The Microbiome Protocol

Building a healthy vaginal microbiome from scratch, in anatomy that nobody's studied.

This is a companion to the Cyclic HRT Protocol. Where that page documents the hormonal framework, this one covers the parallel project: establishing and maintaining a healthy vaginal microbiome in a peritoneal flap neovagina.

The short version: most of what's published about neovaginal microbiomes doesn't apply to me. The literature is almost entirely based on penile inversion vaginoplasty, where the vaginal canal is lined with penile and scrotal skin. That tissue retains cornification, doesn't produce glycogen, and doesn't support Lactobacillus colonization in any meaningful way. My anatomy is different, and that difference changes everything about what's biologically possible.

Why tissue type matters

In a natal vagina, the microbiome story starts with estrogen. Estrogen drives the maturation of vaginal epithelial cells and stimulates those cells to deposit glycogen. When those glycogen-rich cells are shed into the vaginal lumen, human α-amylase breaks the glycogen down into maltose and smaller sugars. Lactobacillus species metabolize those sugars into lactic acid, which drops the vaginal pH into the 3.5-4.5 range. That acidic environment is protective: it inhibits the growth of pathogenic bacteria, reduces the risk of BV, and lowers susceptibility to STIs[1][2].

The entire chain depends on the tissue being capable of glycogen production. Penile skin doesn't do this. One study examining the microstructure of penile skin-lined neovaginas found that cornification was reduced but not eliminated, and no glycogen production was observed, even in participants who were more than nine years post-surgery and had been on estrogen therapy for over a decade[3]. Without glycogen, the Lactobacillus-lactic acid-low pH feedback loop can't establish itself. The tissue simply doesn't provide the substrate.

This is why the existing neovaginal microbiome literature consistently finds polymicrobial communities dominated by skin and gut commensals rather than Lactobacillus. A systematic review covering 458 patients across 13 studies found that neovaginal microflora were generally polymicrobial and shared characteristics with the tissue of origin. Penile skin-lined neovaginas harbored bacteria typically found on skin, in the rectum, and in natal vaginas with bacterial vaginosis[4][5]. A 2025 study of 47 transfeminine participants with penile inversion neovaginas found that while Lactobacillus was detected, Lactobacillus dominance was rare[6].

That said, the picture for PI neovaginas isn't entirely bleak. Petricevic et al. (2014) used PCR-DGGE (a more sensitive molecular method than culture) to profile 63 trans women with PI neovaginas and detected Lactobacillus in 75% of participants, across 13 different species. The dominant species were from the L. delbrueckii group, including L. gasseri, L. crispatus, L. johnsonii, L. iners, and L. jensenii[7]. This doesn't mean Lactobacillus was dominant in those communities, but it was present at detectable levels even in skin-lined tissue. And the same group demonstrated in a follow-up RCT (Kaufmann et al., 2014) that oral administration of L. crispatus, L. rhamnosus, L. jensenii, and L. gasseri for 7 days significantly improved Nugent scores and enriched neovaginal Lactobacillus compared to placebo (p < 0.006 for Nugent improvement; p < 0.0001 for Lactobacillus enrichment)[8]. That's in PI tissue without glycogen. The potential in glycogen-producing peritoneal tissue should, in principle, be even greater.

Peritoneal tissue is a different story

My neovagina was constructed using peritoneal flap vaginoplasty (PPT) with AlloDerm grafting. The peritoneal tissue lining the canal is not skin. It's mesothelium, and it has a documented capacity to undergo metaplasia into stratified squamous epithelium that resembles native vaginal mucosa, including glycogen production.

This has been studied primarily in cis women with Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome, who are born without a vagina and undergo peritoneal vaginoplasty using the Davydov technique. The data is consistent:

  • Origoni et al. (2021) examined peritoneal neovaginas created via the Davydov procedure and found that none of the specimens retained mesothelial cells. The tissue had converted to squamous epithelium with glycogen present in the superficial layers[9].

  • Mhatre et al. (2016) demonstrated that peritoneal tissue undergoes metaplasia into stratified squamous epithelium resembling normal vaginal tissue, confirmed by serial biopsies over a 9-month period. They identified specific progenitor cell markers (OCT4, SOX2) driving the conversion[10].

  • A 2024 longitudinal study in Nature Communications followed MRKH patients after peritoneal vaginoplasty and found that the neovaginal microbiota developed into a community resembling that of a normal vagina by 6-12 months post-surgery, and further matured into a more homeostatic state by 2-4 years[11].

  • Dhami et al. (2025), examining peritoneal neovaginas in transgender women after robotic-assisted peritoneal flap vaginoplasty, confirmed the metaplasia: all five biopsies taken at 12+ months post-surgery showed stratified squamous epithelium with no residual mesothelial cells[12].

And there's direct microecological data from peritoneal neovaginas. Qin et al. (2019) studied 54 MRKH patients after laparoscopic peritoneal vaginoplasty and found that 57.4% had a vaginal pH of 4.5 or below. Dysbiosis rates decreased over time: 64.5% of patients less than 2 years post-op showed dysbiosis, compared to 39.1% of patients at 2+ years. The predominant bacteria were gram-positive macrobacilli in 27.8% of patients[13]. This is the clearest evidence that peritoneal neovaginas can achieve and sustain an acidic, potentially Lactobacillus-supportive environment, and that it improves with time as the tissue matures.

The practical implication: if peritoneal tissue metaplasizes into glycogen-producing squamous epithelium, and if that glycogen can support Lactobacillus colonization, then the natal vaginal microbiome literature becomes a more appropriate reference point for my anatomy than the penile inversion neovaginal literature. The tissue biology is fundamentally different.

The estrogen connection

Estrogen is the upstream driver of the entire glycogen-Lactobacillus chain. This is well established in natal vaginal biology: at puberty, rising estrogen promotes epithelial maturation and glycogen deposition; at menopause, declining estrogen reverses it. Post-menopausal women have significantly lower free glycogen levels and correspondingly lower Lactobacillus colonization[14].

The relevance of menopausal data here is direct. Like postmenopausal cis women, post-gonadectomy trans women are hypogonadal: all sex hormones are exogenous. Without supplemental estrogen, there is no endogenous driver of epithelial maturation or glycogen production. This makes the postmenopausal literature on vaginal estrogen and microbiome recovery the closest available reference for understanding what local estrogen therapy might accomplish in a peritoneal neovagina.

There's also evidence from the other direction. In transgender men on testosterone, vaginal Lactobacillus colonization decreases as the tissue atrophies under androgen influence. Winston McPherson et al. (2019) found that intravaginal estrogen administration was positively associated with the return of Lactobacillus in transgender men on testosterone (p = 0.045), suggesting that local estrogen can restore the conditions that support colonization even in the context of systemic androgens[15].

No study has specifically evaluated the effect of intravaginal estrogen on the neovaginal microbiome in transfeminine patients. The existing systematic reviews note this gap explicitly and call for future research[16]. This is one of the most significant knowledge gaps in the field: providers prescribe vaginal estrogen for trans women with neovaginal complaints without any data on whether it actually helps.

For my protocol, I use vaginal estradiol cream (0.01%, 1g application) twice weekly on Wednesdays and Saturdays. The systemic contribution is negligible (0.1mg per application, most of which acts locally), but the local tissue effect is the point. The goal is to promote epithelial maturation and glycogen production in the peritoneal tissue lining the canal, creating the substrate conditions that Lactobacillus needs to colonize and persist.

The intervention: VagiBiom probiotic suppositories

With the tissue-level groundwork being laid by vaginal estradiol, the next piece is direct introduction of Lactobacillus species.

I'm using VagiBiom probiotic suppositories, which contain a multi-strain complex: Lactobacillus crispatus Bi16, Lactobacillus gasseri Bi19, Lactobacillus acidophilus Bi14, and Bacillus coagulans Bi34, delivered in a coconut fatty acid base with oligofructose prebiotics, hyaluronic acid, and lactic acid. Total count is 10 billion CFU per suppository.

These specific strains matter. L. crispatus is the species most strongly associated with a healthy, stable vaginal microbiome in natal women. L. gasseri is another of the four most commonly found vaginal Lactobacillus species. The inclusion of a prebiotic (oligofructose) and lactic acid in the formulation is intended to provide an immediate environment that supports the introduced bacteria while they establish.

VagiBiom has published data from a randomized, double-blind, placebo-controlled trial (NCT05060029) in perimenopausal women with BV. After four weeks of treatment (5 suppositories per week), the intervention group showed improved Nugent scores, decreased vaginal pH, and improved vaginal health index scores compared to placebo. Microbiome sequencing confirmed that the effect was driven by improved Lactobacillus diversity[17].

To be clear about the limitations: that study was in cis women with natal vaginal tissue and BV. Nobody has studied whether vaginal probiotic suppositories can establish Lactobacillus colonies in a peritoneal neovagina. I'm extrapolating from the tissue biology (peritoneal tissue metaplasizes into glycogen-producing epithelium → glycogen supports Lactobacillus → direct introduction of Lactobacillus into a glycogen-producing environment should allow colonization). The logic is sound. The data doesn't exist yet.

The glycogen piece of the chain has some nuance worth noting. Navarro et al. (2023) showed that glycogen is essential for the growth of L. crispatus and L. jensenii in a medium simulating vaginal fluid, but that glycogen utilization is pH-sensitive: L. crispatus could only metabolize glycogen at pH 5.0, while L. jensenii functioned across pH 4.0-5.0. L. gasseri was the only species that survived without glycogen at all, using glucose instead[18]. This matters because the initial pH of a neovaginal environment is likely higher than 4.5, meaning the early colonizers may need to be pH-tolerant species that can begin the acidification process before L. crispatus can take hold.

It's also worth noting that someone else is asking the same question. A completed clinical trial (NCT05372770) studied whether the Flourish Vaginal Care System could establish a Lactobacillus-dominant neovaginal microbiome in 56 transgender women post-GCS. Results have not been published as of this writing[19]. When those results come out, they'll be the first interventional data on neovaginal microbiome modulation in trans women.

My protocol

Vaginal estradiol cream: 0.01%, 1g applied twice weekly (Wednesdays and Saturdays). Ongoing, not cycled. Applied at bedtime. On mornings where this overlaps with Inito testing, application happens after the test rather than the night before.

VagiBiom suppositories: Loading phase follows the dosing schedule from the VagiBiom RCT: one suppository daily, 5 days on, 2 days off each week, for the first two 28-day cycles. After that, transitioning to a maintenance schedule. Inserted at midday when sharing a day with estradiol cream; inserted at bedtime on days without other vaginal products. Positioned as deep as comfortably possible.

Stacking logic: On days where all three vaginal products are due (progesterone + estradiol cream + VagiBiom), VagiBiom goes in at midday. Progesterone (200mg) goes in at bedtime, with estradiol cream staggered approximately 30 minutes later. On days where only VagiBiom and estradiol cream overlap, VagiBiom is midday and cream is bedtime. The goal is to give each product time to absorb and act locally without competing for tissue contact.

Lubricant compatibility: Good Clean Love BioNude or Almost Naked only. This isn't an arbitrary preference. Lubricant osmolality directly affects both the epithelial barrier and bacterial colonization. Ayehunie et al. (2017) demonstrated that lubricants with osmolality greater than 1500 mOsm/kg caused significant damage to vaginal epithelial barrier integrity, while iso-osmolar lubricants (<400 mOsm/kg) showed no epithelial damage[20]. More directly relevant: Łaniewski et al. (2020) tested Good Clean Love Almost Naked against vaginal Lactobacillus species in vitro and found it did not inhibit the growth of L. crispatus, L. gasseri, L. jensenii, or L. iners, unlike lubricants containing chlorhexidine gluconate[21]. Both the BioNude and Almost Naked products also contain lactic acid, which aligns with the target pH environment.

Monitoring plan

This is where documenting matters. I'm tracking the microbiome at defined intervals using two different sequencing approaches:

Baseline (completed April 2, 2026): Evvy metagenomic vaginal microbiome test. This was taken before any probiotic supplementation. Preliminary PCR results show Prevotella bivia detected at medium load. No Lactobacillus species detected by PCR (L. crispatus/acidophilus, L. gasseri both negative). No Gardnerella, no Mobiluncus, no Mycoplasma, no Ureaplasma. STI panel negative. No antimicrobial resistance genes detected. Full mNGS results pending; these will show the complete community composition and relative abundances, not just the targeted PCR panel.

The Prevotella bivia finding is not surprising. Prevotella is one of the most commonly identified genera in neovaginal microbiomes across surgical techniques, and P. bivia specifically has been found in both healthy and symptomatic vaginal microbiomes. It's classified as "disruptive" by Evvy's framework, but that framework is built on natal vaginal reference data. In the absence of competing Lactobacillus, Prevotella filling available niche space is expected.

The absence of Lactobacillus at baseline is the key finding. It establishes the starting point: this is a pre-intervention measurement of a peritoneal neovaginal environment that has not been exposed to probiotic supplementation. Any Lactobacillus detected in subsequent tests can be attributed to the intervention rather than pre-existing colonization.

Interim (~6 weeks post-baseline): Juno Bio 16S rRNA sequencing. This is a less comprehensive method than Evvy's metagenomics, but it's faster and cheaper, making it useful as a progress check. The question at this timepoint: is Lactobacillus detectable? Is it persisting between suppository doses, or does it disappear within hours of each application?

12-week confirmation (late June/early July 2026): Repeat Evvy metagenomic test. Same methodology as baseline, allowing direct comparison. By this point, the peritoneal tissue will have had approximately three months of consistent vaginal estradiol exposure and probiotic introduction. If the metaplasia-glycogen-Lactobacillus hypothesis holds, this is where I'd expect to see either early signs of Lactobacillus establishment or evidence that the colonization isn't taking hold.

Quarterly Evvy tests through Year 1: After the 12-week test, I'm continuing Evvy metagenomic sequencing every three months for a full year: approximately September 2026 and December 2026 following the July test. Four total Evvy snapshots (including baseline) over 12 months gives enough longitudinal resolution to see whether the microbiome is trending in a particular direction or whether any colonization gains are stable versus transient. This is the kind of dataset that doesn't exist for peritoneal neovaginas.

Ongoing: pH testing with vaginal swabs at depth will be integrated once a consistent monitoring routine is established. A declining pH trend would be an indirect signal that lactic acid-producing bacteria are metabolically active in the canal, even between sequencing timepoints.

What I'm watching for

The honest answer is: I don't know what success looks like here, because nobody has defined it for this anatomy.

In a natal vagina, a "healthy" microbiome typically means Lactobacillus dominance (particularly L. crispatus), low diversity, and a pH below 4.5. But the Frontiers review from Krakowsky et al. raises a point worth sitting with: it's possible that the optimal microbiome for a neovagina isn't Lactobacillus-dominant at all, and that applying natal vaginal standards to surgically constructed anatomy could be misguided[3].

For penile skin-lined neovaginas, that's probably right. Without glycogen, pushing Lactobacillus into an environment that can't sustain it is fighting the tissue biology.

For peritoneal neovaginas, the question is genuinely open. If the tissue is metaplasizing into glycogen-producing epithelium (which the biopsy data says it does), then the substrate exists, and the natal vaginal framework becomes more applicable. Not identical, but closer. My mixed anatomy (peritoneal lining plus AlloDerm grafting) adds another variable: the AlloDerm sections may behave differently than the peritoneal sections in terms of what they can support.

So I'm not aiming for a specific community state type or a target Lactobacillus percentage. I'm watching for trends: is Lactobacillus detectable and persisting? Is diversity decreasing over time (suggesting a shift toward a more stable, less polymicrobial state)? Is pH trending downward? Are there symptomatic changes (discharge consistency, odor) that correlate with the sequencing data?

If none of that happens, that's data too. Knowing that direct Lactobacillus introduction into a peritoneal neovagina under topical estrogen doesn't produce colonization would be just as valuable as knowing that it does. Either way, the answer doesn't exist yet, and it won't exist until someone documents it.

This page will be updated as sequencing results come back and the protocol evolves. See also: The Protocol for the cyclic HRT framework, and the Cycle 1 Log for real-time observations.

References

  1. Amabebe E, Anumba DOC. The vaginal microenvironment: the physiologic role of Lactobacilli. Front Med. 2018;5:181. PMC6008313
  2. Mirmonsef P, et al. Free glycogen in vaginal fluids is associated with Lactobacillus colonization and low vaginal pH. PLOS ONE. 2014;9(7):e102467. PMC4102502
  3. Krakowsky Y, et al. The effect of gender-affirming medical care on the vaginal and neovaginal microbiomes of transgender and gender-diverse people. Front Cell Infect Microbiol. 2021;11:769950. Frontiers
  4. Birse KD, et al. The neovaginal microbiome of transgender women post-gender reassignment surgery. Microbiome. 2020;8:61. PMC7201977
  5. Small AC, et al. Systematic review: the neovaginal microbiome. Urology. 2022;166:12-20. ScienceDirect
  6. Rojas-Vargas J, et al. The neovaginal microbiota, symptoms, and local immune correlates in transfeminine individuals with penile inversion vaginoplasty. Cell Rep. 2025;44:116546. Cell Reports
  7. Petricevic L, et al. Molecular detection of Lactobacillus species in the neovagina of male-to-female transsexual women. Sci Rep. 2014;4:3746. Nature
  8. Kaufmann U, et al. Ability of an orally administered lactobacilli preparation to improve the quality of the neovaginal microflora in male to female transsexual women. Eur J Obstet Gynecol Reprod Biol. 2014;172:102-105. ScienceDirect
  9. Origoni M, et al. The peritoneal neovagina after Davydov's laparoscopic procedure in MRKH syndrome: morphology and ultrastructure investigation of the new epithelium. J Minim Invasive Gynecol. 2021;28(10):1795-1799. ScienceDirect
  10. Mhatre P. Role of progenitor cell producing normal vagina by metaplasia in laparoscopic peritoneal vaginoplasty. J Hum Reprod Sci. 2016;9(4):215-222. PMC5296824
  11. Weiss S, et al. Insights into the assembly of the neovaginal microbiota in Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome patients. Nat Commun. 2024;15:7690. Nature
  12. Dhami JK, et al. The peritoneal neovagina after robotic-assisted peritoneal flap gender-affirming vaginoplasty: a morphologic and histologic investigation of the neovaginal lining. Urology. 2025. ScienceDirect
  13. Qin C, et al. Analysis of the artificial vaginal microecology in patients after laparoscopic peritoneal vaginoplasty. Sci Rep. 2019;9:8482. PMC6560037
  14. Mirmonsef P, et al. An exploratory comparison of vaginal glycogen and Lactobacillus levels in pre- and post-menopausal women. Menopause. 2015;22(7):702-709. PMC4476965
  15. Winston McPherson G, et al. The vaginal microbiome of transgender men. Clin Chem. 2019;65(1):199-207. Oxford Academic
  16. Oliva G, et al. The microbiome of the neovagina: a systematic review and comparison of surgical techniques. BMC Womens Health. 2024. PMC11500519
  17. Vivekanandan V, et al. VagiBIOM Lactobacillus suppository improves vaginal health index in perimenopausal women with bacterial vaginosis: a randomized control trial. Sci Rep. 2024;14:3317. Nature
  18. Navarro S, et al. Glycogen availability and pH variation in a medium simulating vaginal fluid influence the growth of vaginal Lactobacillus species and Gardnerella vaginalis. BMC Microbiol. 2023;23:186. PMC10346149
  19. ClinicalTrials.gov NCT05372770. Pilot study to observe effects of using Flourish Vaginal Care System to establish neovaginal microbiome after gender-confirmation surgery. Vaginal Biome Science. Completed 2024; results not yet published. ClinicalTrials.gov
  20. Ayehunie S, et al. Hyperosmolal vaginal lubricants markedly reduce epithelial barrier properties in a three-dimensional vaginal epithelium model. Toxicol Rep. 2018;5:134-140. PMC5977164
  21. Łaniewski P, et al. Clinical and personal lubricants impact the growth of vaginal Lactobacillus species and colonization of vaginal epithelial cells: an in vitro study. Sex Transm Dis. 2021;48(1):52-58. PMC8793461