endo-protocols

Study 001 — Does pre-operative vaginal microbiome state predict post-surgical recovery in endometriosis?

📋 Status: Protocol v0.1 — open to participant recruitment. Pilot data from one anonymized subject (Participant A) is in place. We’re now looking for 10–30 additional participants with endometriosis who are: (a) planning laparoscopic surgery in the next 6 months, (b) willing to do an Evvy test 2–4 weeks before surgery, (c) willing to wear a WHOOP from at least 2 weeks pre-op through 4 weeks post-op.

💰 Affiliate disclosure: If an Evvy referral / promo code is established, it will appear here with a clear disclosure. At this moment, there is no affiliate relationship. See disclosures.md.

⚠️ Not medical advice. Not a clinical trial. This is an observational, hypothesis-generating citizen-science study. Nothing here replaces care from your surgeon, OBGYN, or functional medicine provider. Participants are responsible for their own medical decisions.

🔬 Part of the EndEndo.io ecosystem.

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The question

Does the composition of the vaginal microbiome at the time of endometriosis surgery predict the trajectory of post-surgical recovery?

Why this study connects to Case Study 001 — and why this study is one part of testing an integrative hypothesis.

The case study at case-studies/001-core-restore-no-bc/ documents one patient’s biomarker response to a liver-side estrogen-clearance protocol (Phase 2 methylation, sulfation, glutathione conjugation work). The biomarker bloodwork confirmed restored methylation cycle capacity. The same patient’s vaginal microbiome data shows a CST-IV community with approximately half the species composition consistent with β-glucuronidase and sulfatase enzymatic potential per published characterization (Gardnerella spp., Prevotella spp., Atopobium / Fannyhessea, Megasphaera, Sneathia).

The integrative hypothesis these two datasets together generate — but do not themselves confirm — is that estrogen clearance in endometriosis depends on both hepatic Phase 2 capacity and microbial handling of cleared estrogens. The case study presents the biomarker side. This Study 001 prospectively tests, at cohort scale, whether vaginal microbiome state predicts a clinically meaningful endpoint (surgical recovery) in additional endometriosis patients. Together, the case study and this study are designed to generate the empirical data that would justify a fully-powered RCT of the integrative thesis. Neither one, alone or together, currently confirms it.

For the full mechanistic synthesis — published research supporting each leg separately, why integrative cohort research has not yet been published, what specific measurements would test the framework directly, and the explicit framing of this thesis as hypothesis-generating rather than confirmed — see microbiome-estrogen-axis.md.

Specifically:

• Do participants with a Lactobacillus crispatus-dominant vaginal microbiome (Community State Type I, or “CST-I”) recover faster than participants with a diverse, Gardnerella-associated microbiome (Community State Type IV, or “CST-IV”)?
• Is the recovery signal visible in WHOOP-measured HRV, resting heart rate, and sleep performance in the 4 weeks after surgery?
• Does subjective pain and fatigue recovery track with the objective autonomic recovery signal?

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Why this matters

1. The clinical detection gap — and why it persists

Endometriosis affects roughly 10% of women and people of reproductive age.¹ Laparoscopic excision is the gold-standard surgical intervention, but:

• Post-surgical recovery varies widely — some people return to baseline activity in 2 weeks, others in 8–12 weeks. The variance is poorly explained by procedure complexity alone.
• Vaginal microbiome state is almost never assessed pre-operatively despite strong evidence that CST-IV (diverse, Gardnerella-associated) communities are associated with elevated local and systemic inflammation,²³ and that chronic inflammation impairs tissue healing, autonomic recovery, and post-surgical symptom resolution.⁴
• Standard clinical BV testing is poorly suited to detect this. Symptomatic BV testing (Amsel criteria, Nugent score, Affirm VPIII DNA probe panel) is calibrated for acute, high-abundance, symptomatic infections. It is not calibrated for the subclinical, asymptomatic CST-IV dysbiosis that an increasing body of research suggests matters for chronic gynecologic conditions including endometriosis.

This isn’t a controversial claim within the microbiome research literature. It IS, however, a claim that hasn’t filtered into routine clinical care — which is the gap this study sits in.

2. Why standard BV testing misses what matters

Three structural reasons standard BV testing produces “negative” results in patients with clinically relevant subclinical dysbiosis:

(a) Detection threshold mismatch. The Affirm VPIII DNA-probe assay (one of the most common clinical BV panels in U.S. primary care, FDA-cleared in the 1990s) requires approximately 2 × 10⁵ to 2 × 10⁷ organisms/mL of Gardnerella vaginalis to flag a positive result. Metagenomic sequencing (mNGS, the technology Evvy uses) detects community composition at relative abundance ≥ 0.75%, independent of absolute load. A vaginal sample with a low overall microbial count but a high relative dominance of Gardnerella (i.e., classic CST-IV at low absolute biomass) can correctly read as Affirm-negative AND Evvy-CST-IV. Both results are technically accurate. They are answering different questions.

(b) The Gardnerella species problem. Until 2019, “Gardnerella” was treated as a single species — Gardnerella vaginalis. Vaneechoutte et al. (2019)⁵ split the genus into four genomically distinct species: G. vaginalis, G. swidsinskii, G. piotii, and G. leopoldii, plus an additional nine genomic species. Standard clinical BV testing (Affirm VPIII, NuSwab, and similar assays cleared before 2019) was designed against the G. vaginalis probe only. A patient with a CST-IV pattern driven primarily by G. swidsinskii, G. piotii, or G. leopoldii — with relatively low G. vaginalis — may be entirely missed by these targeted assays. mNGS detects all four species individually.

(c) The symptomatic-BV framing. Both the U.S. Preventive Services Task Force and ACOG guidelines for BV screening focus on symptomatic, treatment-relevant BV. The clinical question these guidelines answer is: “Should I prescribe metronidazole or clindamycin for this patient right now?” The clinical question subclinical CST-IV dysbiosis raises — “Does this patient’s vaginal community composition predict longer-term reproductive, surgical, or inflammatory outcomes?” — is not what current BV screening was designed for, and is not what most insurance-covered testing is paid to answer.

The result: a patient can have a “BV-negative” record from standard testing while simultaneously carrying a clinically meaningful CST-IV dysbiosis pattern that current research links to elevated reproductive and surgical risk. Pilot Subject A’s records (below) document exactly this pattern.

3. The endometriosis-specific rationale

Two independent lines of evidence link vaginal microbiome composition to endometriosis pathophysiology:

Direct microbial community differences. The Endobiota Study (Ata et al. 2019, Sci Rep) found distinct vaginal, cervical, and gut microbiome signatures in women with stage 3/4 endometriosis vs. healthy controls.⁶ A systematic review (Salliss et al. 2021, Hum Reprod Update) synthesized the broader literature on gut and genital microbiota and the “estrobolome” in endometriosis, infertility, and chronic pelvic pain.⁷

The LPS / TLR4 pathway. Work by Khan and colleagues (multiple papers, 2010–2018) established that bacterial lipopolysaccharide (LPS) from gram-negative organisms in the reproductive tract can promote endometriotic lesion growth via Toll-like receptor 4 signaling.⁸⁹ CST-IV communities are precisely the communities producing more LPS than CST-I/II communities. So a “BV-negative-by-Affirm” patient with a CST-IV community may have more LPS exposure to her own endometriosis lesions than her clinical chart reflects.

4. The diagnostic-and-reimbursement gap — and why it’s a real policy issue

This is the part of the gap that deserves naming clearly:

The older, less sensitive test is covered by insurance. The newer, more sensitive test is not.

Property	Affirm VPIII (and similar clinical assays)	Evvy mNGS
FDA status	FDA-cleared (Affirm VPIII originally cleared in the 1990s)	Laboratory Developed Test (LDT); not currently FDA-cleared
Insurance coverage	Routinely covered under standard CPT codes (e.g., 87510–87512 BV-specific molecular probes; 87480 Candida; 87660 Trichomonas) for any office visit with documented BV suspicion	Generally not covered; patient pays out-of-pocket (typically $129–$379 per test)
Species detected	1 (G. vaginalis probe, pre-2019 species definition) — plus Candida, Trichomonas	700+ vaginal microbes including all 4 Gardnerella species individually
Detection threshold	Requires ~2×10⁵–10⁷ organisms/mL for positive flag	Detects ≥ 0.75% relative abundance
Reports community composition	No (single-organism positive/negative)	Yes (full relative-abundance breakdown)
Designed for	Symptomatic acute BV diagnosis (1990s clinical use case)	Microbiome composition characterization (2020s research-and-clinical use case)

Why does this gap persist? Several structural reasons:

• Insurance coverage is tied to FDA clearance + USPSTF / ACOG guideline endorsement. Affirm VPIII has both. mNGS-based vaginal microbiome panels (Evvy, others) are LDTs operating under CLIA certification but not FDA-cleared as in-vitro diagnostics. Without FDA clearance, insurance coverage is unlikely.
• No standard ICD-10 code for subclinical CST-IV dysbiosis. BV has a billing code (N76.0); “asymptomatic vaginal dysbiosis” does not. Without a billable diagnosis, there’s no clinical reimbursement path even if a test exists.
• Clinical guideline lag. ACOG and CDC BV guidelines focus on symptomatic infections requiring antibiotic treatment. The research evidence base on subclinical CST-IV and downstream reproductive/surgical outcomes is younger than the guidelines and hasn’t been fully incorporated yet.
• No FDA-approved treatment for CST-IV without symptomatic BV. From an insurer’s perspective: even if mNGS detects subclinical dysbiosis, there’s no standardized covered treatment pathway for it. Live biotherapeutics like LACTIN-V (Phexxi-adjacent, CTV-05) are still in clinical development. Insurance is reluctant to pay for a test that doesn’t lead to a covered intervention.

The consequences of this gap:

• Patients with chronic gynecologic conditions are repeatedly told their BV testing is normal, even when subclinical dysbiosis is present and potentially relevant to their condition
• The patients most likely to benefit from microbiome characterization — those with chronic conditions like endometriosis, recurrent UTIs, infertility, recurrent miscarriage — are the patients least likely to access it through standard care, because insurance won’t cover the more sensitive test
• Out-of-pocket cost creates access inequity. $129–$379 for Evvy is meaningful out-of-pocket for many patients. Wealthier patients get answers; lower-income patients don’t.
• Even when patients pay out-of-pocket, many providers don’t know how to interpret the result. mNGS reports are not in standard clinical workflows; CST-IV findings without symptomatic BV often get dismissed as “interesting but not clinically actionable.”
• The standard of care does not currently reflect the research base on subclinical dysbiosis and reproductive / surgical outcomes. Citizen-science studies like this one are a way to accelerate the evidence accumulation needed to change that standard.

This is part of what this study is for: to generate the cohort-level evidence that subclinical CST-IV dysbiosis predicts measurable surgical-recovery outcomes — which is the kind of finding that could eventually change clinical guidelines, FDA-clearance pathways, and insurance coverage decisions.

5. Why citizen science is the right vehicle now

A formal RCT of microbiome-targeted pre-surgical optimization would cost millions and take 3–5 years. In the meantime, tens of thousands of people with endometriosis have surgery each year — many of whom already own a WHOOP and have used Evvy. Structured citizen science can pool that data into hypothesis-level evidence now, using the exact infrastructure the Isala Project (Belgium, 2023) validated for cross-sectional microbiome mapping with ~3,345 participants.¹⁰ This study adapts the Isala model from cross-sectional mapping to a prospective observational cohort design.

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Pilot data — Participant A (anonymized subject)

Why this case motivates the study

Participant A’s documented test history illustrates exactly the clinical detection gap described above:

Documented timeline (anonymized):

Approximate timeframe	Test	Documented result
~7 months before the Evvy test	LabCorp Vaginitis Panel via Affirm VPIII DNA-probe assay (clinician-ordered through a primary care provider)	✅ Negative across all three targets: Gardnerella vaginalis, Candida species, Trichomonas vaginalis. Final-result lab report on file (records held privately).
Spring 2024	Evvy Expanded Vaginal Health Test (mNGS + PCR)	CST-IV pattern with ~46% combined Gardnerella species across 4 species, low overall microbial load
~6 months later	Evvy retest (PCR panel)	L. crispatus still at low microbial load — suggests persistent rather than transient dysbiosis pattern

Pilot Evvy result — full mNGS species breakdown

Selected detections above Evvy’s 0.75% reporting threshold:

Microbe	Relative abundance	Interpretation
Lactobacillus crispatus	35%	Protective species — but not dominant enough for CST-I classification (CST-I requires > 80%)
Gardnerella swidsinskii	21%	BV-associated; novel species not detected by the Affirm probe (Vaneechoutte et al. 2019)5
Gardnerella vaginalis	14%	Classic BV marker — but at this absolute load, may be below Affirm’s detection threshold
Gardnerella piotii	8%	BV-associated; novel species not detected by the Affirm probe
Lactobacillus iners	8%	Transitional species; often present in CST-III, can shift toward dysbiosis
Gardnerella leopoldii	3%	BV-associated; novel species not detected by the Affirm probe
Fannyhessea (Atopobium) vaginae	2%	Strong dysbiosis indicator; synergistic with Gardnerella
Bifidobacterium animalis	1%	—
Megasphaera lornae	1%	Anaerobic dysbiosis marker
Prevotella bivia	1%	BV-associated anaerobe
Prevotella timonensis	1%	BV-associated anaerobe
Sneathia vaginalis (amnii)	1%	BV-associated

Combined Gardnerella across all 4 species: ~46% of detected microbial community.

STIs (PCR panel): all negative (Chlamydia, Mycoplasma genitalium, Neisseria gonorrhoeae, Trichomonas). Antimicrobial resistance genes (PCR panel): none detected across 10 antimicrobial classes. Overall microbial load: LOW.

Community State Type assignment: CST-IV — the diverse, Gardnerella-associated dysbiotic pattern. Not CST-I (would require > 80% L. crispatus), not CST-III (would require dominant L. iners).

How the two results can both be true at the same time

This is the central scientific point of Participant A’s case:

Test	Question it answered	Answer
Affirm VPIII (clinical, insurance-covered)	“Is there enough G. vaginalis DNA above a 1990s-calibrated detection threshold to flag positive?”	No — correctly negative under the test’s design parameters.
Evvy mNGS (LDT, out-of-pocket)	“What is the relative composition of the vaginal microbial community across all detectable species?”	A CST-IV pattern with ~46% combined Gardnerella across 4 species, of which 3 are not covered by the Affirm probe.

Both results are technically correct. They are measuring different things. The Affirm test, designed in an era when “Gardnerella” was a single species and BV was conceptualized as symptomatic and high-load, did its job. The Evvy test, calibrated to current microbiome science, did its job. The clinical interpretation gap is the issue — and it’s a gap a substantial fraction of endometriosis patients may be sitting in without knowing it.

What this pilot case does NOT tell us

• It’s n=1. Nothing here generalizes to anyone else.
• There is no concurrent WHOOP data for Participant A around either test (WHOOP ownership began after the May 2024 test), so we cannot correlate Participant A’s microbiome state with autonomic metrics for the specific Evvy windows. That’s the gap this citizen-science study is designed to fill prospectively.
• We do not have post-surgical recovery data for this case because no laparoscopic surgery has been performed in the study window.

What this pilot case DOES establish

• A worked example of the clinical detection gap — documented BV-negative by a standard insurance-covered DNA-probe assay, simultaneously CST-IV dysbiotic by mNGS, in the same patient, with lab reports for both on file.
• A concrete dataset layout the study will use for all participants (species-level relative abundance, PCR panel, STI/AMR panels, overall microbial load).
• Motivation for the central hypothesis — if subclinical dysbiosis is present in someone with endometriosis who presented as BV-negative by standard insurance-covered testing, how often does this happen in the broader endo population? And does it predict surgical recovery? Those are the questions the cohort will answer.

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Study design (v0.1 — open for critique)

Type

Prospective observational cohort study, citizen-science recruitment, no intervention prescribed by the study.

Primary hypothesis

In participants with endometriosis undergoing laparoscopic surgery, a CST-IV pre-operative vaginal microbiome will be associated with a slower post-surgical HRV recovery trajectory (defined as days to return to within 5% of individual 30-day pre-op HRV baseline) compared to CST-I (or L. crispatus-dominant communities).

Secondary hypotheses

• Higher relative abundance of Gardnerella spp. + Atopobium vaginae pre-op → more reported post-op pain and fatigue in weeks 1–4
• L. crispatus relative abundance correlates positively with sleep performance recovery speed
• Elevated resting HR persistence > 14 days post-op is more common in CST-IV participants
• Participants with a documented “BV-negative” record from standard clinical testing within 12 months pre-op will show non-trivial CST-IV prevalence by mNGS — testing the clinical detection gap directly across the cohort

Participants we are seeking

Inclusion:

• Clinically diagnosed endometriosis (laparoscopic confirmation or imaging/clinical diagnosis per participant’s provider)
• Scheduled for laparoscopic surgery (excision, fulguration, or diagnostic with planned excision) within 6 months
• Own or willing to wear a WHOOP from 2 weeks pre-op through 4 weeks post-op
• Willing to do an Evvy Expanded Vaginal Health Test 2–4 weeks before surgery
• Age 18+
• Willing to provide consent for de-identified data use

Exclusion:

• Currently on antibiotics (within 14 days of the Evvy test) — will bias microbiome state
• Active diagnosed STI at time of testing (these cases need clinical care, not a research study)
• Pregnancy
• Immunocompromised states that might independently affect recovery

What participants provide (de-identified)

1. Evvy results — species-level relative abundance, PCR panel (with STI / AMR results OK to redact if preferred)
2. WHOOP data export — daily recovery, HRV, resting HR, sleep performance, strain for a 10-week window (2 weeks pre-op through 4 weeks post-op plus 4 weeks for baseline)
3. Standard clinical BV-test record (if any) — type of test, date, and result; helps quantify the detection-gap prevalence across the cohort
4. Pre-op survey — surgical plan, current medications (including any hormonal contraception or suppression), endometriosis stage if known, date of last menstrual period
5. Weekly post-op log — pain (0–10), fatigue (0–10), ability to return to normal activity (0–4 scale), any infection or complication, any antibiotics prescribed
6. Post-op Evvy retest (optional) — at 6–8 weeks post-op if the participant wants the longitudinal view

Analysis plan (pre-registered)

• Assign each participant a Community State Type at pre-op (CST-I / II / III / IV / V) using the standard thresholds (Ravel et al. 2011).¹¹
• Compute individual pre-op HRV baseline = median rmssd over the 14 days immediately before surgery
• Define “HRV recovery time” = number of days post-op until a 7-day rolling median of rmssd returns to within 5% of the pre-op baseline
• Compare HRV recovery time across CST groupings using non-parametric tests (Mann-Whitney or Kruskal-Wallis given expected small n)
• Tabulate the detection-gap prevalence: of participants with a documented “BV-negative” standard test in the year pre-op, what fraction show CST-IV by mNGS?
• Report all individual-level data (de-identified) alongside aggregate results
• Explicitly report confounders: surgical duration, procedure type, medications, menstrual cycle phase at sampling, age, hormonal contraception status

Limitations (named up front)

• Self-selection bias — participants who do citizen science are not representative of the general endo population
• Heterogeneous surgeries — lap excision, fulguration, and diagnostic procedures have different recovery trajectories
• Menstrual cycle timing — vaginal microbiome composition varies across the cycle; we’ll attempt to standardize but can’t fully control
• Self-reported data — surgery date, pain scores, and medication use are all self-reported
• Small sample — n=10–30 is hypothesis-generating, not definitive
• Standard clinical BV-test records will not be available for all participants — some will not have been tested; some will not have access to their records; this limits the detection-gap prevalence subanalysis

This is a pilot. A positive signal here is a hypothesis worth formally testing. A null signal means the effect, if it exists, requires larger n or different measurement.

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How to participate

If you meet the inclusion criteria and want to join:

1. Email tblack818@gmail.com with subject “endo-protocols Study 001 interest”
2. Include:
   • Your surgery date (estimated OK)
   • Your time zone (for data handling / cycle phase tracking)
   • Confirmation you own a WHOOP and are willing to do an Evvy test
3. You will receive:
   • A consent form (plain language, one page)
   • A participant ID (your data will only ever be linked to this ID in the public repo)
   • Instructions for timing the Evvy test and WHOOP baseline
   • A link to the weekly post-op survey form
   • [If/when the Evvy referral is established] a discount code

What you get out of participating

• Your own personalized report comparing your trajectory to the cohort (de-identified, graphs only)
• Your de-identified data becomes part of a publicly available dataset that advances evidence-based care for endometriosis
• Cited participation in the final study write-up (by participant ID, not name) if/when we publish

What you do NOT get

• Medical advice
• A prescription to improve your microbiome (any interventions are between you and your provider)
• Any guarantee your surgery will go well

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References

Comprehensive bibliography spanning vaginal-microbiome foundations, the clinical detection gap, Gardnerella species discrimination, vaginal microbiome × endometriosis, LPS/TLR4 pathway, surgical-outcome research, Netherlands- and Europe-based microbiome work, citizen-science precedents, HRV-as-inflammation-proxy, and the reimbursement-policy framing: → ../../research/microbiome-and-endo.md.

Full bibliography including Netherlands-based vaginal microbiome research, citizen-science precedents, and the LPS/TLR4 endo pathway: research.md.

1. Zondervan KT, Becker CM, Missmer SA. “Endometriosis.” N Engl J Med. 2020;382(13):1244-1256. PMID: 32212520 ↩

2. Ravel J, Gajer P, Abdo Z, et al. “Vaginal microbiome of reproductive-age women.” Proc Natl Acad Sci USA. 2011;108 Suppl 1:4680-4687. PMID: 20534435 ↩

3. Anahtar MN, Byrne EH, Doherty KE, et al. “Cervicovaginal bacteria are a major modulator of host inflammatory responses in the female genital tract.” Immunity. 2015;42(5):965-976. PMID: 25992865 ↩

4. Thayer JF, Sternberg E. “Beyond heart rate variability: vagal regulation of allostatic systems.” Ann N Y Acad Sci. 2006;1088:361-372. PMID: 17192580 ↩

5. Vaneechoutte M, Guschin A, Van Simaey L, et al. “Emended description of Gardnerella vaginalis and description of Gardnerella leopoldii sp. nov., Gardnerella piotii sp. nov. and Gardnerella swidsinskii sp. nov., with delineation of 13 genomic species.” Int J Syst Evol Microbiol. 2019;69(3):679-687. PMID: 30648938 ↩ ↩²

6. Ata B, Yildiz S, Turkgeldi E, et al. “The Endobiota Study: A Comparison of Vaginal, Cervical, and Gut Microbiota Between Women with Stage 3/4 Endometriosis and Healthy Controls.” Sci Rep. 2019;9(1):2204. PMID: 30778155 ↩

7. Salliss ME, Farland LV, Mahnert ND, Herbst-Kralovetz MM. “The role of gut and genital microbiota and the estrobolome in endometriosis, infertility and chronic pelvic pain.” Hum Reprod Update. 2021;28(1):92-131. PMID: 34718567 ↩

8. Khan KN, Kitajima M, Hiraki K, et al. “Escherichia coli contamination of menstrual blood and effect of bacterial endotoxin on endometriosis.” Fertil Steril. 2010;94(7):2860-2863. PMID: 20627244 ↩

9. Khan KN, Fujishita A, Hiraki K, et al. “Bacterial contamination hypothesis: a new concept in endometriosis.” Reprod Med Biol. 2018;17(2):125-133. PMID: 29692669 ↩

10. Lebeer S, Ahannach S, Gehrmann T, et al. “A citizen-science-enabled catalogue of the vaginal microbiome and associated factors.” Nat Microbiol. 2023;8(11):2183-2195. PMID: 37884815 (The Isala Project — the methodological precedent for this study.) ↩

11. Ravel J et al. 2011 (see ²). ↩

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