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Injectable Aqueous Suspensions of Sex Hormones and How Depot Injectables Work

By Aly W. | First published June 14, 2019 | Last modified July 1, 2021

Abstract / TL;DR

Depot injectable steroid preparations are formulated mainly as oil solutions and crystalline aqueous suspensions. Oil solutions of steroids are more well-known and widely used but aqueous suspensions also exist and continue to be employed in medicine. For example, depot medroxyprogesterone acetate and estradiol cypionate-containing combined injectable contraceptives are aqueous suspensions. Both oil solutions and aqueous suspensions produce a depot effect. However, they function in very different ways to produce this depot effect. Oil solutions form an oil depot at the site of injection which is slowly absorbed and from which the steroid slowly escapes, whereas aqueous suspensions deliver crystals of pure steroid which slowly dissolve and are absorbed. Aqueous suspensions are essentially like tiny pellet implants that can be injected instead of surgically inserted. Aqueous suspensions tend to have longer durations than oil solutions and can allow for much wider dosing intervals in comparison. The dissolution rates and durations of aqueous suspensions are highly dependent on crystal size. A limitation of aqueous suspensions is that they can cause pain and irritation at the site of injection in a way that oil solutions have not been associated with and this is likely responsible for their less widespread use. Aqueous suspensions of estradiol benzoate (Agofollin Depot) and progesterone (Agolutin Depot) remain limitedly and sporadically available and have much longer durations than the more common oil solutions of these steroids. These preparations may be potential options for use by transfeminine people.


Depot injectable sex steroids are administered by intramuscular injection (into muscle) or subcutaneous injection (into fat) and are formulated in two main ways: 1) as oil solutions; and 2) as crystalline aqueous suspensions. What most transfeminine people are familiar with when it comes to injectable steroid preparations are oil solutions. This is how most common preparations like injectable estradiol valerate (Delestrogen, Progynon-Depot), estradiol cypionate (Depo-Estradiol), testosterone esters, progesterone, and hydroxyprogesterone caproate are formulated. However, injectable aqueous suspensions of sex steroids also exist and are used medically. The most major and well-known formulations include depot medroxyprogesterone acetate (Depo-Provera) and combined injectable contraceptives containing estradiol cypionate/medroxyprogesterone acetate (Cyclofem, Lunelle). Additionally, there exist more obscure preparations of aqueous suspensions such as estradiol benzoate (Agofollin Depot) and progesterone (Agolutin Depot) among others which are marketed in addition to the more common oil solutions of these steroids. These preparations may remain commercially available, including online from sites like EU Aibolit (Reddit). Many transfeminine people are unaware of what aqueous suspensions are or their medical availability. It is notable in this regard that aqueous suspensions are very different from oil solutions in their properties but can have much longer durations in comparison. This is potentially of therapeutic value especially in the case of otherwise shorter-acting injectables like progesterone. The purpose of this article is to shed light on injectable aqueous suspensions, describe how injectable oil solutions and aqueous suspensions work to achieve their depot effect and how they are different, and discuss how relevant aqueous suspensions can be obtained for medical use.

How Depot Injectables Work

Sex steroids are typically lipophilic (fat-soluble or “lipid-loving”) and hydrophobic (water-insoluble or “water-hating”). In other words, they dissolve in and are “attracted to” lipids (e.g., fats), and they are poorly solubility in and repelled by water. From chemistry, this is because sex steroids are very non-polar, whereas water is a quite polar molecule. As a result of their lipophilicity, sex steroids readily form clear homogenous solutions when mixed into oil—that is, they form oil solutions. In contrast, because sex steroids are hydrophobic, they do not easily form solutions when mixed into water—that is, they do not easily form aqueous solutions (like, e.g., salt and water). Instead of aqueous solutions, solid “clumps” or crystal particles of sex steroids can be mixed into and thereby suspended in water—that is, aqueous suspensions of sex steroid crystals can be made. Depot injectables are formulated as oil solutions or aqueous suspensions and these preparations have very different properties.

Injectable Oil Solutions

When an oil solution of a sex steroid is administered by intramuscular or subcutaneous injection, the solution is trapped within the tissue compartment it is injected into and remains there. As the tissue fluid is a water mixture, the oil solution stays together inside the tissue compartment and does not easily separate or distribute. This is because the lipophilic fats and sex steroids within the solution are attracted to each other and are repelled by water. Instead of rapidly dissolving, the fats and sex steroids at the edges of the oil solution are very slowly absorbed into the surrounding water. Once they have escaped the oil depot into the surrounding tissue fluid, they can be distributed into the bloodstream and then into other tissues to exert their biological effects. Eventually, the whole oil solution will be absorbed.

Oftentimes sex steroids that are used by intramuscular or subcutaneous injection are esterified with one or more lipophilic hydrocarbon esters. These esters include fatty acids like propanoic acid (propionate), pentanoic acid (valerate), hexanoic acid (caproate), heptanoic acid (enanthate), decanoic acid (decanote or decylate), and undecanoic acid (undecylate or undecanoate) as well as cyclic compounds like benzoic acid (benzoate), cyclopentylpropanoic acid (cypionate), and phenylpropanoic acid (phenylpropionate), among many others. Examples of these sex steroid esters include the well-known estradiol valerate, estradiol cypionate, estradiol benzoate, hydroxyprogesterone caproate, and numerous others. The attachment of a lipophilic ester (e.g., valeric acid) to a sex steroid (e.g., estradiol) will increase the lipophilicity of the sex steroid compared to merely injecting the unesterified sex steroid in an oil solution. The longer the carbon atom chain in the case of the simple fatty acid esters (e.g., propionate, valerate, enanthate, undecylate), the more lipophilic the resulting esterified sex steroid will be. As a result, the injected sex steroid ester will escape the oil tissue depot more slowly, lengthening the amount of time it takes for the sex steroid ester to be absorbed and therefore its duration in the body. The tables here and here show the lengthening duration of estradiol with longer or bulkier and and more lipophilic esters. Whereas an intramuscular injection of estradiol or progesterone in oil solution has a duration of only around 2 days, an intramuscular injection of an oil solution of estradiol undecylate, an ester of estradiol with a long fatty acid chain, has a duration measured in months. And an intramuscular injection of an oil solution of hydroxyprogesterone caproate, an ester of a derivative of progesterone that has a medium-length fatty acid chain, has a duration measured in weeks.

Most sex steroid esters themselves are biologically inactive. Once they have left the oil tissue depot, they are rapidly cleaved by esterase enzymes into free unesterified steroid (e.g., estradiol, testosterone) and the previously connected ester moiety (e.g., valeric acid). Hence, most sex steroid esters are prodrugs and are otherwise identical to their parent sex steroids in their biological actions. In the case of esters of estradiol and testosterone, this means that they are bioidentical just like the unesterified steroids. Certain synthetic progesterone derivatives like hydroxyprogesterone caproate and medroxyprogesterone acetate are however not prodrugs and are not meaningfully cleaved into the unesterified parent compound. Instead, they have intrinsic hormonal activity of their own and act without bioactivation.

Injectable Aqueous Suspensions

Aqueous suspensions of sex steroids also form an injection-site depot and achieve a long-lasting depot effect when administered by subcutaneous or intramuscular injection. However, they work in a completely different way than oil solutions. Aqueous suspensions of sex steroids consist of tiny crystal particles of pure sex steroid that are suspended in water. These sex steroid particles are highly lipophilic and hydrophobic. When injected, the hydrophilic water vehicle is rapidly mixed into the fluid of the tissue compartment and absorbed by the body. But the hydrophobic sex steroid crystals are not, and instead float about in the fluid of the tissue compartment. As with oil solutions, the sex steroids at the edges of the crystals very slowly dissolve off the surface of the crystals into the surrounding water and are then distributed into the circulation and tissues. Eventually, the crystal will be fully absorbed into the body, but only after a long period of time. Since the rate of absorption is dependent on lipophilicity and hydrophobicity, lipophilic esters lengthen the durations of aqueous suspensions of sex steroids by intramuscular or subcutaneous injection similarly to the case of oil solutions of sex steroids.

In the case of aqueous suspensions, the duration of the sex steroid is additionally highly dependent on particle size. These particle sizes have ranged from nanocrystalline to microcrystalline to macrocrystalline in their range. Almost always however it is microcrystalline particle sizes that have been used in injectable aqueous suspensions of sex steroids. (The present author has seen macrocrystalline preparations described a few times, specifically in research on combined injectable contraceptives (Garza-Flores, Del, & Perez-Palacios, 1992; Newton, d’Arcangues, & Hall, 1994; Sang, 1994), and is fairly sure that no such preparations have ever been marketed. On the other hand, nanocrystalline aqueous suspensions have been used with depot antipsychotics (Spanarello & Ferla, 2014; Correll et al., 2021).) Typically, there is a given particle size range for the formulation, such as 0.01 to 0.1 mm in diameter. The larger the particle sizes, the slower the absorption into the body, and the longer the duration of the preparation; the smaller the particle sizes, the faster the absorption, and the shorter the duration. When microcrystalline aqueous suspensions of sex steroids are manufactured nowadays, the particle sizes are defined and carefully controlled. Particle sizes influence the duration of injectable aqueous suspensions because they result in different surface areas from which sex steroid ester can escape particles. A single large particle has a smaller total surface area and hence dissolution rate than the same particle divided up into many smaller particles.

Particle sizes are manipulated during manufacturing via micronization—the process of decreasing the diameter of larger particles, such as via milling or grinding. Whereas more micronization improves the absorption and bioavailability of estradiol and progesterone with oral administration by increasing the surface area available for absorption into the body (Wiki; Wiki), less micronization decreases the rate of absorption of crystalline aqueous suspensions via depot injection and thereby extends the durations of these preparations by decreasing the total surface area for absorption.

There is a notable similarity of injectable aqueous suspensions of sex steroid to implantable sex steroid pellets, for instance of estradiol, testosterone, and progesterone (Wiki; Wiki; Wiki). Pellet implants are basically just pure crystalline sex steroid compressed into the shape of a small cylinder (Photo; Photo). They are inserted into subcutaneous fat in the body through a small incision using a large needle-like instrument called a trocar (Diagram). Once implanted, pellets slowly dissolve and absorb into the body over time, eventually disappearing completely. As they are nothing but pure crystalline hormone, there is no need for them to be removed or retrieved later on. In other words, implantation of a pellet is in a way the same thing as a subcutaneous injection of an aqueous suspension of sex steroid crystals—a single pellet is just one massive crystal instead of many tiny crystals suspended in water. And with very large crystals comes a very long duration—typically 6 months or more for each subcutaneous pellet of estradiol or testosterone (Kuhl, 2005; Wiki; Wiki). However, though injectable aqueous suspensions are typically much less prolonged than pellet implants, they have the advantages over pellets of being less expensive and not requiring a surgical incision. Due to the similarity between aqueous suspensions and pellet implants, aqueous suspensions have been described and marketed as “micropellets” in the past.

Medical Use of Injectable Aqueous Suspensions

Clinical Durations of Injectable Aqueous Suspensions

Studies that have compared sex steroids in injectable oil solutions versus injectable aqueous suspensions have generally found that the durations are considerably longer with aqueous suspensions than with oil solutions. Whereas injectable estradiol and estrone in oil solution have durations of only about 1 to 2 days, aqueous suspensions of these steroids have durations of 2 to 7 days (Wiki-Table). Moreover, whereas injectable estradiol benzoate in oil solution has a duration of 4 to 6 days, injectable estradiol benzoate as a microcrystalline aqueous suspension has a duration of 2 to 3 weeks (Wiki-Table). Such a duration is on par with the duration of longer-acting injectable estradiol esters like estradiol cypionate in oil solution and estradiol enanthate in oil solution. In addition, whereas injectable progesterone in oil solution has a duration of about 2 to 3 days, injectable aqueous suspensions of progesterone have a duration in the range of 1 to 2 weeks (Wiki-Table). This is a duration that is comparable to that of injectable hydroxyprogesterone caproate in oil solution. The prolonged duration of injectable progesterone suspensions is particularly notable as progesterone cannot be esterified and hence injectable progesterone in oil solution cannot be prolonged except with structural modification (as in progestins like hydroxyprogesterone caproate and dihydroxyprogesterone acetophenide) (Wiki). The duration of injectable testosterone propionate in oil solution is 3 to 4 days, in a suspension of small crystals (0.04–0.1 mm) is 8 days, and as commercial-size crystals (0.05–0.2 mm) is 12 days (Sinkula, 1978). Testosterone isobutyrate as an aqueous suspension is said to have a duration of 2 to 3 weeks (Sinkula, 1978; Wiki-Table). Some injectable aqueous suspensions can have extremely prolonged durations. Depot medroxyprogesterone acetate for instance has a duration of at least 3 months and as long as 6 to 9 months (Wiki). It greatly outlasts the related injectable progestogen norethisterone enanthate in oil solution (Noristerat) (Bassol & Garza-Flores, 1994; Paulen & Curtis, 2009).

Availability of Injectable Aqueous Suspensions

A list of injectable aqueous suspensions of sex steroids that are known to have been marketed can be found here. Most of them were introduced in the 1950s and many of them have been discontinued, but several of the preparations remain available today. These include the single-drug preparations estradiol benzoate (Agofollin Depot, Ovocyclin M), progesterone (Agolutin Depot), testosterone isobutyrate (Agovirin Depot), and medroxyprogesterone acetate (Depo-Provera, Depo-SubQ Provera 104) as well as the combination preparations estradiol benzoate/testosterone isobutyrate (Folivirin, Femandren M) and estradiol cypionate/medroxyprogesterone acetate (Cyclofem, Lunelle). Among the more notable injectable aqueous suspensions that are no longer marketed include estradiol (Aquadiol, Diogyn, Progynon Aqueous Suspension, Progynon Micropellets), estrone (Estrone Aqueous Suspension, Kestrone, Theelin Aqueous), and testosterone (Andronaq, Sterotate, Virosterone) as well as the combination preparation estradiol benzoate/progesterone (Sistocyclin).

Agofollin Depot (estradiol benzoate), Agolutin Depot (progesterone), and Folivirin (estradiol benzoate/testosterone isobutyrate) are all marketed today only in the Czech Republic and Slovakia in Central Europe. However, they all seem to be available from EU Aibolit, a common site that transgender people on do-it-yourself (DIY) hormone therapy obtain their medications from (Reddit). Here are the medication leaflets for all of these products (in Czech and translated into English):

The company that manufactures these products, BB Pharma, is notably also the producer of the well-known Neofollin (injectable estradiol valerate in oil solution) in Europe.

Injection Procedure for Injectable Aqueous Suspensions

Aqueous suspensions require larger needle gauges (e.g., 20 or 21 gauge) than oil or aqueous solutions in order to allow the steroid crystals to pass through the needle lumen. The needle sizes may vary depending on the preparation and its crystal sizes. Injectable aqueous suspensions should be shaken adequately prior to injection as the crystals tend to aggregate. Aqueous suspensions are known to be more irritating and prone to causing pain and injection site reactions like redness and swelling than oil solutions, although this may vary depending on the preparation (Wiki; Wiki). This property is likely responsible for the discontinuation of many injectable suspensions and the greater popularity of injectable oil solutions. On the other hand, an advantage of aqueous suspensions over oil solutions is that in contrast them, there is no oil, and hence there is no risk of allergic reaction to the oil. As with injectable oil solutions, injectable aqueous suspensions are indicated for use specifically by intramuscular injection. Depot medroxyprogesterone acetate, which is available in both formulations for both intramuscular injection (Depo-Provera) and subcutaneous injection (Depo-SubQ Provera 104), is suggestive that aqueous suspensions have similar properties both by intramuscular and subcutaneous injection analogously to the case of injectable oil solutions (Wiki; Wiki). However, it should be noted that depot medroxyprogesterone acetate is formulated differently between these preparations.


Since this article was posted, Agofollin Depot (estradiol benzoate), Agolutin Depot (progesterone), and Folivirin (estradiol benzoate/testosterone isobutyrate) appear to have been discontinued by their manufacturer (Reddit) and seem to no longer be available from online pharmacies like EU Aibolit.