Regarding the references for reference chemicals of alternative methods.

The selection of reference and proficiency chemicals is an important basis for method validation and proficiency evaluations. Reference chemicals are a set of test substances used by a method developer to evaluate the reliability and relevance of a new method, in comparison to reference data (usually to a validated reference method). Proficiency chemicals, as defined in OECD Guidance Document on Good In Vitro Method Practices, are defined post validation as a subset of the reference chemicals or other chemicals with sufficient supporting data that are used by naïve laboratories to demonstrate technical competence with a validated test method. Proficiency chemicals should cover different physical states, several chemical classes within the applicability domain of the method and yield the full range of responses (in the validated reference method and in vivo), they shall be commercially available (at non-prohibitive costs) and have high quality reference data. If reference and subsequent proficiency chemicals are chosen without sufficient evidence for their inclusion, both test method evaluation and demonstration of technical proficiency can be hampered. In this report we present cases in which the selection of reference chemicals led to problems in the reproduction of the reference results and demonstration of technical proficiency: The variability of results was not always taken into account in selection of several reference substances of the LLNA (OECD TG 429). Based on the available reference data one proficiency chemical for the Corrositex skin corrosion test (OECD TG 435) should be replaced. Likewise, the expected in vitro result for one of the proficiency chemicals for the BCOP (OECD TG 437) was difficult to reproduce in several labs. Furthermore, it was not possible to obtain one of the proficiency chemicals for the Steroidogenesis Assay (OECD TG 456) at non-prohibitive costs at a reasonable purity. Based on these, we recommend changes of current proficiency chemicals lists with established OECD Test Guidelines and provide recommendations for developing future sets of reference chemicals.

Establishing reference intervals for sex hormones and SHBG in apparently healthy Chinese adult men based on a multicenter study.

BACKGROUND: Measuring sex hormones is essential in diagnosing health issues such as testicular dysfunction, male infertility and feminization syndrome. However, there are no reports on reference intervals (RIs) in Chinese men. We conducted a nationwide multicenter study to establish RIs for seven sex hormones (luteinizing hormone [LH], follicle-stimulating hormone [FSH], prolactin [PRL], total testosterone [TT], free testosterone [FT], bioavailable testosterone [BAT] and estrogen [E2]), as well as sex hormone-binding globulin (SHBG). METHODS: In 2013, 1043 apparently healthy adult men from five representative cities in China (Beijing, Hangzhou, Guangzhou, Dalian and Urumqi) were recruited; hormones were measured using an automated immunoassay analyzer. Multiple regression analysis (MRA) was performed to identify sources of variation (SVs) that might influence the hormone serum levels. RIs were computed using the parametric method. RESULTS: Dalian and Hangzhou had significantly higher E2 values than other cities; age was a major source of variation for FSH, LH, PRL, SHBG, FT and BAT. FSH, LH and SHBG increased significantly with age, while PRL, FT and BAT decreased with age. TT showed no significant age-related changes. Median (RIs) derived without partition by age were as follows: FSH, 5.6 (1.9-16.3) IU/L; LH, 4.2 (1.6-10.0) IU/L; PRL, 189 (88-450) mIU/L; E2, 85 (4.7-195) pmol/L; SHBG, 29.4 (11.5-66.3) nmol/L; TT, 15.6 (7.4-24.5) nmol/L; FT, 0.31 (0.16-0.52) nmol/L; and BAT, 8.0 (3.7-13.2) nmol/L. RIs were also derived in accordance with between-city and between-age differences. CONCLUSIONS: RIs were established for sex hormones and SHBG in apparently healthy Chinese men in consideration of age.

Biological and chemical standardization of a hop (Humulus lupulus) botanical dietary supplement.

Concerned about the safety of conventional estrogen replacement therapy, women are using botanical dietary supplements as alternatives for the management of menopausal symptoms such as hot flashes. Before botanical dietary supplements can be evaluated clinically for safety and efficacy, botanically authenticated and standardized forms are required. To address the demand for a standardized, estrogenic botanical dietary supplement, an extract of hops (Humulus lupulus L.) was developed. Although valued in the brewing of beer, hop extracts are used as anxiolytics and hypnotics and have well-established estrogenic constituents. Starting with a hop cultivar used in the brewing industry, spent hops (the residue remaining after extraction of bitter acids) were formulated into a botanical dietary supplement that was then chemically and biologically standardized. Biological standardization utilized the estrogen-dependent induction of alkaline phosphatase in the Ishikawa cell line. Chemical standardization was based on the prenylated phenols in hops that included estrogenic 8-prenylnaringenin, its isomer 6-prenylnaringenin, and pro-estrogenic isoxanthohumol and its isomeric chalcone xanthohumol, all of which were measured using high-performance liquid chromatography-tandem mass spectrometry. The product of this process was a reproducible botanical extract suitable for subsequent investigations of safety and efficacy.

Quantitative analysis of conjugated and free estrogens in swine manure: solutions to overcome analytical problems due to matrix effects.

Although animal manure is an important source for environmental estrogens, quantitative analysis of estrogens in manure is complicated due to matrix interference. In the present study, chromatographic methods have been developed for quantification of conjugated and free estrogens in manure samples collected from pig farms. The whole manure samples, immediately after collection, were stored at 4°C, acidified (pH≈2.0) and spiked with (i) (13)C-labeled internal standards to account for possible storage related degradation and (ii) deuterium labeled internal standards for calibration and quantitative analysis. The liquid samples were extracted with ethyl acetate for separating conjugated and free estrogens. The solid samples were eluted with water for desorbing conjugated hormones followed by methanol for desorbing free hormones. The water and extracts were further purified using hydrophilic-lipophilic balance and/or aminopropyl cartridges. The conjugated estrogens were analyzed using high-performance liquid chromatograph-mass spectrometer, while the free estrogens were analyzed using gas chromatograph-mass spectrometer. The extraction and calibration methods used in the present study yielded excellent sensitivity, reproducibility and >85% recovery of both free and conjugated estrogens that was independent of the manure matrix. In general, the total estrogen loads in liquid and solid samples were 5.1mg/l and 4.93mg/kg, respectively. This may represent the hormonal load of approximately 2.3tons estrogen per day.

Evaluation of wetland and tertiary wastewater treatments for estrogenicity using in vivo and in vitro assays.

The effects of wetland wastewater treatment on estrogenic activity and estrogenic activity of water after tertiary treatment were evaluated using in vivo (rainbow trout vitellogenin [VTG] expression) and in vitro (yeast estrogen screening) assays. Juvenile rainbow trout exposed to tertiary-treated wastewater from the Green Acres Treatment Plant in Orange County Water District had increased plasma VTG levels compared with control fish. When trout were exposed to wastewater-dominated water before it entered into Prado Wetland (Riverside County, CA), VTG concentrations were increased above those of controls and were not significantly different from fish exposed to water exiting the wetland. VTG E2-equivalent concentrations (EEQs) of the water samples from the Green Acres Plant were 16.92 +/- 16.49 ng/L. Activity of water entering Prado Wetland was 29.80 +/- 28.41 ng/L EEQ, and water exiting was 24.34 +/- 23.17 ng/L EEQ. In vitro assays estimated that estrogenic activity of water from the Green Acres Plant was <1 ng/L EEQ, whereas water entering and exiting Prado Wetland had yeast estrogen screening EEQs of 2.57 and <1 ng/L, respectively. Our results suggest that environmental estrogens that are not potent estrogen-receptor ligands exist in wastewaters from the Green Acres Plant as well as water entering and exiting Prado Wetland. Wetland treatment did not remove environmental estrogens in the water. Our results also suggest that in vitro assays may underestimate estrogenic activity of sampled water.

Efficacy and safety of a low-dose 24-day combined oral contraceptive containing 20 micrograms ethinylestradiol and 3 mg drospirenone.

The aim of this open-label, multicenter, noncomparative study was to determine the efficacy, safety and bleeding profile of a new low-dose, monophasic combined oral contraceptive containing 20 micrograms ethinylestradiol and 3 mg drospirenone administered daily for 24 days followed by a 4-day hormone-free interval. Contraceptive efficacy was analyzed for 1018 women completing 11,140 treatment cycles. Eleven pregnancies occurred, giving a Pearl Index (PI) of 1.29 (upper limit of the 95% confidence interval [CI], 2.30); of these pregnancies, five were considered due to method failure, giving an adjusted PI of 0.72 (upper limit of the 95% CI, 1.69). A total of 7 (0.7%) women discontinued study medication because of irregular bleeding, suggesting a favorable bleeding profile. Overall, the treatment was well tolerated with an excellent safety profile. The majority of women (86%) stated that they were satisfied or very satisfied with the treatment and over 70% of women would have continued with the study medication.

Estrogen plus progestin therapy: the cardiovascular risks exceed the benefits.

The surprising results of the Women's Health Initiative (WHI) reported in 2002 had a profound effect on women as well as health care practitioners. The WHI was the largest, randomized clinical trial designed to determine if postmenopausal hormone use prevented cardiovascular disease as well as other age-related disorders in women. While observational studies suggested that postmenopausal use of estrogen could decrease cardiovascular risk by 40% to 50%, the WHI demonstrated that use of continuous-combined estrogen plus progestin was not cardioprotective and was even associated with increased health risks. The estrogen alone trial of the WHI is still in progress, leaving practitioners and some women still in a dilemma. This article addresses the WHI in the context of other studies and discusses possible reasons for the unexpected results.

Efficacy, cycle control, and side effects of low- and lower-dose oral contraceptives: a randomized trial of 20 micrograms and 35 micrograms estrogen preparations.

Estrogen content represents a tradeoff between cycle control and side effects, but few direct comparisons of 20 and 30/35 micrograms preparations are available. To address this issue, we conducted a randomized, open-label multicenter clinical trial comparing Alesse (20 micrograms ethinyl estradiol [EE]), Mircette (20 micrograms EE), and Ortho Tri-Cyclen (35 micrograms EE) among 463 OC starters or switchers. Bloating, breast tenderness, and nausea were approximately 50% more common in women using 35 micrograms EE as compared to 20 micrograms EE preparations. Cycle control was similar in all products, although during the first two cycles among starters; users of Mircette and Ortho Tri-Cyclen (Tri-Cyclen) exhibited better cycle control than Alesse users. Discontinuation and pregnancy rates were not significantly higher in 35 micrograms EE users.

PIP: Estrogen content represents a tradeoff between cycle control and side effects, but few direct comparisons of 20 and 30/35 mcg preparations are available. To address this issue, researchers conducted a randomized, open-label multicenter clinical trial comparing Alesse (20 mcg ethinyl estradiol [EE]), Mircette (20 mcg EE), and Ortho Tri-Cyclen (35 mcg EE) among 463 oral contraceptive starters or switchers. Bloating, breast tenderness, and nausea were approximately 50% more common in women using 35 mcg EE as in those using 20 mcg EE preparations. Cycle control was similar in all products, although during the first two cycles among starters, users of Mircette and Ortho Tri-Cyclen (Tri-Cyclen) exhibited better cycle control than Alesse users. Discontinuation and pregnancy rates were not significantly higher in 35 mcg EE users.

Enfermedad Cardiovascular en Mujeres y Terapia Hormonal de Reemplazo / Cardiovascular Disease in Women and Hormonal Replacement Therapy

En la medida en que se comprenden mejor los mecanismos de acción y los efectos benéficos de los estrógenos, la lista de contraindicaciones para su uso se va haciendo cada vez más corta, mientras que el número de usuarias se hace cada vez mayor. En los Estados Unidos hacia 1982 se prescribieron 13.6 millones de recetas con estrógenos y 2.3 millones con progesterona. Diez años mas tarde estas cifras se multiplicaron a 31.7 y 11.3 respectivamente, lo cual muestra una tasa de incremento del 1.6 y 1.4 para cada uno de ellos. Esto significa que se ha tomado con mas responsabilida la promoción de la terapia de reemplazo hormonal en la menopáusica, con la obtención de mayores beneficios para un mayor número de mujeres, una mejor calidad de vida con un menor número de padecimientos y mejoría en las tasas de mortalidad por enfermedad cardiovascular y osteoporosis. Este artículo intenta hacer una revisión sobre los conocimientos hasta ahora obtenidos en cuanto a los factores de riesgo para enfermedad coronaria en la mujer menopáusica, su fisiopatología, mecanismos de acción de los estrógenos y beneficios que con la terapia de reemplazo hormonal se obtiene sobre la paciente con enfermedad cardiovascular

Treatment of bovine retained placenta and its effects.

The literature on the treatment of retained placenta and its effects is reviewed. Manual removal, the oldest and commonest method of treatment, benefits parlour hygiene but may adversely affect the cow. The use of collagenase may allow manual removal without such side effects. Ecbolic drugs are often ineffective, both as prophylaxis and treatment for the condition. They are most effective within one hour of parturition, particularly after a caesarean section in which tocolytic drugs have been used. Endometritis is a very common sequel to retained placenta. Antibiotics and oestrogens have been used to treat, control or prevent the condition, but they are not routinely effective and may have deleterious side effects. Gonadotrophin releasing hormone and/or prostaglandins have been used to reduce the deleterious effect of retained placenta on fertility, but the results obtained have been inconsistent.

Tibolone versus conjugated estrogens and sequential progestogen in the treatment of climacteric complaints.

OBJECTIVE: Tibolone has been shown to alleviate climacteric symptoms. This study was designed to compare the effect of tibolone (Livial, 2.5 mg daily) on different climacteric complaints and its impact on the endometrium, determined by vaginal ultrasound, with that of conjugated estrogens (Premarin, 0.625 mg daily) continuously for 6 months in combination with the progestogen medrogestone (Colpron, 2 x 5 mg daily for 12 days each month). METHODS: One hundred and twenty-nine postmenopausal women were recruited and the severity of climacteric symptoms as well as endometrial thickness were recorded at the pre-trial examination and after 1, 3, and 6 months. RESULTS: With the exception of vertigo, mood depression, mood disorder, loss of libido, and dryness of skin, where tibolone was found to be more effective than conjugated estrogens/medrogestone, climacteric symptoms improved significantly in both groups over the 6-month study period. Endometrial thickness did not increase significantly in the tibolone group, whereas in the conjugated estrogens/medrogestone group there was a highly significant increase after 1 month and still a trend towards significance after 6 months. Recurrence of vaginal bleeding occurred significantly less frequently in the tibolone group than in the comparison group. CONCLUSION: Tibolone seems to offer a complete treatment of the climacteric complaints whilst avoiding some of the problems associated with classical hormone replacement therapy.

Use of genetically identical (clone) steers to determine the effects of estrogenic and androgenic implants on beef quality and palatability characteristics.

Six sets of four genetically identical Brangus steers (n = 24; mean weight = 413 kg, SD = 19.8) were used to study the effects of estradiol and trenbolone acetate on beef quality and palatability characteristics. Steers in each clone set were randomly assigned to the following treatment groups: 1) a nonimplanted control; 2) a single estrogenic implant, containing 20 mg of estradiol benzoate and 200 mg of progesterone; 3) a single androgenic implant, containing 140 mg of trenbolone acetate; or 4) a single combination implant, containing 24 mg of 17-beta estradiol and 120 mg of trenbolone acetate. Following implantation, the steers were fed a high-concentrate finishing diet for a period of 112 d. Compared with control steers, implanted steers had higher (P < .05) average daily gains and heavier (P < .05) finished live weights and carcass weights. However, there were no differences (P > .05) among treatment groups with respect to their effects on growth rate, live weight, carcass weight, dressing percentage, fat thickness, longissimus muscle area, percentage of kidney, pelvic, and heart fat, or USDA yield grade. Moreover, marbling scores for implanted steers were not statistically different from marbling scores for control steers. However, a comparison among implant types showed that steers implanted with the estrogenic implant had significantly lower marbling scores than did steers implanted with the androgenic or combination implants. Use of androgenic and combination implants had no effect (P > .05) on beef tenderness of strip loin, top sirloin, or top round steaks; however, use of estrogenic implants decreased (P < .05) tenderness of top sirloin steaks.

Naltrexone treatment restores menstrual cycles in patients with weight loss-related amenorrhea.

OBJECTIVE: To evaluate whether the efficacy of naltrexone administration in patients with hypothalamic amenorrhea correlates to the response to an acute naloxone test. DESIGN: Thirty patients with hypothalamic amenorrhea associated with weight loss were studied. After naloxone test (4 mg in bolus IV) patients were divided into two groups: group A, nonresponsive (n = 15) and group B, responsive (n = 15). Group A underwent two cycles of hormonal replacement therapy with E2 patches and medroxyprogesterone acetate. Then all patients were administered naltrexone at the dosage 50 mg/d orally for 6 months. A third group of 10 amenorrheic patients were treated with oral placebo with the same schedule. RESULTS: Plasma gonadal steroid levels increased in all patients and in 24 of 30 patients the menstrual bleeding occurred within 90 days from the beginning of treatment. After 6 months from naltrexone discontinuation, 18 of 24 patients still showed the occurrence of menstrual cycles. Luteinizing hormone plasma levels and LH pulse amplitude increased after 3 months of treatment and remained unchanged 6 months after naltrexone suspension. Plasma FSH levels did not show any change in any patient. The body mass index increased after 3 months in all patients who menstruated. Patients treated with placebo did not show any significant change in gonadotropins and gonadal steroid plasma levels. CONCLUSIONS: The present study supports the efficacy of naltrexone therapy for patients with hypothalamic amenorrhea either responsive or nonresponsive to naloxone test.

Incidence of ovulation in perimenopausal women before and during hormone replacement therapy.

Once hormone replacement therapy (HRT) has been commenced, it becomes extremely difficult to advise women approaching the menopause on the need for contraception. In this study of twenty women, neither the regularity of their pre-existing menstrual cycle nor a random FSH concentration predicted the likelihood of subsequent ovulation whilst taking HRT. HRT is not reliably contraceptive and women commencing HRT whilst still menstruating spontaneously must be advised on the need for additional contraception.

PIP: In Scotland, providers determined the incidence of ovulation before and during use of oral hormone replacement therapy (HRT) among 20 women aged 42-52 attending the Family Planning and Well Woman Services menopause clinic in Edinburgh and their need for contraception. Ovulation was defined as a pregnanediol concentration of more than 0.5 mmol/g creatine. HRT consisted of 1.25 mg/day of conjugated estrogens with 150 ug cyclical norgestrel for 12 of 28 days (Prempak-C). Women with irregular cycles were older than those with regular cycles (47.3 vs. 45.5 years; p 0.02). They also had considerably higher mean follicle stimulating hormone (FSH) levels (26 vs. 14.2 IU/l; p 0.001). One woman had a very high FSH level (67 IU/l) before HRT use and ovulated in the subsequent cycle with urinary pregnanediol levels indicating normal ovarian function. All 10 women who had regular cycles ovulated before HRT use. Six still ovulated during HRT use. Among women with irregular cycles, 4 of the 10 women ovulated before HRT use. Three of these women had anovulatory cycles during HRT use while 3 of the 6 who did not ovulate before HRT use ovulated during HRT use. These findings reveal that HRT with the higher dose Prempak-C does not suppress ovulation and that an elevated FSH does not always indicate the absence of ovulation. They also show that neither age nor regularity of menstrual cycles can be used to predict the need for contraception in perimenopausal women using HRT.

Benefits and risks of estrogen replacement therapy.

Estrogen replacement therapy (ERT) has been shown to reduce the risk of cardiovascular disease (CVD) and osteoporosis in postmenopausal women. Studies also indicate a reduced risk of stroke and its consequent mortality among estrogen users, and ERT may also have a role in reducing the risk of Alzheimer's disease and increasing a woman's overall quality of life. On the negative side, some studies show a small duration-related risk of breast cancer with estrogen use and a significant increase in endometrial cancer; the latter is virtually eliminated with the addition of a progestin to the regimen. Although the definitive answer is not yet available, recent epidemiologic data suggest no reduction in protection against CVD and bone fracture with the addition of progestin, which is referred to as hormone replacement therapy, as opposed to using estrogen alone. A woman's potential risks associated with ERT or hormone replacement therapy must be weighed against her lifetime risks of developing CVD, stroke, and bone fracture. The reduction in mortality and morbidity rates with hormone use is generally viewed to be substantial and cost-effective. Health care professionals have an important role in shaping their patients' attitudes. Patients need more information from their physicians about the risks and benefits of estrogen therapy.

Estrogen replacement therapy in practice: trends and issues.

Since their availability in 1926, estrogens and their use in postmenopausal women have met with both acceptance and alarm by the medical profession and potential estrogen users. It was not until the 1980s that long-term research began to demonstrate the beneficial effects of estrogen, thus contributing to a significant increase in use during the past decade. This article provides information on current use of estrogen replacement therapy and describes factors influencing continuance. Poor continuance remains a barrier to the full potential of estrogen replacement therapy for postmenopausal women. The role of the physician in improving continuance is discussed.

Emerging delivery systems for estrogen replacement: aspects of transdermal and oral delivery.

The ideal preparation for estrogen replacement therapy has been the object of intensive research for decades, and the search continues. More than 40 new products are in development in the United States, the United Kingdom, and Europe. Most are transdermal, which reflects the growing acceptance of patch technology at a time when the overwhelming majority of women who use estrogen replacement take oral formulations. Most of the new oral formulations are a combination of estrogen and progestin. Aspects of transdermal and oral estrogen are discussed, including the advantages and disadvantages for use in women with concomitant medical conditions.

Safety of oral contraceptives related to steroid content: a critical review.

In 1980, an FDA advisory committee found inconclusive the evidence that the safety of oral contraceptives was related to their steroid content. In 1988, another FDA advisory committee examining the same evidence concluded that there is a trend to suggest such a relationship. The present review critically examines the published evidence and concludes that there is no scientific support for the suggestion that higher-dose oral contraceptives are less safe. Nevertheless, such products are no longer being made available in the United States.

Generic drugs in reproductive medicine: is the value anticipated the value obtained?

PIP: Potential risks of prescribing generic drugs for oral contraception, ovulation induction and menopausal estrogen substitution are reviewed. In the U.S. all 50 states permit or require generic substitution, unless the prescription states otherwise. F.D.A. guidelines allow the bioavailability to range within 25%, i.e., the circulating level of drug in male test subjects after a single dose must be within + or - 25% of the range reported with the pioneer drug. Thus if a patient renews her prescription monthly she may receive a 50% range in dose level if different generics with both extremes in bioavailability are provided. Potential ranges for women, ill or elderly may vary even more. The potential risks for oral contraceptives are unwanted pregnancy, breakthrough bleeding, blood lipid elevation and less serious consequences such as minor side effects or confusion over package design. Risks for generic estrogen replacement include lack of protection from cardiovascular disease or osteoporosis and lack of therapeutic effect on menopausal symptoms; those from progestins are atherogenesis and failure to stop dysfunctional bleeding, ending in an unnecessary D & C. Possible sequelae of generics for ovulation induction are multiple pregnancy and ovarian hyperstimulation resulting in hospitalization for acute massive ovarian enlargement or rupture, as well as extra months of expense for treatment and monitoring. The most pressing concern is liability for the physician or pharmacist related to provision of generics. In most cases even 1 additional office visit for drug dose adjustment will obviate all savings accrued from taking generics.^ieng