Microbiome in Reproduction: Analysis Methods, Novel Techniques, and State-of-the-Art in Female Reproductive Health

Signe Altmäe, PhD

Microbiota and microbiome, the analysis of microbial genomes, are growing interest for study in the female reproduction field as it would have direct clinical applicability potential. Recent years have witnessed advances in the methodologies in the microbiome research, revealing the potential importance of the microbes in the female reproductive tract and its impact on female health and overall well-being. Understanding its composition in health and disease would open up new avenues for improving fertility and treating infertility. Of special interest is the microbial composition inside the uterus as it holds the potential to influence the process of embryo implantation. Regardless of the exponential research in the field, we do not know what is normal and what is the disease-associated microbial composition. We are just starting to acknowledge the presence of microbes along the female reproductive tract, while the studies are mostly descriptive and the function of the microbes and the host–microbe interplay within the reproductive tract needs to be fully established. The current issue “Microbiome in Reproduction” in Seminars in Reproductive Medicine composes of seven beautiful reviews highlighting the analysis methodologies, novel techniques, and the current knowledge in the female reproductive tract.

The “microbiome revolution” in the microbiome analysis of the female reproductive tract has brought attention to the importance of the study design and data analysis. Different studies are often barely comparable and arrive to distinct conclusions[1]; therefore, there is a need to provide a thorough overview of the methodological possibilities that would lead to consensus guidelines and recommendations for studies exploring microorganisms. The exhaustive review by Salas-Espejo et al[2] provides detailed information and workflows of the current techniques applied, together with their strengths and shortcomings, which altogether serve as an excellent starting point for researchers in understanding and conducting microbe-based studies.

Further, sampling method is crucial in the microbiome studies as it may influence the results, especially when analyzing the female upper reproductive tract such as endometrium that is a low microbial biomass site, being adjacent to high microbial biomass sites like vagina and cervix. Endometrial samples require invasive methods and when obtaining transcervically, there is a high “contamination” risk with bacteria from the vagina/cervix. Different devices are proposed for endometrial sampling, which can minimize the contamination risk but does not avoid it.[1] The article by Lüll and Org[3] beautifully summarizes the current evidence of the different sampling techniques in analyzing uterine microbiome and its influence on the detected microbial composition.

Continuing with the technological challenges, next-generation sequencing techniques that are applied in microbiome studies encounter difficulties when analyzing low microbial biomass sites such as the endometrium, susceptible to sequencing bias from the environment and reagents. Furthermore, the DNA-based sequencing techniques provide knowledge of the possible taxa present and describe the microbiome, while the mere DNA sequence does not necessarily equate with the presence of a live bacteria (DNA molecules can persist for decades, being breakdown products, remaining from dead bacteria, or background contamination).[4] Extensive microbial culturing approach (i.e., culturomics) would provide the knowledge of the alive microbes present and would overcome the shortcomings of the nucleic acid-based sequencing approaches. The engaging article by Vanstokstraeten et al[5] provides a comprehensive review of the culturomics technique for investigating microbiota (alive microbial composition) together with its advantages and shortcomings, and they outline the pioneering culturomics studies in the human endometrium. Renaissance of culturomics, as the authors neatly name, is a promising approach in identifying the alive core microbial composition in the endometrium (and other low microbial biomass sites) in health and disease.[5] This information is crucial as today the microbial dysbiosis is often treated (based on the DNA-sequence analysis) with antibiotics and/or probiotics while the knowledge of the alive microbes is lacking. In short, there is utmost need to first establish the living microbial profiles in the uterus before offering dysbiosis treatment strategies for patients.[6]

Another wave of novel techniques, the so-called organoids, is a highly promising system in advancing our understanding of the functional relationships of the host–microbe in the reproductive tract. The research in our field of microbes in female reproduction is predominantly in the phase of describing microbes present/not present in a specific site in association with healthy/diseased state, and even though the existence of microbiome throughout the female reproductive tract is confirmed by many studies, we lack the knowledge of the host–microbe cross-talk, how the microbes influence the host cells, and how could they lead to a disease development. In fact, we do not know whether the dysbiotic state is the cause or consequence of a disorder. Consequently, we are all hoping that the new organoid models will provide answers to these questions. Kaya et al[7] have provided a refined up-to-date overview of the novel organoid systems developed and the first studies in investigating microbiome–reproductive tract interactions.

When investigating the potential function of the microbes in the female reproductive tract, it is clear that there is a dynamic interaction between the microbes and the immune system (i.e., immunome). The immunome–microbiome interplay is believed to influence the onset and progression of reproductive disorders; nevertheless, the mechanisms are not known and understanding these processes requires innovative methods. A comprehensive review by Lingasamy et al[8] summarizes the current knowledge of the immunome–microbiome interactions and proposes novel techniques such as Phage ImmunoPrecipitation Sequencing (PhIP-Seq) and Microbial Flow Cytometry coupled to Next-Generation Sequencing (mFLOW-Seq) in investigating immunome–microbiome communication with promise for innovative diagnostic and therapeutic strategies in reproductive health.

Now focusing on the microbes detected in the female reproductive health, the existence of the upper reproductive tract microbiome has gained support by numerous studies and has been associated with different gynecological disorders such as endometriosis, chronic endometritis, polycystic ovary syndrome, endometrial polyps, cancer, infertility, and also in vitro fertilization success.[9] [10] [11] However, sites of the female upper reproductive tract as Fallopian tubes and ovaries are scarcely studied. The review by Ganha-Gouveia et al[12] elegantly summarizes the current knowledge of the microbiome studies performed in the Fallopian tubes and ovaries together with the concomitant pathologies and highlight the need for further studies to establish the healthy and pathology associated microbes in these two important sites of the female reproduction such as gamete maturation and oocyte fertilization.

The female lower reproductive tract microbiota is a complex and dynamic ecosystem comprising mostly of Lactobacillus species that have crucial role in protecting and maintaining reproductive health. The compact review by Ruiz-Durán et al[13] focuses on the cervical mucus plug microenvironment during pregnancy and the symbiotic relationship between the vaginal microbes and the cervical mucus plug that result in a natural barrier against ascending infections. Understanding the dynamic interplay at the maternal–fetal interface would provide a step closer to innovative approaches for improving female reproductive health.

Although significant advances are made in the field of microbes in reproductive health, the composition of the core microbes in health and disease still needs to be confirmed and their functions fully established. All reviews under this issue stress that more research in the field is warranted. Meticulously designed studies and bigger sample size should be aimed in the microbiome studies in the female reproduction. As the sample collection is in general invasive, it can be difficult to motivate participants and clinicians to participate; therefore, collaboration between groups would offer the solution for this inherent problem in our field. I hope that this Issue, with a special focus on novel methods and methodological considerations, would stimulate and inspire new approaches for future research of the microbiome in reproduction.

Publication History

Article published online:
06 February 2024

• References

• 1 Molina NM, Sola-Leyva A, Haahr T. et al. Analysing endometrial microbiome: methodological considerations and recommendations for good practice. Hum Reprod 2021; 36 (04) 859-879
• 2 Salas-Espejo E, Terrón-Camero L, Ruiz JL, Molina NM, Andrés-León E. Exploring the microbiome in human reproductive tract: high-throughput methods for the taxonomic characterization of microorganisms. Semin Reprod Med 2023; 41 (05) 125-143
• 3 Lüll K, Org E. Uterine microbiome – does the sampling technique matter?. Semin Reprod Med 2023; 41 (05) 144-150
• 4 Sola-Leyva A, Andrés-León E, Molina NM. et al. Mapping the entire functionally active endometrial microbiota. Hum Reprod 2021; 36 (04) 1021-1031
• 5 Vanstokstraeten R, Demuyser T, Piérard D, Wybo I, Blockeel C, Mackens S. Culturomics in unravelling the upper female reproductive tract microbiota. Semin Reprod Med 2023; 41 (05) 151-159
• 6 Altmäe S, Rienzi L. Endometrial microbiome: new hope, or hype?. Reprod Biomed Online 2021; 42 (06) 1051-1052
• 7 Kaya YA, de Zoete MR, Steba GS. Advanced technologies for studying microbiome-female reproductive tract interactions: organoids, organoids-on-a-chip, and beyond. Semin Reprod Med 2023; 41 (05) 160-171
• 8 Lingasamy P, Modhukur V, Mändar R, Salumets A. Exploring immunome and microbiome interplay in reproductive health: current knowledge, challenges, and novel diagnostic tools. Semin Reprod Med 2023; 41 (05) 172-189
• 9 Molina NM, Sola-Leyva A, Saez-Lara MJ. et al. New opportunities for endometrial health by modifying uterine microbial composition: present or future?. Biomolecules 2020; 10 (04) 10
• 10 Koedooder R, Mackens S, Budding A. et al. Identification and evaluation of the microbiome in the female and male reproductive tracts. Hum Reprod Update 2019; 25 (03) 298-325
• 11 Moreno I, Garcia-Grau I, Perez-Villaroya D. et al. Endometrial microbiota composition is associated with reproductive outcome in infertile patients. Microbiome 2022; 10 (01) 1
• 12 Canha-Gouveia A, Di Nisio V, Salumets A. et al. The upper reproductive system microbiome: evidence beyond the uterus. Semin Reprod Med 2023; 41 (05) 190-199
• 13 Ruiz-Durán S, Tenorio CM, Vico-Zúñiga I. et al. Microenvironment of the lower reproductive tract: focus on the cervical mucus plug. Semin Reprod Med 2023; 41 (05) 200-208