1 RBMO VOLUME 00 ISSUE 0 2020

REVIEW The reproductive – clinical practice recommendations for fertility specialists

BIOGRAPHY Juan García-Velasco, MD, PhD, is Director of IVI RMA Madrid and Professor of Obstetrics and Gynaecology at Rey Juan Carlos University, Madrid, Spain. He studied at Yale University, New Haven, Connecticut, USA, under a Reproductive Endocrinology and Infertility Fellowship. His main research interests are trying to understand difficulties in ovarian stimulation (hyper and poor responders) and complications of ART.

Juan A García-Velasco1,*, Dries Budding2, Hartmut Campe3, Sara Fill Malfertheiner4, Samir Hamamah5, Claudia Santjohanser6, Ina Schuppe- Koistinen7, Henriette Svarre Nielsen8, Sara Vieira-Silva9,10, Joop Laven11

KEY MESSAGE The reproductive microbiome is implicated in female reproductive health and is probably also implicated in outcomes. Understanding how to assess and diagnose microbiome in the female reproductive tract could lead to improvements in reproductive outcomes.

ABSTRACT The interest in and understanding of the has grown remarkably over recent years. Advances in molecular techniques have allowed researchers to identify and study the and also use this information to develop therapeutic solutions for a spectrum of conditions. Alongside the growing interest in the microbiome, societal changes have resulted in many couples looking to start families later in life, therefore increasing the demand for assisted reproductive technologies. Combining these trends, it makes sense that clinicians are eager to understand and exploit the microbiome of their patients, i.e. the reproductive microbiome, in order to help them achieve their goal of becoming parents. This paper aims to provide an overview of the current and future research into the reproductive microbiome in relation to fertility and also share clinical practice recommendations for physicians who are new to this field or unsure about how they can utilise what is known to help their patients.

1 Department of Reproductive Endocrinology and Infertility, IVI Madrid, Rey Juan Carlos University, Madrid 28023, Spain KEYWORDS 2 inBiome B.V., Amsterdam 1098 XG, the Netherlands Clinical assessment 3 MVZ Martinsried GmbH, Martinsried 82152, Germany Fertility 4 Klinik St. Hedwig, Krankenhaus Barmherzige Brüder Universitätsklinik, Regensburg, Germany 5 Centre Hospitalier Universitaire de Montpellier, Service Biologie de la Reproduction, 34295 Montpellier, Inserm U1203, France Pregnancy outcomes 6 Kinderwunsch Centrum München MVZ, München 81241, Germany Probiotics 7 Karolinska Institutet, Department of , Tumor and Cell Biology, Centre for Translational Microbiome Reproductive microbiome Research (CTMR), Stockholm 171 77, Sweden 8 Department of Obstetrics and Gynecology, Hvidovre Hospital, Copenhagen University Hospital, Hvidovre 2650, Denmark 9 Laboratory of Molecular Bacteriology, Department of Microbiology and Immunology, Rega Institute, KU Leuven, Leuven 3000, Belgium 10 Center for Microbiology, VIB, Leuven, Belgium 11 Erasmus MC, University Medical Center Rotterdam, Division of Reproductive Medicine, Rotterdam 3015 GD, the Netherlands

© 2020 The Authors. Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/) *Corresponding author. E-mail address: [email protected] (J A García-Velasco). https://doi.org/10.1016/j. rbmo.2020.06.014 1472-6483/© 2020 The Authors. Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/) Declarations of interest: D. Garcia-Velasco reports lecture fees from Ferring, during the generation of the review paper; research grants and lecture fees from Ferring, Merck, MSD, Gedeon Richter, Roche and Theramex, outside the submitted work. Dr Budding reports personal fees from inBiome B.V. and personal fees from ArtPred BV, outside the submitted work; In addition, Dr Budding has a patent Microbial Community Profiling licensed to ArtPred BV. Dr Campe reports personal fees from Ferring GmbH, during the generation of the review paper; and from MVZ Martinsried, a private medical diagnostic laboratory, where an assay detecting endometrial microbiota of infertile patients was established, validated and is sold. Dr Fill Malfertheiner reports personal fees from Ferring, during the generation of the review paper and personal fees from Ferring, speaker honoraria, outside the submitted work. Dr Schuppe-Koistinen reports funding from Ferring to support the Centre for Translational Microbiome Research. Dr Nielsen reports speaker fees from Ferring Pharmaceuticals, Ibsa and Merck, outside the submitted work. Dr Vieira-Silva reports consulting fees from Ferring NV for participation in the expert panel at the Fertility and the Microbiome Expert Forum, Amsterdam, the Netherlands, February 2019. Dr Laven reports grants from Ferring, grants from the Dutch Heart Association, ZonMw, Danone, Euroscreen, Anshlabs and Titus Healthcare, outside the submitted work. 2 RBMO VOLUME 00 ISSUE 0 2020

INTRODUCTION tract, a healthy microbiota is dominated statements were used as a basis to by Lactobacillus species. Lactobacilli are produce a series of recommendations ince the first IVF baby was born that produce lactic acid and for clinical practice supported by the in 1978, assisted reproductive protect the vagina by sustaining a low pH literature. These recommendations technologies (ART) have become that is prohibitive to the growth of most appear as section titles throughout the safer, more successful and more bacteria. The upper reproductive tract, paper and have been organised into Saccessible; couples are choosing to have long considered sterile, also has its own three sections. First, a brief overview of children later in life and the demand for specific microbiota, which is 100 to 1000 the current literature on the relationship ART is rapidly increasing (Faddy et al., times less dense (Baker et al., 2018), between the reproductive microbiome 2018). However, some women are unable but dominated by a greater variety of and fertility (Part I); second, nine to become pregnant, or experience a bacterial species and also different strains clinical practice recommendations for pregnancy loss or a preterm delivery. A of Lactobacillus than the healthy vaginal fertility experts (Part II); and third, seven new perspective on fertility is emerging microbiota (Moreno and Franasiak, statements about the future of research as a result of increasing knowledge 2017). into the reproductive microbiome (Part about the microbiome – and its role in III). reproductive health. The aim of this work is to briefly discuss the current research on the reproductive Part I: Current literature The first observation of non-pathogenic microbiome of female fertility patients There are strong indications that the bacteria inhabiting the human body in order to provide expert opinion on vaginal and endometrial was of coli in the intestine how to utilise this knowledge in clinical are associated with female reproductive of healthy children in 1885. Since then, practice of infertility diagnosis and health. other commensal bacteria (bacteria that treatment. Bacterial therapies, commonly live in harmony with the host) inhabiting known as probiotics, are also increasingly The vaginal microbiota is of great interest different parts of the human body, such researched, a natural consequence due to its complexity, the increasing as nasal and oral cavities, skin and the of our expanding knowledge of the knowledge of its role in women's health urogenital tract, have been discovered microbiome. Targeted probiotics can and its influence on reproductivity throughout the 20th century. Their role replenish depleted microbiota, restore (Moreno and Simon, 2019). As changes has been underestimated and in general, healthy conditions and consequently in vaginal microbiota homeostasis or microbes were considered a threat help treat infections and illness (Puebla- disrupted microbiota, referred to as to human health. Due to advances in Barragan and Reid, 2019). dysbiosis, can be the cause of infertility sequencing technology, the literature and preterm birth, there is increasing has started to highlight the role of MATERIALS AND METHODS interest in the characteristics of a healthy commensal bacteria (the microbiota) microbiota and in different ways that this in normal physiology. In the mutualistic A panel of 37 fertility experts, including can be re-established (Bedaiwy, 2019; relationship between microbial clinicians from across Europe, met to García-Velasco et al., 2017; Puca and communities and the human host, the generate best practice recommendations Hoyne, 2017). Dysbiosis is also associated host provides the nutrients to support for fertility experts interested in utilising with endometriosis, although it is as bacterial growth, while the microbial the microbiome to improve their clinical yet unclear whether dysbiosis causes communities provide defence against practice. Small groups were formed for endometriosis, or endometriosis induces pathogens, shape the development initial discussion of four key topics: dysbiosis. As of November 2019, there and maturation of the immune system, are 94 clinical trials registered on https:// help to digest food and fibre, produce 1 Clinical assessment: How is the clinicaltrials.gov that include the search vitamins and metabolize xenobiotics reproductive microbial ecosystem terms ‘pregnancy’ and ‘microbiome’. (Puebla-Barragan and Reid, 2019). Of being assessed in women? Which tools Seventeen of these are specifically linked note, the term microbiota refers to all of are being used and in which patients? to the vaginal/endometrial microbiome. the micro-organisms in an environment, 2 and implantation: including bacteria, and single- What is the role of the endometrial The vaginal microbiome plays an cell eukaryotes (and sometimes ), microbiome in endometrial important role in protecting the vagina and the term microbiome refers to the receptivity, implantation and recurrent – the first barrier from the external collection of genomes of the microbiota. implantation failure? environment to the upper reproductive 3 Infertility: How should we evaluate and tract. A better understanding of At present, there are five distinct treat infertile patients and patients the vaginal microbiome is therefore microbiomes according to body site with bacterial vaginosis utilising the mandatory to further our understanding – the oral cavity, nasal cavity, skin, microbiome? of natural fertility and reproductive genitourinary tract and gastrointestinal 4 Birth: What techniques can be used technology. tract (Kumar and Chordia, 2017). Within to evaluate microbial structures and these distinct anatomical locations there evaluate risk for preterm birth and As per our current knowledge, in non- are further microbial habitats, such as pregnancy loss in pregnant women? pregnant, healthy women the vaginal the tongue, cheek and lip of the oral microbiome is dominated by four cavity (Kumar and Chordia, 2017). A full list of the discussion topics was Lactobacillus species: L. crispatus, L. Whereas the has by far compiled (see Supplementary Material). iners, L. jensenii or L. gasseri (Ravel the most dense and diverse microbial The entire group came together to et al., 2011). The vaginal microbiota can community, in the lower reproductive discuss all topics and the agreed be characterized by the presence or RBMO VOLUME 00 ISSUE 0 2020 3

absence of certain bacterial species. vaginosis may be related to poor to be considered, such as the time In 2011, Ravel et al. (2011) identified pregnancy outcomes. Therefore, patients of collection of the vaginal samples, five vaginal microbial community state undergoing IVF should be screened hormonal stimulation, fresh or frozen types (CST); four of them (I, II, III and and eventually treated to enhance the embryo transfer, oocyte donation, and V) are Lactobacillus-dominant and are chances of success. success rate, defined as implantation more commonly found in European and or live birth (Fettweis et al., 2019). Asian women. CST-IV, more frequent Several studies confirm that a vaginal Moreover, novel and more standardized in Hispanic and African-American microbiota rich in Lactobacillus spp. molecular approaches have improved the women, differs due to the increased without bacterial vaginosis, either clinical ability to detect micro-organisms and will abundance of strictly anaerobic bacteria or subclinical, leads to more positive enhance future results. (Gardnerella, Ureaplasma) and reduced outcomes with ART (Babu et al., 2017; presence of Lactobacillaceae (Ravel Eckert et al., 2003; Mangot-Bertrand CST-IV is most commonly associated et al., 2011). Lactobacillus spp. produce et al., 2013; Moore et al., 2000). Haahr with negative ART outcomes lactic acid that helps to keep the vaginal et al. (2016a) studied the microbiota Women with bacterial vaginosis and pH below 4.5 and creates inhospitable of 84 women undergoing IVF and aerobic vaginitis often present with a conditions for pathogens to grow found a strong relationship between microbiota comparable to CST-IV (Smith (Graver and Wade, 2011). The vaginal microbiota composition and pregnancy. and Ravel, 2017). Bacterial vaginosis microbiota composition shows large Of the 22 women who had an altered or a perturbed microbiota are also a ethnic differences and varies throughout composition, only two women (9%) common factor in frequent preterm the woman's lifetime depending on achieved a clinical pregnancy. Out of delivery or miscarriage (Buggio et al., physiological events, like menstrual cycle the 62 women with a normal microbiota 2019; García-Velasco et al., 2017). and pregnancy, and external factors, like composition, 29 women (47%) achieved CST-IV, with or without a diagnosed sexual activity, hygiene habits and medical a clinical pregnancy. Koedooder et al. bacterial vaginosis, has been described treatments (Gajer et al., 2012; Ravel (2019b) performed a prospective study of as a common feature of preterm birth et al., 2011; Zhou et al., 2007). reproductive-age women undergoing IVF in Caucasian women (DiGiulio et al., and found that embryo implantation was 2015; Donders et al., 2009) and African- The vaginal microbiota and its less successful in women with reduced American women (Nelson et al., 2016). relation to infertility Lactobacillus spp. in their vaginal The vaginal microbiota is normally stable, Bacterial vaginosis is one example microbiota. The authors proposed an Lactobacillus-dominated and less diverse of an altered state of the vaginal algorithm, utilising the IS-pro sequencing during pregnancy (Aagaard et al., 2012). microbiome, characterized by depletion method, to predict the IVF success DiGiulio et al. (2015) observed that some of Lactobacillus and augmented diversity rate based on the vaginal microbiota women who presented with a CST-IV of anaerobic bacteria. Bacterial vaginosis composition. They identified women with microbiota, depleted of Lactobacillus affects 20–50% of reproductive-age a low chance of becoming pregnant due spp. throughout the gestational period, women and it represents a risk factor for to reduced Lactobacillus in the vaginal were more likely to deliver preterm. subfertility and infertility (Mastromarino microbiota (18% of the dataset of 192 Another study found that a CST-IV et al., 2014a; Sirota et al., 2014). women). The model was 94% accurate vaginal microbiome was less frequent in Increased presence of specific bacteria (with high specificity [97%], but low women delivering at term even though it (Atopobium vaginae, Ureaplasma sensitivity [26%]): 32 out of 34 women was not possible to define a correlation vaginae, U. parvum, U. urealyticum with an unfavourable profile did not between this vaginal microbiota profile and Gardnerella), normally observed achieve pregnancy. An external validation and preterm delivery (Romero et al., in asymptomatic bacterial vaginosis, cohort study further supported the 2014). together with higher abundance of prediction specificity. According to high and reduced vaginal and versus low Lactobacillus dominance the The endometrial microbiota and its cervical Lactobacillus, is often present women were divided into two subgroups relation to IVF in women with fertility problems with favourable and unfavourable Previously it was believed that the upper (Koedooder et al., 2019a). Moreover, prediction, respectively. None of the reproductive tract was sterile. After the vaginal dysbiosis reduces the local women in the unfavourable group discovery of a specific and independent defences against sexually transmitted became pregnant (Koedooder et al., endometrial microbiome (Chen et al., pathogens (Wiesenfeld, 2003) and 2019b). In another study, Lactobacillus 2017), several studies concentrated on ascension of pathogens up the fallopian crispatus-dominant microbiomes were endometrial dysbiosis (Benner et al., tubes can affect reproductive health associated with higher live birth rate 2018; Moreno et al., 2016). Endometrial (Mastromarino et al., 2014a). (Haahr et al., 2019; Vergaro et al., 2019). dysbiosis can be the cause of These findings suggest that a better implantation failure and lead to infertility The vaginal microbiota and its knowledge about the vaginal microbiota (Moreno and Simon, 2018). relation to IVF outcomes prior to IVF may help both couples and The vaginal microbiota has lately become healthcare professionals decide on the Infertility treatment is intrinsically an important factor in IVF. The lack of timing of their IVF cycles to maximize the complicated; however, the delicacy and consensus on its role comes from the results (Moreno and Simon, 2019). importance of the implantation phase lack of common criteria to cross-check has proven to be critical for positive data, small sample size, and the lack At this point, however, it is very difficult outcomes (Diedrich et al., 2007). A of data due to the novelty of the field. to evaluate and to compare different Lactobacillus-dominant endometrium is Altered vaginal microbiota and bacterial studies because there are many variables more receptive than an endometrium 4 RBMO VOLUME 00 ISSUE 0 2020

with high bacterial diversity and a low by Lactobacilli, with no differences pregnancy. This normalization happens proportion of Lactobacilli. The results of depending on community type, ethnicity in about 50% of women (Hay et al., a study by Moreno et al. (2016) highlight or body mass index (Aagaard et al., 1994). However, there is also a strong that women with a Lactobacillus- 2012). association between pre-pregnancy dominant microbiota have a higher bacterial vaginosis and preterm birth chance of success in implantation, A non-Lactobacillus-dominant vaginal because ascending vaginal infections 60% versus 23% in non-Lactobacillus- microbiota and increased bacterial cause intrauterine inflammation, which is dominant microbiota; in pregnancy, 70% diversity during pregnancy are associated responsible for one-third of preterm birth versus 33%; and in live birth, 60% versus with higher risk of preterm birth (Brown (Buggio et al., 2019; Leitich et al., 2003). 7%. In this study Moreno et al. (2016) et al., 2019). A group of 96 women were also found that women with a receptive divided into three groups according To identify an increased risk of preterm endometrium (Lactobacillus-dominant) to high (38), low (22) or no risk (36) of birth early in pregnancy, despite have a significantly lower miscarriage preterm birth and followed from week the natural evolution of the vaginal rate, 17% versus 60% as compared 6 of pregnancy. Augmented bacterial microbiome during gestation, would with women with a non-Lactobacillus- diversity and a concomitant reduced lead to earlier diagnostics and possible dominant microbiota. These results Lactobacillus spp. abundance were therapeutic interventions. Based on differ from those obtained by Franasiak evident prior to premature rupture of thorough meta-analysis of vaginal et al. (2016), who reported a comparable membrane in 20% and 26% of high- and microbiome datasets, a correlation IVF success rate independent of low-risk women, respectively. Only 3% of between the vaginal microbiome in the the Lactobacillus dominance of the women that finished their pregnancy at first trimester and preterm delivery has endometrial microbiota. Moreno et al. term had an altered vaginal microbiota been established. This correlation and (2016) suggest this difference could be (Brown et al., 2019). related different microbiota profiles have related to differences in the classification been exploited to propose a ‘Taxonomic systems used in the two studies. Black women are more likely to deliver Composition Skew’ metric as a possible preterm, yet the vaginal microbiota of diagnostic tool to predict preterm Hormonal fluctuations, especially pregnant women shows minor to no delivery with high accuracy (Haque et al., in oestrogens, are implicated in the diversity across different ethnicities 2017). regulation of the vaginal microbiota and (Brown et al., 2019; Fettweis et al., in the preparation of the endometrium 2019). The National Institutes of Health's Part II: Recommendations for clinical for implantation and pregnancy (Wessels Integrative assessment and clinical practice1 et al., 2018). It can be expected that includes samples of vaginal microbiome hormones also influence the endometrial from more than 1500 pregnant women For all infertility patients, ask if microbiota. Surprisingly, however, of diverse ancestries and omics data they have clinical symptoms (smell, Moreno et al. (2016) found that the generated from a subset of about 600 discharge, urinary tract infection, endometrial microbiota does not change . Fettweis et al. (2019) Candida) currently and during their under hormonal influence in the period used these data to provide evidence cycle preceding implantation. These results of the role of Lactobacillus crispatus Our understanding of healthy and suggest that we should start to consider in lowering the risk of preterm birth. unhealthy vaginal microbiome is rapidly the endometrial microbiota and its American women of European ancestry developing. When a woman is evaluated health state prior to beginning IVF, in have a vaginal microbiota rich in L. for vaginal dysbiosis, it is important to order to maximize the chance of positive crispatus and a significantly lower risk identify the cause in order to prescribe outcomes. of preterm birth when compared with the proper treatment. Assessment of American women of African ancestry, symptoms combined with microscopic The vaginal microbiota and its who are more likely to have a vaginal and pH evaluation of the vaginal smear relation to preterm birth microbiome poor in Lactobacillus spp. can help distinguish between bacterial The causes of preterm birth are very Additionally, bacterial vaginosis-associated vaginosis, trichomoniasis, candidiasis complex, making it challenging to prevent bacterium 1, a bacterium that is positively and lactobacillosis (Hainer and Gibson, and also difficult to reduce the related associated with preterm birth, is more 2011). However, almost 30% of women infant mortality and the psychological common in Black women (Fettweis with a vaginal disorder do not receive a and economic consequences of et al., 2019). These results correspond proper diagnosis (Sha et al., 2005; van extended hospitalization of the newborn. to those obtained in British women with de Wijgert and Jespers, 2017). As the vaginal microbiota is important Caucasian, Asian and Black ethnicities. for the health of the whole reproductive All Caucasian and Asian British women Bacterial vaginosis is the most common tract and for prevention of pathological who delivered at term had a Lactobacilli- vaginal disorder in reproductive-age infections, it can be expected that it dominant vaginal microbiome, but 12% of women, although 50% of women are affects not only pregnancy, but also Black women who delivered at term were asymptomatic (Haahr et al., 2016a). delivery. The vaginal microbiome Lactobacilli-depleted (Brown et al., 2019; Bacterial vaginosis is characterized by evolves during pregnancy in relation MacIntyre et al., 2015). vaginal dysbiosis (altered microbiome to gestational age and tends to lose composition) presenting as a depletion the diversity observed in non-pregnant Some women with pre-pregnancy of Lactobacillus and a higher diversity women (Freitas et al., 2017). Healthy dysbiosis show a shift toward a healthy of other anaerobic bacteria, such as pregnant women present a distinct Lactobacillus-dominant bacterial Gardnerella, and vaginal microbiota profile dominated community around weeks 15 to 20 of (Turovskiy et al., 2011). Bacterial vaginosis RBMO VOLUME 00 ISSUE 0 2020 5

is positively associated with infertility, comparable symptoms – itching/ bacteria result in a higher Nugent score miscarriage and preterm birth (Donders burning, and abundant vaginal discharge (Nugent et al., 1991). A Nugent score et al., 2009). To confirm diagnosis – to those of bacterial vaginosis and is of 0–3 represents a healthy microbiota, and provide proper treatment, all characterized by the higher abundance 4–6 an intermediate microbiota, and women reporting vaginal symptoms of long segmented Lactobacilli chains 7–10 confirms bacterial vaginosis. The (unusual discharge, burning or itching, (Ventolini et al., 2014). test is inexpensive and more sensitive unusual smell) should have their vaginal than the Amsel criteria, but it takes a secretions tested (van de Wijgert and Vaginal dysbiosis can be measured long time and requires an experienced Jespers, 2017). in multiple ways, there are no gold microscopist to correctly evaluate results standards in assessment. For women that remain subjective (van der Wijgert Urinary tract infections and with symptoms/suspicions of dysbiosis, et al., 2014). infections can be the cause of it is recommended to perform a reproductive tract damage, i.e. tubal speculum exam and pH test. If New molecular biology techniques based damage or pelvic inflammatory disease, available also perform a wet smear, on DNA and RNA analysis, including and result in infertility (Mastromarino Gram Nugent score, and quantitative qPCR, allow a more objective analysis. et al., 2014b). The main clinical polymerase chain reaction (qPCR) Even in the midst of great microbial symptoms of these conditions are Several techniques have been developed complexity, the characterization of DNA/ malodourous vaginal discharge and to evaluate and confirm a diagnosis of RNA sequences allows the identification itching/burning sensations (Klebanoff bacterial vaginosis: the Amsel criteria, of bacteria present in minor amounts or et al., 2004). Good clinical practice Nugent score and more recently qPCR. which are difficult to cultivate (Cartwright requires that women consulting for The Amsel criteria and Nugent score et al., 2012). By utilising qPCR, a larger infertility treatment report whether are inexpensive tests but they cannot number of bacteria can be detected they have suffered from one of these adequately measure microbial complexity compared with bacteria culturing symptoms in the past, during their cycle, and thus may give inaccurate results techniques (Kusters et al., 2015). The or at the moment of consultation. (Mendling, 2016). Amsel criteria and Nugent score can miss a diagnosis of bacterial vaginosis because For women with no symptoms there In 1983, Amsel et al. proposed four they can underestimate the complexity is currently no further testing practical diagnostic criteria: (i) thin, of microbiota alterations (Cartwright recommended in clinical practice white-greyish homogenous vaginal et al., 2012). Recently, the performance Bacterial vaginosis is a deviation from discharge; (ii) vaginal pH >4.5; (iii) a of a novel diagnostic tool, the AmpliSens a healthy Lactobacillus-dominant ‘fishy’ odour after adding 10% potassium assay, was compared with the classical microbiota and several studies correlate hydroxide to vaginal secretions; (iv) methods, and proposed for use in clinical infertility problems with vaginal dysbiosis presence of epithelial cells coated with practice as a more rapid, objective and (van de Wijgert et al., 2014; Weström, bacteria on wet mount microscopy. accurate diagnosis (van den Munckhof 1994). Based on a cross-sectional study Bacterial vaginosis is confirmed if three et al., 2019). of 84 healthy women and 116 women out of four of these criteria are met. The with infertility problems, Babu et al. Amsel criteria is an inexpensive technique There is limited evidence on whether (2017) suggest adding a routine vaginal that may provide the first evidence of uterocervical colonization should be microbiota screening for all women dysbiosis. However, the evaluation of routinely assessed undergoing infertility treatment. At the results relies on microscopy, direct The discovery of a distinct uterine present, however, this evidence is observation and experienced personnel microbiota is recent and there is too limited to recommend a routine (Kusters et al., 2015; Sha et al., 2005). little consensus on the composition screening of all fertility patients. Another limitation of the Amsel criteria of a core healthy uterine microbiota Moreover, molecular diagnostic is that the increased bacterial diversity (Baker et al., 2018). Nevertheless, testing is expensive and there is no causing dysbiosis is consistent with an authors agree on the hypothesis that supporting literature to recommend it altered pH, but might not alter the other uterine microbiota, independent of the in asymptomatic women (van der Veer Amsel criteria, thus resulting in a negative vaginal microbiota, might play a role in et al., 2018). test. In different cohort studies, only infertility (Moreno and Franasiak, 2017). about 40% of women with diagnosed Further research is needed to better If vaginal pH is below 4.5 this could bacterial vaginosis have been found understand the composition and the indicate lactobacillosis positive according to the Amsel criteria role of uterine colonization, keeping in The pH of vaginal secretions is an (Sha et al., 2005; van der Wijgert et al., mind that low-density microbiota are important parameter for an accurate 2014). extremely sensitive to microbial and diagnosis in women presenting with DNA contamination. To date there is no vaginal symptoms such as burning or The Nugent scoring system, proposed in evidence supporting clinical testing to malodourous discharge. Many vaginal 1991, is a Gram-stained scoring system evaluate the uterine microbiome (Baker microbiota pathologies are characterized of vaginal swabs. It is a 0–10 standardized et al., 2018). by Lactobacillus depletion, lower lactic scale evaluating vaginal microbiota based acid production and a consequent on the presence/absence of Lactobacilli Antibiotics should not be used on increase in pH. However, lactobacillosis, a spp., Gardnerella vaginalis and a routine basis to change vaginal less common vaginal condition, presents Mobiluncus spp. bacterial morphotypes. typology with normal or lower pH values (Ventolini A lower abundance of Lactobacilli Antibiotics are not free from secondary et al., 2014). Lactobacillosis presents and an increased presence of other effects, varying from skin rash to 6 RBMO VOLUME 00 ISSUE 0 2020

anaphylaxis, and overuse can increase versus 33% (P = 0.039); and live birth, healthy-weight women to have a higher the chance of developing antibiotic 61% versus 13% (P = 0.02) (Cicinelli et al., Nugent score and bacterial vaginosis resistance. The efficacy of using 2015). Although data do not exist for their (Brookheart et al., 2019). Women who antibiotics in conjunction with IVF use presently, probiotics as a method of smoke are more likely than non-smokers treatment is controversial (Haahr treating chronic endometritis represents a to have a CST-IV vaginal microbiota et al., 2016b; Kaye et al., 2017; Kroon promising approach and requires further depleted in Lactobacillus spp., and a et al., 2011; Moore et al., 2000). Use of study. high Nugent score indicating a risk for antibiotics before embryo transfer could bacterial vaginosis (Brotman et al., 2014). impact the normal healthy colonization Pre-conception counselling is useful of the endometrium and reduce the and lifestyle habits can impact Lifestyle changes, especially those efficacy of the procedure Kroon( et al., reproductive outcomes (possibly by that affect nutrition, can lead to 2011). On the other hand, the risk of modulation of the vaginal microbiota), changes in the gastrointestinal contamination of the upper reproductive therefore weight loss, physical activity microbiome and could have a positive tract by vaginal bacteria is higher during and lifestyle changes should be impact in infertile patients transcervical and transvaginal procedures discussed and encouraged in infertile Diet has a major impact on the gut and antibiotic prophylaxis could be patients microbiome, by far the densest and most preventive (García-Velasco et al., A woman's health is fundamental to a metabolically active human-associated 2017). Based on a systematic review, healthy pregnancy and for the future microbial community (Lozupone et al., the American Society for Reproductive health of the baby (Stephenson et al., 2012; Vieira-Silva et al., 2016; Wu et al., Medicine (2017) strongly recommends 2018). Women who wish to conceive 2016), and so nutritional interventions against the use of antibiotics before are likely to change bad habits, however may improve reproductive outcome embryo transfer. There is an ongoing advice is often inaccurate or incomplete partly through modulating the gut randomized clinical trial of IVF patients (Bookari et al., 2017). Pre-conception diet microbiome composition. A disturbed at Washington University to evaluate the and lifestyle changes can not only lead gut microbiota has been implicated in clinical pregnancy rate when withholding to better general health but also increase many diseases – inflammation, metabolic routine prophylactic antibiotic therapy the chances of a positive reproductive disorders, obesity and cancer (Cani during IVF (NCT03386227). outcome. et al., 2007; Hildebrandt et al., 2009; Le Chatelier et al., 2013; Ridlon et al., 2014) The use of antibiotics can however Obesity represents a major health – most of which can also affect fertility. be of relevance for certain repeated threat with many related pathologies, Therefore, the importance of microbial implantation failure (RIF) patients including metabolic syndrome, diabetes, communities at body sites other than the Chronic endometritis is an inflammatory cardiovascular disease and cancer. The reproductive tract have to be considered state of the uterine endometrium that is estimated reduction in fertility is 18% for as potential actors in human fertility. normally asymptomatic. The inflammatory obese women (Pantasri and Norman, state is the result of an imbalance 2014). Weight loss is strongly related to Part III: Future studies and research between the microbiome and the host improved ovulation, hormonal rebalance techniques to consider immune system, possibly due to micro- and increased pregnancy rate in obese organisms, viruses or parasites, but the women (Clark et al., 1995; Sim et al., Tobacco, alcohol, cannabis, stress, aetiology remains unclear (Park et al., 2014). Diet and mild-to-moderate daily physical activity, infections, lack of 2016; Wu et al., 2017). The role of chronic physical activity should be the first step in sleep, body mass index, antibiotic endometritis in fertility has long been infertility treatments. exposure, drug exposure, sexual underestimated, but it is now recognized activity, diabetes/metabolic disorders as a possible cause of not only infertility Smoking is another major health threat and hygiene habits might impact but also early miscarriage (Johnston- for society as a whole and in particular microbiome composition; modification MacAnanny et al., 2010). Chronic for infertile women. The pregnancy of these factors should be assessed endometritis leads to a non-receptive rate of heavy smokers (>10 cigarettes) in every study in order to assess their endometrium which is a major cause of is significantly lower when compared impact on the microbiota RIF (Margalioth et al., 2006). The fact that with that of non-heavy smokers (<10 The recent discovery of the non-sterility the upper reproductive tract is not sterile cigarettes) (34.1% versus 52.2%, of the upper reproductive tract (Mitchell (Mitchell et al., 2015), and the latest data respectively), and overall women who et al., 2015) and recent debate over showing that both placenta and amniotic smoke are more likely to be subfertile whether even the placenta and amniotic fluid may present bacteria of their own, (Bolumar et al., 1996). However, for fluid may present bacteria of their own, have generated a new awareness to the women who smoke, pregnancy outcomes have brought a new awareness to the role of the microbiota in pregnancy can be quickly changed as women role of the microbiota in pregnancy and and newborn health (Schoenmakers who quit smoking have a time to first newborn health (Schoenmakers et al., et al., 2018). The efficacy of antibiotic pregnancy that is comparable to that of 2018). Moreover, a disturbed vaginal therapy in treating women with certified non-smokers (Bolumar et al., 1996). microbiota has proven to be a major chronic endometritis and RIF has been factor in infertility, miscarriage and established. Women with no infection as Diet and smoking are directly involved in preterm birth (Moreno and Simon, 2019). compared with women with persistent bacterial vaginosis, and bacterial vaginosis infection after antibiotic therapy had is associated with infertility, pregnancy The human microbiota evolves naturally; higher rates of implantation, 37% versus loss and preterm birth. Overweight the vaginal microbiota changes during 17% (P = 0.069); clinical pregnancy, 65% and obese women are more likely than pregnancy (MacIntyre et al., 2015) RBMO VOLUME 00 ISSUE 0 2020 7

and the gut microbiota evolves from to antibiotic therapy. Lactobacillus studies and potential confounding childhood to old age (Woodmansey, spp. supplementation has proven factors. In another study, women 2007) and can be altered due to effective in the prevention of recurrent receiving an oral, eight-strain probiotic exogenous pressures including poor infections after standard metronidazole containing a mixture of Lactobacillus, nutrition, smoking, stress, drug exposure therapy (Menard, 2011). Antibiotics Streptococcus and Bifidobacterium and antibiotic exposure (Gohir et al., are not species-specific, so while they strains in the last 3 months of pregnancy 2015; Falony et al., 2016; Wen and Duffy, fight pathogens, they also decrease had modulated vaginal microbiota 2017). Consequently, lifestyle adjustment Lactobacilli and other commensal and an increase in anti-inflammatory and maybe dietary supplements could be bacteria, thus resulting in a depletion of cytokines (Vitali et al., 2012). Given that proposed to help women with infertility healthy bacteria in the vaginal microbiota inflammation is a major cause of preterm problems, to promote both pregnancy (Macklaim et al., 2015). The concomitant birth (Romero et al., 2014), it is possible outcomes and newborn health. Future use of antibiotics and probiotics might be that reducing vaginal inflammation could research should concentrate on the optimal therapy to fight pathogens have implications in preventing preterm the role of these external factors to and correctly repopulate the vaginal birth. further understand their impact on the microbiota (Moreno and Simon, 2019). microbiota. Microbiota profiling should be used As previously discussed, bacterial in a standardized manner (collection, The use of probiotics to treat atypical vaginosis and unbalanced microbiota analysis, statistics) vaginal microbiota is being studied can reduce success in fertility treatment. The boundaries of the healthy In recent years many probiotic oral Research on the efficacy of supplements reproductive tract microbiota must be supplements have appeared on the in restoring vaginal health is quite new clearly defined before the relationship market, with increased evidence of their and has evolved quickly over the past with fertility can be established. To efficacy, mostly to treat gastrointestinal few years as a result of next-generation date we have some important insights, conditions (Ford et al., 2014). Several techniques (Campisciano et al., 2017). but data are insufficient to definitively oral and vaginal probiotics have been There is yet to be consensus on the characterize healthy versus dysbiotic developed to help women restore healthy efficacy of probiotics Anukam( et al., status. The first step towards this aim vaginal microbiota. 2006; Barrons and Tassone, 2008; is to standardize protocols, sampling Buggio et al., 2019; Homayouni et al., methods and sizes, sequencing Back in 1992, a pioneering study 2014; Reid et al., 2001, 2003), and techniques and bioinformatic pipelines reported that a daily oral dose of yogurt conflicting results may be due to (Koedooder et al., 2019a). This will enriched with Lactobacillus acidophilus probiotic strain choice, or the posology provide comparable data and possibly was effective in treating recurrent adopted to treat specific conditions. lead to general conclusions that can be vulvovaginal candidal infections after To date, available data on probiotics used as a basis for effective therapies 6 months of treatment (Hilton et al., to support infertility treatments are (Koedooder et al., 2019a; Watson and 1992). Since then, many studies have insufficient but promising García-( Reid, 2018). investigated the role of Lactobacillus Velasco et al., 2017). A future challenge spp. supplementation in restoring is to confirm probiotic activity and Prospective studies (randomized, the vaginal microbiota (Falagas to precisely characterize the specific controlled, with large patient et al., 2007). Products are mostly strains with the aim of creating therapies numbers) need to be standardized in formulated with various combinations effective in infertile women. collection, laboratory techniques and of Lactobacillaceae, including L. statistical analyses in order to study crispatus, L. gasseri, L. plantarum, L. The use of probiotics to prevent the microbiota and its relation to reuteri and L. rhamnosus. The presence preterm birth is supported by a large fertility outcomes of Lactobacillus spp. is a recognized body of evidence (Kirihara et al., A healthy reproductive tract microbiota characteristic of a healthy vaginal 2018; Vitali et al., 2012). Kirihara et al. is correlated to fertility and to positive microbiota (García-Velasco et al., 2017), (2018) reported that a combination fertility treatment outcomes. The while a decrease in their abundance of probiotics (Streptococcus faecalis, research is rapidly growing, but is often associated with altered pH Clostridium butyricum and many issues remain unexplored or and dysbiosis, bacterial vaginosis mesentericus) successfully improved controversial. Systematic reviews show or vulnerability to pathogens (Ravel perinatal outcome. A total of 121 women that studies are difficult to compare et al., 2011). Several studies confirm with high risk of preterm delivery were because of varying cohort demographics, the beneficial role of oral or vaginal divided into two groups, one of which inclusion criteria, sample size, laboratory probiotic supplementation in achieving received the probiotics. The women techniques, etc. (Fettweis et al., 2019; and maintaining a healthy microbiota receiving probiotics (n = 45) had longer Peelen et al., 2019). Peelen et al. (2019) (Anukam et al., 2006; Homayouni gestations: only one woman (2%) propose a database of essential and et al., 2014; Reid et al., 2001, 2003). delivered before 32 weeks, while in the desirable items relating to quality, Others question probiotic efficacy, albeit control group 19 women (25%) delivered method and topic to overcome the low confirming the absence of any adverse before 32 weeks. Overall, gestation was quality of information relating the vaginal effect related to their use (Barrons and 36 weeks on average in the probiotic microbiota. Improved study design Tassone, 2008; Buggio et al., 2019). group and 34 weeks on average in the and sampling strategies are needed to control group. This work shows the standardize research and establish causal There is consensus on the efficacy of beneficial effects of probiotics despite relationships between microbiota and vaginal probiotics when used in addition the intrinsic limits of retrospective adverse outcomes. 8 RBMO VOLUME 00 ISSUE 0 2020

For research purposes one might 2019; Koedooder et al., 2019a; Moreno and molecular techniques allows the consider assessing the microbiome et al., 2016). characterization of previously non- in all fertility patients to gain more culturable bacteria in altered microbiota insight into pathophysiology and Other genetic testing methods such as (Lamont et al., 2011). These findings outcomes qPCR, phylogenetic microarrays (e.g. can boost our knowledge about Distinguishing a commensal microbiota V-Chip) (Paily and Agans, 2011) and the microbiota and provide a new from a pathophysiologic microbiota molecular fingerprinting (e.g. IS-pro™) perspective on which bacterial strains without altering standard clinical (Koedooder et al., 2019b), have proved are involved in embryo transfer failure. practice is an important step to better useful in microbiome profiling. There is Some preliminary studies show that understand the reproductive microbiota. an inherent trade-off to these techniques, molecular tests have a high sensitivity in Franasiak et al. (2016) characterized the forcing a choice between the broadness detecting bacterial vaginosis: ATRiDA® endometrial microbiota without altering of the profiling or the resolution and (sensitivity 96.9%, specificity 70.2%) and standard clinical practice. At time of sensitivity of the profiling. Metagenomic AmpliSens® (sensitivity 80.6%, specificity embryo transfer, the 5 mm distal part of sequencing offers broad profiling, >90%) (van den Munckhof et al., 2019; the catheter was sterilely placed in a PCR with very high definition in the case van der Veer et al., 2018). On the basis tube and analysed by next-generation of shotgun metagenomic sequencing, of new findings, future researchers sequencing (NGS). The results of this however low-abundance species will be should aim to incorporate diagnostic study were not statistically significant, missed. IS-pro™ is, similarly, a broad- tools into clinical practice with the goal of but they suggest a direction for future spectrum molecular technique that optimizing the time of embryo transfer to studies and propose a way to analyse depicts the relative composition of the maximize the success rate. the microbiota with very limited starting dominant fraction of the microbiota. As material and without changes to standard discussed in Part 1, an algorithm using DISCUSSION clinical practice. These techniques could the IS-pro™ technique was successfully be applied to all fertility patients and used for IVF failure prediction A more complete understanding of the provide insight into pathophysiology. (Koedooder et al., 2019b). Phylogenetic role of the reproductive microbiome microarrays contain probes that target promises improvements to fertility NGS and other genetic testing specific species. It therefore offers fast treatments and a more nuanced methods are useful in research. More results and high resolution but for a understanding of reproductive health evidence is required to support routine predetermined panel of micro-organisms as a whole. The microbiome is the new use in diagnosing infertility, preterm of interest. qPCR is the technique most frontier; the currently accumulated birth and recurrent pregnancy loss appropriate for low biomass specimens knowledge will propel the discovery of As the microbiota plays a role in or to detect low-abundance species in a new treatment solutions and protocols infertility, recurrent pregnancy loss and community (Haahr et al., 2019). qPCR for superior fertility outcomes. Although preterm birth it is logical to consider it assays are cumbersome if the panel of the field is new, there are already ways to as a counterpart in infertility treatment. interest is extensive, but allow assessment utilise what is known to improve clinical The questions are multiple. How do of quantitatively specific elements of practice and achieve better reproductive we define a healthy microbiome? the microbiota at any level of resolution outcomes. Current and future research Which analyses should we use? How (from strain to full bacterial content) in large clinical cohorts should uncover do we correctly interpret the results? and will even detect bacteria that are more ways in which the microbiota NGS has increased the resolution and present in very small amounts and that can be modulated pre-conception and accelerated the research, allowing full are difficult to cultivate Cartwright( during pregnancy in order to minimize complex microbiota communities to be et al., 2012), which could be important pregnancy loss and preterm birth and monitored. Recent studies show that for the early detection of microbiota maximize implantation, pregnancy rate small changes in vaginal microbiota perturbations. and at-term delivery of healthy babies. can greatly affect fertility, therefore we need more specific tests in order to Perhaps in the future, NGS and other create accurate therapeutic protocols molecular techniques could be used ACKNOWLEDGEMENTS for infertility treatments (Campisciano prognostically for counselling to et al., 2017). NGS has detected a new optimize the time of embryo transfer This work received funding from Ferring bacterium, Atopobium vagina, highly Embryo transfer is a crucial step Pharmaceuticals. represented in the vaginal microbiota in fertility treatments and can fail of women suffering from idiopathic for many reasons including uterine infertility. With a less specific analysis, anatomy, immunological factors or the Nugent score, no difference was embryo genetics. The reproductive detected between fertile and infertile tract microbiota is one variable that women (Campisciano et al., 2017). This has recently been associated with result demonstrates that we have only embryo transfer failure. Several touched the surface and we must fully studies concentrate on the vaginal and exploit new technologies to deepen the endometrial microbiota to confirm knowledge. Moreover, as we continue to their role in embryo transfer, and to move from research to clinical practice, better characterize the bacteria involved this analysis is required in order to (Cicinelli et al., 2015; Koedooder et al., improve ART success rates (Haahr et al., 2019b). The introduction of NGS RBMO VOLUME 00 ISSUE 0 2020 9

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