Genetic Polymorphism of CYP2D6 and Clomiphene Concentrations in Infertile Patients with Ovulatory Dysfunction Treated with Clomiphene Citrate.

CYP2D6 is primarily responsible for the metabolism of clomiphene citrate (CC). The purpose of the present study was to investigate the relationship between CYP2D6 genotypes, concentrations of CC and its major metabolites and drug response in infertility patients. We studied 42 patients with ovulatory dysfunction treated with only CC. Patients received a dose of 100 mg/day CC on days 3-7 of the menstrual cycle. CYP2D6 genotyping and measurement of CC and the major metabolite concentrations were performed. Patients were categorized into CC responders or non-responders according to one cycle response for the ovulation. Thirty-two patients were CC responders and 10 patients were non-responders with 1 cycle treatment. The CC concentrations were highly variable within the same group, but non-responders revealed significantly lower (E)-clomiphene concentration and a trend of decreased concentrations of active metabolites compared to the responders. Nine patients with intermediate metabolizer phenotype were all responders. We confirmed that the CC and the metabolite concentrations were different according to the ovulation status. However, our results do not provide evidence for the contribution of CYP2D6 polymorphism to either drug response or CC concentrations.

Genetic Polymorphism of CYP2D6 and Clomiphene Concentrations in Infertile Patients with Ovulatory Dysfunction Treated with Clomiphene Citrate

CYP2D6 is primarily responsible for the metabolism of clomiphene citrate (CC). The purpose of the present study was to investigate the relationship between CYP2D6 genotypes, concentrations of CC and its major metabolites and drug response in infertility patients. We studied 42 patients with ovulatory dysfunction treated with only CC. Patients received a dose of 100 mg/day CC on days 3-7 of the menstrual cycle. CYP2D6 genotyping and measurement of CC and the major metabolite concentrations were performed. Patients were categorized into CC responders or non-responders according to one cycle response for the ovulation. Thirty-two patients were CC responders and 10 patients were non-responders with 1 cycle treatment. The CC concentrations were highly variable within the same group, but non-responders revealed significantly lower (E)-clomiphene concentration and a trend of decreased concentrations of active metabolites compared to the responders. Nine patients with intermediate metabolizer phenotype were all responders. We confirmed that the CC and the metabolite concentrations were different according to the ovulation status. However, our results do not provide evidence for the contribution of CYP2D6 polymorphism to either drug response or CC concentrations.

Quantification of clomiphene metabolite isomers in human plasma by rapid-resolution liquid chromatography-electrospray ionization-tandem mass spectrometry.

Since the 1960s, clomiphene citrate is used for ovulation induction. Since nonresponse to clomiphene therapy is still not well understood, interindividual variability of clomiphene metabolism has been considered to be a plausible explanation. Therefore, a comprehensive, rapid, sensitive, and specific analytical method for the quantification of (E)- and (Z)-isomers of clomiphene and their putative N-desethyl, N,N-didesethyl, 4-hydroxy, and 4-hydroxy-N-desethyl metabolites, and the N-oxides in human plasma has been newly developed, using HPLC-tandem mass spectrometry and stable isotope-labeled internal standards. All standards other than the parent drug were synthesized in our laboratory. Following protein precipitation analytes were separated on a ZORBAX Eclipse plus C18 1.8 µm column with a gradient of 0.1% formic acid in water and 0.1% formic acid in acetonitrile and detected on a triple quadrupole mass spectrometer with positive electrospray ionization in the multiple reaction monitoring mode. Lower limit of quantification for metabolites ranged from 0.06 ng/mL for clomiphene-N-oxides to 0.3 ng/mL for (E)-N-desethylclomiphene. The assay was validated according to FDA guidelines. Plasma levels of clomiphene and its metabolites were measured in two women after single-dose treatment with clomiphene.

Evaluation of the relationship between plasma concentrations of en- and zuclomiphene and induction of ovulation in anovulatory women being treated with clomiphene citrate.

OBJECTIVE: To investigate the relationship between the plasma concentrations of clomiphene citrate (CC) isomers zu- (Zu) and enclomiphene (En), and ovulation outcome. DESIGN: Prospective, cohort study. SETTING: Reproductive medicine and fertility center in a university teaching hospital, United Kingdom. PATIENT(S): Forty-two women with World Health Organization type 2 infertility. INTERVENTION(S): The clinical and biochemical features of patients who were about to start CC for induction of ovulation were recorded. Plasma concentration of Zu and En were monitored at three points (days 2, 8, and 21) throughout the treatment cycle(s). MAIN OUTCOME MEASURE(S): Ovulation. RESULT(S): Thirty-nine patients completed the study. Both En and Zu accumulated throughout treatment. Among the 36 responders, there was no statistically significant relationship between the clinical and biochemical characteristics of the patients, En or Zu concentrations, and the dose required to induce ovulation. Moreover, the Zu and En concentrations were not different in the three patients who failed to respond. CONCLUSION: The concentrations of En and Zu in plasma, on their own or in combination with other covariates (e.g., weight, body mass index, free androgen index), are not a predictor of the ovulation response to CC or of the dose requirement. Further studies are needed to explore the role of additional covariates, including the presence of active metabolites, and the balance of the effects of En and Zu.

Determination by liquid chromatography-mass spectrometry of clomiphene isomers in the plasma of patients undergoing treatment for the induction of ovulation.

A rapid, sensitive and selective LC-MS method is described for the simultaneous determination of zuclomiphene and enclomiphene in plasma from patients undergoing treatment with clomiphene citrate for the induction of ovulation. Samples spiked with N-didesmethyltamoxifen, the internal standard, were extracted into methyl tertiary butyl ether. The compounds were separated on a Luna C(18) analytical column, and a mobile phase of methanol-water (70:30 v/v) containing 0.05% trifluoroacetic acid at a flow rate of 1ml/min. The limits of determination were 35pg/ml and 7pg/ml for zu- and enclomiphene, respectively. Within-day coefficients of variation ranged from 2.1% to 7.2%.

Monitoring plasma concentrations to individualize treatment with clomiphene citrate.

OBJECTIVE: Recent reports have indicated that a number of individual patient characteristics are responsible for the success or failure of clomiphene citrate treatment. However, a priori individualization of doses in different patients has not been investigated. We examined the thesis that wide variability in the metabolism of the active component (zuclomiphene) contributes to variability in response. METHODS: The dose-response relationship of clomiphene was established from a meta-analysis of data from 13 published reports. Limited data relating plasma drug concentrations to treatment outcome were examined to determine whether insufficient systemic exposure at a fixed dosage might contribute to therapeutic failure. RESULTS: A fixed-dosage regimen of 50 mg clomiphene per day is likely to cause ovulation in only 46% of patients; subsequent increment in dosage increases the number of responders but at the expense of considerable delay in individualization of treatment. Case reports indicated that dosage based on plasma drug concentration monitoring could improve patient management, and an algorithm is proposed to facilitate treatment. CONCLUSIONS: Prospective studies of clomiphene citrate should be performed to confirm the hypothesis that the monitoring of plasma zuclomiphene concentrations can significantly accelerate dose individualization and improve the therapeutic outcome with this "orphan" drug.

Serum concentrations of enclomiphene and zuclomiphene across consecutive cycles of clomiphene citrate therapy in anovulatory infertile women.

OBJECTIVE: To determine the serum concentrations of enclomiphene and zuclomiphene across consecutive cycles of clomiphene citrate treatment in anovulatory infertile women. DESIGN: Prospective cohort. SETTING: Tertiary institutional infertility clinic. PATIENT(S): Fourteen consenting anovulatory infertile women receiving standardized, cyclic, incremental treatment with clomiphene citrate for ovulation induction. INTERVENTION(S): Clomiphene citrate treatment (50-150 mg/d, cycle days 5-9), titrated to the minimum effective ovulation-inducing dose, was administered for three to six total cycles. Blood samples were obtained on cycle days 3 and 10 in each treatment cycle. MAIN OUTCOME MEASURE(S): Serum concentrations of enclomiphene and zuclomiphene. RESULT(S): Cycle day 3 zuclomiphene levels were below assay limits in all initial cycles, increased progressively across three consecutive cycles, and thereafter plateaued. Cycle day 3 enclomiphene concentrations were uniformly undetectable. Cycle day 10 enclomiphene levels increased with dose administered, but these observations were not statistically significant. CONCLUSION(S): Clomiphene citrate induction of ovulation results in an isomer-specific systemic accumulation of zuclomiphene across consecutive cycles of treatment. The combined maximum concentration of enclomiphene and zuclomiphene attained in practice approximates 100 nmol/L and is generally well below levels previously demonstrated to have adverse effects in vitro.

A má resposta à estimulaçäo ovariana controlada com gonadotrofinas em fertilizaçäo in vitro / The low response to ovulation induction with gonadotropins for in vitro fertilization

Existe um grupo especial de pacientes dentro da fertilizaçäo assistida chamadas de más respondedoras, que apresentam uma resposta diminuída à estimulaçäo ovariana controlada com gonadotrofinas, juntamente com uma baixa taxa de gravidez nestes ciclos. Além do elevado custo financeiro, os resultados desapontadores provocam um desgaste físico e emocional acentuados. Neste trabalho os autores procuram definir esse grupo de pacientes de acordo com a literatura médica mundial, bem como expor os métodos para se tentar prever uma má resposta e as alternativas de tratamento para essas pacientes.

Simple and rapid determination of clomiphene cis and trans isomers in human plasma by high-performance liquid chromatography using on-line post-column photochemical derivatization and fluorescence detection.

A validated, sensitive and rapid high-performance high-performance liquid chromatographic method has been developed for the analysis of both cis and trans isomers of clomiphene in human plasma. The method involves a new off-line pre-column solid-phase extraction for sample preparation of clomiphene from human plasma in the presence of an internal standard. Analysis was performed by isocratic elution on a LiChrospher 100 RP-18 5-microns column using on-line post-column photochemical derivatization, with fluorescence detection at 247 nm (excitation) and 378 nm (emission). The limit of quantitation was 0.75 ng/ml for the cis isomer and 1.25 ng/ml for the trans isomer. The precision and accuracy of method were between good laboratory practice (GLP) required limits.

Effect of clomiphene citrate treatment on endometrial estrogen and progesterone receptor induction in women.

A direct adverse effect of clomiphene citrate on the endometrium has been presumed, and interference with estrogen receptor-mediated endometrial estrogen receptor and progesterone receptor induction has been implicated as the mechanism responsible for an increased incidence of luteal phase deficiency in association with clomiphene citrate treatment. To clarify the net influence of clomiphene administration on endometrial steroid receptor induction, we studied five normal ovulatory women, in both a spontaneous and clomiphene-induced (150 mg/day, cycle days 5 to 9) ovulatory cycle. From cycle day 11 blood samples were obtained daily and urinary luteinizing hormone determinations were performed twice daily. Endometrial biopsy was performed on the day of the urinary luteinizing hormone surge and again 13 days after the surge. Serum levels of follicle-stimulating hormone and luteinizing hormone were determined by immunoradiometric assay, estradiol and progesterone by radioimmunoassay, and clomiphene citrate isomer concentrations in treatment cycles by reversed-phase high-performance liquid chromatography and fluorescence detection. Total, cytosolic, and salt-extracted nuclear endometrial estrogen receptor and progesterone receptor concentrations were determined by enzyme-linked immunoassay. Serum estradiol was threefold to fivefold higher (p less than 0.05) in clomiphene-induced than in spontaneous cycles 8 and 10 days before the luteinizing hormone surge, and progesterone was increased (p less than 0.05) from the day of the surge to end of the cycle. Serum enclomiphene rose to plateau between 12 and 6 days before the luteinizing hormone surge (4.1 +/- 0.8 ng/ml, mean +/- SE, n = 19) and fell thereafter to less than 1.0 ng/ml. Zuclomiphene levels increased rapidly between 14 and 8 days before the surge (53.9 +/- 2.8 ng/ml, mean +/- SE, n = 5) and then decreased gradually but remained elevated throughout the luteal phase (29.0 +/- 1.2 ng/ml, mean +/- SE, n = 33). Late luteal endometrial histology was abnormal in one of four available treatment cycle specimens, but the endocrine characteristics and number and subcellular distribution of estrogen receptor and progesterone receptor in the abnormal cycle were not different from those of normal, in-phase cycles.(ABSTRACT TRUNCATED AT 250 WORDS)

Ovulation-inducing drugs versus progesterone therapy for infertility in patients with luteal phase defects.

The efficacy of ovulation-inducing drugs (OVI) for treating infertility related to luteal phase defects (LPD) was compared with the efficacy of progesterone vaginal suppositories (PVS). Patients were divided into two groups: (1) LPD secondary to immature follicles and (2) pure LPD, in which the follicle was mature. Twenty-four of 31 women (77%) with pure LPD conceived (one aborted) during the first 6 months, compared with only 3 of 27 (11%) treated with OVI--and 2 of 3 aborted. However, in women with LPD secondary to immature follicles, 14 of 20 (70%) treated with OVI and PVS conceived (and one aborted) compared with 7 of 10 conceiving (70%) with OVI only (four aborted), and 3 of 12 conceived (25%) with PVS only (none aborted). Thus, both PVS and OVI are effective in treating LPD; follicular maturation studies help determine the proper choice. PVS appears to decrease the risk of abortion in both categories.

Estrogen receptor binding material in blood of patients after clomiphene citrate administration: determination by a radioreceptor assay.

The aim of the current study was to assess whether clomiphene citrate (CC) and/or active metabolites are present at presumed time of ovulation, nidation, or steroid-sensitive organogenesis, in serum of patients receiving CC for induction of ovulation. A radioreceptor assay, based on competitive replacement of 3H-estradiol on the rat uterus estrogen receptor, by ligands present in serum of patients after CC administration, was developed. Ligands reached maximal concentration 4 to 5 hours after a single dose of CC was administered, and declined with a half-life of 4.5 to 10 hours. In patients receiving CC on day 5 to day 9 in the cycle, ligands are still present on day 14 in the cycle and in some patients on day 22 of the cycle, but no ligands were detected 60 days after CC treatment.

Single-dose pharmacokinetics of clomiphene citrate in normal volunteers.

Twenty-four healthy adult female volunteers participated in a randomized, three-phase double-blind crossover trial comparing the single-dose (50 mg) pharmacokinetics of three formulations of clomiphene citrate (CC). Plasma levels of both the Z(cis) and E(trans) isomers of CC, as well as principal metabolites, were determined at periodic intervals; and no differences between formulations were observed. The active Z isomer attained peak blood levels later than the inactive E isomer and was eliminated much more slowly, significant plasma concentrations still being detected up to 1 month after treatment. The results of this study demonstrate that three commercially available formulations of CC are bioequivalent.

[Endocrine fertility-restricting factors in clomiphene therapy]. / Endokrine fertilitätsein-schränkende Faktoren bei der Clomiphen-Therapie.

A marked discrepancy between a high ovulation rate (70-80%) and a low pregnancy rate (30-35%) is generally observed during clomiphene therapy in female infertility. Disturbances in follicular maturation, inadequate secretory transformation of the endometrium and influence on the quality of cervical mucus are discussed as cause for this phenomenon. The purpose of this study was to investigate whether changes in endocrine patterns might lead to these changes thus limiting the success of clomiphene therapy. In 7 patients gonadotropins, prolactin, ovarian and adrenal androgens, sexual hormone binding globuline (SHBG) and plasma levels of clomiphene were measured at short intervals during a stimulated cycle. The increase of testosterone and androstendione promoted by clomiphene during the follicular phase is in accordance with previous observations. The decline in SHBG due to an antioestrogenic effect on its hepatic production leads to an additional increase in free, biologically active androgens. These androgens interfere with follicular maturation, causing atresia and premature luteinisation. As a direct effect of clomiphene on the adrenal gland, we observed an elevation of DHEAS. The significance of this finding remains unclear. During the follicular phase prolactin remained suppressed inspite of supraphysiological oestrogen levels. In the late luteal phase the diminished antioestrogenic influence on lactotrophic cells causes a 80% rise in prolactin that in some cases may impair luteal function. The average plasma half-life of clomiphene was found to be 12 days. This is in accordance with the half-life of the closely related compound tamoxifen. Clomiphene, also present in the luteal phase of the cycle, may cause inadequate secretory transformation of the endometrium by blocking endometrial oestrogen receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

High-performance liquid chromatographic determination of clomiphene using post-column on-line photolysis and fluorescence detection.

A method has been developed for the extraction and quantitation of the ovulatory stimulant drug clomiphene from plasma. The cis- and trans-isomers were separated by normal-phase chromatography using chloroform-methanol as the mobile phase. After eluting from the column, the clomiphene was passed through a PTFE photolysis coil irradiated by a powerful UV lamp, resulting in conversion of the isomers to highly fluorescent species. The derivatised material was then detected using a fluorescence spectrometer. Use of this method enables a substantial improvement of sensitivity over UV detection and has permitted the measurement of plasma clomiphene levels in patients receiving clomiphene therapy.