The Human Microbiome and Gender Medicine

Abstract

Discoveries in molecular genetics over the last two decades have broadened our information about the genomics of complex microbial communities. As in all other fields of medicine, there is an undeniable need to explore the microbiome and the way it is impacted by biological sex. A number, although small, of recent studies have demonstrated that women and men have striking differences in the species that constitute their microbiomes. This effects pathological physiology in fields such as hepatology, oncology, autoimmune disease (most notably diabetes mellitus), autism, and obstetrics. There is still an unfortunate lack of research being done on the “microgenderome”: the interaction between microbiota, sex hormones, and the immune system. This review will highlight some of the main areas to be affected by microgenderome physiology, with an in depth focus on obstetrics.

In 2001, Hooper coined the term ‘microbiome’ which refers to the collection of genes of the microbes in a community ¹ “The Human Microbiome Project,” sponsored in 2008 by the NIH Common Fund, was established to characterize the colonies of microbes that inhabit human nasal passages, oral cavities, skin, the gastrointestinal tract, and the urogenital compartment. ² In 2011, Aquirre de Carcer et al authored one of the first papers to explore the gender-specific characteristics of the microbiome. ³ Flak introduced the term microgenderome’ in 2013: this term refers to the interaction between microbiota, sex hormones and the immune system. ⁴ Current literature supports scientific evidence that gut microbiota differ significantly between men and women. ⁵ Some of these are the consequences of hormones: Twin studies have demonstrated that the once similar microbial composition of opposite-sex twins becomes different after puberty when compared to same-sex twins that remain compositionally similar. ^6,7

The portal system has been studied to detect differences in microbiota. In particular, bile acids (BAs), which aid in digestion and absorption of fats and fat-soluble vitamins in the small intestine, have been found to show sex-specific differences. Bile acids are critical for the production of waste products, including bilirubin, which are eliminated from the body by secretion into bile and elimination in feces. ⁸

Another study demonstrated a causative relationship between sex-specific gut microbiota and malignancy. This study provided a link between microbiota and hepatocellular carcinogenesis: Male mice with increased intrahepatic retention of hydrophobic BAs had decreased hepatic expression of tumor-suppressive microRNAs and as a result a higher incidence of hepatocellular carcinoma (HCC). ⁹ A metagenomic analysis showed differences in gut microbiota involved in BA metabolism between male and female mice, and such differences were amplified when mice of both sexes were exposed to Streptozotocin-High-Fat Diet (STZ-HFD) model. The study proved that treating STZ-HFD male mice with 2% cholestyramine led to significant improvement in hepatic BA retention, tumor-suppressive microRNA expressions, microbial gut communities, and prevention of HCC. ⁹ This study may indicate possible new approaches for preventing and controlling human obesity-related gastrointestinal cancers. These cancers are characterized by sex differences that are found in gut microbiota. ⁹ Xie et al provide evidence that pointed to an association between sex and gut microbiota, BAs, and gastrointestinal cancers.

There are sex-specific and host-regulated circadian rhythms that govern microbiome function. One study demonstrated that the absolute amount of fecal bacteria and the abundance of Bacteroidetes exhibited circadian rhythmicity, which was more pronounced in female mice. ¹⁰

Another prevalent issue that is influenced by microgenderome is obesity. In a 2016 study, 16S sequencing in 39 men and 36 postmenopausal women with similar dietary backgrounds, matched by age, were stratified according to body mass index (BMI). The results portrayed an abundance of Bacteroides genus in females with BMI >33, while the amount of Bacteroides was decreased in men with increased BMI. Another finding was a higher number of Veillonella and Methanobrevibacter genera in fecal samples in men compared to women (84.6% vs 47.2%; P = .002, Q = 0.026), and an abundance of Bilophila was lower in men compared to women; these findings were independent of BMI. This study illustrated that the divergence in gut microbiota observed between men and women might have a dominant role in the definition of biological differences in the prevalence of metabolic and intestinal inflammatory diseases. ⁵

Sex differences in the gut microbiome may drive hormone-dependent regulation of autoimmunity. Historically, males are generally more prone to infections and women are known to suffer more autoimmune diseases. ¹¹ This is due to hormonal differences, since sex steroids can influence the function of immune cells, as well as genetic differences, since the X chromosome is known to harbor more genes involved in immunity. ^12,13 Recently, this has been further tested using nonobese diabetic (NOD) mice; Markle et al successfully proved that sex-biased autoimmunity depends on the microbiome. This 2013 study investigated early-life microbial exposures to determine sex hormone levels and autoimmunity in the NOD mouse model of type 1 diabetes. Markle et al observed that colonization by commensal microbes increased serum testosterone and protected NOD male mice from type 1 diabetes. Transferring gut microbiota from adult males to immature females altered the recipient’s microbiota, resulting in elevated testosterone, reduced islet inflammation, autoantibody production, and type 1 diabetes protection. ⁴ In a similar study, germ-free NOD mice were divided by gender. ¹⁴ Females had a 1.3 to 4.4 times higher incidence of type 1 diabetes. Furthermore, the differences of microbiota trends in gut microbiota of males and females were reversed by male castration, confirming that androgens influence gut microbiota, which in turn predicts susceptibility to autoimmune disease.

Differences in the impact of gut microbiota on the brain have been hypothesized to be involved in the pathogenesis of autism spectrum disorders (ASDs). The theory of autism-associated microbiota–gut–brain axis alteration states that microbial dysbiosis plays a role in the development or presentation of ASD symptoms. ¹⁵ Coretti et al in 2017 demonstrated sex-specific alteration of gut microbiota in mice showed sex-associated differences that supported the theory of autism-associated microbiota–gut–brain axis alteration. ¹⁶ The inbred BTBR T+tf/J (BTBR) mouse strain has been used as an animal model of core behavioral deficits in autism. The findings indicate that alteration of gut microbiota in BTBR inbred mice shows relevant sex-associated differences and supports the use of BTBR mouse model to dissect autism-associated microbiota–gut–brain axis alteration. The observation of high comorbidity between intestinal inflammatory diseases and psychiatric symptoms such as anxiety and stress and the frequent occurrence of gastrointestinal dysfunctions in autistic patients strongly support the hypothesis of a role for gut microbiota in psychiatric conditions including ASD. Although evidence of a deviation of gut microbiota composition in patients with ASD with respect to normally developing individuals has been reported, to date no clear-cut conclusion was reached on the association of specific bacterial profiles with ASD [Note 1]. ¹⁷

Fetal–Maternal Medicine

Molecular microbial interactions at the interface between vaginal epithelia and resident microflora emerge as a “new frontier” in the study of invasive and noninvasive pathologies. ¹⁸ The sterility of the fetal environment and the possibility of in utero microbiome transfer have been debated for almost 150 years and have been referred to as the sterile womb paradigm, microbes are acquired both vertically (from the mother) and horizontally (from other humans or the environment) during and after birth. ¹⁹ Nevertheless, there is now a multitude of recent studies using modern sequencing technologies that have challenged the traditional view of human microbiome acquisition. ²⁰ These studies propose that neither the fetus, the placenta, nor the amniotic fluid is sterile and that acquisition and colonization of the human gastrointestinal tract begins in utero. ²¹ If this “in utero colonization hypothesis” proves correct, there would be major repercussions on our understanding of the establishment of the pioneer human microbiome, its role in human health, and the role of environmental, lifestyle, and clinical factors that affect its assembly and function. This concept would also have significant implications on how we view the fundamental aspects of host–microbial symbiosis in humans as well as clinical practices such as cesarean sections, which are currently thought to disrupt transmission of microbes. ^21,22 The relatively nonsterile neonate rapidly harvests microorganisms from the environment, and the newborn microbiota are inoculated by his mother during and after delivery. ²³ Dominguez-Bello et al reported that the type of delivery (vaginal or cesarean) impacts newborn’s microbiota and development. ²⁴ The study carried out a comparison of the microbiota of 4 vaginal and 6 cesarean born babies in Venezuela and revealed that cesarean born neonates acquired microbiota closely resembling their mother’s skin microbiota, while vaginally born neonates acquired microbiota that resembled their mother’s vaginal microbiota. ²⁵

The vaginal microbiome plays an integral role in a woman’s reproductive health and is typically characterized predominately by several Lactobacillus species, which are best understood for their ability to produce lactic acid from glycogen released by the vaginal epithelium. ²⁶ Ravel et al focused on the vaginal microbiota of asymptomatic reproductive-age North American women of different ethnicities. The most common species were Lactobacillus: L iners, L crispatus, L gasseri, and L jensenii. ²⁵ The observation that anaerobic organisms, rather than Lactobacillus, dominated in one of Ravel et al’s 5 groups of bacterial clusters indicates that Lactobacillus is not a staple of healthy vaginal flora. Ravel concluded that the detection of bacterial genera that have been associated with abnormal vaginal microbiota, such as Prevotella, Atopobium, Gardnerella, Megasphaera, and Mobiluncus, in asymptomatic reproductive-age women challenge the traditional distinction between healthy and abnormal flora. Additionally, groups of bacterial clusters were found in different ethnic groups. Asian participants had a higher prevalence of CST III (L iners), non-Hispanic Caucasians had a higher prevalence of CST I (L crispatus), while African-American and Hispanic participants had a higher prevalence of CST III (L iners) and IV (decreased Lactobacillus species). ²⁷ In pregnancy, overall diversity of the vaginal microbiome tends to decrease until in the third trimester when Lactobacillus species tends to become the dominant member. ²³

Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. Although the etiology is not fully understood, intrauterine infection may account for 25% to 40% of preterm deliveries. ^{28 -30} Understanding the microbiology of the female urogenital tract, and the role that the microbiome might play in preterm deliveries, is crucial to advance our understanding of the relationship between the microbiome and the “great obstetrical syndromes. ^26,23,31 ” This term was coined over 20 years ago by Roberto Romero and refers to common obstetrical pathologies about whose etiologies we know very little. Examples of frequent pathologies include preeclampsia, premature labor, placental abruption, premature rupture of membranes, and fetal growth restriction. The only fact we can attribute to the etiology for many of these pathologies are that they result from adaptive responses of the fetal–maternal unit to pathological insults. Today, there are efforts to explore the gut, cervical–vaginal, placental, and oral microbiota in the further search of etiologies of preterm birth. To date, however, there are not enough data to link a specific microbiome community or microorganism with preterm birth. ²⁶ In the past, polymerase chain reaction (PCR) has been used to investigate uteroinvasive bacteria and revealed ureaplasma cultures positively associated with preterm birth. ³² Mysorekar and Cao found that Ureaplasma and Gardnerella vaginalis were the most commonly detected organisms in chorioamniotic membranes, whereas Mycoplasma were most commonly detected in amniotic fluid. Additional bacterial species associated with amniotic fluids include Leptotrichia, Sneathia, and Bergeyella. ^30,33 Digiulio et al found that less Lactobacillus and more high-diversity vaginal microbes were correlated with preterm birth. This finding further implicated the commonly bacterial vaginosis-associated taxa was actually due to Ureaplasma and Gardnerella as causative agents. ^26,33 Although Lactobacillus species have been known to be a predominant colonizer of the vaginal microbiome, not all Lactobacilli influence host biology in the same way. ²⁶

To challenge the notion that delivery is the first exposure to microbiota, recent publications have suggested that the placenta is the earliest source for microbiota colonization in the fetus. The placenta appears to harbor a low biomass microbiome that is presumptively established in early pregnancy and varies in association with a remote history of maternal antenatal infection and preterm birth. ³⁴ The placenta has long been considered sterile in normal gestation. However, the presence of bacteria in placental membrane (in the absence of histological infection) has been discovered repeatedly over the last few decades and is associated with preterm birth. ³⁴ A strong case for the importance of the placental microbiota is that in the first week of life, the full-term neonatal gut microbiome is largely colonized by the phyla Actinobacteria (including Bifidobacterium), Proteobacteria, Bacteroides, and, much less, Firmicutes (including the Lactobacillus spp., which dominate the vaginal flora). ³⁵ In contrast, neonates who weigh <1200 g are found to be colonized by both Firmicutes and Tenericutes phyla, with much less dominance of Actinobacteria. ³⁶ These collective observations raise the possibility that the infant may be first seeded in utero by a commonly shared source with a relatively sparse microbiome, such as the placenta, and that this seeding may vary by length of gestation. ³⁷ Aagaard et al studied placental specimens collected under sterile conditions from 320 participants and analyzed (16S ribosomal DNA–based and whole-genome shotgun metagenomic studies) and compared to other human body sites from nonpregnant controls. The study described a placental microbiome, composed of nonpathogenic commensal microbiota from the Firmicutes, Tenericutes, Proteobacteria, Bacteroidetes, and Fusobacteria phyla. A noteworthy finding from this study was that the placental microbiome profiles resembled the human oral microbiome. 16S-based operational taxonomic unit analyses revealed associations of the placental microbiome with a remote history of antenatal infection such as urinary tract infection in the first trimester, as well as with preterm birth <37 weeks. ³⁷ In addition, characterization of the vaginal microbiome of pregnant women revealed a substantial reduction in taxonomic diversity as pregnancy advances. Among Caucasian women, a greater diversity of the vaginal microbiome was found for those with term compared with preterm births. ^25,26 Lauder et al demonstrated that the bacterial DNA observed by Aagaard et al was due to contamination. Lauder et al quantified total 16S ribosomal RNA (rRNA) gene copies using quantitative PCR and found that placental samples and negative controls contained low and indistinguishable copy numbers. The study results demonstrated 16S rRNA gene sequencing and community analysis and found no difference between communities from placental samples and contamination controls. ³⁸

Fetal microbiomal programming has been of great interest and refers to a stimulus or insult at a critical, sensitive period of fetal life which has permanent effects on structure, physiology, and metabolism. The characterization of urogenital microbiota can shed light on etiologies of obstetrical syndromes and fetal development that lead to pathologies in adulthood. A recent study showed that stress during pregnancy altered the temporal and spatial dynamics of maternal and offspring microbiome in mice in a sex-specific manner. The hypothesis of this study was that maternal stress disrupts gut and vaginal microbial dynamics during critical prenatal and postnatal windows. The microbiome changes naturally during pregnancy and, more importantly, is disrupted by stress. ³⁹ A comparison of maternal and offspring microbiota revealed that similarities in bacterial community composition could be attributed to a complex interaction between maternal body part and offspring age and sex. It was concluded that early prenatal stress influenced the bacterial community of offspring in a sex-specific manner. This compelling study underlines the need to expand this frontier of fetal–maternal medicine research and its connection with the human microbiome. ^29,28 Kozyrskyj discovered correlations between lower infant fecal immunoglobulin A (IgA) levels and greater maternal distress, and between lower infant fecal IgA levels and more frequent Clostridium difficile colonization of the infant stool, a marker for gut dysbiosis. ⁴⁰ Maria Jenmalm’s group in Sweden has published on the binding of gut microbes to fecal IgA and future risk for allergic disease. ⁴¹

Although new research in the field of vaginal and cervical microbiota is emerging, an equally important field that needs to be investigated more is the vaginal virome. Not many studies have been done on the vaginal virome, but one worth noting is by Wylie et al, who performed a nested case–control study within a prospective longitudinal cohort of pregnant women receiving prenatal care. Data were collected using vaginal swabs obtained by speculum examination in each trimester. For the analysis of viruses, DNA sequencing libraries were constructed using the Swift Biosciences Accel NGS kit of Ann Arbor, Michigan. Although many viruses were detected, no specific virus or group of viruses (eg, papillomaviruses, high-risk papillomaviruses, herpesviruses) was associated with preterm delivery. However, when categorized by race, the African American subgroup had a higher viral richness that was statistically significantly associated with preterm delivery. The study concluded that the first trimester appears to have the highest magnitude of difference in viral diversity between term and preterm birth patients. Therefore, examinations during the first trimester could be clinically useful to identify women at risk or not at risk for preterm birth. ⁴² Vaginal virome studies are scant and there is a need to further investigate its properties during pregnancy because first trimester is a critical period for fetal development.

The microbiome has a great impact in the cause and treatment of vaginal infections. Lamont et al illustrated that the population of vaginal flora is not constant but changes as a response to many factors. ⁴³ They confirmed the importance of Lactobacillus and other acid-producing bacteria in promoting an optimal vaginal pH and in combating bacterial vaginosis by the production of hydrogen peroxide. The implications of this study are that by using molecular-based techniques to diagnose vaginal flora, treatments for persistent infections may improve in the future.

Conclusion

The future of medicine lies in gender medicine. Pharmacology, oncology, cardiovascular medicine, and many other fields have shifted their approach toward treating patients by sex. The microbiome is a new terrain for scientists and clinicians. To quote the leading pioneer in this field, Dr M. Legato, “if we can decode and understand the contribution to our individual lives of this huge cache of extra genes, we will reap a whole world of new information that will help us conquer stubborn, debilitating, and even fatal illnesses” ¹ (pp. 339–340). A number, although small, of recent studies have demonstrated that women and men have striking differences in the species that constitute their microbiomes. This influences hepatology, oncology, autoimmune disease (most notably diabetes mellitus), autism, and obstetrics. There is still an unfortunate lack of research being done on the “microgenderome”: the interaction between microbiota, sex hormones, and the immune system. Knowing the differences in colonization between men and women will benefit diagnosis and treatment of patients and could potentially create prediction models for disease in the future. Hopefully, more and more clinicians will see the critical need to investigate the human microbiome and gender.