Contraceptive efficacy and clinical performance of Nestorone implants in postpartum women.

The objective of this study was to evaluate the contraceptive efficacy and clinical performance of a Nestorone subdermal implant (NES) in the postpartum period. NES (n = 100) and Copper T intrauterine device (T-Cu; n = 100) acceptors initiated contraception at 8 weeks postpartum and were followed at monthly intervals during the first year and at 3-month intervals thereafter. Pregnancy rates, breastfeeding performance, infant growth, bleeding pattern, and side effects were assessed. Blood and milk NES concentration were measured. No pregnancy occurred in 2195 and 2145 woman-months of NES implant and T-Cu use, respectively. No effect of NES on lactation and infant growth and no serious adverse events were observed. Lactational amenorrhea was significantly longer in NES users (353 +/- 20 days) than in T-Cu users (201 +/- 11 days). More NES users (55.8%) experienced prolonged bleedings than did T-Cu users (36.2%). Concentrations of NES in breast milk ranged between 54-135 pmol/liter. The Nestorone implant is a highly effective contraceptive, safe for breastfed infants because the steroid is inactive by the oral route.

Effects of androgen on androgen receptor expression in rat testicular and epididymal cells: a quantitative immunohistochemical study.

Androgen is essential for maintenance of spermatogenesis in the testis and for maturation of spermatozoa in the epididymis. The effects of androgen are mediated through its receptor (AR), the levels of which are, in turn, regulated by androgen. Previous studies have shown that AR concentrations in Leydig and Sertoli cells are differentially regulated during development. The aim of the present study was to determine if cell-type-specific regulation of AR by androgen occurs in testicular and epididymal cells during adulthood. Adult male rats were treated with the LHRH-antagonist Azaline B (100 g/day) by osmotic pump for 1, 2, 3, 4, or 8 wk to suppress endogenous androgen, with identical numbers of intact control animals at each time period. An androgen replacement group was simultaneously treated with the antagonist and a synthetic androgen, 7 alpha-methyl-19-nortestosterone (MENT), during the final 4 wk of the experiment. Levels of nuclear AR protein in specific cell types were quantified by immunohistochemistry in conjunction with computer-assisted image analysis. Levels of AR in testicular cells declined sharply after treatment with the LHRH antagonist. In Sertoli cells, nuclear AR levels decreased to 8% of control (P < 0. 01) after 4 wk treatment; and to 12% and 17% of control (P < 0.01) in Leydig and myoid cells, respectively. Androgen replacement resulted in complete recovery of nuclear AR levels in Sertoli cells (93%, P > 0.05) but in only partial recovery in myoid (69%, P < 0. 01) and Leydig cells (56%, P < 0.01). In the epididymis, tubular epithelial cells and stromal cells differed in their responses to the LHRH antagonist. After 1 wk, nuclear AR levels in caput stromal cells decreased dramatically to 34% of control (P < 0.01) and in cauda stromal cells to 43% (P < 0.01). In contrast, the decline of AR levels in epididymal epithelial cells was not as dramatic as that in stromal cells. After 1 wk, the decline in the caput and cauda was to 87% and 76% of control, respectively. After 8 wk, nuclear AR levels in stromal cells further declined to 1.1% in caput and 1.4% in cauda, whereas in the epithelial cells, a smaller decline in nuclear AR was noted (to 30% in the caput and 45% in the cauda). After androgen replacement with MENT, nuclear AR levels recovered to more than 90% of control in both epididymal cell types. These results indicate that AR levels in the nuclei of adult Sertoli cells depend mainly on the level of androgen, whereas in the adult Leydig and myoid cells, the androgen dependency is more limited. The results also indicate that in the epididymis, stromal cells are more sensitive than epithelial cells to the regulation of AR levels by androgen.

Ovarian function during use of Nestorone(R) subdermal implants.

Nestorone(R) progestin (NES) is a potent 19-nor-progesterone derivative which is biologically inactive when administered orally; however, it is an excellent option for implant contraception. The objective of this study was to evaluate ovarian function during use of either one 4-cm or two 3-cm NES implants for 24 months. A total of 60 volunteers were enrolled in each dose group. Vaginal ultrasound (VUS) and blood sampling for determinations of estradiol (E(2)), progesterone (P) and NES serum levels were carried out twice a week for 6 consecutive weeks, beginning in months 1, 6, 12, 18, and 24 of implant use. Serum levels of NES declined with time, with a more pronounced decrease during the first 18 months of implant use; thereafter, NES levels remained stable until the end of the study at 24 months. Luteal activity was very infrequent during the first year of use (<3%) but increased during the second year, occurring in 27% and 35% of the sampling periods in the 1-implant group, and 2% and 16% of the sampling periods in the 2-implant group, at months 18 and 24 of use, respectively. No luteal activity was observed with NES levels above 80 pmol/L. Serum P levels in periods of luteal activity were significantly lower than those of controls. Persistent anovulatory follicles were the most common VUS finding and this was associated with E(2) levels that remained within the normal range (101-1500 pmol/L) in the majority of the sampling periods studied. Considering that a single implant offers advantage for insertion and removal, a new single NES implant is being developed with a slightly higher release rate, to reduce effectively the incidence of ovulation and provide a greater margin of safety beyond 2 years.

7Alpha-methyl-19-nortestosterone maintains sexual behavior and mood in hypogonadal men.

The synthetic steroid 7alpha-methyl-19-nortestosterone (MENT) is a potent androgen that is resistant to 5alpha-reductase. It thus has decreased activity at the prostate and may have advantages over testosterone-based regimens in long term treatment or as part of a male contraceptive. Administration to eugonadal men results in suppression of gonadotropins, but its ability to support androgen-dependent behavior has not been investigated. For sustained release administration, MENT acetate was used, because its diffusion characteristics were more suitable for use in implants. However, upon release the acetate is rapidly hydrolyzed, and MENT is the biologically active moiety in circulation. We studied the effects of MENT on sexual interest and activity, spontaneous erection, and mood states in comparison with testosterone enanthate (TE) in 20 Caucasian and Chinese hypogonadal men recruited in Edinburgh and Hong Kong (n = 10 in each center). Outcomes were measured using a combination of daily diaries, semistructured interviews, and questionnaires. Nocturnal penile tumescence (NPT) was also recorded in the Edinburgh group. After withdrawal of androgen replacement treatment (wash-out phase) for a minimum of 6 weeks, subjects were randomized to two groups in a cross-over design. Drug treatment regimens were of 6-week duration and consisted of two implants, each containing 115 mg MENT acetate, inserted s.c. into the upper arm and removed after 6 weeks and two injections of TE (200 mg, i.m.) 3 weeks apart. MENT treatment resulted in stable plasma MENT concentrations of 1.4 +/- 0.1 nmol/L after 3 weeks and 1.3 +/- 0.1 nmol/L after 6 weeks (mean +/- SEM; all men). Nadir testosterone concentrations were 3.6 +/- 0.6 nmol/L at the end of the wash-out phase and 9.4 +/- 0.6 nmol/L 3 weeks after each injection. There were no differences in hormone concentrations between centers. There were no adverse toxicological effects. There were only minor differences between the two treatments. Both MENT and TE treatment resulted in significant increases in sexual interest and activity, spontaneous erection (both by self-report and NPT measurement), and increases in positive moods, with decreases in negative moods in the Edinburgh group. In the Hong Kong group, both treatments increased waking erection, with a trend toward increased sexual interest and activity. Mood states appeared to be less affected during the wash-out phase than in Edinburgh men and showed no significant response to either treatment. These results demonstrate that MENT has similar effects on sexual activity and mood states as testosterone in hypogonadal men. As NPT is a physiological androgen-dependant outcome, these data provide further evidence for the androgenicity of MENT. The lack of detected effect of either androgen in Hong Kong men other than on waking erection illustrates the importance of the cultural context of symptomatology and its measurement. The appropriate dose of MENT remains to be determined, but these results support its development as a potential androgen replacement therapy.

Gonadotrophin and testosterone suppression by 7alpha-methyl-19-nortestosterone acetate administered by subdermal implant to healthy men.

The synthetic androgen 7alpha-methyl-19-nortestosterone (MENT) is a potent suppressor of gonadotrophin that has several advantages for long term administration to normal or hypoandrogenic men. The aim of this study was to examine MENT serum concentrations following subdermal insertion of MENT acetate (MENT Ac) implants and their effects on gonadotrophins, testosterone, dihydrotestosterone (DHT), sex hormone-binding globulin, prostate specific antigen and insulin-like growth factor-1 serum concentrations in normal men. A total of 45 healthy men were recruited at three clinics. Each subject received one, two or four implants for 28 days. Serum samples were obtained before insertion and on days 8, 15, 22, 29, 36 and 43 after implant insertion. The average daily dose delivered in vivo by one implant was approximately 500 microg. One, two or four MENT Ac implants produced dose dependent and sustained serum MENT concentrations for the entire duration of treatment of 0.7 +/- 0.1, 1.2 +/- 0.1 and 2.0 +/- 0.1 nmol/l respectively. This treatment induced a dose dependent decrease in gonadotrophin and androgen serum levels. Two and four implants induced maximal suppression that was maintained throughout treatment and was completely reversed after removal of the implants. The mean decreases were 93 +/- 1% for testosterone, 80 +/- 3% for DHT, 97 +/- 1% for luteinizing hormone and 95 +/- 1% for follicle stimulating hormone. No serious adverse reactions were reported by the volunteers and no consistent changes in clinical chemistry and haematology were found. These results indicate that MENT Ac implants are an efficient way of MENT administration and confirm the potent gonadotrophin and androgen suppressive effect of this drug.

Changes in the distribution of intermediate filaments in rat Sertoli cells during the seminiferous epithelium cycle and postnatal development.

BACKGROUND: Intermediate filaments (IFs) are components of the cytoskeleton. In mammalian Sertoli cell, IFs are formed by vimentin. Previous studies have shown some characteristics of its distribution in Sertoli cells, however, very little is known of its distributional changes during the seminiferous epithelium cycle and during postnatal development. METHODS: Immunohistochemical and electron microscopic methods were used to determine the distribution of vimentin-type IFs in rat Sertoli cells during the seminiferous epithelium cycle and postnatal development. RESULTS: The distribution of IFs in adult rat Sertoli cell showed distinct cyclic changes during the seminiferous epithelium cycle. At stages I-VI, bundles of IFs extend from the perinuclear region to the supranuclear and apical regions of the Sertoli cell. These apical extensions became shorter at stage VII, and at stages VIII-X IFs were observed only in the perinuclear region. Short apical extensions reappeared at stages XI-XII; and at stages XIII-XIV, they extended again into the apical region. During this cycle, IFs were always closely associated with the heads of elongate spermatids. IFs were also shown to be in close apposition to some specialized structures on the cell membrane, such as the ectoplasmic specialization between adjacent Sertoli cells. During postnatal (p.n.) development, IFs were mainly observed at the basal nuclear region on p.n. day 7. The IFs in the supranuclear or apical regions first appeared at p.n. day 14 and gradually increased during the development. The perinuclear IFs network was fully established by p.n. day 28 and the adult distribution pattern of the IFs was established by p.n. day 42. CONCLUSIONS: Vimentin-type IFs in rat Sertoli cells are a delicate endocellular network, which is centered in the perinuclear region and extends to the apical region of the cell. During the seminiferous epithelium cycle, the distribution of IFs changes in a stage-dependent manner and is closely related to the location of the heads of elongate spermatids. During postnatal development, IFs gradually increase in numbers and the main distribution area is transferred from the basal nuclear to the perinuclear and supranuclear regions.

Accelerated stability studies on 16-methylene-17 alpha-acetoxy-19-nor-pregn- 4-ene-3,20-dione (Nestorone).

The stability of the contraceptive steroid, Nestorone (16-methylene-17 alpha-acetoxy-19-nor-pregn-4-ene-3,20-dione) in the solid state and in aqueous solutions, was investigated using reverse-phase high-performance liquid chromatography. In the solid state, whether as a powder or when it is incorporated into Silastic implants, the steroid does not undergo detectable degradation even under severe experimental conditions. In solution, the drug undergoes slow degradation that is dependent on temperature and pH of the medium. The decomposition is defined by first-order mechanism. As expected, the reaction rate increases with increasing storage temperature. The linearity of the Arrhenius plot indicates that there is no change in the reaction mechanism within the temperature range studied. In alkaline media, the drug degrades at a faster rate through hydrolytic rather than an oxidative mechanism. The major hydrolytic degradation product, 16-methylene-17 alpha-hydroxy-19-nor-pregn-4-ene-3,20-dione, was separated and identified by mass spectrometry.

Clinical trial with Nestorone subdermal contraceptive implants.

The clinical performance and the in vivo release rate of a single 4-cm Nestorone subdermal implant were investigated. Implants manufactured by two different procedures were compared. Volunteers were 70 healthy women of proven fertility. Forty women provided blood samples twice a week in the pretreatment cycle and for 5-6 weeks at 6-month intervals during treatment. Additional control cycles (n = 31) were studied in 19 Copper T users. No pregnancy occurred in 1570 woman-months. Nestorone plasma levels (x +/- S.E.) declined from 112 +/- 8 to 86 +/- 3 pmol/L (Implant A) and from 145 +/- 8 to 57 +/- 5 pmol/L (Implant B) from the first to the 24th month. Progesterone levels were < 9.5 nmol/L in 166 (93%) of 178 blood samplings taken during treatment. Progesterone levels > 16 nmol/L were found in only 7 sampling periods (3.9%) in treated women and in 70 (98.6%) out of 71 control cycles. No ovulation occurred with Nestorone plasma levels above 105 pmol/L. No abnormal changes were observed in plasma lipoproteins or other clinical chemistry parameters during treatment. The implants were well tolerated. The most frequent complaint was the occurrence of irregular bleeding. Enlarged follicles found during pelvic examination in 8 subjects (11.4%) disappeared spontaneously in 10 days to 6 weeks. Implants were removed because of medical (n = 10, 14.3%) or personal reasons (n = 6, 8.6%) or at the 24th month of treatment (n = 54, 77.1%). The estimated average daily in vivo release rate of Nestorone was 45-50 micrograms/day. A single Nestorone subdermal implant affords efficient contraceptive protection during two years.

Clinical trial with 3-keto-desogestrel subdermal implants.

The study was done to assess the clinical performance and in vivo steroid release rate of 3-keto-desogestrel subdermal implants designed to deliver 5 different doses of the progestin. Volunteers were healthy women of proven fertility who provided blood samples at scheduled intervals during treatment. No pregnancy occurred in 514 woman-months in users of implants delivering 30 and 40 micrograms per day of 3-keto-desogestrel. Three pregnancies, one ectopic, were observed in 109 woman-months recorded with implants delivering 20 micrograms per day or less. Ovulation was inhibited, as judged by depressed progesterone levels, in 57 of 59 (97%) blood samplings in women whose 3-keto-desogestrel plasma levels were greater than 0.28 nmol/L and in 39 of 75 (52%) of cases with lower levels. Users of 4 cm implants manufactured by The Population Council, New York, showed mean levels above 0.28 nmol/L until 18 months of use. Levels achieved with 4.4 cm implants manufactured by Organon, Oss, Holland, were less consistent. No changes were observed in the plasma lipoprotein pattern or clinical chemistry during treatment. The main complaint was the occurrence of bleeding irregularities, particularly with the lower doses. Ovarian cysts found during pelvic examination in 11 (22%) subjects disappeared spontaneously within 7-90 days. 3-keto-desogestrel implants releasing around 40 ug/day and providing plasma levels around 0.28 nmol/L afford efficient contraceptive protection.

Influence of a levonorgestrel-containing contraceptive vaginal ring on plasma lipids and lipoproteins in cynomolgus monkeys.

The effect of a contraceptive vaginal ring (CVR) containing levonorgestrel on plasma lipid and lipoprotein concentrations and characteristics was assessed in ten cynomolgus monkeys. The animals were fed a diet similar to the average American diet in fat (40% of calories) and cholesterol (0.2 mg/kcal) content. The objective of this study was to determine if changes in lipids and lipoproteins caused by progestogen administration parallel those seen in human females. A parallel pattern would recommend the cynomolgus monkey as a model for studying the effects of progestogens on the atherosclerotic process. Treatment with the CVR resulted in significant decreases in total plasma, VLDL + ILDL + LDL, and HDL cholesterol concentrations and a decrease in the percentage of HDL2 in total HDL. Plasma triglyceride concentrations were low throughout the study and consistent effects of the CVR were not seen. CVR treatment resulted in increases in TPC:HDL-C ratios and in the flotation rate of the LDL particle. The patterns of effects on HDL cholesterol, total plasma cholesterol, and HDL2 concentrations were similar to the progestogen-induced changes observed in human plasma lipids and lipoproteins. Based on these effects, the cynomolgus monkey appears to be a suitable model for the study of progestogen-induced changes in plasma lipids and lipoproteins and their consequent influences on coronary artery atherosclerosis.

Subdermal norethindrone pellets -- a method for contraception?

The mode of action of compressed pellets containing 85 per cent norethindrone (NET) and 15 per cent cholesterol was studied. Four pellets were inserted subcutaneously, in each of five healthy volunteers and left in place for 200--229 days. The NET content of the pellets varied between 23.9 mg and 25.6 mg; and the cholesterol content between 4.2 mg and 4.5 mg. Plasma levels of NET, estradiol and progesterone were determined by radioimmunoassays. Plasma levels of NET varied mostly between 1--2 ng/ml the first month after insertion. After two months plasma levels of NET ranged between 0.5 ng/ml and 1 ng/ml in all volunteers and there was a gradual decrease of the plasma NET levels throughout treatment. Pronounced day-to-day variations in plasma NET levels were recorded. The release rates of NET was calculated to be between 187 micrograms/day and 243 micrograms/day among the five volunteers. Ovulations occurred in four out of five subjects during treatment. This study indicates that the release of gestagen from four NET pellets was only initially high enough to completely inhibit ovulation and that to accomplish full contraceptive efficacy, a higher dose, i.e. more pellets, would have to be inserted.

PIP: Pellets containing norethindrone (NET)-cholesterol (85%:15%, weight to weight) used for long-term subdermal implant for contraception were studied to elucidate their mode of action. Plasma levels of NET, estradiol, and progesterone were measured by radioimmunoassay; bleeding pattern was recorded; and daily release of NET was calculated after pellet removal from 5 healthy volunteers, each of whom had 4 subdermal pellets implanted for 200-229 days. NET content of pellets varied from 23.9 mg-25.6 mg; cholesterol content varied from 4.2-4.5 mg. Plasma levels of NET varied mostly between 1 and 2 ng/ml the 1st month after insertion. After 2 months, plasma levels of NET ranged from .5-1 ng/ml in all volunteers; there was a gradual decrease of the plasma NET levels throughout treatment. Pronounced day-to-day variations in plasma NET levels were recorded. Release rates of NET were calculated as between 187 and 243 mcg/day among the 5 volunteers. Ovulation occurred in 4/5 subjects, as determiend by hormonal profiles, during treatment. Overall, the release of NET was only high enough during the initial phase to completely inhibit ovulation, and therefore, to accomplish full contraceptive efficacy, a higher dose, i.e., more pellets, must be inserted subdermally.

Copper levels in maternal and fetal tissues of rabbits bearing intrauterine copper wires.

PIP: A study was undertaken to determine if copper wire, placed within the uterine lumen after implantation and kept in situ throughout pregnancy, results in an excessive accumulation of the metal in maternal and/or fetal tissues; and if so, what effect if any, such an accumulation may have on both mother and offspring. Copper wires were inserted into the uterine cavities, near the utero-tubal junction, of rabbits on Day 7 of pregnancy and were allowed to remain in situ until Day 28, at which time the animals were sacrificed. Tissues from mother and offspring were subjected to routine histological examinations and their copper content was determined. A parallel series of sham-operated animals served as the control. Copper was released from the intrauterine copper wires at an average daily rate of 38.81 plus or minus 7.26 mcg. Histological examination of maternal adrenal, brain, heart, kidney, liver, lung, ovary, spleen, and uterus revealed no anatomical abnormalities. There was no increase in copper in any of the tissues except the uteri and placentae of animals bearing intrauterine copper wires. There were no anatomical abnormalities in either fetal brain, heart, kidney, liver, or lung. There was an increase in the amount of copper found in the liver of fetuses obtained from animals bearing intrauterine copper wires. As there were no histological differences between livers of the control and experimental groups, and no evidence of teratological effects due to copper, it is assumed that this small but significant increase in fetal liver copper is insufficient to produce any toxic effects on the offspring.^ieng