ARTICLE https://doi.org/10.1038/s42003-021-02394-6 OPEN Vaginal microbiome Lactobacillus crispatus is heritable among European American women Michelle L. Wright 1,2,10, Jennifer M. Fettweis3,4,5,10, Lindon J. Eaves6, Judy L. Silberg6,7, Michael C. Neale 6, Myrna G. Serrano 3,5, Nicole R. Jimenez3,5, Elizabeth Prom-Wormley8, Philippe H. Girerd4,5, ✉ Joseph F. Borzelleca Jr.4, Kimberly K. Jefferson3,5, Jerome F. Strauss III4,5, Timothy P. York 4,6,11 & Gregory A. Buck 3,5,9,11 The diversity and dominant bacterial taxa in the vagina are reported to be influenced by multiple intrinsic and extrinsic factors, including but not limited to pregnancy, contraceptive use, pathogenic states, socioeconomic status, and ancestry. However, the extent to which 1234567890():,; host genetic factors influence variation in the vaginal microbiota is unclear. We used a biometrical genetic approach to determine whether host genetic factors contribute to inter- individual differences in taxa from a sample of 332 twins who self-identified as being of African (44 pairs) or European ancestry (122 pairs). Lactobacillus crispatus, a major deter- minant of vaginal health, was identified as heritable among European American women (narrow-sense heritability = 34.7%, P-value = 0.018). Heritability of L. crispatus is consistent with the reduced prevalence of adverse reproductive disorders, including bacterial vaginosis and preterm birth, among women of European ancestry. 1 School of Nursing, The University of Texas at Austin, Austin, TX, USA. 2 Department of Women’s Health, Dell Medical School, The University of Texas at Austin, Austin, TX, USA. 3 Department of Microbiology and Immunology, School of Medicine, Virginia Commonwealth University, Richmond, VA, USA. 4 Department of Obstetrics and Gynecology, School of Medicine, Virginia Commonwealth University, Richmond, VA, USA. 5 Center for Microbiome Engineering and Data Analysis, Virginia Commonwealth University, Richmond, VA, USA. 6 Department of Human and Molecular Genetics, School of Medicine, Virginia Commonwealth University, Richmond, VA, USA. 7 Mid-Atlantic Twin Registry, Virginia Commonwealth University, Richmond, VA, USA. 8 Family Medicine and Population Health, Division of Epidemiology, Virginia Commonwealth University, Richmond, VA, USA. 9 Department of Computer Science, School of Engineering, Virginia Commonwealth University, Richmond, VA, USA. 10These authors contributed equally: Michelle L. Wright, Jennifer M. ✉ Fettweis 12These authors jointly supervised this work: Timothy P. York, Gregory A. Buck. email: [email protected] COMMUNICATIONS BIOLOGY | (2021) 4:872 | https://doi.org/10.1038/s42003-021-02394-6 | www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-021-02394-6 ifferences in microbial diversity and predominant bacteria Table 1 Reproductive health characteristics of twin of the vaginal microbiome have been reported to be participants by self-reported ancestry. D 1 2,3 related to pregnancy , vaginal infections , contraceptive 4 5–8 9 use , preterm birth , menopause , and ethnic/racial N P background10–13. Women harboring more complex vaginal Variable European ancestry African ancestry ( value (N = 244) = 88) microbiomes are more likely to have bacterial vaginosis, higher susceptibility to sexually transmitted diseases, pelvic inflamma- Zygosity (%) tory disease, and premature birth14. Yet, the contribution of host MZ 168 (69) 48 (55) DZ 76 (31) 40 (45) 0.0224 genetic factors to shaping the vaginal microbiota is largely BMI unknown. Minimum 18.00 17.00 Overall, studies published to date suggest there are not broad Median (IQR) 25.10 (21.00, 31.02) 30.90 host genetic influences, but rather genetic factors contribute to (25.30, 36.00) presence of specific microbes associated with clinical conditions. Mean (sd) 26.84 ± 6.74 30.95 ± 7.02 0 Twin studies of gut microbiome composition in monozygotic Maximum 50.00 49.00 (MZ) and dizygotic (DZ) twin pairs show15,16 that some bacteria Missing 32/244 (13) 11/88 (12) appear to be heritable and associate with a clinical phenotype15. Age One study reports that the vaginal microbiomes of MZ twins (n Minimum 19 18 = 26, 13 pairs) are more similar to each other than to their Median (IQR) 36.00 (29.00, 49.00) 36.50 (29.00, 51.75) mothers (n = 8) or sisters (n = 8)9. Yet, this study does not Mean (sd) 39.58 ± 13.90 38.98 ± 13.40 0.7199 adequately distinguish host genetic from other shared familial Maximum 78 65 Bacterial vaginosis (%) influences. Another study evaluating the vaginal microbiome = No 213 (89) 65 (77) among Korean twins and selected relatives (N 542, 111 MZ and Yes 19 (8) 18 (21) 28 DZ pairs), reports Prevotella as the most heritable bacterial Not sure 6 (3) 1 (1) 17 taxon in the vaginal microbiome . However, most of the taxa are Missing 6/244 (2) 4/88 (5) 0.0021 reported at the genus level. Given the importance of species-level Pregnant status (%) differences to women’s health outcomes, further studies with No 231 (96) 85 (97) improved resolution among more diverse cohorts are warranted. Yes 4 (2) 1 (1) The estimation of a genetic contribution to inter-individual Not sure 6 (2) 2 (2) differences in phenotypic trait measurements was proposed over Missiing 3/244 (1) 0/88 (0) 1 a century ago18. The subsequent development of methods to Nulliparous (%) partition a trait of interest into separate genetic and environ- No 164 (69) 62 (70) mental contributions using genetically informative twin and Yes 75 (31) 26 (30) 19 Missing 5/244 (2) 0/88 (0) 0.8543 family samples remains an important tool in the genomics era . Hormone therapy (%) Heritability is a technical term that refers to the proportion of No 64 (78) 34 (92) phenotypic variance of a trait measured in a population that can Yes 18 (22) 3 (8) be accounted for by genetic sources. A meta-analysis of over Missing 162/244 (66) 51/88 (58) 0.1155 17,804 human traits from 2748 twin studies report an average Hormonal birth control (%) heritability of 49%, and a subset of reproductive traits at an No 162 (75) 61 (77) average heritability of 31%20. The utility of this summary ratio of Yes 54 (25) 18 (23) genetic variance to the total phenotypic variance not only Missing 28/244 (11) 9/88 (10) 0.811 expresses the extent of genetic influence but also allows for Sample pH (%) meaningful comparisons across traits. An accurate representation Minimum 4.00 4.00 of the genetic architecture of species-level vaginal microbiota Median (IQR) 4.50 (4.00, 5.50) 4.70 (4.40, 5.50) requires heritability estimates across different populations. Mean (sd) 5.02 ± 1.13 5.08 ± 1.03 0.6369 Maximum 7.00 7.00 Evaluating host genetic and environmental contributions to the Missinig 4/244 (2) 0/88 (0) vaginal microbiome composition has unique challenges com- Current smoking (%) pared to other microbiome sites due to the sparsity of data and No 142 (67) 64 (83) relatively few numbers of taxa present across all women within Yes 71 (33) 13 (17) the vagina. Vaginal microbiomes of ~60–90% of reproductive Missing 31/244 (13) 11/88 (12) 0.0099 aged women are predominantly composed of Lactobacillus species10,11,13. High proportions of specific species within vaginal microbial communities are associated with vaginal health (e.g., Lactobacillus crispatus) and adverse clinical conditions (e.g., Results Gardnerella vaginalis with bacterial vaginosis). Previous studies A total of 380 mid-vaginal wall swabs were obtained from self- consistently report different proportions of predominant taxa by identified MZ or DZ twin participants (Table 1). Thirty-four samples self-reported ancestry10,11,13. For example, L. crispatus is more were collected from only one member of a twin pair and not included prevalent among women of European ancestry, whereas Lacto- in these analyses. There were 332 twins of self-identified African (44 bacillus iners is the most prevalent Lactobacillus species among pairs) or European ancestry (122 pairs). There was a higher pro- women of African ancestry10,11. portion of African American DZ than MZ twins (chi-square = 5.21, The goal of this study was to estimate the contribution of host P value = 0.022). Participants ranged in age from 18 to 78 years old genetic factors to species-level variation in microbial taxa of the (median = 36). The median BMI of all participants was 27 (range = vagina using a biometrical genetic approach. Differences in these 17–50), and the mean was higher among African American parti- estimates by self-reported ancestry were considered due to con- cipants (t-test = 4.45, P value < 0.001). In this sample African sistently reported heterogeneity in vaginal microbiome composi- American participants were more often diagnosed with bacterial tion between ancestry groups, including our previous studies10,12. vaginosis (chi-square = 9.48, P value = 0.002), while more European 2 COMMUNICATIONS BIOLOGY | (2021) 4:872 | https://doi.org/10.1038/s42003-021-02394-6 | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-021-02394-6 ARTICLE Fig. 1 Taxa distribution across zygosity and ancestry. Twin pairs are represented as one horizontal line within a pane with each member separated by the vertical dashed line. Monozygotic twin pairs are presented in the left panels, with dizygotic twin pairs on the right. The upper panels are twin pairs of African ancestry, and European ancestry twin pairs are the bottom two panels. Panels summarize taxa proportions for A. Monozygotic/African ancestry; B. Dizygotic/African ancestry; C. Monozygotic/Eurpean ancestry and; D. Dizygotic/European ancestry twin pairs. American participants reported active smoking at the time of crispatus abundance among women of European ancestry may assessment (chi-square = 6.66, P value = 0.010). partially explain why a higher prevalence of L. crispatus is con- After initial quality control screening of samples containing at sistently reported among this population.