Anabolic-androgenic steroids (AAS) stimulate protein synthesis, leading to increased skeletal muscle mass and strength (anabolic effect). They are also responsible for the development of male secondary sexual characteristics (androgenic effect) (Hoffman and Ratamess, 2006). AAS have selected therapeutic uses, including male hypogonadism, anemia, and osteoporosis treatment. However, their anabolic effects have led to widespread non-medical use, especially among young adults engaged in athletic activities as performance-enhancing drugs (PEDs) (de Ronde and Smit, 2020; McCabea et al., 2007). Furthermore, AAS are banned according to the List of Prohibited Substances and Methods of the World Anti-Doping Agency (WADA) (World Anti-Doping Agency, 2024c).

Uncontrolled use of substances from the AAS group for non-medical purposes is associated with a high risk of adverse effects on the liver and the reproductive, musculoskeletal, cardiovascular, and central nervous systems. Exogenous AAS inhibit endogenous testosterone production, and discontinuation of their use is often associated with a prolonged decrease in testosterone secretion (Kanayama et al., 2015; Rasmussen et al., 2016; Shankara-Narayana et al., 2020). AAS-induced hypogonadism is a serious problem, and complete recovery is difficult in most cases (Vilar Neto et al., 2021). It can take months or years for the testosterone levels to return to normal after AAS cessation (Kanayama et al., 2015; Shankara-Narayana et al., 2020). Men who stop taking AAS may experience decreased libido, erectile dysfunction, fatigue, and depression symptoms (Kanayama et al., 2015; Rasmussen et al., 2016). Since no evidence-based recommendations for safe AAS withdrawal management currently exist, some users self-administer non-approved post-cycle therapy (PCT) to stimulate endogenous testicular function (Griffiths et al., 2017; Grant et al., 2023a).

The substances used in PCT are administered in the treatment of male hypogonadism unrelated to androgen abuse (Grant et al., 2024). However, PCT is recommended in bodybuilding forums for men who have discontinued testosterone or other androgenic hormones. Moreover, the PCT protocol differs depending on the individual group. Recreational AAS users (men without hormone therapy indications) predominantly start with optimal testosterone levels, aiming to quickly improve their physique. However, they experience a decrease in endogenous testosterone production owing to AAS use, which manifests, among others, as erectile dysfunction and spermatogenesis suppression, resulting in testicular atrophy, and infertility. PCT is believed to prevent testicular atrophy and inhibits feminization processes, such as gynecomastia. PCT can become even more important when high AAS doses are used or when multiple AAS are administered simultaneously.

To restore endogenous testosterone, mainly aromatase inhibitors (AIs), selective estrogen receptor modulators (SERMs), and human chorionic gonadotropin (hCG) are used (Karavolos et al., 2015; Bonnecaze et al., 2021). AIs and SERMs are relatively new doping agents, similar to selective androgen receptor modulators (SARMs) (Hackney, 2018).

In the 2024 WADA Prohibited List (World Anti-Doping Agency, 2024c) Section 4: “hormone and metabolic modulators,” AIs (Subsection S4.1) and anti-estrogenic substances, including anti-estrogens and SERMs (Subsection S4.2), are prohibited at all times (in- and out-of-competition). In Subsection S2.2.1, chorionic gonadotropin is listed as a testosterone-stimulating peptide in males.

Aromatase is an enzyme that converts testosterone to estradiol (the most potent estrogen form) and androstenedione to estrone (Kang et al., 2017). As aromatase mediates estrogen production, its inhibition with chemical molecules is considered an effective treatment for estrogen receptor (ER)-positive breast cancer. Moreover, AIs are used off-label to reduce circulating estradiol levels, thereby inhibiting the negative feedback of AAS use, limiting excessive testosterone release. Therefore, AIs play a role in minimizing the side effects associated with elevated estradiol levels in men, including gynecomastia (Basaria, 2010). AIs can be divided into two groups based on their chemical structure: steroidal (analogs of androstenedione, exemestane, formestane, and testolactone), which provide irreversible aromatase inhibition, and non-steroidal (anastrozole and letrozole), which provide reversible inhibition (Ahmad, 2015). The main AIs currently used include the new-generation letrozole, anastrozole, and exemestane, which can be administered orally.

The anti-estrogenic compounds mentioned in the WADA list can be divided into two groups: SERMs (bazedoxifene, clomiphene, cyclofenil, ospemifene, raloxifene, tamoxifen, and toremifene) and selective estrogen receptor degraders (SERDs), of which fulvestrant is a representative. Fulvestrant is the first SERD approved for clinical use to treat advanced and metastatic breast cancer. SERMs are non-steroidal estradiol analogs with mixed agonist-antagonist effects on ERs and are used in osteoporosis and breast cancer treatment (Jordan, 2004). Tamoxifen was the first SERM used to treat ER-positive breast cancer (Hackney, 2018). Clomiphene and cyclofenil are also older SERMs (although perhaps less selective than tamoxifen) (Heuberger and Cohen, 2019). In approved medicines, clomiphene is a mixture of two isomers: cis (Z-isomer or zuclomiphene) and trans (E-isomer or enclomiphene). Enclomiphene is anti-estrogenic, whereas zuclomiphene manifests moderate estrogenic and anti-estrogenic effects (Girase et al., 2023). Raloxifene is a new SERM developed for osteoporosis that appears to prevent breast cancer (Lewis and Jordan, 2005). Similar to AIs, SERMs are used off-label to alleviate the side effects of excess estradiol. The main antiestrogenic substances can be administered orally.

In addition, other medicines are recommended on bodybuilding forums during PCT. hCG is used by bodybuilders and athletes at the end of an AAS cycle to prevent muscle tissue breakdown. hCG directly stimulates Leydig cells to produce and release intratesticular testosterone by binding to the luteinizing hormone/choriogonadotropin receptor (Rizzuti et al., 2023). Moreover, hCG maintains spermatogenesis during gonadotropin suppression (Bond et al., 2022). However, according to bodybuilder forums, hCG is also important in restoring testicles to their normal size, as often, after a period of AAS use, an athlete’s testicles can shrink. Chorionic gonadotropin is a heterodimeric glycoprotein composed of two non-covalently bound subunits. The α subunit of hCG is structurally identical to the α subunits of gonadotropins secreted by the pituitary gland. The β subunit differs in its amino acid sequence from other gonadotropins and is hCG-specific. However, posttranslational modifications with eight potential glycosylation sites in the hCG structure result in a wide range of hCG glycoforms.

When administering 19-nortestosterone derivatives with significant progestogenic activity (nandrolone and trenbolone), cabergoline is recommended. Cabergoline is an ergot alkaloid derivative used to treat hyperprolactinemia, prolactinoma, and Parkinson’s disease. It effectively inhibits prolactin secretion and is therefore used by men during PCT to prevent gynecomastia. Bromocriptine, an ergot alkaloid derivative used to treat hyperprolactinemia, has a structure and activity similar to cabergoline. Mesterolone is used not only to increase androgen levels in athletes as an AAS but also for its supposed intrinsic aromatase enzyme antagonist properties as an anti-estrogen (Llewellyn, 2017). To the best of our knowledge, there is only one available source (outside of sports blogs and forums) that provides information on aromatase inhibition by mesterolone (Llewellyn, 2017).

Medicines used in PTC may cause serious side effects, including blood clots, stroke, and endometrial cancer in women taking SERMs (Hackney, 2018). The main adverse events reported by male AAS users to SERMs (clomiphene and tamoxifen) were decreased libido and erectile dysfunction (24%), acne (21%), fatigue (19%), mood disorders (13%), visual disturbances (5%), arthralgia (4%), insomnia (4%), and hot flushes (2%). Two men reported serious adverse drug reactions (hepatic cytolysis requiring hospitalization and abdominal pain requiring a visit to the emergency department) (Rochoy et al., 2022). In another study based on website data, AAS users reported tamoxifen side effects such as blurred vision, dizziness, headaches, and reduced libido (Karavolos et al., 2015). The main adverse events reported on AIs (anastrozole, letrozole, and exemestane) were decreased libido (27%), arthralgia (24%), mood disorders (12.5%), asthenia (10%), headaches (6%), anxiety (4.5%), and palpitations (3%). Three men reported serious adverse drug reactions requiring an emergency department visit (gross hematuria, palpitations, and urticaria) (Rochoy et al., 2022). According to other study AAS users reported arthralgia as an AI side effect (Karavolos et al., 2015). Furthermore, hCG can cause or aggravate gynecomastia (Rahnema et al., 2014). Adverse effects of chronic dopamine agonist (cabergoline) use include headaches, orthostatic hypotension, nausea, and sometimes cardiac valvular disease (Bonnecaze et al., 2021).

Owing to restrictions on the purchase of AAS and PCT products, an international black market for such preparations has developed. Unfortunately, because AAS cycles and PCTs are carried out outside the supervision of the doctor, most often using illegally traded products, they can be considered a potential threat to consumer health. The availability of illegally sold medicines has become a challenge in recent decades. For many years, falsified medicines have been traded worldwide; however, this has become increasingly easier owing to the increased availability of medicines and open channels of trade, including online sales. In addition to drug trafficking, pharmaceutical crime is of interest to organized criminal groups, and many factors contribute to the falsification of medicines. It is generally stated that over 50% of medical products sold through websites other than established online pharmacies can be classified as falsified medical products (Deconinck et al., 2021). The World Health Organization distinguishes between three categories of poor-quality medical products: substandard, unregistered/unlicensed, and falsified (World Health Organization, 2018).

The necessity of identifying unknown and undeclared substances in illegal and falsified products requires the constant development of techniques used for analytical research in forensic or control laboratories to perform analyses for public authority needs. Mass spectrometry (MS) has long been used for the identification and determination of the molecular structure of organic compounds (Khalikova et al., 2024). Different analyzers used in high resolution MS, including time of flight (TOF), and Orbitrap enable the determination of the elemental composition (assignment of the most probable sum formula) of a compound by measuring the exact mass and comparing the isotopic profile of the obtained spectrum with the theoretical profile. In many cases, the analysis of fragmentation spectra allows for the determination of the structural formula of the compound or elements of its structure. In the identification of unknown compounds, the use of high-resolution hybrid quadrupole (Q)-TOF, additionally coupled with high performance liquid chromatography (LC-QTOF-MS/MS) is irreplaceable (Johansson et al., 2014; Rebiere et al., 2017). However, to confirm the structure of the unknown compounds, especially those for which reference materials are not available, nuclear magnetic resonance (NMR) spectrometry is recommended (Johansson et al., 2014; Rebiere et al., 2017; dos Santos Ribeiro et al., 2021). X-ray powder diffraction (XRPD) enables the identification of crystalline substances, including both active pharmaceutical ingredients and excipients, that can be lost during dissolution and filtration when preparing samples for other analytical techniques. It also allows the determination of the form in which a compound is present, whether as a salt or a co-crystal. The Polish Official Medicines Control Laboratory (OMCL), upon request by police, customs, or other enforcement groups, regularly tests AAS and other PEDs submitted to the laboratory using accredited methods such as LC-QTOF-MS/MS and XRPD.

Although a growing body of literature on PCT and user characteristics associated with PCT use exists (Griffiths et al., 2017; Bonnecaze et al., 2021; Grant et al., 2023a), data on the quality of medications used within PCT are limited, in contrast to AAS quality (Graham et al., 2009; Frude et al., 2020; Magnolini et al., 2022). This study aimed to estimate the prevalence of medicines used for non-approved PCT among other PED samples and to identify declared and undeclared substances in illicit samples to check whether they were in accordance with the declaration. In this report, we describe the results of these studies to raise awareness that illegal and falsified products that pose a risk to consumer health exist in the European market.

Between January 2020 and the end of August 2024, 601 PED samples seized by the police and prosecutor’s office from the illegal market were submitted for testing at the Polish OMCL. The fewer samples in 2021 were mainly due to the COVID-19 pandemic in 2020, as samples that reached the laboratory were usually seized much earlier. In contrast, the number of samples received in 2024 was lower; however, this was because the samples were counted only until the end of August. New samples are continually tested in the laboratory, and we typically receive more samples in the second half of the year than in the first half. More than a hundred samples were seized and sent to the Polish OMCL, where they are waiting for analysis.

It is not surprising that AAS products constituted the largest group in each year between 2020 and 2024 (average: 68.4%); however, the number decreased from 80.8% in 2020 to 58.2% in 2023. The year 2024 can only be assessed after completion. Nevertheless, a significant number of AAS products are expected to be analyzed in 2024 because illicit sites that distribute huge quantities of anabolic agents (located in Poland) have recently been tracked down by the police (Centralne Biuro Sledcze Policji, 2023; Centralne Biuro Sledcze Policji, 2024). AAS were also the most dominant group among all analyzed products used as PEDs in other studies (Begley et al., 2017; Deconinck et al., 2021; Magnolini et al., 2022).

Conversely, the number of other PED samples increased (mean 21.1%) from 7.5% in 2020 to 32.9% in 2023. This could be due to a significant increase in interest in SARMs (Deconinck et al., 2021; Gaudiano et al., 2024; Holubeck et al., 2024) or peptide hormones and their releasing factors, growth factor modulators, and metabolic modulators. SARMs have become particularly popular recently among PED users because they are not “steroids” and, therefore, have less stigma attached to them, have lower risks of needle sticks and blood-borne diseases, and appear more legal (Chakrabarty et al., 2021).

Since AAS use is still very popular, the users self-administer post-cycle drugs after an AAS cycle to mitigate the side effects. Self-administration of PCT medicines was reported by 73% of men discontinuing AAS in Scotland between 2015 and 2022 (Grant et al., 2023a) and by 80% of 100 male athletes in the Netherlands between 2015 and 2018 (Smit et al., 2021). However, in an online survey conducted between December 2021 and February 2023, 56.5% of the 470 respondents recruited through advertisements on websites related to AAS use reported PCT use when stopping AAS (Grant et al., 2023b).

In our study, PCT medications constituted 6.5% of samples in 2021, 14.5% in 2022, and 8.5% in 2024 (average, 10.5%). There was a stagnant interest in PCT drugs, as recently demonstrated by other researchers (Holubeck et al., 2024). However, they will not disappear from the market when AAS are still used.

Of the 63 samples, SERMs were declared in 21 (33.3%), AIs in 16 (25.4%), and other PCT substances in 26 (41.3%). Nine different substances were declared or identified as undeclared PCT drugs in the samples and, in order of occurrence frequency, they were clomiphene, hCG, tamoxifen, exemestane, anastrozole, cabergoline, letrozole, mesterolone, and raloxifene (Figure 9). The compounds most frequently encountered in our study were consistent with those described by other researchers. Grant et al. demonstrated that the most popular drugs used for PCT were clomiphene (77%), tamoxifen (75%), and hCG (74%) (Grant et al., 2023a). Notably, hCG was also used during AAS cycle and as a PCT drug together with tamoxifen and clomiphene in 47%, while anastrozole was self-reported by only 4% of respondents. However, according to results of a web-based survey between August 2019 and April 2020 within 2,385 AAS users (Bonnecaze et al., 2020), the main PED used during AAS cycle were anastrozole (48%), tamoxifen (31%), exemestane (30%), and hCG (29%), whereas tamoxifen (40%), clomiphene (32%), hCG (26%), anastrozole (16%), exemestane (9%), letrozole (3%), and cabergoline/bromocriptine (1.6%) were used as PCT; however, 33% of them reported no PCT medicine use. In another study (Smit et al., 2021), tamoxifen (70%), clomiphene citrate (54%), and hCG (55%) were mostly used as PCT drugs. Other researchers analyzed 1792 posts published between 2013 and 2019 on the bodybuilding forum among AAS users, and 845 posts concerned SERMs while 571 concerned AIs (Rochoy et al., 2022).

All the analyzed PCT samples looked like medicines, which may give users confidence in the product quality despite coming from the illegal market. Some evidential product packaging contained anti-counterfeit markings and hints on how to verify product authenticity on the manufacturer’s website. Notably, this is a common “anti-counterfeiting” procedure for manufacturers who illegally produce medicinal products. However, data on the packaging of these products often contain inaccurate information on their composition, origin, performance, and use, which might mislead consumers.

Only over half of the samples were consistent with the declaration. The discrepancies between the label and content can be divided into whether the product contained a similar PED, a completely different API, an additional API, or contained no API. Undeclared pharmacologically active substances in illegal and falsified products can cause extreme harm to public health and pose a serious threat to human life. It is not surprising that there are counterfeits that do not contain API, especially since pharmaceutical API, such as hCG, is expensive compared to other doping agents such as AAS.

Although only three anti-estrogenic substances banned by WADA were identified in this study, clomiphene, tamoxifen, and raloxifene, it should be remembered that other banned substances from the S4.2 group, such as bazedoxifene, cyclofenil, fulvestrant, ospemifene, or toremifene, may also be present in illegal samples purchased by athletes (Okano et al., 2022). Bazedoxifene, ospemifene, and toremifene are newer-generation SERMs that are expected to soon become more popular in the illegal market. Bazedoxifene and ospemifene have been included in the WADA Prohibited List since January 2020. According to blogs, toremifene under the Fareston brand name is even preferred as a modern and safer SERM than tamoxifen and clomiphene; however, it is not yet commonly sold in the black market. In contrast, cyclofenil (Fertodur) is not as commonly used as clomiphene and tamoxifen, which have dominated this drug category for many decades. The SERD fulvestrant is also not yet widely available in the black market (Llewellyn, 2017). This may be due to the fact that it is very expensive, administered as an injection, and must be stored in a refrigerator. Therefore, it is important to note that falsifications without an API can be expected.

Similar to the anti-estrogenic group, only three AIs banned by WADA were identified in this study: letrozole, anastrozole, and exemestane. Notably, other banned substances from the S4.1 group, such as aminoglutethimide, arimistane, formestane, or testolactone, may also be present in illegal samples purchased by athletes. However, letrozole, anastrozole, and exemestane are newer-generation AIs that dominate the drug category. Aminoglutethimide and testolactone (Teslac) were the first generation of non-selective irreversible AIs; however, they are no longer commonly used in clinical medicine. Formestane is a newer AI used as an injection solution and is not widely available in the athletic community (Llewellyn, 2017).

Substances from the newer generations have started appearing among athletes. Within the class S4 of “hormone and metabolic modulators” of the WADA Prohibited List, 39% of the adverse analytical findings (AAF) reported in 2020 resulted from SERMs (tamoxifen and clomiphene) and another 29% from AIs (letrozole, anastrozole, exemestane, and adrostatrienedione) (World Anti-Doping Agency, 2022). In 2021, the numbers of AAF were 33% from SERMs (tamoxifen and clomiphene) and 15% from AIs (anastrozole, letrozole, adrostatrienedione, armistane, and 2-androstenone) (World Anti-Doping Agency, 2024a). Further, the numbers were 35% from anti-estrogenic substances (tamoxifen, clomiphene, fulvestrant, toremifene, and raloxifene) and 20% from AIs (letrozole, anastrozole, exemestane, adrostatrienedione, and armistane) in 2022 (World Anti-Doping Agency, 2024b).

Among the other PCT drugs, bromocriptine, which is similar to the popular cabergoline, an ergot alkaloid derivative used to treat hyperprolactinemia, is less common than cabergoline among AAS users during PCT owing to its many side effects, such as low blood pressure, dizziness, confusion, and nausea.

The effectiveness and purity of PCT drugs are concerns for many users (Grant et al., 2023b). Although many AAS users believe that PEDs are safe for long-term use, the vast majority would stop using AAS if they experience a serious health problem (Bonnecaze et al., 2020). In another study was reported that respondents believed that confirming substance legality would encourage safer practices and reduce potentially dangerous side effects (Piatkowski et al., 2023).

Polypharmacy associated with AAS and PCT drugs may have serious harmful effects on those engaged in such practices, which constitutes a serious public health concern (Sagoe et al., 2015). Moreover, illegal and falsified products used in PCT may interact with other medications or affect preexisting disease development or treatment.

Although these medications are legally available only with the prescription of a doctor, they can be bought without a prescription on the Internet. In another study researchers analyzed posts on a bodybuilding forum concerning SERMs and AIs, revealing that a minority of users were able to obtain PCT medication through a pharmacy (5% clomiphene, 5% tamoxifen, 6% anastrozole, 8% exemestane, and 22% letrozole) (Rochoy et al., 2022). Only 5% of AAS users reported consulting physicians. However, many of them (25% of SERM and 33% of AI users) reported having a blood test to monitor levels of gonadotropins, estradiol, and total, free, and bioavailable testosterone and to adjust AI doses during AAS treatment. It should be emphasized that in another survey (Grant et al., 2023b), most respondents believed that PCT should be prescribed under the supervision of a physician. However, no recommended treatment for AAS cessation currently exists. PCT use after discontinuing AAS treatment reduced the desire to reuse AAS, withdrawal symptoms, and suicidal thoughts by 60%, 60%, and 50%, respectively (Grant et al., 2023b). However, SERM, AI, or prolactin inhibitor use during PCT remains an unproven practice (Bond et al., 2022). Some researchers were unable to demonstrate any beneficial effect of PCT medications; therefore, the use of these agents should be discouraged (Smit et al., 2021).

Owing to the high prevalence of illicit steroid use, the self-administration of unapproved PCT using illegal and falsified medicines is dangerous and can be considered a potential threat to consumer health. Pharmaceutical and drug-related crimes are problems that the modern world struggles with. The increasing scale of this phenomenon, dynamic changes in the market for falsified and illegally traded products, and threats to the health and life of people receiving such products imply the urgency of systematic monitoring of both legal and illegal chains of medicinal products.

We have shown that the illicit drugs used in PCT are of poor quality; they may be substituted, contain no active ingredients, or be adulterated. Our results indicate that in view of the high prevalence of illicit AAS use, the self-administration of unapproved PCT using illegal and falsified medicines is dangerous and can be considered a potential threat to consumer health.