Dydrogesterone disrupts lipid metabolism in zebrafish brain: A study based on metabolomics and Fourier transform infrared spectroscopy.

Brain is a potential target for neuroprogestogens and/or peripheral progestogens. Previous studies reported that expression of genes about steroidogenesis, reproduction, cell cycle, and circadian rhythm in zebrafish brain could be affected by progestogens. However, there are limited information from metabolites or biomacromolecules aspects, leaving an enormous gap in understanding toxic effects of progestogens on fish brain. In this study, we exposed zebrafish embryos to 2.8, 27.6, and 289.8 ng/L dydrogesterone (DDG, a synthetic progestogen) until sexual maturity (140 days). LC-MS and GC-MS based untargeted metabolomics and Fourier-transform infrared (FTIR) spectroscopy were then performed to investigate the metabolic profiles and macromolecular changes of brain of these zebrafish. The results from multivariate statistical analysis of metabolite features showed a clear separation between different treatment groups of both female and male zebrafish brains. DDG exposure increased the levels of cholesterol, saturated fatty acids, and nucleoside monophosphates, but decreased the contents of polyunsaturated fatty acids (PUFAs), lysophosphatides, and nucleosides in dose-dependent manner. FTIR results indicated that DDG exposure led to accumulation of saturated lipids, reduction of nucleic acids and carbohydrates, and alteration of protein secondary structures. The findings from this study demonstrated that DDG could affect contents of metabolites and biomacromolecules of zebrafish brain, which may finally lead to brain dysfunctions.

Lack of analytical interference of dydrogesterone in progesterone immunoassays.

OBJECTIVES: Progesterone, a sex steroid, is measured in serum by immunoassay in a variety of clinical contexts. One potential limitation of steroid hormone immunoassays is interference caused by compounds with structural similarity to the target steroid of the assay. Dydrogesterone (DYD), an orally active stereoisomer of progesterone, is used for various indications in women's health. Herein, we report a systematic in vitro investigation of potential interference of DYD and its active metabolite 20α-dihydrodydrogesterone (DHD) in seven widely used, commercially available progesterone assays. METHODS: Routine human plasma samples were anonymized and pooled to create three graded concentration levels of progesterone (P4 high, P4 medium, P4 low). Each pooled P4 plasma sample (6-7 mL) was spiked at high, medium, and "none" concentration with DYD/DHD and was divided into 0.5 mL aliquots. The blinded aliquots were analyzed by seven different laboratories with their routine progesterone assay (six different immunoassays and one liquid chromatography-tandem mass spectrometry assay, respectively) within the Dutch working group on endocrine laboratory diagnostics of the Dutch Foundation for Quality Assessments in Medical Laboratories. RESULTS: The sample recovery rate (P4 result obtained for sample spiked with DYD/DHD, divided by the result obtained for the corresponding sample with no DYD/DHD × 100) was within a ±10% window for the medium and high P4 concentrations, but more variable for the low P4 samples. The latter is, however, attributable to high inter- and intra-method variability at low P4 concentrations. CONCLUSIONS: This study does not indicate any relevant interference of DYD/DHD within routinely used progesterone assays.

Effects of Progesterone, Dydrogesterone and Estrogen on the Production of Th1/Th2/Th17 Cytokines by Lymphocytes from Women with Recurrent Spontaneous Miscarriage.

Anti-inflammatory Th2 cytokines have been shown to be associated with healthy, successful pregnancy while pro-inflammatory Th1 and Th17 cytokines are associated with pregnancy loss due to recurrent spontaneous miscarriage. This nexus between unexplained recurrent spontaneous miscarriage (uRSM) and maternal inflammatory has led to the possibility of using pregnancy-related hormones to modify the maternal cytokine bias in a manner that is conducive to successful pregnancy. We investigated the ability of progesterone, dydrogesterone and estrogen to modulate cytokine production by peripheral blood lymphocytes from women undergoing uRSM. Peripheral blood mononuclear cells (PBMC) from females with uRSM were stimulated in vitro with phytohemagglutinin (PHA) in the presence and absence of progesterone or dydrogesterone or 17ß-estradiol. Culture supernatants were assayed for IFN-α, TNF-γ, IL-2, IL-6, IL-10, IL-13, IL-17A, and IL-23 by ELISA. Progesterone and dydrogesterone significantly down-regulated the secretion of the Th1 cytokines IFN-α and TNF-γ, and the Th17 cytokine IL-17A, and IL-23. Additionally, the secretion of the Th2 cytokine IL-6 was up-regulated. Estrogen, on the other hand, decreased the production of IFN-α and IL-2, increased the production of IL-6 but did not affect IL-17A and IL-23 secretion. Progestogens and estrogen can decrease the production of some Th1/Th17 inflammatory cytokines secreted by lymphocytes from uRSM and upregulate the production of anti-inflammatory cytokines. These data support the notion that progestogens can be used for altering maternal cytokine profiles to manage pregnancy complications.

Microbial transformation of progesterone and dydrogesterone by bacteria from swine wastewater: Degradation kinetics and products identification.

Natural and synthetic progestogens in livestock environments have become a concern due to the frequent presence and potential adverse effects on aquatic organisms. Here we investigated the biotransformation of progestogens by wastewater-borne bacteria in the field and laboratory under oxic and anoxic conditions. The results showed that all progestogens dissipated faster under oxic conditions than under anoxic conditions, and natural progesterone transformed faster than synthetic progestogens. Meanwhile, dozens of bacterial strains capable of degrading progestogens were successfully isolated from the swine wastewater, and Bacillus sp. P19 and Bacillus sp. DGT2 were found the best for progesterone and dydrogesterone transformation, respectively. In the degradation experiments using a single bacterial strain, progesterone and dydrogesterone dissipated under oxic conditions with half-lives of 11.6 h and 18.2 h, respectively. The transformation pathways were proposed based on the identified transformation products. The findings from this study showed that progestogens can be biotransformed, but not fully mineralized in the environment.

Global metabolite profiles of rice brown planthopper-resistant traits reveal potential secondary metabolites for both constitutive and inducible defenses.

INTRODUCTION: Brown planthopper (BPH) is a phloem feeding insect that causes annual disease outbreaks, called hopper burn in many countries throughout Asia, resulting in severe damage to rice production. Currently, mechanistic understanding of BPH resistance in rice plant is limited, which has caused slow progression on developing effective rice varieties as well as effective farming practices against BPH infestation. OBJECTIVE: To reveal rice metabolic responses during 8 days of BPH attack, this study examined polar metabolome extracts of BPH-susceptible (KD) and its BPH-resistant isogenic line (IL308) rice leaves. METHODS: Ultra high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QToF-MS) was combined with multi-block PCA to analyze potential metabolites in response to BPH attack. RESULTS: This multivariate statistical model revealed different metabolic response patterns between the BPH-susceptible and BPH-resistant varieties during BPH infestation. The metabolite responses of the resistant IL308 variety occurred on Day 1, which was significantly earlier than those of the susceptible KD variety which showed an induced response by Days 4 and 8. BPH infestation caused metabolic perturbations in purine, phenylpropanoid, flavonoid, and terpenoid pathways. While found in both susceptible and resistant rice varieties, schaftoside (1.8 fold), iso-schaftoside (1.7 fold), rhoifolin (3.4 fold) and apigenin 6-C-α-L-arabinoside-8-C-ß-L-arabinoside levels (1.6 fold) were significantly increased in the resistant variety by Day 1 post-infestation. 20-hydroxyecdysone acetate (2.5 fold) and dicaffeoylquinic acid (4.7 fold) levels were considerably higher in the resistant rice variety than those in the susceptible variety, both before and after infestation, suggesting that these secondary metabolites play important roles in inducible and constitutive defenses against the BPH infestation. CONCLUSIONS: These potential secondary metabolites will be useful as metabolite markers and/or bioactive compounds for effective and durable approaches to address the BPH problem.

Biotransformation of progestonic hormone dydrogesterone with Macrophomina phaseolina, and study of the effect of biotransformed products on phagocytes oxidative burst.

Biotransformation of a synthetic progestonic hormone dydrogesterone (1), C21H28O2, with a plant pathogenic fungus Macrophomina phaseolina yielded two new 2 and 3, and a known 4 metabolites. These analogues were identified as, 3ß,11α-dihydroxy-5ß,9ß,10α-pregna-7-ene-6,20-dione (2), 15ß-hydroxy-9ß,10α-pregna-4,6-diene-3,20-dione (3), and 8α-hydroxy-9ß,10α-pregna-4,6-diene-3,20-dione (4). Major structural changes were observed in metabolite 2. New metabolite 3 showed anti-inflammatory potential, and was found to be the potent inhibitor of intracellular reactive oxygen species (ROS) from whole blood phagocytes (IC50 = 4.2 ±â€¯0.3 µg/mL), as compared to standard drug Ibuprofen (IC50 = 11.2 ±â€¯1.9 µg/mL). The metabolites 2, 3, and 4 were found to be non-toxic to NIH-3T3 (CRL-1658) normal cell line. This indicated anti-inflammatory potential of resulting metabolites.

Dydrogesterone affects the transcription of genes in GnRH and steroidogenesis pathways and increases the frequency of atretic follicles in zebrafish (Danio rerio).

Dydrogesterone (DDG) is a synthetic progestin broadly used in human and veterinary medicine and has been widely detected in aquatic environments. However, its potential effects on aquatic organisms are little documented. Here we investigate the short-term effects of DDG on the transcriptional and histological responses in adult zebrafish (Danio rerio). Adult zebrafish were exposed to 32.0, 305 and 2490 ng L-1 of DDG for 14 days. Real time quantitative PCR analysis showed that DDG significantly increased transcripts of most genes involved in the gonadotropin-releasing hormone (GnRH) pathway in the brain of female. In contrast, apparent down-regulation of these gene transcriptions was observed in the brain of males. The transcription of cyp19a1a in the ovary had a 2.3 fold increase at 2490 ng L-1 of DDG and the transcription of hsd17b2 at 305 and 2490 ng L-1 in the testis was enhanced by approximately 2.0 fold and 2.4 fold, respectively. Histopathological analysis revealed exposure to 2490 ng L-1 DDG significantly increased the percentage of atretic follicles in the ovary. The results of this study suggest that DDG has potential endocrine disrupting effects and affects the ovarian development in zebrafish.

Dydrogesterone metabolism in human liver by aldo-keto reductases and cytochrome P450 enzymes.

1. The metabolism of dydrogesterone was investigated in human liver cytosol (HLC) and human liver microsomes (HLM). Enzymes involved in dydrogesterone metabolism were identified and their relative contributions were estimated. 2. Dydrogesterone clearance was clearly higher in HLC compared to HLM. The major active metabolite 20α-dihydrodydrogesterone (20α-DHD) was only produced in HLC. 3. The formation of 20α-DHD by cytosolic aldo-keto reductase 1C (AKR1C) was confirmed with isoenzyme-specific AKR inhibitors. 4. Using recombinantly expressed human cytochrome P450 (CYP) isoenzymes, dydrogesterone was shown to be metabolically transformed by CYP3A4 and CYP2C19. 5. A clear contribution of CYP3A4 to microsomal metabolism of dydrogesterone was demonstrated with HLM and isoenzyme-specific CYP inhibitors, and confirmed by a significant correlation between dydrogesterone clearance and CYP3A4 activity. 6. Contribution of CYP2C19 was shown to be clearly less than CYP3A4 and restricted to a small group of human individuals with very high CYP2C19 activity. Therefore, it is expected that CYP2C19 genetic variations will not affect dydrogesterone pharmacokinetics in man. 7. In conclusion, dydrogesterone metabolism in the liver is dominated primarily by cytosolic enzymes (particularly AKR1C) and secondarily by CYP3A4, with the former exclusively responsible for 20α-DHD formation.

[Relative binding activity of dydrogesterone with blood mononuclear cells progesterone receptors of women included in the IVF program].

It has been shown that specific binding of dydrogesterone with progesterone receptors in blood mononuclear cells of the patients included in the programs of in vitro fertilization, varies from 0 to 298%, thus at 38% of women dydrogesterone concedes to a progesterone in the competition for receptors of a progesterone, and 62%--exceed. The revealed variations of specific binding dydrogesterone with receptors of a progesterone are a reserve of increase of efficiency of auxiliary reproductive technology at the expense of individual selection of hormonal agents.

Expression of human aldo-keto reductase 1C2 in cell lines of peritoneal endometriosis: potential implications in metabolism of progesterone and dydrogesterone and inhibition by progestins.

The human aldo-keto reductase AKR1C2 converts 5α-dihydrotestosterone to the less active 3α-androstanediol and has a minor 20-ketosteroid reductase activity that metabolises progesterone to 20α-hydroxyprogesterone. AKR1C2 is expressed in different peripheral tissues, but its role in uterine diseases like endometriosis has not been studied in detail. Some progestins used for treatment of endometriosis inhibit AKR1C1 and AKR1C3, with unknown effects on AKR1C2. In this study we investigated expression of AKR1C2 in the model cell lines of peritoneal endometriosis, and examined the ability of recombinant AKR1C2 to metabolise progesterone and progestin dydrogesterone, as well as its potential inhibition by progestins. AKR1C2 is expressed in epithelial and stromal endometriotic cell lines at the mRNA level. The recombinant enzyme catalyses reduction of progesterone to 20α-hydroxyprogesterone with a 10-fold lower catalytic efficiency than the major 20-ketosteroid reductase, AKR1C1. AKR1C2 also metabolises progestin dydrogesterone to its 20α-dihydrodydrogesterone, with 8.6-fold higher catalytic efficiency than 5α-dihydrotestosterone. Among the progestins that are currently used for treatment of endometriosis, dydrogesterone, medroxyprogesterone acetate and 20α-dihydrodydrogesterone act as AKR1C2 inhibitors with low µM K(i) values in vitro. Their potential in vivo effects should be further studied.

Biotechnological production of 20-alpha-dihydrodydrogesterone at pilot scale.

The human sex hormone progesterone plays an essential and complex role in a number of physiological processes. Progesterone deficiency is associated with menstrual disorders and infertility as well as premature birth and abortion. For progesterone replacement therapy, the synthetic progestogen dydrogesterone is commonly used. In the body, this drug is metabolized to 20α-dihydrodydrogesterone (20α-DHD), which also shows extensive pharmacological effects and hence could act as a therapeutic agent itself. In this study, we describe an efficient biotechnological production procedure for 20α-DHD that employs the stereo- and regioselective reduction of dydrogesterone in a whole-cell biotransformation process based on recombinant fission yeast cells expressing the human enzyme AKR1C1 (20α-hydroxysteroid dehydrogenase, 20α-HSD). In a fed-batch fermentation at pilot scale (70 L) with a genetically improved production strain and under optimized reaction conditions, an average 20α-DHD production rate of 190 µM day(-1) was determined for a total biotransformation time of 136 h. Combined with an effective and reliable downstream processing, a continuous production rate of 12.3 ± 1.4 g 20α-DHD per week and fermenter was achieved. We thus established an AKR-dependent whole-cell biotransformation process that can also be used for the production of other AKR1C1 substrates (as exemplarily shown by the production of 20α-dihydroprogesterone in gram scale) and is in principle suited for the production of further human AKR metabolites at industrial scale.

Progestins as inhibitors of the human 20-ketosteroid reductases, AKR1C1 and AKR1C3.

The human aldo-keto reductases 1C1 and 1C3 (AKR1C1 and AKR1C3) are important 20-ketosteroid reductases in pre-receptor regulation of progesterone action. Both AKR1C1 and AKR1C3 convert progesterone to the less potent metabolite 20α-hydroxyprogesterone, although AKR1C1 has a higher catalytic efficiency than AKR1C3. Recently, we reported significant up-regulation of AKR1C1 and AKR1C3 in ovarian endometriosis, a complex estrogen-dependent disease. The typical characteristics of endometriosis are increased formation of estradiol, which stimulates proliferation of endometriotic tissue, and disturbed action of the protective progesterone. Although progestins have been used for treatment of endometriosis since the 1960s, their detailed mechanisms of action are still not completely understood. In the present study, we evaluated the potential inhibitory effects of progestins on the pre-receptor regulatory enzymes AKR1C1 and AKR1C3. We examined the following progestins as inhibitors of progesterone reduction catalyzed by recombinant AKR1C1 and AKR1C3: progesterone derivatives (dydrogesterone, its metabolite, 20α-hydroxydydrogesterone; and medroxyprogesterone acetate), 19-nortestosterone derivatives (desogestrel, norethinodrone and levonorgestrel), and the androgen danazol. Dydrogesterone, medroxyprogesterone acetate, 20α-hydroxydydrogesterone and norethinodrone inhibited AKR1C1 and AKR1C3 with K(i) values of 1.9 µM, 7.9 µM, 20.8 µM and 48.0 µM, and of 0.5 µM, 1.4 µM, 18.2 µM and 6.6 µM, respectively. Levonorgestrel and desogestrel preferentially inhibited AKR1C3 with K(i) values of 5.6µM and 39.1µM, respectively. Our data thus show that dydrogesterone, medroxyprogesterone acetate, 20α-hydroxydydrogesterone and norethinodrone inhibit AKR1C1 and AKR1C3 in vitro, although their physiological inhibitory effects still need to be evaluated further. Additionally, we investigated whether progestin dydrogesterone can be metabolized to its active 20α-hydroxymetabolite by AKR1C1 and AKR1C3. AKR1C1 converted dydrogesterone with a high catalytic efficiency while AKR1C3 was less active, which suggests that in vivo dydrogesterone is metabolized mainly by AKR1C1. Docking simulations of dydrogesterone into AKR1C1 and AKR1C3 also support these experimental data.

Antinociceptive profile of ring A-reduced progesterone metabolites in the formalin test.

The present study investigated the effect of acute systemic administration of six progesterone metabolites on formalin-induced pain in the rat. The 3alpha-hydroxylated metabolites allopregnanolone and pregnanolone are highly potent positive modulators at the GABA(A) receptor and produced a biphasic effect on pain in the formalin test. Dose-dependent antinociception was observed at lower doses (maximal antinociception at 0.16mg/kg) and was reversed at higher doses. Bicuculline abolished the antinociceptive effect. The 3beta-hydroxylated epipregnanolone and isopregnanolone are inactive or only weekly active at the GABA(A) receptor, and did not affect formalin-induced pain. 5alpha- and 5beta-dihydroprogesterone have also been shown to have low affinity for the GABA(A) receptor, but can be rapidly metabolized to their 3alpha-hydroxylated counterparts. In the formalin test, they produced a biphasic effect on pain similar to that of pregnanolone and allopregnanolone, but with lower potency. The effect was reversible by bicuculline, showing involvement of the GABA(A) receptor, and was blocked by indomethacin, implying that the antinociceptive effect is dependent on their conversion to allopregnanolone or pregnanolone. The results indicate that GABA-ergic progesterone metabolites modulate nociception. A change in levels of GABA-ergic progesterone metabolites, such as is observed in depression, chronic fatigue and premenstrual dysphoric disorder could, therefore, contribute to the pain complaints associated with these disorders.

Fungal transformation of dydrogesterone and inhibitory effect of its metabolites on the respiratory burst in human neutrophils.

The biotransformation of the synthetic hormone dydrogesterone (1) by a number of fungal strains - including Cephalosporium aphidicola, Rhizopus stolonifer, Cunninghamella elegans, and Fusarium lini - afforded ten different metabolites, compounds 2-11. From a structural point of view, these transformations involved a combination of hydroxylation, oxidation, reduction, and/or epoxidation. The pregnane-based metabolites 10 and 11 are new compounds. All the known compounds were obtained for the first time from 1 by fungal transformation. The metabolites 3, 5, and 8 showed more-potent respiratory-burst inhibitory activity than the substrate 1 in a neutrophil-based cellular assay (Table 3).

Effects of dydrogesterone and of its stable metabolite, 20-alpha-dihydrodydrogesterone, on nitric oxide synthesis in human endothelial cells.

OBJECTIVE: To investigate the effects of P, medroxyprogesterone acetate (MPA), and dydrogesterone (DYD) and its metabolite, 20-alpha-dihydrodydrogesterone (DHD) on endothelial synthesis of nitric oxide (NO) and characterize the signaling events recruited by these compounds. The Women's Health Initiative trial reports an excess of heart disease in postmenopausal women receiving MPA. DESIGN: Cell culture. SETTING: Research laboratory. PATIENT(S): Human endothelial cells from umbilical vein. INTERVENTION(S): Treatments with P, MPA, DYD, or DHD. MAIN OUTCOME MEASURE(S): Measure of NO release, endothelial nitric oxide synthase (eNOS) activity and expression, and activation of ERK 1/2 and Akt. RESULT(S): The administration of DYD alone or in combination with estrogen to endothelial cells results in neutral effects on NO synthesis and on the activity and expression of eNOS. In parallel, the stable metabolite DHD acts similarly to natural P, enhancing the expression of eNOS and inducing rapid activation of the enzyme through the regulation of the ERK 1/2 mitogen-activated protein kinase cascade. 20-Alpha-dihydrodydrogesterone and P also potentiate eNOS induction by E2. On the contrary, MPA does not trigger eNOS enzymatic activation and decreases the extent of eNOS induction by E2. CONCLUSION(S): These findings support the concept that synthetic progestins act differently on vascular cells and that hormonal preparations may differ as to their cardiovascular effects.

Oral dydrogesterone versus intravaginal micronised progesterone as luteal phase support in assisted reproductive technology (ART) cycles: results of a randomised study.

The objective of this prospective, randomised study was to compare the efficacy, safety and tolerability of vaginal micronised progesterone with oral dydrogesterone as luteal phase support after in-vitro fertilisation (IVF). A total of 430 women underwent IVF/intracytoplasmic sperm injection (ICSI) treatment. Long protocol gonadotropin releasing hormone analogue down-regulation was followed by gonadotropin stimulation. Human chorionic gonadotropin was given when two or more follicles reached > or = 17 mm. After 36 h, oocytes were retrieved and IVF was performed. Embryo transfer was done at the 4-8 cell embryo stage. Luteal support was initiated from the day of embryo transfer and continued for up to 14 days. Patients were randomised to luteal supplementation with either intravaginal micronised progesterone 200 mg three times daily (n=351) or oral dydrogesterone 10 mg twice daily (n=79). In cases of a positive pregnancy test, luteal support was continued for 12 weeks. Both dydrogesterone and micronised progesterone were associated with similar rates of successful pregnancies. Vaginal discharge or irritation were reported by 10.5% of patients given micronised progesterone. Significantly (p<0.05), more patients given dydrogesterone than micronised progesterone were satisfied with the tolerability of their treatment. There were no differences between the treatments with regard to liver function tests.

Maternal drug treatment and human milk banking.

We studied prospectively during one year temporary drug use by mothers donating breast milk to assess the problem of drug treatment of donors. Sixty-four of the 284 mothers (22.5%) had to abstain from donating due to medication. The indication was infection in 50/56 treatments (89.3%). Antimicrobial agents were prescribed 44/52 times (84.6%). The channelling of milk from mothers in early phases of lactation to premature and newborn infants was identified as a special risk situation, if mothers on medication are not excluded. The limited number of such donors leads to use of milk unpooled or pooled to small volumes with increased risk for adverse effects to babies as a consequence. We recommend a wash-out period of 5 half-lives of the drug after the last ingested dose. For the majority of drugs in this study, with some important exceptions, a wash-out period of 1 day was sufficient.

Determination of 20 alpha-hydroxy-9 beta,10 alpha-pregna-4,6-dien-3-one in plasma by selected ion monitoring.

A selected ion monitoring (SIM) method has been devised for the determination of metabolites of dydrogesterone, 20 alpha-hydroxy-9 beta,10 alpha-pregna-4,6-dien-3-one (DHD) and DHD glucuronide, in plasma. Using testosterone as an internal standard (IS), DHD and IS were extracted with n-hexane and were purified by means of magnesium oxide column chromatography. The purified DHD and IS were converted to their diheptafluorobutyryl derivatives (DHD diHFB and testosterone diHFB) with heptafluorobutyric anhydride in acetone for analysis by SIM. SIM was carried out with a 2% OV-17 column (1 m) at 230 degrees C by monitoring the molecular ions of the derivatives (m/z 706 for DHD diHFB, m/z 680 for testosterone diHFB). DHD was determined from a calibration curve using a peak area method. The determination limit of the devised method was about 5 ng DHD per ml of plasma and the reproducibility was within +/- 6% of the coefficient of variation for 30 ng of DHD per ml of plasma or above.

Dydrogesterone: metabolism in animals.

The metabolic pattern of dydrogesterone was investigated in the rat, dog, mouse, rabbit and rhesus monkey. The drug was administered orally in 3H-labelled form. Following enzymatic hydrolysis of conjugates the radioactive metabolites were extracted from the urine, and in rat and dog also from bile. The separation method used for the development of the metabolite patterns was reversed-phase high performance liquid chromatography. Dydrogesterone and 4 derivatives, known or suspected to be metabolites, were used as marker substances. In all the species a substantial portion of the urinary or biliary radioactivity was too polar to be extracted, or it was not resolved in the chromatographic system used. The radioactivity which did develop into a pattern coincided with two or more of the marker substances. Only in the monkey, the pattern contained a peak of some substance which did not coincide with any marker. The urinary patterns of rat, dog, and mouse differed substantially, from each other as well as from those of rabbit and monkey. The patterns for the latter two animals showed certain similarities, both to each other and to the human urinary pattern as reconstructed from previous studies. It is concluded that with regard to the metabolic fate of dydrogesterone, the rabbit resembles man more than does any other species.