamental part of the disease. Therefore, increased exposure to estrogen and progesterone could influence the onset of endometriosis. A Scientific American article argued that environmental estrogen could influence endometrial growth outside the uterus[7]. As discussed in this section, the primary ideas around endometriosis forming are hormonal or menstrual blood related; this leaves an opportunity for genetic and microbiome exploration, among other angles of biology.
The Bottleneck of Diagnosis
The vague explanations for endometriosis’s etiology contribute to our limited and inefficient diagnostic methods. It is a chicken-and-egg scenario: if our understanding is limited, our diagnostics will reflect a limited understanding. But without concrete diagnostics, we cannot adequately compare people with endometriosis to people without to further our understanding. Hence, the questions of why endometriosis happens and how we can diagnose it are intertwined.
Up until recently, diagnosing endometriosis was only possible through a laparoscopic surgery. This is a minimally invasive surgery where a doctor can view the abdominal region with a small incision. A doctor can see if endometrial tissue is growing in the pelvic region beyond the uterus through this procedure. If there is abnormally placed endometrial tissue, it fits our definition of endometriosis. The merits of this method focus on analyzing the presence of endometrial tissue. Naturally, because our theories merely focus on hormones, endometrial cells and menstruation, that is the focus of our diagnostic test. However, recently doctors have tried using imaging tools like ultrasounds and MRIs to diagnose endometriosis[8]. But they often lack high enough resolution to be useful, and none have meaningfully contributed to updating our theories. This chasm in diagnostics is where lab tests could come in: what if genomics, blood tests, or biopsies could be diagnostic tools for endometriosis?
The Genetic and Microbiome Introduction to Endometriosis
An Oxford University study stated that endometriosis has 50%heritability. Hence, the etiology of endometriosis is partially genetic[9]. Identifying the genes that correspond to endometriosis does two things. One, we can test people’s genomes for their risk of endometriosis. Two, knowing which genes correlate with endometriosis can give us clues on the mechanisms behind its etiology. “Genomics” is the branch of biology that studies the sequence of genes. With a “genomics” approach, we can more thoroughly decode endometriosis.
On the surface, endometriosis seems like a reproductive illness since it affects the reproductive organs of females. However, although it originates with endometrial tissue, which has a fundamentally reproductive function, the disease itself is more likely to be related to the immune system. One key aspect of the immune system is the microbiome which is the collection of non-human cells in your body. This paper will also explore recent advances in microbial studies on endometriosis patients as there may be the potential for microbiome-related diagnostic tests.
Genetic Targets for Endometriosis
In August 2021, researchers from Oxford9 found evidence for neuropeptide S receptor 1 (NPSR1) as a target gene for future non-hormonal therapies through genetic analyses of endometriosis in rhesus macaques and humans. They found NPSR1 expressed in endometrial tissues and within immune cells, implying a connection between endometriosis and the immune system. Previous studies identified areas of the chromosome that are associated with endometriosis risk. For example, Sapkota et al[10]. identified five new loci (physical regions on the chromosome) that are correlated with endometriosis. However, these genes were all related to sex steroid hormone pathways or blood pathways. These genetic results suggest that endometriosis is closely linked to the immune system and hormone function. Burns et al further confirm this in their study analyzing the early onset of endometriosis. They identified two stages in the early onset of endometriosis: first, an immune phase and then, a hormone phase. In their conclusion, they argue that their “findings strongly support the need for detailed studies that focus on the innate immune system.”[11] Overall, genetic research supports that endometriosis should be analyzed from the hormonal and the understudied immune system perspective.
The genetic connection between the immune system and endometriosis is particularly interesting because it is a new way of understanding the disease. For decades, retrograde menstruation has been the leading theory, and it fails to recognize a potential role for the immune system. The discovery of NPSR1 cements the modern idea of recognizing endometriosis as an immune and reproductive system disease. In addition, there is more evidence than this one promising immunoregulatory gene to support this claim: a study found that 396 out of 579 immune/inflammation genes varied significantly between endometriosis tissue and controls[12]. Another paper analyzed cytokines (proteins in charge of growth and regulation of immune cells/blood cells) to uncover more evidence supporting a difference in immune responses between endometriosis patients and the control group[13]. They found increased expression of pro-inflammatory type 1 cytokines which, as the name suggests, promote inflammation. Notably, this study was the first to identify the increased expression of TSG-6 (tumor necrosis factor-stimulated gene 6), which could connect to female infertility, one of the main endometriosis symptoms[13]. The discovery of TSG-6 expression shows another example linking endometriosis, the reproductive system and the immune system.
A literature review on endometriosis biomarkers created a “meta-signature” with 57 genes that they suggest as future biomarkers for the illness[14]. The review highlights five innate (first line of defense) immune system genes: C1R, SERPING1, CD55, C4BPA and CFD. All these genes have an immune role; for example, CD55 and C4BPA have an immune role in protecting the embryo, and both were found to be present at lower levels in people with endometriosis. The aforementioned genes illustrate the immune discrepancies between endometriosis patients and controls and could be used as biomarkers for non-invasive diagnostic tests. Additionally, these results emphasize the need to study the mechanisms behind the immune system’s connection to endometriosis. Additionally, since this study had a large sample size, we can be confident in its results. Since multiple studies found that immunoregulatory genes correlated with endometriosis, we can confidently hypothesize a strong connection to the immune system and the disease. However, more studies will be needed to confirm the specific genes.
Another genetic perspective that could further our knowledge of endometriosis is studying the genetic overlap between diseases similar in nature or similar in their region. For example, the genetic overlaps between endometrial cancer and endometriosis are insightful: both diseases involve the endometrium, and both involve foreign cell invaders. A 2018 study took a genetic approach to investigate the etiology of both illnesses and they found 13 loci associated with both diseases[15]. The most significant of the genetic factors was the PTPRD gene. It is part of the protein tyrosine phosphatase (PTP) family which are signaling molecules essential for cellular processes such as differentiation (one cell transforming into another cell)[16]. The genetic relation to cellular differentiation is relevant because an alternative endometriosis theory suggests that it originates from extrauterine cells (cells outside the uterus) that abnormally differentiate into endometrial cells.
Overall, the genomic research done on endometriosis is a useful start in identifying its link to the immune system, infertility and related diseases. However, there is a multitude of genes and combinations of genes that correlate with endometriosis so a simple gene test will not be an adequate diagnosis. We can use these genes as clues towards the mechanisms behind the disease. For instance, the genes in this review suggest that there is a connection between pro-inflammatory cytokines and endometriosis. This induces the question: is there a concentration or quantitative number of cytokines that could indicate diagnosis? If there was a distinct connection, endometriosis diagnosis could start with flow cytometry, a technique that uses bodily fluid (like blood from a blood test) to rapidly test individual cells. This test could identify cytokines within the body, and it could assess their relation to endometriosis. Ultimately, studying the genomics of endometriosis is a means towards advancing diagnostic testing and advancing our fundamental knowledge of why it happens.
Endobiota: How Microbes Differ in Endometriosis Patients
The microbiome is the collection of non-human cells in our bodies that are crucial to our survival. There are multiple microbiome regions within the human body, each with very different microorganisms and conditions[18]. The most well-known region is the gut microbiome: the encompassing organisms that aid in digestion. However, other microbial regions are the mouth, the skin, urogenital regions, eyes and lungs. Just like microorganisms in the gut help our digestive system perform its function of digesting food, microorganisms in other parts of the body help those systems function smoothly. Therefore, it is logical to assume that the microorganisms in the female reproductive tract play a role in maintaining healthy, regular periods or fertility; since endometriosis is a type of irregular menstrual period, it could be the case that an irregular microbiome is a cause of this malfunction.
A paper published in 2020 established the connection between the microbiome and the immune system: “The microbiome plays critical roles in the training and development of major components of the host’s innate and adaptive immune system, while the immune system orchestrates the maintenance of key features of host-microbe symbiosis.”[19] Earlier in this review, I discussed the immunoregulatory gene NPSR1 and its relationship between endometriosis and the immune system. Therefore, because the immune system relates to both endometriosis and the microbiome separately, there must also be a relationship between the microbiome and endometriosis.
There is, indeed, literature to support a divergence in microbiome data between endometriosis patients and non-endometriosis control groups. For instance, a 2019 research paper published in nature from the University of Koç in Turkey compared stool (gut), vaginal and cervical microbiome samples of 14 endometriosis patients and 14 control patients[20]. In their introduction they summarized that there is an association between dysbiosis (malfunctioned microbiome) and IBS, psoriasis, arthritis among other immune related diseases implying that the microbiome relates to the immune system’s inflammatory responses. Their study analyzed the diversity and genera of microbes to conclude that although diversity between the endometriosis group and control group were similar, there are differences at the genus level on the types of microbiota present. Both the control and endometriosis groups had the same dominant genus, Lactobacillus; however, the samples diverged when looking at the non-dominant species. For example, Gardnerella was a higher proportion of the leftover microbiota when the majority (Lactobacillus) was removed (vaginal: 72.9% endometriosis vs 36.8% control, cervical: 67.7% vs 36.8%). Furthermore, the paper reports the absence of typical microorganisms in their samples, such as the complete absence of Gemella and Atopobium in the vaginal microbiome. A balanced microbiome contributes to typical bodily function; hence, these results show us that endometriosis patients do not have the typical microbial makeup of a healthy person.
Additionally, a 2018 paper focused on endometriosis’s effects on the gut microbiome in mice replicated these results: while the diversity among endometriosis and non-endometriosis microbiomes is similar, the types of microbes differed[21]. Although these results point to an interesting conclusion that microbes are different in endometriosis patients and, therefore, are a contributing factor in the disease, it still questions whether the microbial differences are a symptom of the disease or a cause of the disease. We must confirm how directly microbiota impact endometriosis. Seeing as most of these studies focus on the quantitative contrasts in diversity and genera within the microbiome of endometriosis patients it is unclear as to why these differences occur. Thus, we cannot draw an exact conclusion to the microbial influence on the disease, only that there is a statistically significant difference among patients indicating that it is a useful path of research to explore. Furthermore, the study sizes were too small and not diverse enough to conclude the widespread implications on the disease.
The microbiome studies mentioned earlier found that endometriosis patients had a similar number of microbial species (i.e., diversity) than controls, but that the types of microbes differed; there was a lack of some families of microbes and a big increase in others. However, a more recent 2021 paper found that diversity in the endometrial microbiome does vary[22]. In their analysis, endometriosis patients had more diversity in their endometrial microbiome.
Whilst these three papers conclude slightly different results, they have one idea in common: based on their experiments, endometriosis patients have differences in their microbiomes compared to the study controls. We still need to conclude if endometriosis patients have more diverse microbiomes and if they have varying compositions of microbiota.
From these results, microbes differ among patients and control groups, and therefore could be a vital part of endometriosis. These results imply that the microbiome could be used to test for endometriosis by identifying the dominant genera in endometriosis patients. Or, there is the opportunity to use microbial supplements to rebalance the endometrial microbiome. Replicating the results from these studies to confirm trends in endometrial microbes could confirm future treatments. For example, Gardnerella is found in excess in endometriosis patients; we could find microbiome treatments that target reducing Gardnerella in endometriosis patients to “rebalance” the microbiome.
The contents of this paper indicate that endometriosis has multiple influences, thus, it is unlikely that there is one easy microbiome fix. The implication from this research is that it is one piece of the puzzle and bringing the microbiome up to the typical contents of a non-endometriosis patient is a step in the right direction pertaining to a healthy, disease-free body.
Finally, we should ponder how the microbiome leads to this change. Is it environmental? Is it hormonal? Is it immune-related? Likely, it is a combination of many things. If there is one thing the study of endometriosis differences, like the microbiome, have emphasized it is that multiple systems are interrelated to the disease: it is impossible to adequately study endometriosis from a single angle.
Conclusion
Endometriosis impacts millions of people, and although our understanding is limited right now, genetic and microbial research indicate that there is potential to transform our scientific approach around endometriosis. The literature reviewed in this study highlighted promising genes and microbes at could be used for future diagnostic tests or treatments. Specifically, NPSR1 is the immunoregulatory gene with the most identified clinical potential. Moreover, some microbial genera were found to be absent or unconventionally abundant in endometriosis patients, suggesting opportunities to rebalance the endometrial microbiome.
Most importantly, this literature review highlighted the importance of studying this complex disease from interdisciplinary angles. Conventionally, endometriosis was classified as a reproductive or infertility disorder, and thus, research analyzed it from that narrow perspective. Hence, theories focus on menstrual periods or hormones, the two most associated factors with female reproduction. From the genetic and microbiome research reviewed in this study, we revealed that there are non-reproductive factors of the disease. Genetics, the immune system and the microbiome are not limited to the procreation functions of the body and studies have proven their significant links to endometriosis.
On the surface, it might seem that genetic and microbial research open up a new can of worms related to endometriosis, providing more questions about the disease than answers. But more so, they highlight the complexity of the body, and prove the inevitable truth that diseases are multifaceted, and our approach to curing them should mimic their multipronged nature. If we analyze the illness from an immunology, genetic and microbial perspective, we can break away from conventional theories grounded in reproductive health. These techniques could become tools for 21st-century diagnostics through genetic or microbiome testing. Also, these tests could be tools that offer new tests: analyzing genetic results might point towards common enzymes associated with endometriosis, and enzymic testing could become a tool for earlier diagnostics. Finally, using these perspectives for studying the disease could reveal more truth to where it comes from, why it happens, and by extension: how to solve this medical mystery.
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