Trans HRT · History of Science · Evidence Deep Dive

A century of E1S evidence

From Doisy's 1929 isolation of estrone to Tebbens 2022 finding that estrone is not associated with breast development in 212 trans women — every paper that built or undermined the "estrogen sulfate reservoir" framing, with verbatim quotes from primary literature, contestation noted, and a candid audit of community lore that overstates the evidence.

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00 Headline answers
Q: Is there a published E₁S → E₁ → E₂ pathway? How strong is the evidence?

Yes, in two distinct forms. Plasma-level back-conversion was quantified by Ruder, Loriaux & Lipsett 1972 at ρ = 0.014 for E₁S → E₂ and ρ = 0.21 for E₁S → E₁. Tissue-level intracrine conversion via STS → HSD17B1 is documented in postmenopausal breast cancer tissue (Pasqualini 1996; Reed 2005) but is much weaker in normal adult breast (Lønning 2015) and has not been measured directly in trans-feminine breast tissue.

Q: Has E₁S been administered to humans and the resulting E₁S/E₁/E₂ measured?

Yes — multiple times across 54 years. Ruder 1972 (IV + oral in 2 subjects), Longcope 1972 (IV in men), Schindler 1982 (pure oral E₁S vs E₁ vs E₂ vs Premarin head-to-head), Englund 1990 (piperazine E₁S vs oral E₂V head-to-head), the Bhavnani 1981–2000 Premarin program, and many Premarin / Ogen / Estropipate PK studies in postmenopausal women. What still does not exist: a modern LC-MS/MS PK study of pure oral E₁S in trans-feminine subjects. Doll 2022, the only modern trans LC-MS/MS PK study, deliberately did not measure E₁S.

Q: Does the E₁S "reservoir" actually drive more feminization?

The dispositive trans cohort data so far says no. Tebbens et al. 2022 (ENIGI, n=212): "Change in fat percentage and breast development in trans women were not associated with estrone concentrations nor with administration route." Oral users had ~8× the E₁ of transdermal users; breast volume changes were 62 vs 69 mL — statistically identical. E₁S itself was not measured in the cohort, but if its proximal product (E₁) does not predict outcomes, the strong-form reservoir argument loses most of its force.

01 The timeline

Color band on the left maps to era. Every event card has a year, the primary paper or milestone, a verbatim quote where one was retrievable from the abstract or accessible text, and a "contemporary context" line noting what was contested or what the surrounding discussion looked like.

Era 01 · Discovery — 1920s to mid-1960s
1923

Allen & Doisy: the rat-ovariectomy bioassay

Allen E, Doisy EA. JAMA 81:819. [QUOTE UNAVAILABLE — JAMA archive paywall]

The "estrus-producing hormone" gets a quantitative bioassay (the Allen-Doisy test). Becomes the standard for hunting and purifying estrogens through the late 1930s.

1929

Doisy and Butenandt isolate estrone — independently, in the same year

Doisy EA, Veler CD, Thayer SA. Am J Physiol 90:329–330 (Aug). Butenandt A. Naturwissenschaften 17:879 (Nov–Dec).

Initially called "theelin" (Doisy) and "Progynon" (Butenandt). Doisy presented in Boston first; Butenandt printed first. The priority dispute is real and contemporaneous, not retroactive.

Butenandt later: "I was beaten to publicity, not to science." Butenandt receives the 1939 Nobel; Doisy receives the 1943 Nobel (for vitamin K).

1930

Marrian isolates estriol from pregnancy urine

Marrian GF. Biochem J 24:435. [QUOTE UNAVAILABLE — PMC image-only]

The third major natural estrogen is identified. Marrian becomes the dominant figure in conjugate chemistry through the 1930s–40s.

1934–1936

Cohen & Marrian develop conjugate-hydrolysis chemistry

Cohen H, Marrian GF. Biochem J 28:1603 (1934); subsequent papers through 1936.

The acid-hydrolysis methodology that lets urinary estrogens be cracked open. The presence of glucuronide and sulfate conjugates is established. The dominant interpretation: conjugation is an inactivation step — the body's way of preparing estrogens for excretion.

This framing — conjugates as terminal end-products, not active reservoirs — will dominate clinical thinking for 30+ years.

1936

Estradiol isolated from sow ovaries — the ovary secretes E₂, not E₁

MacCorquodale DW, Thayer SA, Doisy EA. J Biol Chem 115:435. [QUOTE UNAVAILABLE — JBC archive 403]

Earlier urinary work had suggested estrone was the primary estrogen. The sow-ovary work demonstrates that what the ovary actually secretes is estradiol, and estrone is a metabolite. Reframes the picture: E₂ is the source, E₁ is downstream.

1938

Schachter & Marrian isolate estrone sulfate from pregnant-mare urine

Schachter B, Marrian GF. J Biol Chem 126:663. [QUOTE UNAVAILABLE — JBC archive 403]

The foundational identification of estrone-3-sulfate as a distinct chemical entity. The mare's urine context is not accidental — the high concentration in pregnant-mare urine is what makes purification tractable. This paper enables Premarin four years later.

1942

Premarin (conjugated equine estrogens) launches without efficacy proof

Ayerst (Canada 1941 → US 1942). Wyeth historical documentation.

An oral menopause-symptom drug, mostly E₁S by mass, with additional equilin sulfate, equilenin sulfate, and Δ⁸-E₁-sulfate. Clinically deployed in millions of women decades before the "E₁S as reservoir" framing is formalized.

Throughout the 1950s–60s, the dominant mental model in the menopause-medicine community is that Premarin works because some conjugates get hydrolyzed to active estrogens during/after absorption — but the kinetic picture is hand-waved until Bhavnani's 1980s program.

1946–1957

The "conjugates are inactivation" consensus (Szego, Roberts) — and the first crack

Szego CM, Roberts S. Recent Prog Horm Res 1948. Sandberg AA, Slaunwhite WR. J Clin Invest 36:1266 (1957). [QUOTE UNAVAILABLE — PMC image-only]

Szego/Roberts solidify the "liver inactivates estrogens via conjugation" view. Sandberg & Slaunwhite begin documenting enterohepatic recirculation of conjugates, which is the first hint that conjugates aren't strictly terminal.

Era 02 · Tracer studies — 1960s to 1980s
1963

Tait, Gurpide, Mann & Lieberman formalize the multi-pool / "no return" model

Tait JF. JCEM 1963. Gurpide E, Mann J, Lieberman S. JCEM 23:1155 (1963).

The mathematical machinery — transfer constant ρ, blood production rate, metabolic clearance rate (MCR) — that all subsequent steroid kinetic studies use. The "no return" axiom (radiolabel doesn't recycle back to the pool it came from) makes the math tractable at the price of being slightly wrong at the tissue level.

1968

Longcope, Layne & Tait measure free-E₁ / E₂ MCRs and E₁ ↔ E₂ transfer

Longcope C, Layne DS, Tait JF. J Clin Invest 47:93.

Free E₁ MCR ~1990 L/day/m²; free E₂ MCR ~1600 L/day/m². E₂ → E₁ transfer constant ~15%; E₁ → E₂ ~5%. The kinetics of unconjugated estrogen interconversion are pinned down.

1968

Piperazine estrone sulfate (estropipate, Ogen) — FDA-approved oral E₁S drug

Abbott Laboratories NDA. Marketed as Ogen.

A second oral E₁S product joins Premarin. Unlike CEE, Ogen is "clean" — just E₁S as a piperazine salt, no equine extras. Subsequent PK studies of estropipate become the cleanest data on what oral E₁S itself does to serum.

1969

Hembree notices a slow-equilibrating tritium pool

Hembree WC et al. JCEM 1969.

In tritiated-estrogen infusion studies, some radiolabel ends up in a slowly-equilibrating compartment that doesn't fit the free-E₁/E₂ kinetics. Hembree suspects but doesn't identify the responsible species. Ruder 1972 will demonstrate it's E₁S.

1972

Ruder, Loriaux & Lipsett — the canonical E₁S transfer factors

Ruder HJ, Loriaux DL, Lipsett MB. J Clin Invest 51:1020–1033. DOI 10.1172/JCI106862.

IV-infused radiolabeled E₁S in 5 men + 6 women; also orally administered to 2 subjects. Measures the three numbers that the entire modern conversation runs on.

The calculated transfer factors were: ρE1SE1 = 0.21, ρE1SE2 = 0.014. … Essentially, similar data were obtained when E1S was given by mouth to two subjects.

MCRE1S = 157 L/day. About 1 in 70 plasma E₁S molecules makes it to plasma E₂; about 1 in 5 makes it to E₁. Oral and IV behave kinetically the same way.

This 1.4% number has never been re-measured by modern LC-MS/MS in the 54 years since. It is the single most-cited transfer factor in the entire estrogen-pharmacology literature.

1972

Longcope independently measures E₁S kinetics in normal males

Longcope C. J Clin Endocrinol Metab 34:113–122. DOI 10.1210/jcem-34-1-113.

Same year, independent cohort, slightly different numbers. ρE1S→E1 = 0.15 (95% CI 0.12–0.18); ρE1S→E2 = 0.022–0.044 in two subjects. MCR ~90 L/day/m².

Already in 1972, the "1.4% back-conversion" number has 1.5–3× scatter when measured in a different cohort. Modern reviews that cite "ρ = 0.014" as if it's a settled physical constant are doing the literature a small injustice.

1973

Grodin, Siiteri & MacDonald — peripheral aromatization of androstenedione

Grodin JM, Siiteri PK, MacDonald PC. JCEM 36:207.

Establishes that postmenopausal estrogen comes substantially from peripheral aromatization of androstenedione, not just ovarian secretion. The systemic-pool model is supplemented with a tissue-source model.

1975

Yen et al. — oral 2 mg E₂ PK in postmenopausal women

Yen SSC, Martin PL, Burnier AM, Czekala NM, Greaney MO, Callantine MR. JCEM 40:518.

Peak E₂ 110 pg/mL at 5 h; peak E₁ 467 pg/mL at 6 h. E₁:E₂ ratio 3–6 (vs < 1 by IV). The textbook oral-vs-IV asymmetry — first-pass hepatic metabolism inflates E₁ — is empirically nailed down.

1978

X-linked ichthyosis mapped to STS deficiency

Webster D, France JT, Shapiro LJ, Weiss R. Lancet 1978.

A skin disease turns out to be a steroid-sulfatase enzyme defect on the X chromosome. The STS gene gets a chromosomal address. Sets up the 1987 cloning.

1981–1990

Bhavnani's Premarin / equilin-sulfate program at Western Ontario

Bhavnani BR et al. JCEM 1981, 1983, 1989; J Soc Gynecol Investig 2000.

A 20-year program of CEE PK. 1981: oral vs IV Premarin; PO peak E₁ 1.4 ng/mL at 5 h (vs IV peak 11.2 ng/mL at 20 min). 1983–1989: dual-isotope ([³H]equilin-[³⁵S]sulfate) work demonstrates that some orally administered sulfate survives intact through the gut wall, while some is hydrolyzed first and re-sulfated post-absorption. "Oral E₁S" is two parallel kinetic routes, not one.

1982

Schindler et al. — pure oral E₁S vs E₁ vs E₂ vs Premarin, head to head

Schindler AE, Bolt HM, Zwirner M, Hochlehnert G, Göser R. Arzneim-Forsch 1982.

At equimolar doses (9.74 µmol of each), conjugated estrogens — including pure E₁S — produce a "much more protracted influx of oestrogens from the intestine into the plasma compartment," with a clear second-bump enterohepatic recirculation event at 10–12 hours. Free E₁ gives the highest serum E₁/E₂ ratio. This is the closest 1980s reference for "what does oral pure E₁S look like in serum" — and it shows the protracted, smoothed profile that the reservoir framing predicts.

1984

Santner et al. — breast tumor sulfatase activity is ~10× aromatase

Santner SJ et al. JCEM 1984.

The first quantitative demonstration that breast tumor tissue runs its own E₁S → E₂ chain locally. Sulfatase activity in tumor >> aromatase activity, suggesting most tumor E₂ comes from the conjugate pool, not from in-tumor androgen aromatization. The first crack in the "systemic pool only" model.

This breast-cancer-tissue finding will be heavily cited and over-generalized for the next 30 years.

Era 03 · STS molecular biology — late 1980s through 2000s
1987

The STS gene is cloned

Yen PH, Allen E, Marsh B, Mohandas T, Wang N, Taggart RT, Shapiro LJ. Cell 49:443–454. PMID 3471362.

Steroid sulfatase becomes a defined gene product, not just an enzyme activity. The molecular era of intracrine biology begins.

1991

Labrie coins "intracrinology"

Labrie F. Mol Cell Endocrinol 78:C113–C118. PMID 1838014.

A new framework: in many tissues, local enzymes — not just plasma steroid concentration — set the active hormone concentration that the receptor sees. Endocrine (gland → blood → target) vs paracrine (cell → adjacent cell) vs intracrine (within-cell synthesis from circulating precursors).

Initially controversial; gradually adopted as the standard framing for postmenopausal estrogen biology, prostate androgen biology, and the breast-cancer sulfatase-inhibitor program.

1994

Reed/Purohit team synthesize EMATE — the first STS-inhibitor lead

Howarth NM, Purohit A, Reed MJ, Potter BVL. J Med Chem 1994.

Estrone 3-O-sulfamate — an irreversible STS inhibitor. The starting point of the Reed/Purohit Imperial College London STS-inhibitor program. The therapeutic logic: if breast tumor STS supplies most of tumor E₂ from the E₁S reservoir, disabling STS should be therapeutic.

1996

Pasqualini — breast tumor sulfatase 50–200× aromatase

Pasqualini JR, Chetrite G, Blacker C, et al. JCEM 1996.

Massive enzyme-activity ratio in tumor tissue. Becomes the quantitative anchor for the "the intracrine pathway dominates" framing in breast cancer.

Quantitative anchor for the strong-form reservoir argument. Will be cited (often without the "in tumor tissue" qualifier) for decades.

2001

OATP-B identified as the major peripheral E₁S transporter

Tamai I, Nezu J, Uchino H, Sai Y, Oku A, Shimane M, Tsuji A. Biochem Biophys Res Commun 2001.

E₁S doesn't passively cross cell membranes — it needs an organic-anion transporter. OATP1B1, OATP2B1 (OATP-B), OATP4A1 get characterized as the main carriers. A third rate-limiting step in the reservoir → E₂ chain (after STS hydrolysis and HSD17B1 reduction): the steroid has to get into the cell first.

2005

Reed, Purohit, Newman & Potter — the canonical sulfatase review

Reed MJ, Purohit A, Woo LWL, Newman SP, Potter BVL. Endocr Rev 26:171–202. DOI 10.1210/er.2004-0003.

Steroid sulfatase catalyzes the hydrolysis of aryl and alkyl steroid sulfates… STS, especially in postmenopausal women with hormone-dependent breast cancer, may contribute substantially to the estrogenic stimulation of tumor growth.

The standard reference for the molecular biology of STS, its regulation, its tissue distribution, and the rationale for STS inhibitors. 200+ pages, extensively cited.

2005

Kuhl's encyclopedic route-comparison review

Kuhl H. Climacteric 8 Suppl 1:3–63. PMID 16112947.

Becomes the standard reference for oral / sublingual / transdermal / injectable PK comparison. Among other things, states that on oral 2 mg E₂V at steady state, "E₁ is 4–6× E₂; E₁S is about 200× E₂."

2006

First-in-human STS inhibitor — STX-64 / irosustat

Stanway SJ et al. Clin Cancer Res 12:1585.

Reed/Purohit's lead compound reaches a phase I trial in advanced breast cancer. 98–99% tumor STS inhibition achieved. The strong-form intracrine model predicts substantial clinical benefit.

Era 04 · HSD17B isoforms — the direction of conversion
1988

HSD17B1 cloned — the reductive isoform

Peltoketo H, Isomaa V, Mäentausta O, Vihko R. FEBS Lett 239:73–77.

First cloning of an HSD17B from human placenta. 327-aa cytosolic protein. Prefers the reductive direction (E₁ → E₂). Concentrated in placenta and ovary. The "estrogen-activating" isoform.

Until this paper, "17β-HSD" was treated as a single enzyme. Now the field starts to grasp that it's a family.

1993

HSD17B2 cloned — the oxidative isoform

Wu L, Einstein M, Geissler WM, Chan HK, Elliston KO, Andersson S. J Biol Chem 268:12964.

A distinct enzyme. Microsomal. Broader substrate range. Prefers oxidation (E₂ → E₁), the opposite direction. Expressed in liver, gut, secretory endometrium. The "estrogen-inactivating" isoform.

1996

Miettinen — the tissue-direction rule crystallizes

Miettinen MM, Mustonen MVJ, Poutanen MH, Isomaa VV, Vihko RK. Biochem J 314:839.

17HSD type 1 acts as a reductase converting oestrone into oestradiol, whereas 17HSD type 2 possesses oxidative activity inactivating oestradiol by converting it into oestrone… in cultured cells the direction of 17HSD activity is exclusively determined by the expression of these distinct isoenzymes.

Whether a tissue runs E₁ → E₂ or E₂ → E₁ is set by which HSD17B isoform it expresses. Estrogen-target tissues (ER+ breast, ovary, endometrium) tend toward HSD17B1/7/12 (reductive); estrogen-inactivating tissues (liver, GI, placenta-fetal interface) tend toward HSD17B2/4/8 (oxidative). This is the fact that determines whether a high circulating E₁S pool actually produces local E₂ in any given tissue.

1994–2010s

HSD17B3, 4, 5, 7, 12 characterized — at least 14 isoforms identified

Geissler WM 1994 (HSD17B3); Adamski J 1995, 2001 (HSD17B4); Penning TM (AKR1C3 = HSD17B5); Luu-The 2006 (HSD17B12).

The "17β-HSD" field bifurcates by structural family — most are SDRs (short-chain dehydrogenase/reductase, NAD-preferring); HSD17B5 is an AKR (aldo-keto reductase, NADPH-preferring, cytosolic). The field is messy through ~2000.

Era 05 · Direct E₁S administration — the user's question
1990

Englund & Johansson — head-to-head: piperazine E₁S vs oral E₂V

Englund DE, Johansson EDB. Maturitas 1990.

8 postmenopausal women, crossover, 2.5 mg/day piperazine E₁S vs 2.0 mg/day estradiol valerate, 21-day arms.

The levels of all the other oestrogens analyzed were consistently higher and the area under the curve significantly greater (except in the case of E3S) following PE1S administration than those recorded after E2V ingestion.

Dose-for-dose, oral E₁S raises serum E₁ and E₁S more than oral E₂. The 21-day arm accumulated E₁S without saturation. Translation: if you actually want a big E₁S pool, oral E₁S is more efficient at building one than oral E₂ is.

1992

Lobo & Cassidenti — the clinical-consensus review

Lobo RA, Cassidenti DL. J Reprod Med 37:77–84.

Crystallized the framing for menopause medicine. Premenopausal E₂:E₁ ~1:1; transdermal ~1:1; oral E₂ ~1:5, sometimes 1:20. Oral E₂ raises E₁S by 8–20× over premenopausal baseline. Oral E₂, oral E₂V, and oral E₁S/PE₁S/estropipate all converge on a similar first-pass-dominated serum fingerprint.

1997

Price et al. — sublingual vs oral E₂ (the foundational SL paper)

Price TM, Blauer KL, Hansen M, Stanczyk F, Lobo R, Bates GW. Obstet Gynecol 89:340. DOI 10.1016/S0029-7844(96)00513-3.

6 postmenopausal women, multiple doses of micronized E₂ given orally or sublingually, E₂ / E₁ / E₁S measured.

Estrone sulfate levels correlated with the dosage administered and also tended to be higher with sublingual administration.

SL gave a 13× higher peak E₂ (451 vs 34 pg/mL for 1 mg). SL E₁:E₂ ratio was lower over 24 h. BUT E₁S correlated with dose, not route: at 4 hours, mean E₁S was 11.8 ng/mL after 1 mg SL. Sublingual administration did NOT avoid the E₁S pool.

This is the single most important fact for the "sublingual is just better" community claim. The Cmax narrative is right; the "avoids first-pass" narrative is partial — swallowed saliva delivers a substantial fraction through the gut anyway.

1998

Bhavnani — the encyclopedic CEE review

Bhavnani BR. Proc Soc Exp Biol Med 217:6–16.

Estrogen sulfates can be absorbed directly from the gastrointestinal tract; however, hydrolysis of the sulfates also occurs in the gastrointestinal tract, and the unconjugated estrogens formed are readily absorbed. After absorption, these estrogens are sulfated rapidly and circulate in this form. The pharmacokinetics of these estrogens indicate that the unconjugated estrogens are cleared from the circulation at a faster rate than their sulfate ester forms.

The clearest published summary of what "oral E₁S as a drug" actually does in the body.

2001

Slater — long-term serum E₁S on oral E₂

Slater CC, Hodis HN, Mack WJ, Shoupe D, Paulson RJ, Stanczyk FZ. Menopause 8:200–203. PMID 11355042.

Postmenopausal women, oral 1 mg E₂ daily for 16 months vs transdermal patches. Oral E₂ → serum E₁S 38.8 ng/mL at 15 months (RIA). Transdermal → 1.8–3.2 ng/mL. A ~10–20× difference at matched serum E₂.

This 38.8 ng/mL is the single most-cited "E₁S elevates a lot on oral E₂" number in the literature. Note: it is RIA. The Zumenon SmPC (LC-MS/MS-based) gives Cavg 5.28 ng/mL for the same 1 mg oral dose — a 7× discrepancy that has never been fully reconciled, and is almost certainly assay methodology.

Era 06 · Modern LC-MS/MS, trans cohorts, and community lore
2007–2013

Stanczyk, Rosner — the assay-methodology revolution

Stanczyk FZ, Clarke NJ. J Steroid Biochem Mol Biol 2010; Rosner W et al., Endocrine Society position 2007 / 2013.

A wave of methodology critiques. Direct E₂ immunoassays are unreliable below ~50 pg/mL — useless at transdermal serum ranges. Median postmenopausal E₂ reference values differ by 6× across assays (Stanczyk 2007). Older RIA conjugate values, especially E₁S, are systematically biased high due to cross-reactivity.

Implication for everything before ~2010: numbers should be treated as approximations whose absolute calibration is uncertain. The Slater 2001 vs Zumenon SmPC discrepancy is the textbook example.

2014

First large ENIGI publications — the modern trans-feminine cohort era

Wierckx K, Van Caenegem E, Schreiner T, et al. J Sex Med 11:1999.

The European Network for the Investigation of Gender Incongruence cohort begins publishing. For the first time, large trans-feminine HRT outcome data exists, with multiple routes (oral, transdermal, injectable), modern assays, and rigorous outcome tracking.

2015

Lønning — normal breast tissue ≈ serum E₂

Lønning PE, Helle H, Duong NK, Ekse D, Aas T, Geisler J. PMID 25721699.

LC-MS/MS measurement of E₂ in normal breast tissue and matched serum in cycling women. Median tissue E₂ ≈ median serum E₂ (~30 pg/g vs ~30 pg/mL).

The strong-form intracrine framing inherited from breast-cancer-tissue literature does not generalize to healthy adult breast. Tissue does not concentrate E₂ above serum levels in normal physiology. The "tissue is 5–50× serum" community claim is a misapplied tumor-tissue number.

2017

Palmieri — irosustat phase II: STS inhibition adds little to AI

Palmieri C et al. — IRIS phase II trial of irosustat (STX-64) added to aromatase inhibitor.

Phase II in advanced postmenopausal breast cancer. STS inhibitor added to AI gives a clinical benefit rate of only 18.5%. The strong-form prediction — that disabling the E₁S reservoir would substantially augment AI therapy — is largely not borne out.

If the E₁S reservoir were as load-bearing in tumor biology as the 1980s–90s in-vitro work suggested, IRIS should have shown a much larger effect. The 2017 clinical signal is the strongest empirical pushback on the strong-form reservoir argument.

2022

Tebbens / de Blok — estrone is NOT associated with trans feminization

Tebbens M, Heijboer AC, T'Sjoen G, Bisschop PH, den Heijer M. J Clin Endocrinol Metab 107:e458. DOI 10.1210/clinem/dgab638. PMID 34632510.

Prospective 1-year ENIGI cohort, n=212 trans women, two arms: oral vs transdermal E₂.

Change in fat percentage and breast development in trans women were not associated with estrone concentrations nor with administration route. Therefore, measurement of estrone concentrations does not have a place in the monitoring of feminization in trans women.

Oral users had ~8× the E₁ of transdermal users. Breast development was 62 vs 69 mL volume change — statistically not different. E₁S itself was not measured. This is the dispositive trans-cohort paper for the "reservoir → feminization" claim.

The strong-form community claim — that high E₁S pools drive more breast growth or more feminization — is contradicted by the best trans data we have.

2022

Doll et al. — first modern LC-MS/MS trans PK (oral vs SL)

Doll EE, Shoger KE, Hooper MJ, Patel R, Tangpricha V, Sarvaideo JL. Endocr Pract 28:237. PMID 34781041.

10 trans women, 1 mg oral vs 1 mg SL E₂, LC-MS/MS. SL Cmax 144 vs PO 35 pg/mL; AUC₀-₈ ratio 1.8×; E₂:E₁ ratio 1.1 ± 1.0 (SL) vs 0.7 ± 0.4 (PO).

E₁S was not measured. The single biggest missed opportunity for the reservoir question in the modern era.

2022

Cirrincione — the Beckman immunoassay reads ~40% low on oral E₂

Cirrincione LR, Smith K, McAuley J, et al. PMC8859944.

The Beckman Access Sensitive Estradiol direct immunoassay under-reports E₂ by ~40% specifically in patients on oral E₂, almost certainly via estrone cross-reactivity. Pre-2022 "oral E₂ trough is low" clinical reports are systematically biased.

02 Direct answer — every human study that administered E₁S and measured serum estrogens

This is the explicit answer to "has E₁S been put into humans?" Compiled from era-05's deep dive. Multiple studies across 54 years — but no modern LC-MS/MS pure-oral-E₁S PK in trans-feminine subjects exists.

YearAuthors / journalProductRoute, dosenKey result
1972Ruder, Loriaux, Lipsett / J Clin InvestRadiolabeled E₁SIV infusion + PO in 2 subjects11 + 2ρ E₁S→E₁ = 0.21; ρ E₁S→E₂ = 0.014; MCR 157 L/d; "essentially similar" oral vs IV
1972Longcope / JCEMRadiolabeled E₁SIV infusionmalesρ E₁S→E₁ = 0.15; ρ E₁S→E₂ = 0.022–0.044; MCR ~90 L/d/m²
1975Yen et al. / JCEMOral micronized E₂PO 2 mg9 postmenopausalPeak E₂ 110, E₁ 467 pg/mL; E₁:E₂ ratio 3–6
1981Bhavnani / JCEMPremarinIV 10 mg + PO 10 mgpostmenopausalPO peak E₁ 1.4 ng/mL at 5 h vs IV peak 11.2 ng/mL at 20 min
1982Schindler et al. / Arzneim-ForschPure oral E₁S vs E₁ vs E₂ vs PremarinPO 9.74 µmol eachwomen, small n"Protracted influx" + 10–12 h enterohepatic bump for conjugates
1989Bhavnani et al. / JCEM[³H]equilin-[³⁵S]sulfatePO + IV (dual-isotope)postmenopausalSome oral sulfate absorbs intact; some hydrolyzed and re-sulfated post-absorption
1990Englund, Johansson / MaturitasPiperazine E₁S vs E₂VPO 2.5 vs 2.0 mg8 postmenopausalPE₁S → higher AUC for E₁ and E₁S than E₂V; 21-day E₁S accumulation
1992Lobo & Cassidenti / J Reprod MedOral E₂ (review)PO 1 mgreviewOral E₂ raises E₁S 8–20× over baseline
1997Price et al. / Obstet GynecolMicronized E₂ SL vs POSL 1, 0.5, 0.25 mg; PO 1, 0.5 mg6 postmenopausalSL Cmax 13× PO; E₁S correlated with dose not route
2000Bhavnani et al. / J Soc Gynecol InvestigPremarin vs synthetic CESPO single dose41Not bioequivalent; total estrone AUC >> unconjugated
2001Slater et al. / MenopauseOral E₂ vs transdermal E₂PO 1 mg vs patch33E₁S 38.8 ng/mL (oral) vs 1.8–3.2 ng/mL (transdermal) at 15 months
2022Doll et al. / Endocr PractOral vs SL E₂PO 1 mg vs SL 1 mg10 trans womenSL Cmax 144 vs PO 35 pg/mL; E₂:E₁ different; E₁S NOT measured
03 Community lore audit

What r/TransDIY, r/Estrogel, Discord servers, and DIY-HRT compendia say vs what the primary literature actually supports. Note: Aly's writeups at Transfeminine Science are usually careful and do NOT make the strong-form claims; those are folk extrapolations from Wikipedia's PK page and selective Reddit reading.

ClaimVerdictEvidence
"Sublingual is just better."refinedSL gives higher Cmax and modestly higher early-window AUC (Doll 2022, 1.8×), but no breast-development advantage in any modern cohort. SL does not avoid the E₁S pool (Price 1997).
"E₁S is a reservoir that drives extra feminization."refutedTebbens 2022 (n=212): "breast development in trans women were not associated with estrone concentrations nor with administration route." E₁S unmeasured but its proximal product E₁ is null.
"Premarin is a slow-release E₁S reservoir."refinedPharmacokinetically true that CEE is ~50–70% E₁S by mass and has a slow-release profile (Bhavnani 1981–2000). But CEE contains equilin sulfate, equilenin sulfate, Δ⁸-E₁-sulfate which are not pharmacologically equivalent to micronized E₁S. "Premarin = pure oral E₁S" is wrong.
"Sublingual generates less E₁S."refinedPrice 1997 found E₁S correlated with dose, not route. SL still delivers a substantial dose through the swallowed-saliva GI route, and recycled E₁/E₂ still get sulfated. Cirrincione 2021 found SL still drives high E₁.
"Tissue E₂ is 5–50× serum E₂ in target tissues."overstatedThe 5–50× figure is from postmenopausal breast cancer tissue (Pasqualini, Geisler). In normal breast tissue (Lønning 2015 LC-MS/MS), tissue E₂ ≈ serum E₂ in cycling women.
"Oral E₂ is 'really' 15–25% bioavailable when you count E₁S."refutedNot a published PK metric. Bioavailability is a plasma metric for intact drug. Tissue activation via reservoir doesn't compress to one percent.
"Anastrozole + oral E₂ proves the STS bypass."overstatedSTS bypasses aromatase mechanistically. But the IRIS phase-II trial showed STS inhibition added to AI gives only 18.5% clinical benefit rate in postmenopausal breast cancer — much less than the strong-form intracrine model predicts. No trans oral-E₂ + AI study supports the clinical claim.
"E₁S half-life is up to 30 hours."refinedKuhl 2005 and Lobo & Cassidenti 1992 both give 10–12 h. The 30 h anchor wasn't located in any primary source.
04 Remaining gaps in the literature
  • No modern LC-MS/MS PK of pure oral E₁S in trans-feminine subjects. Doll 2022 came closest and chose not to measure E₁S. Would clarify the post-Slater-vs-SmPC calibration discrepancy.
  • Ruder 1972's ρ = 0.014 has never been re-measured by LC-MS/MS in 54 years. The most-cited transfer factor in the literature still rests on a 1972 isotope-dilution measurement in 11 humans.
  • HSD17B1 / 7 / 12 expression has not been measured in trans-feminine breast tissue under exogenous E₂. Determines whether the local reductive E₁ → E₂ step actually runs in transfemme breast.
  • OATP transporter expression in trans-feminine target tissues is unstudied. Determines whether circulating E₁S can actually get into target tissues.
  • Sublingual PK with explicit E₁S measurement in a trans cohort. Price 1997 did it in postmenopausal women; nobody has repeated it in trans women with LC-MS/MS.
  • The IRIS-style question for trans HRT: does an STS inhibitor or HSD17B1 inhibitor co-administered with oral E₂ change feminization outcomes? Nobody will run this trial for ethical and commercial reasons. So the strong-form intracrine reservoir claim for trans HRT may never be directly tested.
05 Sources

The full evidence map sits at darkarts.wiki/research/oral-vs-sublingual-e2/historical/:

  • brief.md — research scope
  • era-01-discovery-1920s-1960s.md — 36 KB
  • era-02-tracer-studies-1960s-1980s.md — 33 KB
  • era-03-sts-pathway-1990s-2000s.md — 29 KB
  • era-04-hsd17b-isoforms.md — 31 KB
  • era-05-direct-e1s-administration.md — 20 KB
  • era-06-modern-reframing-trans-community.md — 32 KB
  • timeline.md — chronological synthesis
  • quotes/ — 91 individual verbatim-quote files, one per paper

Each era file cites primary papers with PMID/DOI and links to a verbatim quote file in quotes/. The full era-by-era research output runs to about 180 KB of structured analysis, plus the quote library.