Here you can find ongoing work at REBLAB. Our most important finding so far is the connection between a type 1 pullulanase and Lactobacillus crispatus glycogen metabolism. Find the post here, and accompanying protocols and data here. Find an introductory lecture to the field and my work here. I also send out a newsletter, if you want stay up to date subscribe here
Happy new year everyone! To start this year off I would like to share the work of Alexander Woudstra, a 2nd year master student at the Vrije Universiteit Amsterdam. As part of his internship he has studied glycogen metabolism of the vaginal bacterial species Lactobacillus crispatus and Gardnerella vaginalis. This host-derived glycan is the most abundant vaginal carbohydrate source and thought of as vital as a acarbon and energy source.
Previously, Deborah Jekel and Ritesh Panchoe carried out growth experiments with L. crispatus and G.vaginalis on glucose and glycogen. They noticed that in the presence of glucose, L. crispatus does not break down glycogen . Furthermore, a putative pullulanase type I gene was identified in L. crispatus strains that is involved in breakdown of glycogen. Lastly, it was shown that G. vaginalis has a longer lag phase when grown on either glucose or glycogen.
Alexander further studied growth of these species on glycogen and glucose, looking specifically at lag phase, and preferred carbon source when presented with both glucose and glycogen. He also studied inhibition of growth by the alpha-glycosidase inhibitor acarbose, which inhibited growth on glycogen of both Gardnerella and Lactobacillus but also – surprisingly- showed inhibiton of Gardnerella growth on glucose.
Find his full thesis here. We wish Alexander all the best on his future steps!
Earlier this year, I contributed to the farewell symposium of prof. Hans Westerhoff, a giant in the systems biology field. I did not speak about my research but shared my view on the role of scientific research in society and politics. The whole symposium was taped and posted online. My talk starts around 2 hour and 30 minutes. It was an impressive line up with speakers like Nick Lane (starting at 1:26) and John van der Oost (2:55) and my own supervisor Remco Kort (2:20).
I have recently been reading about the role of the vaginal microbiota and fertility. Multiple reports have now emerged that the microbiome affects succes rates in fertility treatment. Women colonized by lactobacilli seem to have higher success rates when using artificial reproductive techniques such as in vitro fertilization treatment.
One of the papers showing this is this recent publication by the people behind https://artpred.com/ who use an alternative technique for microbiome analysis (IS-PRO) and find low Lactobacillus to be unfavourable for pregnancy outcomes.
Next question is of course: How does this work? What is the mechanism?
There are lots of possible answers coming from immunological, endocrine and gynaecological perspective that will all have some grain of truth. The vaginal microbiome may be a secondary outcome of a host endocrine factor which may result both in lowered Lactobacillus colonization as well as reduced fertility.
I hypothesize that there may be a metabolic mechanism too in which the bacterial members of the dysbiotic microbiome can contribute autonomously to subfertility. Specifically, colonization of high numbers of Gardnerella vaginalis, a glycogen-degrading species, may render the endometrium less conceptive to embryo transfer due to bacterial breakdown of endometrial glycogen stores.
There are a few phenomena on which this hypothesis is based, that I will outline using the respective literature:
- Firstly: our own research shows that Gardnerella vaginalis can breakdown glycogen efficiently. It will most likely secrete its enzymes to access external glycogen.
- Secondly: This is one of several older papers demonstrating an association between subfertility and lowered endometrial glycogen levels.
- This recent review by Matthew Dean stresses the importance of these endometrial glycogen stores for implantation (although most research is done in rodents).
- Women with dysbiotic microbiota have reduced levels of vaginal glycogen. Could this be similar for endometrial glycogen?
- Lastly, at the Keystone conference in Cape Town, Caroline Mitchell reminded us all of the open access between uterus and vagina in women who are not pregnant. Many bigger particles can freely diffuse from the vagina into the uterus, so why would the glycogen-degrading enzymes of Gardnerella not access endometrium glycogen?
However, there is one big phenomenon opposing this hypothesis. Women colonized by Gardnerella do not generally show subfertility (as fas as I am aware), so it may be true only in certain conditions or only in the specific case of artificial reproductive techniques. (or it may not be true at all…)
In short, I am very curious to look at the link between endometrial glycogen and the vaginal microbiome. Could bacterial enzymes reach endometrial glycogen stores and render the endometrium less suitable for embryo transfer or implantation?
To be continued…
Just like last year I gave a talk as part of the Lecture Series ‘Human Microbiome in Health and Disease ‘. Find the slides here.
Ritesh Panchoe and Rosanne Hertzberger, REBLAB / Group of Prof. Dr. Remco Kort, Systems Biology Lab, AIMMS, Vrije Universiteit Amsterdam, O|2 location De Boelelaan 1085, NL-1081HV Amsterdam, The Netherlands
Find the corresponding data file here…
Summary of the finding
After a pregrowth step on glucose, Lactobacillus crispatus strain RL_010 shows a significant lag phase on glycogen compared to glucose. This is consistent with the differential regulation of the glycogen degradation machinery which we reported before. Gardnerella vaginalis does not show such a difference in lag phase but does exhibit a long pause before starting growth on either glucose or glycogen.
Previously we focused on growth of various strains of Lactobacillus crispatus on glycogen and glucose. We identified the role of a type 1 pullulanase, and we observed how glycogen breakdown ability is downregulated in the presence of glucose. Since a few months we have our anaerobic chamber up and running and we are now able to include Gardnerella vaginalis in our growth studies. Gardnerella belongs to the family Bifidobacteriaceae and is one of the prominent members of the dysbiotic vaginal community.
By studying glycogen metabolism in members of both the Lactobacillus-dominated and dysbiotic community we try to understand the fluctuations in vaginal glycogen content in reproductive-age women from a microbial perspective. In general, it is hypothesized that Lactobacillus utilizes glycogen as its primary carbon source to acidify the vagina. However, in the absence of a dominant Lactobacillus in the vaginal community (‘dysbiotic state’ or community state type IV) vaginal glycogen is reduced. We hypothesize that members of the dysbiotic vaginal community may be responsible for this enhanced glycogen degradation. The strong increase in absolute numbers of bacteria in the dysbiotic state increases carbon and energy requirements of the vaginal microbial community. During Ritesh’ internship we analyzed growth of Gardnerella vaginalis and Lactobacillus crispatus on glycogen to growth on glucose.
Gardnerella vaginalis DSM 4944 and Lactobacillus crispatus RL_10 were pregrown in liquid NYCIII medium [PDF] with 0,5% glucose and incubated anaerobically for 48 hours in an anaerobic chamber filled with a gas mix of 95% N2 and 5% CO2. Find full inoculation details here [PDF], in these experiments we inoculated anaerobically.
This preculture was tenfold diluted in NYCIII medium containing either 0,5% glucose, 0,5% glycogen or water as a control in flat-bottom transparent 96 well plate (Greiner 655161). The volume in each well was 380 uL to leave as little headspace as possible. The plates were sealed-off with adhesive film (VWR 60941-072).
This experiment should not be considered as anaerobic. Although the 96-well plate was prepared in the anaerobic chamber, it was subsequently taken to the plate reader outside of the chamber. It is safe to assume that part of this growth experiment should be considered aerobic.
We performed this experiment on three separate occasions and show the results of two individual experiments in the graph.
We find that Lactobacillus crispatus shows a considerable longer lag phase on glycogen compared to glucose after it has been pregrown on glucose (Figure 1A). Although growth of Gardnerella vaginalis resulted in a very long lag phase (>10 hours) on both carbon sources, we did not detect any difference in lag phase between growth on glycogen compared to glucose (Figure 1B).
The lag phase of Lactobacillus crispatus may be explained by the repressed expression of the type 1 pullulanase in the presence of glucose. There are several other findings that support carbon catabolite regulation of this gene associated with glycogen metabolism: 1) previously, we were unable to detect glycogen degradation activity in L. crispatus pellets after growth on glucose. 2) The type 1 pullulanase gene of L. crispatus has a palindromic region upstream of its start site showing similarities to carbon responsive elements in Lactobacillus plantarum https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986886/. 3) The expression of the ortholog pullulanase gene in L. acidophilus is also repressed by glucose. None of these findings provide conclusive evidence but they are all in line with repressed expression of the type 1 pullulanase in presence of glucose.
The ~10 hours lag phase of Gardnerella vaginalis on glycogen is likely due to other circumstances, for instance oxidative stress due to influx of oxygen through the microplate seal. The results do not exclude that Gardnerella regulates its genes involved in glycogen degradation, but under these experimental conditions Gardnerella grows as readily on glycogen as on glucose.
-Warning, wild speculation ahead- This difference in regulation of the glycogen degradation machinery of these bacterial species, that are associated with distinct microbial signatures of the vagina, may have implications on the bacterial dynamics in the human vagina. If a L. crispatus culture encounters glucose in the vagina, it is likely that it will continue growth on glucose and thereby stop expressing its enzymes needed for glycogen breakdown.
After depletion of glucose the bacteria will have to switch back to using vaginal glycogen but need some time to accomplish this metabolic switch. In contrast: if we find in further experiments that Gardnerella vaginalis constitutively expresses its glycogen degradation enzymes, these bacteria can continue growing on glycogen and may even be capable of utilizing both glycogen and glucose in paralel. It may even be beneficial for Gardnerella in bacterial competition to leave trace amounts of glucose during glycogen degradation (“chew more than you eat”), to provoke this metabolic shift in Lactobacillus crispatus.
-Alexander Woudstra is currently repeating these experiments in the anaerobic chamber. Although we do not have a plate reader in there, we do have a 37° heating block.
-A simple experiment that would confirm the role of glycogen in these growth dynamics is by pre-growing on glycogen. This should eliminate the L crispatus lag-phase on glycogen.
-Lastly, Alexander is also attempting to grow these bacteria on both glucose and glycogen. If our hypothesis is correct, Lactobacillus crispatus should degrade glucose first and then switch to glycogen. Gardnerella vaginalis should be capable of utilizing both substrates simultaneously.
Practical notes on the experiments:
The data file has several other experimental conditions that were included. For instance: growth of Lactobacillus iners, growth on galactose and maltose and growth of Gardnerella vaginalis supplemented with supernatants from previous growth experiments. None of these experimental conditions provided results that we would like to discuss extensively at this point.
Ritesh has repeated this experiment on one more day (July 16th), but during this experiment the controls were contaminated. Also, for some unknown reason L crispatus did not show growth on glycogen during this experiment. We have omitted these lines from the graph but the data can be found in a separate tab in the data file.
The increased OD600 measurements at the onset of some growth experiments were likely due to air bubbles under the plate seal.
Some more practical findings from his internship:
-the anaerobic chamber is key in growing Gardnerella vaginalis and Lactobacillus iners. Previously we were attempting to grow them in anaerobic jars, but this means that the handling and pipetting is happening in the presence of oxygen and this means rapid decline of viability. In this project we used the anaerobic chamber for the first time and it worked out well.
-However, we are still experiencing problems with reproducibility of growth of Lactobacillus iners. We still have to find the best way to capture the exponential phase and the optimal inoculation conditions.
-Starch degradation was in this project much more heterogeneous as previously. We need to look into the origins of this heterogeneity, and we think it may have something to do with handling of the cultures. I believe that the findings from Deborah’s internship about starch degradation are still credible and reproducible, but the difference between experiments in this project were much larger and we do not feel confident enough to share them.
-Due to these practical reasons we chose to only share this one observation on growth of Lactobacillus crispatus and Gardnerella vaginalis.
We would like to express our gratitude to Jurgen Haanstra for supervision, advise and positive vibes.
Hi everyone! My name is Ritesh Panchoe and Rosanne was so kind to offer me an internship position. While I am here I will work on the characterization of bacterial enzymes involved in glycogen degradation. The species I will focus on are Gardnerella vaginalis, Lactobacillus iners and Lactobacillus crispatus. That being said, I also want to mention that I have a lot of fun doing my internship while being able to work with Rosanne. Since my specialization is in infectious diseases, and not necessarily in glycobiology, I learn a lot from her while working on this subject.
I am working with these bacteria since early April and already have some findings which we want to share with you. In our assays we use acetate buffer with varying pH (4.0-4.5-5.0) to try to resemble the vaginal pH. We have been using α-amylase (read: my saliva) as a positive control next to B. subtilis α-amylase and for now It looks like α-amylase from both origins has a higher activity in pH 5.0 compared to pH 4.0.
We cultured G. vaginalis in liquid NYClll medium supplemented with glycogen or glucose. After incubation G. vaginalis shows something that looks like film formation in the 10 ml tubes (we incubated roughly 96 hours at 37C). Fun fact: G. vaginalis colonies on plates smells like potatoes!
Also, amyloglucosidase (Sigma product# A7095-50ml) does not work anymore if it is stored at -20 ℃. While this might seem as an open door because it is stated on the product label we should store at 2-8 ℃, we thought an extra warning could be of use (since some enzymes can be stored at freezer temperature).
Currently we’re trying new methods for analysis of glycogen degradation enzymes. Ultimately, we would like to be able to differentiate between the enzymes of various vaginal bacteria, since this allows us to identify them in human specimens.
The starch-iodine assay we use only gives information about the total starch breakdown after a set incubation time. We want to be able to measure enzymatic rates over time in our samples, therefore we included the AZCL-amylose assay (protocol can be found here). We had some challenges with this assay.
Our positive controls (human saliva 100x diluted in PBS and B. subtilis amylase (60u/ml) do show amylose breakdown (see figure). We have performed this test three times, all times with the same result. But the bacterial pellets of L. crispatus give some weird results. Despite the fact that starch (which is composed of amylose and amylopectin) can be degraded, the bacterial samples included in the AZCL amylose assay showed no breakdown of amylose. This is weird.
We hypothesized that the xanthan gum applied to our buffer might be the problem for the bacterial enzymes. Rosanne has added this xantan gom as a bulk ingredient to prevent pelleting of the insoluble granules. We tested this by omitting the xanthan gum in the buffer. As a consequence, the AZCL-amylose granules precipitate as expected. Therefore, we decided to do an OD measurement at 590 nm before the start of incubation. We then incubated our samples (in a shaker at 30 ℃, we currently do not have the tools to shake at 37 ℃) and measured OD after 21 hours. There was no increase in OD values (again, controls showed correct signals), although the positive controls did show some breakdown of the granules. Conclusion: it is unlikely that the xanthan gum interferes with the assay. We hypothesize now that it may have to do something with the structure of the amylose (which is very dense).
In the coming weeks I will perform growth experiments on G. vaginalis and L. iners, followed by HPLC of these samples. I will follow-up as soon as I have interesting findings. I will keep you posted!
I am Deborah Jekel and I am a third-year bachelor student Health and Life sciences at the Vrije Universiteit in Amsterdam. The last couple of months I have had the pleasure to work with Rosanne and do an experimental internship. My project comprised of researching the regulation of glycogen metabolism of Lactobacillus crispatus – a prominent member of the vaginal microbiome. Teaser, it might possibly be carbon catabolite repression!
Because this internship was part of an Open Kitchen Science research project, all figures, data, methods and protocols are shared. Find the entire dataset at FigShare:
First, I spend some time on finding the optimal growth conditions for L. crispatus. I compared two methods, the use of Eppendorf tubes versus the Anaerobic jar. I cultured L. crispatus in NYCIII medium supplemented with glucose (find protocol here) and used Eppendorf tubes that I filled completely to reduce oxygen levels to a minimum and an Anaerobic jar that I pulled vacuum and filled with CO2 + N2 gas. Results presented that L. crispatus grows faster and better in the Anaerobic jar than in the Eppendorf tubes (figure 1). Of course, this is sort of an open door since we know that L. crispatus is an anaerobic bacterium, but these experiments were also good for me to get acquainted with working in the lab and to start off easy.
As you may know glycogen is an abundant carbohydrate in the vagina, can L. crispatus utilize glycogen to colonize the vagina? Earlier experiments completed by Rosanne already showed results that some strains can grow on glycogen-supplemented growth media (see blogpost here). I did growth experiments with L. crispatus strain 9 and 10 cultured in NYCIII medium 1.1x supplemented with glycogen, glucose or demi water (see last paragraph of NYCIII protocol). Results showed that strain 9 cannot grow on glycogen while strain 10 can, see Figure 2. This corresponds with the results of Rosanne. Could the lack of growth on glycogen by strain 9 be caused by the frameshift mutation of strain 9 in the putative type I pullulanase gene? We don’t know yet. Unfortunately, I have not been able to calculate growth rates as a result of a lack of measurements during the exponential phase of growth.
We also know that the vagina has a low pH which may be caused by lactate possibly produced by bacteria. I looked at lactate concentrations in the supernatant of L. crispatus strain 10 after growth on glucose and glycogen using HPLC (find the protocol here). We see that growth on glucose shows a higher lactate concentration than growth on glycogen, see Figure 3, but this may also be explained by the difference in cell density (optical density at 600nm) that was on average 0.4. Could it be that L. crispatus also utilizes glycogen to produce lactate and acidify the vagina?
In addition, I wanted to investigate the enzymatic activity of L. crispatus. I used starch as a proxy for glycogen and carried out enzymatic assays with an iodine staining (find protocol here). I incubated the pellets and supernatants of L. crispatus strain 9 and 10 in a starch solution after growth on glycogen, glucose and demi water. I discovered that only strain 10 could degrade starch after growth on glycogen.
After seeing the results of the starch assay, I was curious whether growth on other carbon sources could also induce starch degradation activity. I examined maltose, maltotriose and galactose. Maltose and maltotriose are of interest because they are breakdown products of glycogen and galactose has the same structural formula as glucose but a different metabolic pathway. Interestingly, the presence of galactose during growth of L. crispatus strain 10 also induces starch degradation activity while maltose or maltotriose did not! So, some carbon sources induce starch degradation and others do not, could this be carbon catabolite repression? This would mean that L. crispatus uses the carbon sources that are most easily accessible and allow for fastest growth while inhibiting the synthesis of enzymes involved in the metabolism of secondary carbon sources. This makes sense, right? Why would L. crispatus waste energy on grinding (glycogen) when there are also mashed and grounded nutrients available (glucose, maltose or maltotriose)?
That is all we have on the speculation of carbon catabolite repression in L. crispatus for now.
One more result I wanted to share: I have tried growing on two carbon sources simultaneously and performing a starch assay after 48 hours (find data here), but had some trouble interpreting the results. What I intended was to try to catch the switch from one carbon source to the other. I was hoping to find the degradation activity to correspond with the second carbon source that I added in addition to glycogen. This would prove glucose repression, instead of other regulatory mechanisms, since in the presence of glycogen and glucose the activity would remain repressed. Unfortunately, I wasn’t able to get a meaningful result, possibly because I couldn’t distinguish between growth on the different carbon sources. Experimental design needs improvement! In the future I hope we can measure glycogen concentrations and determine starch- and growth rates to be able to pinpoint exactly the moment this activity is switched on or off. Nevertheless, data of the different experiments are shown in the FigShare file and are also mentioned in my thesis (name: “double carbon sources”).
I have learned a lot these past 4 months and I want to thank Rosanne for giving me this opportunity. If you’re interested in reading my thesis, you can find it with all other data files on FigShare. Enjoy!
After visiting the Keystone conference I was wondering if genetic factors of the hostess could be related to her vaginal microbiome. Janneke van de Wijgert pointed me towards the HELIUS study, that has both SNP data and vaginal microbiome available for a few hundred participants. I wrote a proposal to compare these datasets. I hope we will be able to perform this study this year. If you are working on something comparable, let me know and perhaps we can join forces! If you have any other proposals of promising SNP’s that we should take into account in the analysis, I would be very interested to hear about this.
Summary Evidence is accumulating that glycogen released by endometrium and the cervicovaginal epithelium functions as an important carbohydrate for vaginal bacteria. Luminal glycogen is found to vary both with hormonal status and with the bacterial make-up of the reproductive tract. Here we propose to study whether host genetic factors that are involved in glycogen metabolism regulation show correlation with vaginal microbial signatures.
The multi-ethnic HELIUS cohort of Amsterdam is uniquely positioned for this analysis . The vaginal microbiome of several participants was characterized previously , showing a variation in Lactobacillus-dominated microbiota and Lactobacillus-depleted dysbiotic states. This latter microbial state is associated with an increased risk of preterm labor and acquisition of sexually transmitted infection. Of a subset of these women (346 in total) genome wide SNP data will become available this year. We propose to analyze whether women with Lactobacillus-dominated vaginal microbiota are significantly more likely to carry certain alleles in the most prominent SNP’s of the TCF7L2 gene that are associated with type 2 diabetes. This preliminary analysis data could inform novel studies within this same HELIUS cohort to study the correlations between host vaginal glycogen synthesis, lactate concentration and vaginal microbiome.
Hypothesis: Glycogen functions as a carbon source for Lactobacillus species colonizing and acidifying the human vagina. We hypothesize that genetic changes in the glycogen synthesis pathway and its regulatory factors can affect the ability of Lactobacillus species to acidify the vagina and prevent changing to a bacterial vaginosis microbial state. Moreover, unpublished experiments have shown that bacterial members of this dysbiotic state such as Gardnerella and Prevotella also benefit from glycogen to grow to high numbers, produce biogenic amines (odor), cause inflammation (recruiting target cells for HIV to the mucosal surfaces), desialylation of the mucosa and exfoliation of the vaginal epithelium (discharge). Lower glycogen stores may partially explain why certain women with this microbial state have less symptoms, such as odor, discharge and inflammation, than other women.
Background During reproductive years glycogen is synthesized by the tissues of the reproductive tract [3, 4] and is essential for early embryo implantation and development. Glycogen shed into the vaginal lumen functions as a carbon source for vaginal lactobacilli such as Lactobacillus crispatus [5, 6] and iners but also for Gardnerella vaginalis and Prevotella bivia (unpublished data). Glycogen levels are reduced in women who have Lactobacillus-depleted microbial states  which could be explained by the interplay between host factors (glycogen synthesis) and bacterial factors (glycogen breakdown) .
It has been well-established that ethnicity is an important factor determining the odds of a woman to have bacterial vaginosis. Women of African descent are found to have more Lactobacillus iners than Lactobacillus crispatus and are more often colonized by a Lactobacillus-depleted community [9-11]. These women often have symptoms such as discharge and odor, and are at higher risk of acquiring sexually transmitted infection [12, 13], are more likely to have persistent HPV colonization [14, 15] and are more likely to give birth prematurely [15-17].
There has been considerable effort to understand the variation of health outcomes and symptoms amongst women with comparable vaginal microbiota. Most studies were directed at understanding this variation from a bacterial perspective, for instance by looking at genetic variation amongst bacterial isolates of Gardnerella vaginalis [18, 19] and Lactobacillus crispatus [6, 20]. We believe that the dataset we propose to analyze provides a unique opportunity to also take host factors into consideration.
Variations in glycogen metabolism due to host genetic factors may affect both hepatic glycogen accumulation as well as endometrial and vaginal glycogen accumulation. For a long time it was thought that progesterone was the main controlling hormone for glycogen synthesis in the endometrium [21, 22]. A recent study has now found that the progesterone/glycogen link is only indirect. Insulin is the hormone that inactivates glycogen synthase kinase 3β thereby activating glycogen synthase, similarly in liver tissue as well as in endometrial cells .
These commonalities lead to our hypothesis that there may be a genetic relationship between bacterial vaginosis and type 2 diabetes. In genome-wide association studies into genetic risk factors for type 2 diabetes several SNP’s have been identified. The most robust and consistent SNP’s are found in the TCF7L2 gene. Certain SNP’s in this transcription factor (elsewhere referred to as a TCF/LEF or TCF7) confer up to twofold increased risk of developing type 2 diabetes . TCF7L2 knockout mice had reduced glycogen stores in the liver . People carrying the risk allele synthesized less insulin in response to a glucose challenge test [26, 27]. This lower insulin production may have consequences for glycogen accumulation in the reproductive tract. Less glycogen synthesized means less carbohydrate for vaginal Lactobacillus species to acidify and Gardnerella to cause symptoms, odor and discharge. As of yet the ethnic differences in type 2 diabetes (where African ethnicity are at higher risk) were not able to be traced back to any SNP’s. The link between one specific TCF7L2 SNP’s (rs7903146) and type 2 diabetes was found in various regions of the world.
This would be the first study into host genetic factors and vaginal colonization.. The strength that this analysis offers is that it does not only include people from European heritage but includes a variety of groups with Asian, African and European roots. Clues from this study may inform bigger studies where we could look at the influence of genetic factors on lactate and glycogen taking into account the different ethnic groups and vaginal colonization patterns. If glycogen metabolic commonalities are found between diabetes and BV, this opens up a wide range of new treatment options for BV and related conditions, and possibly novel applications for established diabetes drugs including amylase inhibitors, insulin and metformin.
To study the SNP’s of the TCF7L2 gene and their relationship to vaginal microbiome. The HELIUS cohort will this year have both datasets available for 346 women. The vaginal microbiome of these women was previopusly studied and the authors are willing to share these data too. We propose to look at three SNP’s of the TCF7L2 gene, specifically rs7903146, rs 4506565 and rs12255372 and study whether their relationship with having a Lactobacillus-deplete (case) or Lactobacillus-dominated (control) microbiome. In case the study cohorts permit (and the odds ratio), we could differentiate further into ethnicity. In case we find a strong signal that would warrant further study, we could look at glycogen concentration, lactate concentration, vaginal microbiome and ethnicity. There are many more vaginal swabs available and SNP data for 12,000 participants in total.
Collaboration and transparency This is an Open Kitchen Science project, meaning that the aim is to maximize collaboration and transparency. All data files, methods and results, either positive or negative, will be released and published prior to peer review (either on a preprint-server or on blog). Regular information about proceedings will be shared (including this proposal, without financial details) on blogs.
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