This presentation is Impact of Vaginal Estrogen on the Urogenital Microbiome in Postmenopausal Women with Recurrent Urinary Tract Infection by Joan et al. Thank you. These are our disclosures. It is well known that UTIs affect up to 50% of women in their lifetime, and 2 to 5% of those experience recurrent UTIs. We also know that postmenopausal women are disproportionately affected as menopause changes the vaginal hormonal milieu, leading to a change in bacterial flora with decreased lactobacillus, increased pH, creating a hospitable uropathogen environment. We can replace vaginal estrogen, and this can restore the healthy environment, and this can be used to prevent UTI. Our objective is to describe the effects of vaginal estrogen on the urogenital microbiome in postmenopausal women with recurrent UTI. We wanted to look at alpha diversity, or diversity within the samples, beta diversity, diversity between the samples, and lactobacillus predominance. We hypothesized that there would be significant changes across these parameters, in both the paired baseline and six-month groups, as well as between treatment successes and failures. This was a nested pilot microbiome study of postmenopausal women with recurrent UTI enrolled in an RCT of vaginal estrogen versus placebo for treatment of their recurrent UTI. There were 17 women from the parent study who were both compliant with therapy and had baseline and six-month postvaginal estrogen treatment specimens available for analysis. The average age was 68, and they were mostly overweight, Caucasian, and non-smokers. We defined the treatment failure as having a UTI after at least three months of vaginal estrogen treatment, and 35% of this group failed treatment. Our samples were clean-cache, they were preserved in a DNA preservative, they were frozen and stored, and then they were sequenced with 16S rRNA gene sequencing and analyzed with QIIME 2. Our outcome was changes in diversity and lactobacillus, and we looked at the first group from baseline to six months post-treatment, as well as between treatment successes and failures. We measured alpha diversity with FATE's phylogenetic diversity index and beta diversity with unweighted unifrac distance metric, and we looked at percent lactobacillus from taxonomy bar plots. Here we see the changes in alpha diversity between the paired baseline and six-month samples, or the diversity within the samples, and we see that there's no significant difference between alpha diversity in these groups. This graphic demonstrates how beta diversity, or beta diversity which is between the samples, is measured. You can see the red dots are representing the baseline samples, and the blue dots are representing the six-month vaginal estrogen samples. The lines between the two are representing the differences in beta diversity. One can also appreciate from this graphic the differences in the individuals even at baseline. This box plot shows the previous data in box plot form, and shows that there's no significant differences in beta diversity between these two groups. Here we are looking at overall lactobacillus proportions in the baseline and six-month groups, with lactobacillus being represented by the light purple bars. The average proportion of lactobacillus was greater in samples after six months of vaginal estrogen when compared to baseline, with baseline having 7.3% of bacteria being lactobacillus, as opposed to 32.9% after six months of vaginal estrogen. And this represented a 25.6% proportional increase, which was statistically significant. Switching gears now to look at the paired successes and failures, again looking at alpha diversity, we did not find a significant difference. And again looking at beta diversity, now with the successes represented in the purple and the failures represented in the orange, we see there was no significant difference in beta diversity either. Looking at overall lactobacillus proportions between the failures and successes and baseline in six months, we see that there was lower baseline proportional lactobacillus concentration seen in failures at only 0.8% at baseline compared to successes that had 10.9% lactobacillus at baseline. Looking at the increases over zero to six months, there was a greater increase from baseline in lactobacillus in failures, which increased by 45.5% compared to successes, which increased by 15.8%. The strengths of our study include that we had paired samples from women in a randomized controlled trial. Our study was limited by a small sample size of only 17 women, and we did use non-catheterized urine specimens, which we know can have contributions from both vaginal and urethral flora. In conclusion, lactobacillus concentrations in the urogenital microbiome increased after six months of vaginal estrogen in postmenopausal women with recurrent UTI. Higher baseline lactobacillus may be associated with successful treatment of recurrent UTI in vaginal estrogen after six months. However, due to our small sample size and pre-specified significance cutoffs, we were not able to identify significant changes in diversity, but hope this study may help design future studies. Thank you. First off, I'd like to thank my co-author, Dr. Chen, for allowing me to present the work today. So as many people in the rest of this room probably know, the work to characterize the urinary microbiome has been largely done using transurethral catheterized urine. However, since collecting urine by this method is still minimally invasive, it's largely restricted us to patient populations. Therefore, to move into doing research with general populations or conducting longitudinal studies, it would benefit us to switch to using voided urine. However, it's been shown that voided urine does not resemble bladder urine. And for this reason, our group set out to characterize the urethral microbiota of women so that we could better interpret voided urine. I have no disclosures. Okay. So first we aimed to characterize the microbiota of urethral specimens from female participants. And then we used those microbiota characterizations that we saw to compare the microbiota of the urethra to that of the bladder and vulvovaginal skin. Finally, we used voided urine specimen and compared that to the microbiota of the three anatomical sites to see which niche was being represented more often. In this study, we recruited 50 female participants who contributed four sample types. We used extreme voided urine, a periurethral swab which represented vulvovaginal microbiota, a urethral brush specimen, and urine taken by transurethral catheterization. All samples were cultured via EQIC and bacterial isolates were identified via MALDI-TOF mass spectrometry with our primary outcome being the measure of microbiota, comparison of microbiota between the four sample types. We had one participant who was excluded from analysis because she was unable to provide all four sample types. So all the analysis that follows is with 49 participants. The median age was 55. These women were mostly Caucasian and mostly postmenopausal. After obtaining culture results, we first compared overall diversity between each of these samples. And to do this, we used Ray Curtis Dissimilarity Index. This index compares microbiota presence and abundance between two sample types within an individual. And it's ranked on a score of 0 to 1, with 0 meaning the two sample types are completely similar and 1 meaning they are dissimilar. So for this first comparison, we have the bladder compared to the urethra, signified by B versus U. And you see that the bladder is most often dissimilar from the urethral microbiota. We see this trend again in comparing the bladder to the periurethral microbiota, which is in yellow, and the bladder to the voided microbiota, meaning that the bladder represents a distinct niche within the lower urinary tract. However, when comparing the urethral microbiota to the periurethral microbiota, we see a moderate dissimilarity, meaning there's no difference between overall diversity between the two sample types. The urethral microbiota compared to the voided urine microbiota shows us that the urethral microbiota are more often dissimilar than the voided urine microbiota. However, the voided urine microbiota are often similar to the periurethral microbiota, suggesting to us that voided urine is most often representative of the periurethral niche or bovinal vaginal niche. To further analyze these differences we are seeing, we compared microbiota frequency and abundance for the top ten most prevalent genera in each specimen type. You see here the comparison of frequency of each microbiota with the bladder in blue, the urethra in orange, periurethra in gray, and voided urine in yellow. The general trend, I'm sure you can see, is that each genera is isolated at lower frequencies from the bladder, but similar frequencies in the other three sample types. However, this trend breaks when you look at the Escherichia, Gardnerella, and Lactobacillus, suggesting that these three genera are found evenly throughout the lower urinary tract. However, when looking at the abundance of these genera, we see the Escherichia breaks from this trend of being a generalist, and instead is found in much higher abundance in the bladder, denoted by the yellow bar here, than the urethra or periurethra. It's picked back up again in the voided urine. Conversely, Gardnerella and Lactobacillus are found generally throughout these samples. And again, with this abundance comparison, you see that voided urine is not representative of the bladder, or a mixture of the urethra and periurethra. To summarize, we found that the bladder and urethra represent distinct niches within the lower urinary tract. The urethral microbiota resemble periurethral microbiota, and midstream voided urine resembles periurethral skin. Some microbiota prefer to reside in the bladder, Escherichia, while others prefer to reside in the urethra or periurethra, with some microbiota being found at similar levels throughout the lower urinary tract. And in conclusion, the urethra represents a distinct microbial niche within the lower urinary tract. Clinical implications of this should be studied, and past methods to deconstruct voided urine mathematically are not feasible from this study, so we should focus on developing cleaner catch methods for voided urine. Thank you, and we have no disclosures. Mesh-augmented prolapse repairs result in good anatomical outcomes at the expense of possible mesh erosion. Currently, suture is the only modality used to anchor mesh to the vagina. However, suture material magnifies foreign body response and may increase material burden and potential risk factor of mesh erosion due to mesh wrinkling. Gelatin methacryl hydrogel, also known as GelMA, resemble native extracellular matrix with cell-attaching and metalloproteus-responsive peptide motifs that allow cells to proliferate and spread. GelMA has been extensively studied as an adhesive compound with hemostatic properties involving the lung, kidney, and vascular systems, but has yet to be studied as an adhesive agent for use in the vagina. The objects of this study were to evaluate the effectiveness of GelMA as an adjunct to vaginal mesh, to examine the inflammatory response and extracellular matrix composition associated with GelMA compared to sutures, and to examine the biomechanical properties of mesh-implanted vagina when mesh is anchored with GelMA compared to sutures. Guinea pigs are randomized to undergo anterior coporaphy with mesh augmentation using either GelMA or suture for mesh fixation and epithelial re-approximation. The photo on the left side of the screen depicts a guinea pig vagina with an anterior wall coporaphy immediately after mesh fixation with GelMA. The photo on the right side of the screen demonstrates mesh fixation using suture. Vaginal tissues were harvested for analysis at 4 days, 1 month, and 3 months after mesh implantation was performed. An additional 5 guinea pigs were used as control specimens and did not undergo a surgical procedure prior to tissue harvesting. Tissues harvested at 3 months also underwent biomechanical testing. These are cross-sections of vaginal tissue with mesh implant stained with hematoxylin and eosinophil. Both slides are from tissue harvested 1 month after the assigned procedure. The slide on the left depicts a guinea pig that was randomized to the suture group, while the right slide was randomized to receive GelMA. Please note the mesh fibers as illustrated by the asterisks. No mesh erosions were seen in any animal specimen and mesh remained anchored to the underlying vaginal tissue regardless of modality of mesh fixation used. These two images are of tissue samples harvested 3 months after mesh fixation. The tissue slide on the left underwent mesh fixation with suture and GelMA was used in the tissue sample shown on your right. The externally erosional sphincter is outlined in yellow. GelMA and sutures were both present at 4 days and 1 month, however, both materials were completely dissolved by the 3-month time period. This slide illustrates the increase in angiogenesis in the suture compared to the GelMA group based on blood vessel count. The top row depicts mesh fixation with suture while the images on the bottom row were from the GelMA group. The first column depicts tissue samples harvested after 4 days, the middle column are tissue samples harvested 1 month, and the last column are tissue samples harvested 3 months after their assigned procedure. The arrows demonstrate blood vessels. Note the larger amount of blood vessels in the slides from the earlier time points on the left side of the screen. There was also increased angiogenesis group in the suture group compared to the GelMA group. To evaluate the inflammatory response, we immunostained sections with CD45, a marker of leukocytes, shown here in green. In this representative photomicrograph, the suture filaments also stain green. The important aspect, however, is that numerous CD45 positive leukocytes represented by yellow arrows aggregate near the suture in mesh pores. To further evaluate the inflammatory and foreign body response, we immunostained sections with CD68, a marker of macrophages, shown here in green. In this representative photomicrograph, the mesh pores are depicted with white asterisks. Numerous CD68 positive macrophages aggregate near the suture in mesh pores. To examine the collagen content of extracellular matrix, slide were stained with Picrosirius red. Note the increased intensity of red stain in the GelMA group on the left side of the screen compared to the suture group on the right. As demonstrated by this graph, mesh implanted vaginas using suture as an anchoring mechanism had increased vaginal stiffness compared to the mesh implanted vaginas which used GelMA to anchor the mesh to the underlying vaginal tissue. The small sample size, in addition to biological variability, limits the results of our biomechanics. The absence of longer term data beyond three months may have limited our ability to evaluate for mesh erosion. The strengths of our study include the examination of a novel material which could be used to facilitate mesh anchoring for vaginal prolapse repairs. We included evaluation of biomechanical properties, inflammatory responses, and composition of extracellular matrix. In conclusion, GelMA is a safe alternative to suture for anchoring of prolapse meshes to the vagina. Studies including longer time points are needed to evaluate for the long term durability of repairs using GelMA. Further studies including large animal preclinical models are necessary to assess the feasibility of use in humans. Thank you. Thank you. These are my disclosures, and I should point out that this was funded by a PFD grant, the Interstitial Cystitis Foundation. And I'm giving this talk for the first author, Kristen Jacobs. So most of you, or probably all of you, know that interstitial cystitis, painful bladder syndrome, is a significant medical problem and that it is a diagnosis of exclusion. And this is typically by the standard urine culture approach. But over the past several years, we and others have shown that the bladder is not a sterile environment and large proportions of women have detectable bacteria in their bladders. So we aimed to determine whether the female urinary microbiome differed between women with ICPBS and those that did not. And our secondary aim was to ask whether or not the microbiome of the urinary tract was associated with disease severity or any particular symptoms. I'm going to give you the bottom line. We have no compelling evidence that bacteria play a role in the etiology of ICPBS. So here's why we think that. First of all, here's our recruitment criteria. So over the age of 18 years, they identified as having ICPBS and the absence of any infection. Some of the exclusion criteria, what I'd like to point out is that we gave a four-week washout for antibiotics along with all the other obvious exclusion criteria. The women that participated filled out lots of different ICPBS questionnaires. The ICSI, which asked questions about symptoms over the past month. ICPI, which does the same. The GUPI, which asked questions about symptoms over the last week. And UTISA, which asked questions over the last 24 hours. So we detected microbes in catheterized urine using the expanded quantitative urine culture that Bailey mentioned. Compared to the standard urine culture, it uses 100 times more volume, a variety of different media compositions, a variety of different atmospheric conditions, and we incubated for twice as long, 48 hours instead of 24. So this is an example. On the left is the standard urine culture test for a urine sample. On the right is the same sample done by one of the conditions by EQUA. Now, the ClinLab would have said no growth, and you might have interpreted it as sterile. But that urine's not sterile. One other factor is that EQUA can detect yeast. It can detect candida. We detect it relatively often. We also used 16S ribosomal RNA sequencing on the same samples. I'm going to show you only the data from the EQUA analysis because of time. All right. So demographics, there were 89 individuals, 49 in the IC cohort, 40 in the control. It turns out that the IC cohort was a little bit older than the controls. They were essentially the same in terms of race and ethnicity. The control cohort was a little bit more premenopausal than the IC cohort. No difference in parity. So after we identify the organisms by the method that Bailey mentioned earlier, we then cluster the different communities in each individual using the same Bray-Curtis dissimilarity index. So a couple things you need to notice is that a lot of... It doesn't work. Well, okay. There are a lot of individuals that were negative. We didn't detect bacteria. But in both groups we saw the most common organism was Lactobacillus. There's Gardnerella in both. There's E. coli in both. The major difference is that the IC cohort had a streptococcus. More on that in a moment. So when we looked at... We name the community a ureotype by the most common member of that community. So if it's mostly Lactobacillus, it's put in the Lacto ureotype. So the negative individuals, as you can see, that the individuals that had IC that had no symptoms, they were less likely to be negative. And all of these comparisons are significant. So what you see is that individuals with severe and moderate symptoms are negative at about the same rate as individuals that were in the control. Lactobacillus, there was no difference. Streptococcus was present in the IC with no symptoms, but the control and the individuals with severe symptoms didn't have streptococcus. Something that's interesting is that the urine pH of the control group was higher than that of those with IC. And there was no difference in biomass. So the pH was significantly lower in the IC cohort, and that's consistent with treatments that involve deacidifying urine. When bacteria were isolated, Lactobacillus was the most common ureotype in both cohorts, but Lactobacillus was not associated with symptomology. Streptococcus was the only ureotype that differed significantly, in ICPBS. And I'd like to thank Luric and our mascot who speaks for the piece. Applause This will be presented by Dr. O'Shaughnessy. Good afternoon. Thank you for allowing us to present our research. These are the disclosures. The U.S. Food and Drug Administration has removed transvaginal mesh for pelvic organ prolapse repair and has removed transvaginal mesh products from the market. Polypore hydroxybutyrate is a fully absorbable material that resembles synthetic polymers and can be configured to have similar handling properties P4HB is a natural polymer produced by E. coli bacteria by fermentation. Despite P4HB being a natural polymer, it strongly resembles the functionality of synthetic polymers. P4HB is fully absorbed in vivo by 18-24 months post implantation and is primarily degraded P4HB is subsequently cleared by the prep cycle resulting in water and carbon dioxide. The safety of P4HB has been well established in other soft tissue applications such as hernia repair and plastic and reconstructive surgery. The objective of this study was to evaluate the histological relationship between the abdomen and vagina. This was an IACAC approved protocol 8 New Zealand white retired breeder rabbits were used. Animals were divided into 2 groups. Group 1 had polypropylene implanted in the anterior vagina and P4HB implanted in the posterior vagina. Group 2 had the opposite P4HB implanted in the anterior vagina Half of the rabbits had abdominal samples and the abdominal samples were 2 polypropylene and 2 C4HB. Rabbits were sacrificed at 18 months post implantation. Gross necropsy was performed and the mesh exposures were noted. Specimens were divided equally between histological and biomechanical testing. They were same with H&E, Mason's trichome, and scored by a blinded pathologist. The collagen 1 to 3 ratio was calculated using ImageJ. An Instron machine was used to perform biomechanical low to failure testing. And then we calculated statistics. This is a gross examination at the time of 18 months of the sacrifice. The first picture shows the abdominal placement of the mesh the second shows the anterior vaginal Gross exam revealed full reabsorption of the P4HB mesh vaginally. There were 2 polypropylene and 1 P4HB vaginal exposure and with the vaginal exposure for P4HB it was only the piece of mesh that was left and there were no abdominal exposures noted. These are the histology scores and they were very similar for both groups as expected there was no acute inflammation. The collagen 1 to 3 ratio was calculated and it's important because a lower collagen 1 to 3 ratio may lead to reduced stability and subsequently increase the risk of recurrence. And vaginally there was no difference between polypropylene and P4HB. Biomechanically there was a difference in the abdominal versus vaginal biomechanics but when you compared vaginal polypropylene to P4HB there was no difference noted. In conclusion, the P4HB graph demonstrated less vaginal exposures than the polypropylene and a comparable histologic response in the rabbit vagina at 18 months post implantation. The P4HB graph appeared to result at a faster rate when implanted vaginally than abdominally. P4HB placed abdominally as well as polypropylene placed abdominally performed different than vaginally while polypropylene and P4HB vaginally were similar. Overall the data suggest that P4HB is a promising biocompatible alternative graph material to polypropylene for the surgical correction of prolapse. Thank you. Good afternoon everybody. I'd like to thank the committee for this opportunity to present our work. We are grateful to the NIH support. Previously we have shown that pore geometry is a critical factor contributing to the outcomes of mesh implantation. Diamond pores and hexagonal pores are highly unstable configurations that lead to pore collapse and unloading. In contrast, the square pore is relatively stable pore configuration, maintaining its shape when loaded. However, if the square pore is rotated 45 degree, as shown here, into a diamond configuration, the pore becomes highly unstable and collapsed with loading. This indicates the importance of pore geometry. We hypothesized that altered mesh properties associated with pore collapse would negatively impact the host response with increased inflammation and tissue degradation. To define the impact of pore geometry on the host immune and matrix remodeling response, the rasterial mesh was implanted in recessed macaque with three different pore configurations, as shown here on the left. R0 is a stable square pore configuration, R45 is a diamond pore configuration that becomes unstable and collapsed with loading, and RPD is a pre-deformed pore that was loaded and collapsed prior to implantation. Sham-operated animals were used as controls. The mesh were implanted with acrylopopexy and loaded to 10 newton. The mesh tissue complexes were harvested at 12 weeks and prepared for histomorphological and biochemical analysis. The H.E. staining showed that all mesh groups were associated with mild to moderate infiltration of inflammatory cells around mesh fibers. No difference in growth morphology were observed between mesh groups. With Luminex multiplex assay, we found that relative to Sham, all mesh groups showed increased levels of pro-inflammatory cytokines and chemokines, while the anti-inflammatory response was not significant. In terms of matrix remodeling outcomes, the pre-deformed group showed 30% less of collagen with increased NLP activities relative to Sham, consistent with tissue degradation. Meanwhile, the level of TGF-beta-1 was also increased in this group, suggesting repetitive injury repair and fibrosis. All groups had similar levels of elastin content and sulfated glycosaminoglycan content. In conclusion, all mesh induced a persistent predominant pro-inflammatory response. Pre-deformed mesh with collapsed pores had the most negative impact on vagina with increased matrix remodeling favoring degradation. Meshes with stable pore configurations under loading conditions in vivo were associated with improved outcomes. And thank you for your attention. This will be presented by Mark Kipcho from the University of Toronto. Thank you very much for giving us the opportunity to present our data at this AYUGA meeting. We have no conflict of interest. So cell-based therapies hold the potential to be one of the next big things in treatment of pelvic floor disorders. However, the first thing you have to think about is what cell type do you use to cure what condition, and as important, from what part of the patient's body you can get the cell type to have enough autologous cells to treat a patient. So in the past, people used muscle biopsies or liposuction to get adipose or muscle-derived stem cells. The problem is these biopsies can be accompanied by severe side effects, like pain, inflammation, or infection. So what we presented on last AYUGA is a novel approach. We thought, why can't we use IPS technology that is available since 2006 now to derive sufficient amount of autologous cells to treat patients with pelvic floor disorders. So what we showed on the last AYUGA is we took a void UN sample of women with gestational incontinence, isolated these cells, and reprogrammed them into IPS stem cell lines. So these IPS stem cell lines, when you once have them, you can freeze them, you can expand them to unlimited amounts, and when the direct differentiation protocols are available, you can potentially differentiate them into any cell type you need to cure pelvic floor disorders. And what we showed on last AYUGA was we differentiated them into fibroblasts via an embryoid body formation assay, which we have published several times. It's very easy. You get 100% patient-specific fibroblasts from the IPS lines. The other arm is we performed myogenic differentiation towards skeletal muscle cells. And several protocols are available since 2014. We adapted them, and we showed that we can differentiate them into patient-specific myoblasts and myocytes. And what we did then, we said, okay, we can in theory do it, but now we need to get sufficient numbers, so we have to improve the isolation and expansion protocols. We need a way to track them when we inject them into a RAD model, and we used immunocompromised R and U RADs to test these cell lines. So the first thing we put a lot of effort in is how do we track cell lines when they are injected into RADs? Because very often in publications you get, after three months, you get a functional assay like FEP assay, and you get histology of a treated urethra, but it's not very often, it's not so clear, if this healing is due to the injected cell lines or to spontaneous regeneration, for example. So we need a way to trace our cells. And the easiest way to do it is to label them with iron rhodamine B, that the colloids of supermagnetic particles used in MRI applications, which is taken up by these cells, and which is non-toxic. You don't have to transfect the cells, there's no transgene induced, and you can, which I'll show in the next slide, you can detect them in histologies. So and then we injected our fibroblasts and myocytes into the periureal region of R and U immunocompromised RADs. And this shows what it looks like, an injection of fibroblasts after four weeks. The blue stain on the left side shows our labeled cells. It's a very easy, very sensitive, and highly specific way to detect your cells in the periureal region of these RADs. And we also showed, by using human-specific proteins against mitochondria and human nucleoproteins, that these are really human cells injected into the RAD. And very important, using Ki-67, these cells stay proliferated. So when we used the same with myocytes, that was a little bit tricky. We had first to enrich them, and we used the CD56 high, CD57 negative sorting strategy. So from 5 to 10 percent, we can enrich them to 50 to maximum 75 percent in cultures. That's the maximum you can get. And when we inject these cells into the RADs, again, you can see, we can easily track them after labeling with the iron colorate. This, after two weeks incubation, you can detect them. They are humans, these are the human-specific cells, and they are proliferative. So that means we have now a way to test all our cell lines into stress-unary incontinence models of RAD models. So the injections have been done, the RADs are on the way, and we hope that in the future we can use this model and this way to differentiate cells and label them to test different combinations of cells, fibroblasts, muscle cells, maybe smooth muscle cells, to see what combination of cells is best to treat stress-unary incontinence or other pelvic floor disorders in the future. Thank you. Good afternoon. I'd like to thank the program committee and Dr. Lukatz in particular for the kind invitation to come and speak with you this afternoon. I'm from the University of Texas Southwestern Medical Center, which is in Dallas, Texas, and today I'll be speaking about the use of vaginal estrogens in women with a history of breast cancer. These are my disclosures. Of note, Pfizer Incorporated, which does provide me some research support, is the manufacturer of Primarin and Primarin vaginal cream, and I also receive funding from the National Institute on Aging and the National Institute of Child Health and Human Development. So my objectives. First, to evaluate vaginal estrogens' possible impact on the risk of breast cancer recurrence. Next, to detail the strengths and weaknesses of various alternatives to vaginal estrogen therapy for the treatment of urogenital symptoms. And last, I really hope to provide you data to aid you when you are guiding your patients through an informed decision-making and consent process, which is necessary for these cancer patients who are considering the benefits and the potential risks of low-dose vaginal estrogen therapy. I suspect everyone in this room knows what GSM is. This is the new recommended terminology for vulvovaginal atrophy, but as GSM would suggest, this is a condition that affects more than just the vulva and the vagina. Patients commonly complain of urinary symptoms such as urgency, dysuria, or they have recurrent urinary tract infection, all of which is associated with estrogen deficiency. About 50% of postmenopausal women complain of GSM symptoms, and interestingly, even those who are using systemic estrogen therapy still often complain of GSM symptoms, about 40%. But 70% of survivors of breast cancer have symptoms of GSM. And the reasons are probably pretty obvious to most of you all. This is, they'll go through menopause earlier if they've had chemotherapy-induced ovarian insufficiency or perhaps they've had a surgical removal of the ovaries or radiation therapy. Also adjuvant endocrine therapy, which is GnRH agonists, aromatase inhibitors, or CERMs, selective estrogen receptor modulators, the use of these agents has increased from about 70% in 2004 to 82% nine years later. And it's common that these are used in patients for longer than they were previously. Now it's not uncommon to use a CERM for 10 years, for example. Also, these patients are commonly not candidates for the use of estrogens, or certainly these patients are more reluctant to try estrogens as their local hormone therapy. And if most long-term survivors of breast cancer were to ultimately describe normalization of their physical and emotional function, a large number of these women have persistent decreased sexual function and satisfaction five years after treatment. There was a large retrospective review of 800 women that were all breast cancer survivors, and they had charts to look at, and they could see documented description of GSM complaints. And only about 40% of them actually received therapy or were offered referral. So I would say the unintended consequence of not asking your patient about these symptoms is that they'll seek treatment somewhere. They may try an untested or unapproved therapy. So it's important that we ask about GSM. Clearly, vaginal estrogen is an established treatment for GSM, and we have three formulations available to us, vaginal creams, a vaginal ring, and vaginal inserts or tablets. Of the creams, we have 17-beta-estradiol and conjugated equine estrogens available to us in the United States. Estriol cream is commonly used when we send patients to get a compounded estrogen made, but it's also available outside of the U.S., in some countries even without a prescription. The vaginal ring delivers 17-beta-estradiol at about 7.5 micrograms per day. And finally, vaginal inserts, which also use estradiol, deliver 4 or 10 microgram doses, and these are usually inserted vaginally twice weekly. Importantly, yes, estrogen can fuel breast cancer. Estrogen does increase breast epithelial cell and breast cancer cell proliferation under a variety of in vitro and in vivo conditions, and we know that lowering estradiol or lowering tissue responsiveness to it has proven to reduce the development and recurrence of breast cancer. But what about local therapy, just vaginal estrogen? As one might imagine, it's quite difficult to design a large enough study where you would have a placebo-controlled trial of vaginal estrogen and find vaginal breast cancer patients wanting to buy in. So most of our studies are of small sample size or have other design flaws, but it is assumed that the less systemic absorption you have, the lower the risk of breast cancer. And absorption varies by these different activities, different active ingredients, the amount of the ingredient, its location of application and the condition of the vagina. It's thought that conjugated equine estrogens are more readily absorbed than estradiol, more so than estrone, and more than estriol. Also it appears it's logical that if you're using a cream, it's applied to a greater surface area of the vagina, and there may be larger amounts of absorption there. There's greater absorption in the upper two-thirds versus the lower third of the vagina. And finally, the condition of the vagina is very important. If it's atrophic and thin, there's not much distance between the skin of the vagina and the vasculature. As a patient is on estrogen for weeks and weeks and they plump up that vaginal epithelium, there tends to be less systemic absorption. And I apologize for the busyness of this slide, but I do want to review three large observational studies that do argue all for the safety of vaginal estrogen therapy. The first comes from the Finnish Cancer Registry, published in 2006. This was a study that followed 85,000 women who were using systemic estradiol, and in most cases this was transdermal estradiol. And there was also oral estriol patients and about 18,000 women who were using vaginal estrogen therapy. Everyone was using their therapy for at least six months, and they were followed for varying durations. It was a registry, but for some women more than five years. And the important finding was that systemic estradiol use, when it was used for less than five years, or oral estradiol, or vaginal estrogens, none of these were associated with an increased risk of breast cancer. The second important study, which was published last year by Crandall et al., is a study of about 45,000 women who were participants of the Women's Health Initiative observational study. None of these women were using systemic estrogens, but about 4,000 of them were using vaginal estrogen therapy. Women were followed for a median of seven years, and you can see the findings here. Among women who had an intact uterus, the risks of stroke, invasive breast cancer, colorectal cancer, endometrial cancer, and venous thromboembolism were not significantly different between the estrogen users and non-users. Whereas the risks of coronary artery disease, fracture, and all-cause mortality, interestingly, were actually lower in the users than in non-users. Finally, the last study of interest, different patient population that comes from the United Kingdom General Practice Research Database. These were women with a history of breast cancer, all with estrogen receptor-positive breast cancer, and they were on either tamoxifen or an aromatase inhibitor, and they followed these women. And those who developed a recurrence of their breast cancer were the cases, and they were compared to the other women who did not develop breast cancer. And the crude recurrence rate was 25.9 per 1,000 women per year, and overall, the use of vaginal estrogen was not associated with an increased risk of recurrence. So if you're considering the use of vaginal estrogen, and you want the lowest dose possible that would perhaps be systemically absorbed, these are the data that—the data most support using an estradiol tablet, where typically the serum estradiol level might be 4.6 picograms per mil, and the estradiol vaginal ring may increase—I shouldn't say increase. You may find a serum estradiol level of about 8 picograms per mil. The important reference point is that the postmenopausal estradiol serum range is less than 20 picograms per mil. Aromatase inhibitors, however, can decrease those rates to essentially nil, 1 to 3 picograms per mil. And it is important, again, to note that when you are first giving a vaginal estrogen, there may be a temporary uptick in the systemic estradiol levels, and it's probably because of the thin epithelium and the efficient uptake of those estrogen receptors. And then over time, that will be kind of tapped down, and you'll have less systemic estrogen. I think anecdotally, patients will sometimes call us and say, I think you prescribed too much. I'm feeling some breast tenderness. What's happening? And we have to explain this, again, that—just stick with it. And I want to now credit—give credit where credit is due. You can see at the bottom of the screen here that a lot of these suggestions come from the North American Menopause Society and the International Society for the Study of Women's Sexual Health, or ISWSH. In 2018, they came up with a combined consensus document about how to treat these women. And as I was reviewing the literature, trying to find the most helpful articles to share with you all, I kept coming back to this review. It's excellent, and I would recommend it to anyone who wants to get further information about this topic. First of all, their suggestion in women with breast cancer history is to start with non-pharmacologic alternatives, such as moisturizers, lubricants, and pelvic floor physical therapy. Moisturizers are given every day or every few days on a regular basis. This is independent of sexual activity. They mimic normal secretions, but they do not restore or reverse cellular or pH changes of GSM. And they can easily be coupled with other therapies. We have patients that might use something every other day, a vaginal estrogen and then a moisturizer and then a vaginal estrogen and then a moisturizer and so forth. Lubricants, which come in water, silicone, or oil base, are used as needed for sexual activity to increase comfort and pleasure. We should encourage patients to avoid possible irritants, such as glycerin, parabens, propylene glycol. These are also commonly coupled with other therapies. And then finally, I think many of us recognize that these patients with dyspareunia that have pain and dryness often will help pelvic floor spasm and tightness of their levator and eye. So it's very important to couple with a physical therapist in your vicinity who's comfortable specializing in these pelvic floor disorders and helping patients downtrend the tightness of these muscles. Other commonly used non-pharmacologic options are vaginal dilators, self-stimulators, or vibrators. And patients really just appreciate being educated about the bulbovaginal changes that they're experiencing with the low estrogen states. And it's also good to have a sex therapist or counselor available to you to refer these patients. When we think about prescription alternatives to estrogen, we have some vaginal inserts, DHEA or dehydroepiandasterone, or Prasterone, which goes by the product name Intrarosa. This is dosed vaginally every day, and it has been shown to improve secretions, improve lubrication, help with sexual dysfunction. It's important to note, though, that when highly sensitive assays are used of the blood, we do see a slight increase in plasma estradiol and testosterone, and it has not been studied in breast cancer survivors. Ospemiphene is a CIRM that is taken orally, and it is FDA-approved for dyspareunia. And it's good to note that it has some anti-estrogenic effects on the breast in preclinical trials, but it has not been studied specifically in breast cancer patients. Interestingly, it is not contraindicated in Europe for women who have a history of breast cancer who have completed their therapy. Finally, topical lidocaine is a good agent to have in patients who are sexually active who experience pain with intercourse. You can apply 4% aqueous lidocaine to the vestibule a few minutes before sexual activity. Many patients, or many of y'all, I'm sure, are going to be interested in what about energy-based devices, energy-based therapies. Here we have microablative fractional CO2 laser, erbium lag laser, or radiofrequency energy. All of these therapies purport to stimulate new collagen, new elastin, and an increase in glycogenated cells. This is not meant to be a comprehensive talk on a review of energy-based therapies, but suffice it to say there are multiple, multiple trials underway, and if you go to clinicaltrials.gov, you'll see numerous studies that are actually studying the breast cancer population with energy-based devices. And the evidence that we have to date, if I could summarize it, would say that CO2 and the erbium lag lasers, in particular, show promise in the treatment of vulvovaginal atrophy, vaginal dryness, and menopausal dyspareunia, with benefits that last up to a year. The long-term safety data, though, is lacking. We do have short-term favorable safety profiles. So this may be a very good option for these patients. This slide tries to summarize broadly who would be the most desirable versus your least desirable candidates for estrogen therapy. Obviously you won't have a patient that ticks every box in each column, but if we look at the more desirable patients, these are women with lower stage disease, lower intermediate grade of breast cancer, no lymph node involvement, negative hormone receptor status. It's helpful if they're on tamoxifen and anti-estrogen therapy. And if they're deemed to have a low risk of recurrence, if it's been remote since they've had their disease, a remote time since their disease, and if they have not responded well to non-hormone therapies and have severe GSM symptoms that are severely affecting their quality of life. Conversely, the least desirable candidates would be, as you expect, those women with higher stage and grade of disease, positive lymph node status, those who are requiring aromatase inhibitors as part of their breast cancer therapy, and perhaps not worth considering vaginal estrogen if they could or have responded well to a non-estrogen alternative. My last two slides give specific recommendations for certain subpopulations. First, general guidelines. Of course, like anything else, we need to individualize our treatment to the patient that's in front of you, balancing the risk of recurrence of her breast cancer, her symptom severity, and GSM's impact on her quality of life. First-line therapy would be moisturizers, lubricants, pelvic floor physical therapy or dilator therapy. It's important early on to involve the patient's treating oncologist in your decision-making or in the patient's decision-making, I should say, when you're considering a hormonal treatment. Again, laser therapy may be considered, although we do not have long-term safety data, and this will be commonly limited by cost and availability. At our institution, we don't have a laser therapy available to our patients unless it's in a research setting. Women who are at high risk for breast cancer, so these are not women with breast cancer, but who are deemed to have a greater than 20 percent lifetime risk of cancer, such as BRCA carriers, those with a personal history of atypical ductal hyperplasia, lobular or ductal carcinoma in situ, in these women, local hormone therapy, I would say, is a reasonable option after failed non-hormonal treatment, and observational data do not suggest an increased risk of breast cancer with systemic or local estrogen therapies. This next category is who we're really thinking about when we talk about our breast cancer patients. Those women who typically have estrogen receptor-positive breast cancers often fall into two camps. Those who are probably taking tamoxifen and those who are on aromatase inhibitors. For those women taking tamoxifen, I would say that local hormone therapy may be a reasonable option after failed non-hormonal treatment if GSM symptoms persist and are severe and her risk of breast cancer recurrence is deemed low. Tamoxifen continues to have a competitive interaction with estrogen receptors, which would argue for the possible safety of using a low-dose vaginal estrogen therapy. But for those women who are on aromatase inhibitors, that's a little trickier. Low-dose estrogen therapy could be considered as an option in concert with the patient's oncologist, but I've seen one recommendation would be to consider switching that patient to tamoxifen for several weeks or a month while there is that slight increased systemic dose. Again, if you start estrogen therapy, you may have a systemic bump in estrogen levels. And so thereafter, you could return that patient to her aromatase inhibitor. And DHEA, which recall can have slight increases in testosterone, I would be cautious in someone with an androgen receptor-positive cancer. How about women with triple negative breast cancers? Here data are lacking, but at least in theory, it would seem reasonable to give local hormone therapy. And finally, women with metastatic disease. Here treatment should be tailored to the patient. If she has a severe impact on quality of life and comfort and intimacy, some would argue that go ahead and proceed with local hormone therapy to try to improve her quality of life. And so with that, I would welcome your questions. I hope that's helpful for you. I'd like to start. That was an excellent review. You acknowledged that there's different, there's more systemic absorption if you use conjugated estrogen cream or estradiol cream as opposed to VagFM or Estrin. Is there any data that's shown that there's a difference in outcome between those two? In other words, is there a higher incidence of recurrence if you use the higher dose absorption method of treatment? No, I'm not aware of data that given conjugated equine estrogen versus a lower dose alternative that there would be an increased actual risk in breast cancer. We are using serum estradiols, estradiol levels as a surrogate for that outcome. Can I come back on that, Harold? Yes, please. Premarin was developed as a non-oral hormone replacement therapy, not as a low-dose local estrogen therapy. And there is definitely more bleeding and more endometrial proliferation with Premarin cream. And so theoretically, breast cancer sufferers should never use Premarin cream. They should use, we have Ovestin, which is estriol, you have Estrace, or they should use Vagifem or an Estrin, but they should never use Premarin. Very good point. Thank you. Thank you very much.

vaginal estrogen therapy

postmenopausal women

breast cancer

urogenital symptoms

observational studies

treating oncologist

alternative treatment options

moisturizers

laser therapy