The Cyclical HRT & Microbiome Protocol

Two projects, one body, running in parallel.

This page covers the short version of both halves of what I'm doing: a cyclical hormone protocol designed to approximate a cis female menstrual cycle, and a microbiome intervention aimed at establishing Lactobacillus in peritoneal flap neovaginal tissue. The full protocol document (v2.0, May 2026) is attached at the bottom. This page is the orientation.

The premise

Standard trans-feminine HRT holds estradiol and progesterone flat. Same dose, same schedule, week after week. It hits therapeutic ranges, but it doesn't reflect how these hormones actually behave in a cycling body. Cis women have a 28-day (approximate) rhythm: estrogen rises and falls, progesterone surges in the luteal phase, and the whole thing crashes into a withdrawal bleed. Mood, energy, immune function, bone metabolism, breast tissue. All of it evolved around that fluctuation.

This protocol tries to approximate that rhythm using injectable estradiol valerate (EV) and vaginal bio-identical progesterone on a fixed 28-day cycle. Perfectly reproducing a cis female hormone curve isn't achievable. I don't have ovaries, and exogenous dosing only lets you do so much. The goal is to approximate the shape, hit the key inflection points, and observe what happens.

Running in parallel: a microbiome protocol aimed at establishing Lactobacillus in peritoneal flap neovaginal tissue. Most of the neovaginal microbiome literature is based on penile inversion, where the tissue biology is fundamentally different. The peritoneal case has barely been studied, and there is no interventional data for this anatomy in trans women at all.

These two projects aren't unrelated. Estrogen is the upstream driver of both the menstrual cycle and the vaginal microbiome. Doing them together is partly efficient and partly hypothesis-generating.

Baseline

Trans woman, several years on estrogen-based HRT.

Vaginoplasty using peritoneal flap with AlloDerm grafting. Tissue type matters (a lot) for both halves of this protocol. See the section on tissue biology below.

No gonads. All sex hormones are exogenous, adrenal contribution is negligible. Same endocrine category as postmenopausal cis women, which is why most of the PK and safety literature I reference comes from that population.

Prior steady-state: 6mg EV SubQ weekly + 200mg progesterone nightly (switched from oral to vaginal).

Symptomatic low-E2 threshold: below ~50–60 pg/mL for more than a few days and I start feeling it (mood, physical symptoms).

During the pre-protocol washout, my FSH rose to 1.25 mIU/mL as E2 dropped to trough (~65 pg/mL). The HPG feedback loop is intact and responsive, which matters for a protocol that deliberately creates low-E2 phases every month.

The 28-day cycle

Four EV injections per cycle, totaling ~11mg. That's significantly less than the ~24mg/month I was taking on weekly 6mg dosing. The tradeoff is intentional because this protocol relies on timing rather than volume.

Days 1–4 are deliberately unmedicated. Estradiol is at its lowest point, decaying from the previous cycle's final injection. That trough is what drives the withdrawal bleed.

Progesterone is absent during the follicular and ovulatory phases, introduced at the start of the luteal phase, escalated to mimic peak luteal levels, then abruptly stopped to trigger withdrawal.

The abrupt cessation on Day 27 is what creates the withdrawal event. Both estradiol and progesterone decline simultaneously, which is the hormonal trigger for menstruation.

Target serum levels for each cycle phase are drawn from published reference ranges for normally cycling cis women (primarily Verdonk 2019 for estradiol, Stricker 2006 for progesterone):

Why tissue biology changes what's possible

In a natal vagina, the microbiome story starts with estrogen. Estrogen drives epithelial maturation and glycogen deposition in vaginal epithelial cells. Those cells are shed into the lumen, human α-amylase breaks the glycogen down into smaller sugars, Lactobacillus species metabolize those sugars into lactic acid, and the pH drops to 3.5–4.5. That acidic environment is protective against BV, pathogens, and some STIs. The entire chain depends on tissue that can produce glycogen.

Penile skin can't. Krakowsky et al. (2022) examined penile skin-lined neovaginas and found no glycogen production, even in participants 9+ years post-surgery on long-term estrogen. Without glycogen, the Lactobacillus–lactic acid–low pH feedback loop can't establish itself. That's why the existing neovaginal microbiome literature, which is almost entirely based on penile inversion, consistently shows polymicrobial communities dominated by skin and gut commensals rather than Lactobacillus.

Peritoneal tissue is a different substrate. It's mesothelium, and it has a documented capacity to undergo metaplasia into stratified squamous epithelium resembling native vaginal mucosa, including glycogen production. This has been studied primarily in cis women with MRKH syndrome, who undergo peritoneal vaginoplasty via the Davydov technique:

Origoni et al. (2021): Davydov peritoneal neovaginas had converted entirely to squamous epithelium with superficial glycogen; no residual mesothelial cells.

Mhatre et al. (2016): serial biopsies over 9 months confirmed metaplasia, with specific progenitor cell markers (OCT4, SOX2) driving the conversion.

Chen et al. (2024): MRKH patients after peritoneal vaginoplasty developed neovaginal microbiota resembling a normal vagina by 6–12 months, maturing further by 2–4 years.

Dhami et al. (2026): confirmed metaplasia in transgender women specifically after robotic peritoneal flap vaginoplasty. All biopsies at 12+ months showed squamous epithelium with no residual mesothelial cells.

The clearest direct evidence comes from Qin et al. (2019): 54 MRKH patients after laparoscopic peritoneal vaginoplasty, 57.4% had a vaginal pH of 4.5 or below. Dysbiosis dropped from 64.5% in patients under 2 years post-op to 39.1% at 2+ years. The tissue can sustain an acidic, potentially Lactobacillus-supportive environment, and it improves with time as the tissue matures.

The practical implication: if peritoneal tissue metaplasizes into glycogen-producing squamous epithelium, the natal vaginal microbiome literature becomes a more appropriate reference for my anatomy than the penile inversion neovaginal literature. That's a significant departure from how this is usually discussed in community spaces, where PI assumptions get generalized across surgical techniques they don't actually describe.

The microbiome intervention

Three pieces working together:

Vaginal estradiol cream (0.01%, 1g twice weekly, Wednesdays and Saturdays). Systemic contribution is negligible (~5–15 pg/mL), but that's not the point. The point is local: promoting epithelial maturation and glycogen production in the peritoneal tissue. In transgender men on testosterone, intravaginal estrogen was positively associated with the return of Lactobacillus (p = 0.045). No study has evaluated this intervention in trans women. The existing systematic reviews explicitly call out the gap.

VagiBiom probiotic suppositories, a multi-strain formulation containing L. crispatus Bi16, L. gasseri Bi19, L. acidophilus Bi14, and B. coagulans Bi34 in a coconut fatty acid base with oligofructose prebiotics, hyaluronic acid, and lactic acid (10 billion CFU per suppository). The VagiBiom RCT (NCT05060029) in perimenopausal women with BV showed improved Nugent scores and vaginal health index with 5 suppositories per week over 4 weeks, with the effect driven by improved Lactobacillus diversity. That study was in cis tissue with BV. Nobody has studied whether this can establish colonies in a peritoneal neovagina.

Loading phase: one suppository daily, 5 days on / 2 off, for the first two 28-day cycles. VagiBiom is inserted at midday on every administration day, regardless of what other vaginal products are scheduled that day. This fixed midday slot was adopted in Cycle 2 after the original plan (bedtime on days without other products, midday only on overlap days) created a consistent collision with evening progesterone during the BID luteal window. Maintenance frequency after loading depends on what sequencing shows.

Lubricant discipline: Good Clean Love BioNude or Almost Naked only. This isn't an arbitrary preference. Ayehunie et al. (2018) showed that lubricants above 1,500 mOsm/kg damage vaginal epithelial barrier integrity. Łaniewski et al. (2021) tested Almost Naked against vaginal Lactobacillus species in vitro and found it didn't inhibit growth of L. crispatus, L. gasseri, L. jensenii, or L. iners. Both products also contain lactic acid. If you're trying to establish a microbiome, hyperosmolar lubricants are actively working against you.

Monitoring

Hormones. I use an Inito fertility monitor with first morning urine (FMU) to track urinary metabolites (E3G, PdG, LH, FSH) throughout the cycle. Inito is designed for cis women tracking fertility, and its ovulation predictions are meaningless here because there will never be an LH surge. I ignore the algorithmic interpretations and use it strictly as a metabolite measurement device. The Inito API (not the consumer app) is the source of truth for all readings, since it preserves two-decimal precision the app rounds off.

One caveat worth flagging: the value Inito reports as "FSH" is not straightforwardly measured. The test strip has four lateral-flow lines for E3G, PdG, LH, and a fourth line. There's no published evidence the fourth line uses a dedicated FSH-beta-subunit antibody, and Inito's validation paper (Pattnaik et al., 2023) doesn't include FSH in its analytical validation. The most likely explanation is that the reported FSH value is algorithmically derived from the alpha subunit shared by LH and FSH, not independently measured. The protocol refers to it as "ifsh" and treats it as a directional indicator only, not equivalent to serum FSH. Calibrating ifsh against serum FSH is itself one of the secondary endpoints.

Urinary metabolites lag serum levels in a way that isn't captured by any single conversion ratio. When serum hormones are rising, urine hasn't caught up yet and underestimates. When serum hormones are falling, urine still reflects the recent peak and overestimates. There is no static multiplier. Paired Inito FMU plus serum draws are what actually pin down what the numbers mean: the two paired draws so far (one post-peak, one at trough) produced apparent E3G-to-E2 ratios of 1.12 and 0.89, respectively, illustrating the direction-dependent lag rather than a concentration-dependent conversion factor.

Paired draws are pursued opportunistically across cycle phases rather than committed to fixed cycle days. The phases targeted for calibration coverage are: rising mid-follicular, peak ovulatory, steady-state mid-luteal, declining late-luteal or withdrawal, and trough early-follicular (with concurrent elevated ifsh expected). Specific draw days within each cycle are determined by insurance coverage, out-of-pocket budget, and Inito trajectory at the time. Cycle 1 captured only the trough early-follicular phase (April 2, Day 2). Cycle 2 has added the early-follicular/pre-injection window and the ovulatory peak; mid-luteal and late-luteal draws are planned. All paired draws contribute to the longitudinal calibration map.

Testing frequency shifted in Cycle 2. Cycle 1 hit 13 of 28 days, with significant gaps in the late luteal and withdrawal windows. Starting Cycle 2 Day 8, I'm testing daily through the end of Cycle 3 to build a continuous curve and capture ifsh behavior across all phases. Frequency from Cycle 4 onward will be reassessed based on what the daily data reveals.

Bleeding and symptoms are logged in Clue throughout each cycle.

Microbiome. Tracked at defined intervals using two sequencing approaches.

Baseline (April 2, 2026): Evvy metagenomic sequencing. Taken before any probiotic supplementation. The preliminary PCR results (reported April 2) came back showing no Lactobacillus, no Gardnerella, no Mobiluncus, no Mycoplasma or Ureaplasma, no antimicrobial resistance genes, and Prevotella bivia at medium load. The full metagenomic results (reported April 11) told a substantially different story, and the discrepancy between the two is itself informative.

The mNGS reported a polymicrobial anaerobic community with a diversity score of 2.9 and approximately 96% disruptive species by relative abundance. The dominant taxa: Prevotella at ~40% combined, Peptoniphilus ~14%, Atopobium/Fannyhessea ~11%, Porphyromonas ~10%, and three Gardnerella species totaling ~10%. A tetracycline resistance gene was detected; all other resistance classes (carbapenem, macrolide, methicillin, quinolone, vancomycin) were clear. STI panel negative.

The most important finding: Lactobacillus species were detected at low relative abundance. L. crispatus at 2.29% and L. iners at 1.53%. PCR didn't pick them up because the absolute copy number was below its quantitative threshold; mNGS caught them because it reports relative abundance and detects organisms at lower absolute concentrations. The same detection-threshold dynamic explains the Gardnerella discrepancy.

This reframes the microbiome intervention question. It's not "can L. crispatus be introduced into a Lactobacillus-zero environment?" but "can the existing L. crispatus population be expanded to a clinically meaningful relative abundance against the established anaerobic community?" One mechanistic note worth making: the VagiBiom suppositories contain L. crispatus strain Bi16, so the supplementation is plausibly reinforcing a population that has already demonstrated viability in this anatomy rather than introducing a foreign organism. Whether the supplemented strain integrates with, displaces, or transiently augments the resident L. crispatus isn't resolvable with relative-abundance sequencing alone.

Planned follow-up:

End of Cycle 2 / start of Cycle 3 (approximately May 27, 2026): Juno Bio 16S rRNA sequencing as an interim check. This was originally planned for ~6 weeks post-baseline but shifted to the inter-cycle transition window. Sampling during the BID progesterone window of mid-to-late luteal Cycle 2 would have introduced confounds from progesterone-driven epithelial changes and high vaginal product residue. Sampling post-withdrawal, pre-Day 5 injection provides a cleaner specimen. VagiBiom dosing is paused for 48 hours before the swab. The question at this timepoint: has the baseline L. crispatus of 2.29% expanded, held, or contracted under sustained loading?

12 weeks post-baseline (late June / early July 2026): repeat Evvy metagenomic test for direct comparison to baseline.

Quarterly Evvy metagenomic tests through Year 1, for four snapshots over 12 months.

pH testing with swabs at depth is conducted opportunistically rather than on a fixed schedule. Absolute pH values are confounded by residual vaginal product activity and tissue maturation stage, so the data is treated as exploratory and supplementary to sequencing rather than standalone evidence. A declining trend over time would be an indirect signal that lactic acid-producing bacteria are metabolically active between sequencing timepoints.

What success looks like

For the hormone protocol, v2.0 of the full protocol document reframes the primary endpoints from concordance (how close individual measurements land to target ranges) to characterization (describing what the protocol actually produces). The original v1.0 used a 25% concordance threshold, which was the wrong framing for an N=1 protocol pushing exogenous hormones through anatomically distinct tissue. The protocol should describe what it produces, not score itself against an arbitrary tolerance band.

The shape evaluation asks whether each cycle produced the intended hormonal rhythm: did the mid-cycle E2 peak exceed both the early-follicular and withdrawal E2? Did mid-luteal P4 exceed follicular P4? Did both E2 and P4 decline in the withdrawal window? A cycle that produces the right shape is reported as such even if individual measurements land outside the target ranges. This captures the rhythm-replication intent independent of absolute calibration.

Numbers alone aren't enough, though. A handful of secondary markers matter for whether this actually feels like a cycle rather than just looking like one on paper:

Cyclical symptoms mapping to cycle phase. Cramping, breast tenderness, bloating, mood changes, fatigue, vasomotor symptoms. If the protocol is working, these should show up in phase-appropriate windows rather than randomly.

Calibration of the Inito-to-serum relationship across cycle phases, including ifsh-to-serum FSH, is itself an endpoint. No one has mapped the urinary metabolite lag behavior across a full exogenous cycle, and the ifsh derivation question means that even the directionality of that value against serum FSH needs to be established rather than assumed.

Failure looks like trough days that are functionally intolerable despite dose adjustment, or serum levels that stay out of clinically safe ranges. Either of those across enough consecutive cycles and the protocol gets abandoned.

For the microbiome, the honest answer is I don't know, because nobody has defined it for this anatomy. In a natal vagina, "healthy" means Lactobacillus dominance (particularly L. crispatus), low diversity, and a pH below 4.5. For penile skin-lined neovaginas, applying that standard probably doesn't make sense. Without glycogen, you're fighting the tissue biology.

For peritoneal neovaginas, the question is genuinely open. 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 target Lactobacillus percentage. I'm watching for trends. Is the existing Lactobacillus expanding and persisting? Is diversity decreasing over time? Is pH trending down? Do symptomatic changes (discharge, odor) correlate with sequencing data?

If none of that happens, that's data too. Knowing that direct Lactobacillus reinforcement in a peritoneal neovagina under topical estrogen doesn't produce expansion of an existing low-abundance population 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.

A few notes if you're thinking about replicating

Monthly estradiol dose is significantly lower than typical static protocols (~11mg vs my prior ~24mg). The trough days (1–5) are real, and if your symptomatic low-E2 threshold is higher than mine, those days may be rough. Doses can be scaled proportionally while keeping the same ratios and timing.

Injection site matters. A thigh injection early in my pre-protocol phase created a SubQ depot that released slowly for weeks and contaminated my tracking data. Abdominal SubQ has been more predictable.

Vaginal progesterone absorption in a peritoneal neovagina likely differs from what literature reports for cis women, but the direction and magnitude haven't been directly measured. My doses were calibrated using my own paired serum data, not literature estimates. Different anatomy will absorb differently.

You can abort at any time. Resuming steady-state dosing is always available. This is an experiment, not a commitment.

The full protocol

Everything above is the short version. The full protocol document (v2.0, May 2026) covers the complete PK observations, protocol modification and abandonment criteria, failed-cycle rules, the limitations section, and the full reference list. It's attached below.

Cycle data, monitoring logs, Inito readings, and serum results will be kept and archived. Narrative observations and cycle-specific posts will be published here. Sequencing data from Evvy and Juno Bio will be reported as they come in.