A New Strategy for the Analysis of Steroid Hormones in Industrial Wastewaters by Paper Spray Ionization Mass Spectrometry.

A new approach using paper spray ionization mass spectrometry (PSI-MS) for the analysis of steroid hormones in wastewater samples has been demonstrated. Triangular papers containing paraffin barriers as microfluidic channels were used to direct the sample solution to the paper tip, preventing the sample from spreading over the corners of the paper. The method was used to analyze the hormones levonorgestrel and algestone acetophenide in industrial wastewaters. Analytical curves presented a correlation coefficient (R2) above 0.99. Limits of quantification were below 2.3 ppm and limits of detection below 0.7 ppm. Values of precision (coefficient of variation) and accuracy (relative error) were less than 15% for all analyses. Recovery results ranged from 82% to 102%. Levonorgestrel was also analyzed by high-performance liquid chromatography coupled to mass spectrometry in order to compare the analytical performance with PSI-MS. No statistically significant differences were found between both methods. This study demonstrates the usefulness of PSI-MS for rapid analysis of hormones in industrial wastewater samples and also indicates its potential to be employed as a simple and reliable analytical method in environmental sciences.

Effect of anovulatory drugs on the human urinary tract and urinary tract infections.

PIP: Over a 1-year period, 44 women using either Demulen, Deladroxate or the Lippes loop were studied at the University of Pennsylvania for urinary tract disorders. Monthly catheterized urine specimens were cultured and examined cytologically. Excretory urograms and serum creatinine examinations were performed 1 month prior to and 6 and 12 months after contraceptive initiation. No effect on the urinary tract was observed by X-ray studies. All serum creatinines remained normal. Urine sediments showed cytologic patterns consistent with phase of cycle. Incidence of asymptomatic bacteriuria was 6.8% initially and 11% during the course of study. Only 1 Demulen patient developed asymptomatic tract infection. Urographic abnormalities were found in a certain percentage of women of reproductive age irrespective of contraceptive use. Study results, are confirmed by urograms of family planning clinic patients, indicate that estrogen-progestogen contraceptives appear to have little significant effect on human tract anatomy or infection rate.^ieng

Conception control by a single monthly injection.

PIP: 73 women of childbearing age and proven fertility were injected with dehydroxy-progesterone acetophenide, 150 mg and estradiol enanthate, 10 mg on Day 7, 8, or 9 of a new cycle and on Days 7-9 in succeeding cycles for a total of 929 cycles (1-24 cycles/patient). Patients missing 1 or more injections because of failure to menstruate were started as new patients and no patient was restarted more than 3 times. 69% received 1 series of injections; 24% received 2 series; and 7% received 3 series. There was a moderate prolongation of monthly bleeding. Cycles averaged 2 days shorter than before therapy. There was absolute pregnancy protection. Discontinuation was 45% for this study, most during the first 5 cycles, 1/2 of drug related discontinuation due to abnormal bleeding. Cytology smears, breast examinations and endometrial biopsies repeated before and during therapy were unremarkable.^ieng

Endometrial histology and parentereal estrogen-progestogen administration.

PIP: During the sixth, seventh, eighth, or ninth cycle of use, endometrial tissue was obtained through suction biopsy from 40 patients (mean age 26 years, mean number of pregnancies 3.5) receiving Deladroxate at the Hospital of the University of Pennsylvania Family Planning Clinic. Tissue was obtained from Day 1-29 after the most recent injection. Deladroxate is composed of 150 mg of 16-alpha, 17-alpha dihydroxyprogesterone acetophenide and 10 mg of estradiol enanthate in an oil vehicle. Injections were parenteral and administered on the seventh, eighth, or ninth day of each cycle. In the 36 specimens considered adequate for evaluation, the histologic features studied and scored were glandular morphology, stromal morphology, and vascular morphology. During the first 9 days of the cycle, the time interval before, during, and immediately after each injection, glandular activity attributable to estrogenic action was evident. After the first cycle, throughout each entire cycle, including the brief interval of estrogenic effect, glandular vacuolation and predecidual stromal reaction secondary to progestational effect were seen. The endometrial histologies may have been affected by variabilities in the absorption of the parenterally administered prepartion or by selective absorption of the estrogen or progestogen. In the discussion comparisons are made to typical sequential and combination preparations.^ieng

In vitro steroid metabolic studies in human testes I: Effects of estrogen on progesterone metabolism.

A technique of incubation of testicular tissue in vitro with radiolabeled precursors was applied in the investigation of the steroid biosynthesis by testes of four young men after long-term, high-dose estrogen treatment. A positive correlation between plasma and testicular steroid levels, and in vitro capacity of the testes to metabolize progesterone was demonstrated. Estrogen administration produced a very significant inhibition of plasma and testicular levels of testosterone. The in vitro synthesis of testosterone from progesterone was very severely impaired; especially 17alpha-hydroxylation of progesterone. 20alpha-hydroxysteroid-dehydrogenase activity was found to be increased after estrogen treatment, both in vivo and in vitro. These findings suggest that testicular 17alpha-hydroxylase activity (and possibly also 17-20 lyase activity) is either under gonadotropin regulation, or is directly suppressed by estrogen. This could result by decreased enzyme synthesis, direct enzyme inhibition or affectation of the cofactors or cytochromes necessary for the enzymatic activity. 20alpha-reduction of C21-steroids would represent an alternative pathway for their catabolism, not regulated by gonadotropin or not affected by estrogen, that would be significant in situations with reduced 17alpha-hydroxylase activity.

PIP: 4 male transsexuals, aged 23-46 years, were treated with ethinyl estradiol (1-2 mg daily) for at least 12 months prior to orchiectomy and sex-change surgery. The pattern of in vitro steroid biosynthesis by testicular tissue before and after this therapy was examined histologically and hormonally. Serum follicle stimulating hormone and luteinizing hormone were measured by radioimmunoassay as were plasma and testicular concentrations of testosterone (T). Plasma estradiol (E) and testicular progesterone (P) were also measured. In vitro steroid biosynthetic studies were made with radiolabeled precursors. Histology showed a uniform picture of spermatogenic arrest at the primary spermatocyte level after treatment. Concentrations of T, P, E, and 20alpha-dihydroxyprogesterone in the testes of the treated men showed that all hormones except E decreased markedly. Biosynthesis studies demonstrate that radioactive P substrate was poorly metabolized. In vitro synthesis of T from P was severely impaired; 17alpha-hydroxylation decreased substantially while 20alpha-hydroxy-steroid-dehydrogenase activity increased after estrogen treatment. It is suggested that 17alpha-hydroxylase activity is either under gonadotropin regulation or is directly inhibited by estrogen treatment. The increase in 20alpha-reduction might represent an alternate pathway unaffected by the treatment.

Review of ovulation return upon discontinuation of once-a-month injectable contraceptives.

Once-a-month combined injectable preparations draw their contraceptive efficacy from continuous ovulation suppression. When their use is discontinued, ovulation resumes within a few weeks or a few months, depending on the formulation. After use of the dihydroxyprogesterone acetophenide 150 mg/estradiol enanthate 5 mg combination for one to two years, ovulation returns in most subjects 3-4 months after discontinuation of treatment. Similarly, recent data show that after 2-year use of the depot-medroxyprogesterone acetate 25 mg/estradiol cypionate 5 mg or the norethisterone enanthate 50 mg/estradiol valerate 5 mg combination, approximately 70% women have resumed ovulation by the third month post-treatment. This is shorter than the time for return of ovulation experienced by ex-users of progestogen-only injectable contraceptives.

PIP: This paper reviews the progress made in the determination of ovulation and specifically addresses the effects of returning ovulation after discontinuance of once-a-month injectable contraceptive preparations. Correlation between ovulation and the hormones estrogen, progesterone, and luteinizing hormone (LH) is well documented. It has served as the basis for many studies on determining ovulation mid-point and in evaluating the efficacy, safety, and time of returned ovulation when using various contraceptive methods and preparations. Current monthly injectable contraceptive formulations are discussed and used as comparison for the new generation injectables. New generation contraceptives in this study are preparations (combinations) of several compounds. The depot microcrystalline form of medroxyprogesterone acetate (DMPA) in combination with estradiol cypionate (E2-Cy) was studied. The authors conclude that these initial studies on the new generation combination monthly injectables indicate that these new contraceptives are highly effective in inhibiting ovulation, as well as allowing for predictable return of ovulation.

Monthly combined injectable contraceptives and neoplasia.

Monthly injectable contraceptives, containing a combination of a long-acting progestogen and an estrogen, have been used in Latin America and China for many years. While knowledge about the effects of other hormonal contraceptives on cancer risk is relevant, close analogies with monthly injectables cannot be made. The relation between use of these preparations and cancers of the breast and cervix has been examined in case-control studies, but no firm conclusions can be drawn because of limitations in sample size. Adequate studies of the influence of monthly injectable contraceptives on risk of neoplasia need to be carried out.

PIP: Monthly injectable contraceptives containing long-acting progestogen and estrogen have been used in Latin America and China for many years. In Mexico and some other Latin American countries, several proprietary preparations containing dihydroxyprogesterone acetophenide and estradiol enanthate are available, with injections often administered by pharmacists. It is estimated that in the early 1980s more than one million ampoules per year were sold privately through pharmacies in Mexico. A large body of evidence exists, however, about the relationship between oral contraceptives and, to a lesser extent, DMPA and the risk of various tumors. The nature of the relationship between using the above monthly preparations and the risk of breast and cervical cancer remains to be determined. Toxicological studies in animals have proved to be of limited value in predicting the effects of contraceptive steroids on cancer risk in humans, but results have nonetheless delayed the introduction of monthly injectables in the US and other developed countries. The only studies which have examined associations between the use of monthly injectable contraceptives and cancer risk in women have been handicapped by limitations in sample size. There is clearly a significant and urgent need to conduct studies on the influence of monthly injectable contraceptives on the risk of cancer.

Pharmacodynamic assessment of dihydroxyprogesterone acetophenide plus estradiol enanthate as a monthly injectable contraceptive.

The pharmacodynamics of the combination of dihydroxyprogesterone acetophenide (DHPA) and estradiol enanthate (E2-EN) following its intramuscular administration at two doses were studied in 16 healthy women of reproductive age. Subjects were randomly allocated in two groups: group I (n = 9) received the combination DHPA 150 mg + E2-EN 10 mg on three consecutive monthly injections, while group II (n = 7) received half-dose of the same formulation. Ovarian function and endometrial bleeding patterns were investigated in all participants for one pre-treatment cycle, three treatment intervals and two post-treatment cycles. The results disclosed that ovulation was inhibited for at least 30 days following DHPA/E2-EN administration in all participants from both groups. The circulating estradiol levels 30 days after last injection were slightly elevated as compared with those observed in normal early follicular phase. Return to ovulatory cycles was documented within 90 days after treatment. The length of the bleeding-free intervals during treatment was shortened in both groups, particularly in group II. No significant changes in HDL-cholesterol levels were observed throughout the study. It is envisaged however, that large modification of the formulation and additional long-term safety studies will be required prior to its recommendation.

PIP: The pharmacodynamics of the combination of dihyroxyprogesterone acetophenide (DHPA) and estradiol enanthate (E2-EN) following its intramuscular administration at 2 doses were studied in 16 healthy women of reproductive age attending the Family Planning Clinic at General Hospital in Mexico City. Subjects were randomly allocated in 2 groups: group I (n=9) received the combination DHPA 150 milligrams and E2-EN 10 milligrams on 3 consecutive monthly injections, while group II (n=7) received 1/2 dose of the same formulation. Ovarian function and endometrial bleeding patterns were investigated in all participants for 1 pretreatment cycle, 3 treatment intervals and 2 post-treatment cycles. The results disclosed that ovulation was inhibited for at least 30 days following DHPA/E2-EN administration in all participants from both groups. The circulating estradiol levels 30 days after last injection were slightly elevated as compared with those observed in normal early follicular phase. Return to ovulatory cycles was documented within 90 days after treatment. The length of the bleeding-free intervals during treatment was shortened in both groups, particularly group II. No significant changes in HDL-cholesterol levels were observed throughout the study. It is envisaged however, that large modification of the formulation and additional long-term safety studies will be required prior to its recommendation.

Efficacy, acceptability, and clinical effects of a low-dose injectable contraceptive combination of dihydroxyprogesterone acetophenide and estradiol enanthate.

A total of 1,904 women, aged 15-38, used an injectable contraceptive combination of 90 mg dihydroxyprogesterone acetophenide with 6 mg estradiol enanthate, given once during each menstrual cycle between the 7th and 10th day, and preferably on the 8th day of the cycle, for a total of 17,576 cycles. Of these 1,904 women, 1,197 completed 12 cycles of use of the injectable combination. One subject became pregnant during the trial, resulting in a cumulative pregnancy rate of 0.07%. Principal reasons for discontinuation were personal, non-medical reasons, such as lost to follow-up, no longer wished to continue, protocol violation, desire to change to another contraceptive method, moved away, or other personal reasons. Mean weight of 1,901 subjects at admission to the trial was 53.5 +/- 0.2 kg and this increased to 54.3 +/- 0.3 kg after 12 cycles of use. Approximately 50% of subjects experienced menstrual bleeding similar to normal throughout the study period. The most frequent menstrual abnormality was irregular bleeding, experienced by approximately one-third of subjects.

Estrogen-progestogen once-a-month injectable contraceptives and serum prolactin.

To assess the effect of hormonal monthly injectable contraceptives upon the serum values of immunoreactive prolactin (Prl), three groups of women of reproductive age exposed to different estrogen-progestogen injectable formulation for a minimum of one year were studied. The first group (n = 10) received dihydroxyprogesterone acetophenide 150 mg and estradiol enanthate 10 mg (DHPA/E2-EN), Group 2 (n = 21) received medroxyprogesterone acetate 25 mg and estradiol cypionate 5 mg (MPA/E2-C) and Group 3 (n = 19) was exposed to norethisterone enanthate 50 mg and estradiol valerate 5 mg (NET-EN/E2-V). A group of IUD users (n = 16) served as the control group. Serum Prl and 17 beta-estradiol (E2) concentration were determined in blood samples (0 and 15 min.) on days 0 (day of last injection), 10, 20 and 30 after last contraceptive injection. The results demonstrated a slight though not significant increase (p greater than 0.05) in serum Prl in the three experimental groups as compared with the IUD control group. This increase in Prl levels observed on day 10 post-last injection never exceeded the upper limits of the normal range (20 ng/ml). Overall, the data demonstrated that the chronic administration of these estrogen/progestogen once-a-month injectable contraceptives does not affect the Prl baseline secretion in women.

Cervical mucus in estrous ewes after treatment with estrogen, progestogens and intrauterine devices.

PIP: The influence of estradiol, 6-alpha-methyl-17alpha-acetoxyprogesterone (MAP), and a unilaterally placed IUD on cervical mucus production was studied in estrous ewes. The animals received either 60 mg MAP from Days 9 to 21 of the extended estrous cycle, 25 mcg estradiol from Day 9 until estrus, or an IUD. MAP treatment significantly (p less than .05) increased cervical mucus production at estrus compared with the other treatments. However, the percentage of dry matter, chloride concentration, and spinnbarkeit did not markedly vary between groups. The protein content was markedly increased by estradiol. In a 2nd experiment, ewes received intravaginal sponges containing either 60 mg MAP, 30 mg 9-fluro-11beta-17-dihydroxyprogesterone-17-acetate (Cronolone), or 60 mg Cronolone from Day 9 to 21 of the extended estrous cycle. All 3 treatments significantly (p less than .01) increased cervical mucus production at estrus. There was little difference in the measured chemical and physical properties of cervical mucus between controls and treated animals. The increased production of cervical mucus in progesterone-treated animals may account for the reported reduction in the number of sperm in progesterone-treated ewes.^ieng

Steroid contraceptives: neuro-psychiatric & electroencephalographic complications.

PIP: The neuropsychiatric and electroencephalographic (EEG) complications of oral contraceptive (OC) use were investigated in 8 groups of 10 patients each. 61 of the women (76.2%) reported neuropsychiatric side effects, and changes in EEG were found in 48 patients. The reported symptoms were tension and anxiety states (45%), neurasthenic states (62.5%), and depressive states (52.5%). The administration of progesterone or estrogens alone increased the incidence of side effects. It is concluded that the ideal OC formulation provides a balance of both components in the lowest effective concentrations.^ieng

[Response of peripheral receptors to the parenteral administration of an association of dihydroxyprogesterone acetophenide and estradiol-3 benzoate-17-n-butyrate. Preliminary note]. / La risposta dei recettori periferici alla somministrazione parenterale dell'associazione diidrossiprogesterone acetofenide estradiolo-3-benzoato-17n-butirrato. Nota preventiva

PIP: The basal temperature curves, cervical mucus crystallation, endocrin e colpocytology, and endometrial cytology (Endo-Cyte) in 11 women over 30 cycles was determined following the im administration on the 8th day of the cycle of an estroprogestinic combination of 150 mg of dihydroxyprogesterone acetophenide and 10 mg of estradiol-3-benzoate-17-n-butyrate. The findings confirmed the antiovul atory action of the preparation, the absence of unwanted side effects, s ecretory transformations in the endometrium, the normality of the menstrual cycle, and the advantages associated with a single monthly administration.^ieng

[Clinical analysis and evaluation of various hematologic and metabolic constants with the use of monthly microdoses of parenteral contraceptives]. / Análisis clínico y valoración de algunas constantes hematologicas y metabólicas con el uso de un anticonceptivo parenteral mensual en microdósis.

PIP: 75 women of proven fertility were treated as a contraceptive measure with an injection of 75 mg. of duhydroxyprogesterone acetophenide, and of 5 mg. of estradiol enanthate. Doses were half of what regularly used, and were injected between the 7th and the 9th day of the cycle. Total number of cycles studied was 859. Most important side effects of the treatment was headache in 28.3% of patients, spotting in 15.5%, and emotional instability in 10.5%. Metabolic and hematologic data were unchanged, and vaginal cytology was negative. There were no pregnancies. It must be remembered that, in every contraceptive treatment, lower doses are always preferable when equally effective. (Summary in ENG).^ieng

Clinical trial of intramuscular injection as contraceptive compound.

PIP: 99 women of proven fertility received 778 intramuscular contraceptive injections. An injectable compound containing 150 mg dihy droxyprogesterone acetophenide and 10 mg of estradiol enanthate (Deladroxate) in castor oil was prepared in a 1-cc disposable syringe. The injections were administered intramuscularly in the upper outer glut eal region on Day 8 of each menstrual cycle. The study was initiated in September 1965 and was completed by February 1969, with all data being recorded on a computer for future analysis. No pregnancies occurred. No serious side effects were noted. There were improvements in cases of dysmenorrhea and premenstrual tension. Minor complaints were not a problem in the use of the contraceptive except for intermenstrual spotting, which prompted 8 patients to discontinue the method. It is concluded that the long-acting intramuscular injection of estrogen-progesterone given once a month is effective and useful in difficult contraceptive patients in a public health clinic.^ieng

Ovulation inhibition with a log-acting injectable. II. The cycling effects of varying progestagen-estrogen combinations.

PIP: The cycling effects of long-term injectable progestational compounds were studied to determine the most satisfactory dosage level. The compound Deladroxate (Deladroxone and estradiol enanthate) was administered to 38 subjects in the 5 following doses: 150 mg Deladroxone with 5 mg, 10 mg, 15 mg estradiol enanthate; and 200 mg Deladroxone with 5 mg or 20 mg estradiol enanthate. The compounds Deladroxate 150-10 and Deladroxate 200-5 showed the most satisfactory cycling and aftereffects. Further study of Deladroxate 150-10 showed mean cycle lengths of 26.2 and 28.3 days in 143 cycles, a 16% deviation of plus or minus 2 days from a pretreatment means, minimal side effects, and ovulatory cycles that returned within 5 to 13 weeks after withdrawal of the drug.^ieng

Steroid excretion in users of the injectable combined oestrogen progestogen preparation, deladroxate.

PIP: Steroid excretion analysis before and after monthly injections of deladroxate (150-mg dihydroxyprogesterone acetophenide combined with 10-mg estradiol enanthate per ml of oily solution) was compared in 9 healthy 20-35 year old women. Injections were given on Day 8 of the cycle. 24-hour urine collections were made on Day 23 of the cycle, both before and after injections. Assays were made for pregnandiol, estriol, 17-keto, and 17-ketogenic steroids. Analyses were repeated at 3 and 6 months after the injections were begun. Pregnandiol determinations after 3 and 6 months of treatment showed values below 1 mg/24 hours, indicating anovulatory cycles. After 3 months of injections, estriol levels declined significantly from 18.05 mcg/24 hours to 13.02 mcg at 3 months and to 7.23 at 6 months. 17-kestosteroids declined after 3 months and declined further after 6 monthly injections, i.e., from 11.05 mcg/24 hours before treatment to 6.5 at 3 months and 5 mcg at 6 months. Despite this decrease, the values of 17-ketosteroids even after 6 months were still on the borderline of low normal levels for women of their ages. The 17-ketogenic steroids showed a general tendency to decrease i .e., from 8.66 mcg/24 hours to 6.67 after 3 months and 6.5 mcg after 6 months. This decrease was not considered due to low adrenocortical functional reserve capacity, rather it was thought to be due mainly to the increased binding capacity of the plasma proteins caused by the estrogenic component of the preparation.^ieng

Effect of two long acting injectable progestogens on lactation in the human.

The effects of Depoprovera and Deladroxone were studied in humans, on certain milk components as well as on the growth of the nursed infants. Both drugs caused reduction in milk yield. Both drugs caused an increase in the concentration of milk total proteins, however. Depoprovera caused an increase while Deladroxone caused a decrease in the total amount of milk proteins per feed. Depoprovera showed no effect while Deladroxone caused an increase in the concentration of milk lipids; however, both drugs caused reduction in the total amount of milk lipids per feed. Both drugs showed no effect on the concentration of milk lactose, but caused reduction in the total amount of milk lactose per feed. The percentage increase in weight of nursed infants was decreased by Depoprovera, but not affected by Deladroxone.

PIP: The effects of Depo Provera and Deladroxone, both long-acting inject able contraceptive agents, on lactation and infant growth was studied in 76 lactating women. Milk yield was decreased by both drugs, though the concentration of total milk proteins was increased. Depo Provera increased the total amount of milk proteins per feed, while a decrease was observed with Deladroxone. Deladroxone increased the concentration of milk lipids, but Depo Provera had no effect. Nonetheless, both agents reduced the total amount of milk lipids per feed. Concentrations of milk lactose were unaffected, but the total amount of milk lactose per feed was reduced by both drugs. Depo Provera decreased the rate of infant growth, while Deladroxine had no effect.

Blood coagulation and fibrinolysis with an injectable long-acting progestogen-oestrogen contraceptive.

PIP: To determine the effect of a long-acting injectable progestogen-estrogen contraceptive (Deladroxate) on blood coagulation and fibrinolysis, 67 multiparous women receiving the contraceptive for 12 to 14 cycles aged 18-40 years were followed-up monthly for 1 year. The prothrombin consumption index, prothrombin percentage, whole blood clotting time, plasma fibrinogen, clot retraction, bleeding time, and fibrinolytic activity of all patients were all within normal limits both 6 and 12 months after beginning of treatment. Research on the relationship of contraceptive use and occurrence of thrombophlebitis is contradictory and no valid test for measuring blood coagulability exists that is satisfactory for these investigations. This study concludes nevertheless that no aberrations in blood coagulation occurred from this progestogen-estrogen treatment.^ieng

Ovarian changes during and after long term steroid contraceptive therapy.

PIP: This Egyptian study made use of ovarian wedge biopsies from 5 patients (aged 30-37, parity greater than 5) undergoing laparotomy. The contraceptives and durations of treatment were Lyndiol 2.5 (2.5 mg lunestrenol, .075 mg mestranol) 3 years; Gynanovlar 21 (3 mg norethisterone acetate, .05 mg ethinyl estradiol) 1 year; Deladroxate monthly injections (150 mg 16, a 17a dihydroxyprogesteorne acetophonid, 10 mcg estadiol enanthate) 1 year; Lyndiol 2.5, 5 years stopped 2 months before operation; and 1 unidentified contraceptive stopped 5 months before operation. except in the 2 cases receiving Lyndiol 2.5, follicular development and maturation had proceeded normally. The tunic a albugenia generally was more thickened than in full-term pregnancy but markedly less thickened than in Stein-Leventhal ovaries. Hyperthecosis was unobserved. Fibrotic and cystic changes were less in extent than in Stein-Leventhal ovaries. Primary damage after long-term therapy appears to be effected at a higher level than the ovary. The authors warn against long-term therapy given freely to young nulliparas and advise interrupted courses (i.e., 1-2 years followed by a periof of rest of 1-2 ovulations) for patients in general.^ieng