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Objective: An effective vaginal microbicide against sexual HIV transmission remains elusive, with requirements for adherence to appropriate application of effective, nontoxic products being a major deterrent. We explored methods to enable sustained release of combinations of antiretroviral microbicides, utilizing intravaginal rings composed of biosoluble Acacia gum or nonbiodegradable hydrogel of 2-hydroxyethyl methacrylate and sodium methacrylate, materials approved for use by the US Food and Drug Administration., Design and Methods: The reverse transcriptase inhibitors TMC120, PMPA, 3'-azido-3'-deoxythymidine, and a newly characterized anti-HIV agent, Boc-lysinated betulonic acid, were incorporated into vaginal rings with different combinations. Daily and cumulative release rates of these inhibitors in ring eluates were determined by high-performance liquid chromatography, gas chromatography, or immunoassay. Anti-HIV effects were measured by assessment of p24 Gag antigen in T-cell cultures exposed to HIV-1 isolates., Results: Drug release rates were sustained at concentrations higher than the minimum effective dose for HIV inhibition. The release was maintained for no less than 15 and 28 days from the Acacia gum and 2-hydroxyethyl methacrylate and sodium methacrylate rings, respectively. Boc-lysinated betulonic acid showed more than 90% inhibition of HIV-1 infection in H9 cells, with little toxicity to normal cells., Conclusion: The intravaginal rings described here are capable of efficacious drug delivery. Incorporation of several antiretroviral agents, including betulinol derivatives, which act at multiple levels of the HIV life cycle, may provide a synergistic effect to achieve higher efficacy on the inhibition of HIV infection.

Objectives: Construction of vaginal rings to deliver nonhormonal contraceptives and 3'-azido-3'-deoxythymidine (AZT) as an anti-HIV agent and determination of their daily release and efficacy in vitro., Materials and Methods: Intravaginal rings of 0.5-0.7 cm rim and 5-5.5 cm in diameter were cast in the molds. The rings were composed of biosoluble acacia gum or nonbiodegradable hydrogel of 2-hydroxyethyl methacrylate (HEMA) and sodium methacrylate (SMA) [P(HEMA-co-SMA)]. The rings were impregnated with nonhormonal contraceptives such as ferrous gluconate to cause spermiostasis, l-ascorbic acid to increase the viscosity of the cervical mucus, and pharmalytes of pH 4-5 or AZT., Results: The daily release rate of nonhormonal contraceptives as well as AZT from the rings was efficacious in vitro. Cumulative effect of nonhormonal contraceptives showed complete spermiostasis within 30 s up to 10 and 28 days, respectively. Daily release of AZT from both rings was also likely to be efficacious to inhibit HIV proliferation in vitro for 10 and 28 days, respectively., Conclusion: The data indicate that the rings described here can be developed for intravaginal delivery of nonhormonal contraceptives and/or anti-HIV agents.

Betulonic acid, derived from betulinol, a pentacyclic styrene, has shown a highly specific anti-prostate cancer activity in in vitro cell cultures. However, due to the lack of solubility of betulonic acid in aqueous medium, its potent anti-cancer activity in vivo has not been determined to the fullest extent. The present study describes the chemical synthesis of hydrophilic Boc-lysinated-betulonic acid, which has improved its solubility in an aqueous biocompatible solvent. Evaluation in cytotoxicity assays, Boc-lysinated-betulonic acid dissolved in phosphate-buffered saline (PBS) containing 22% ethanol and 4% human serum albumin, has shown 95.7% inhibition of LNCaP prostate cancer cells in culture after 72 h incubation at a concentration of 100 microM, but with little effect on normally proliferating fibroblast cells. In the in vivo assay, male athymic mice transplanted with human prostate LNCaP xenografts were injected with Boc-lysinated-betulonic acid intraperitoneally at a dose of 30 mg/kg daily for 17 days. The treated mice exhibited 92% inhibition of tumor growth as compared to controls. Histological sections of the tumors showed that Boc-lysinated-betulonic acid arrested mitosis and induced apoptosis, which was confirmed by TUNEL assay, Yo-Pro-1 staining, and the release of cleaved caspase-3 from the ex vivo in tumor culture. These studies, for the first time, demonstrate that a non-toxic hydrophilic lysinated derivative of betulonic acid and its solubility in a biocompatible aqueous medium has enhanced the bioavailability of the drug and has thus unleashed its full anti-prostate cancer activity.

This investigation describes the synthesis of a biodegradable hydrogel composed of a core surrounded by four concentric sheaths containing dextran, copolymers of polylactide and epsilon-caprolactone. The hydrogel was impregnated with iron (II) d-gluconate dihydrate, which causes complete spermiostasis due to lipid peroxidation, ascorbic acid to increase the viscosity of the cervical mucus and mixtures of polyamino and polycarboxylic acids to sustain vaginal pH close to 4.5. The combined effects of the agents in the daily eluates of the hydrogel were efficacious up to 16 days, within 30 s, as shown by sperm penetration tests. For in vivo studies, rabbits were chosen as the experimental model because they are easy to handle and the female is always in estrus. The anterior vagina of estrous female rabbits was instilled with the hydrogel, and then inseminated with the semen from a fertile male. Postinsemination flush from the female rabbits showed that all of the sperm were dead. These observations demonstrate the potential for the development of a biocompatible, nonhormonal, intravaginal contraceptive device.

Objective: To determine the effect of immunization with bovine luteinizing hormone receptor (LH-R) on ovarian function of cats., Animals: 9 adult female domestic cats., Procedure: 7 cats were immunized with 0.5 mg of LH-R encapsulated in a silastic subdermal implant (3 x 10 mm); 2 served as control cats. Receptors had 80% specific binding to 125I-human chorionic gonadotropin with a binding capacity of 2,682 pM/mg. Cats received booster injections of LH-R. Cats were induced to ovulate with luteinizing hormone (LH) releasing hormone on day 345. Samples of venous blood and vaginal cells were collected through day 395. Observation of estrus behavior continued until day 516. Serum concentrations of estradiol, progesterone, thyroid gland hormones, LH, and LH-R antibody were determined., Results: LH-R antibody was detected in the sera of immunized cats within 21 days after implantation. Detection of LH-R antibody was associated with suppression of serum progesterone to < or = 0.5 ng/mL during the study period, compared with concentrations of 5 to 10 ng/mL in control cats. Immunized cats did not display signs of estrus. Release of LH after administration of LH-releasing hormone indicated an intact hypothalamic-pituitary axis but poor corpus luteum function. Serum estradiol concentrations remained between 30 to 40 pg/mL in immunized and control cats. With the decrease antibody titers, hormone concentrations returned to a pattern consistent with that during fertility., Conclusions and Clinical Relevance: Active immunization with LH-R suppressed corpus luteum function in cats. The effect was reversible. An LH-R-based antifertility vaccine may have clinical application in other vertebrates.

Context: Noninvasive magnetotherapeutic approaches to bone healing have been successful in past clinical studies., Objective: To determine the effectiveness of low-amplitude, extremely low frequency magnetic fields on patients with knee pain due to osteoarthritis., Design: Placebo-controlled, randomized, double-blind clinical study., Setting: 4 outpatient clinics., Participants: 176 patients were randomly assigned to 1 of 2 groups, the placebo group (magnet off) or the active group (magnet on)., Intervention: 6-minute exposure to each magnetic field signal using 8 exposure sessions for each treatment session, the number of treatment sessions totaling 8 during a 2-week period, yielded patients being exposed to uniform magnetic fields for 48 minutes per treatment session 8 times in 2 weeks. The magnetic fields used in this study were generated by a Jacobson Resonator, which consists of two 18-inch diameter (46-cm diameter) coils connected in series, in turn connected to a function generator via an attenuator to obtain the specific amplitude and frequency. The range of magnetic field amplitudes used was from 2.74 x 10(-7) to 3.4 x 10(-8) G, with corresponding frequencies of 7.7 to 0.976 Hz., Outcome Measures: Each subject rated his or her pain level from 1 (minimal) to 10 (maximal) before and after each treatment and 2 weeks after treatment. Subjects also recorded their pain intensity in a diary while outside the treatment environment for 2 weeks after the last treatment session (session 8) twice daily: upon awakening (within 15 minutes) and upon retiring (just before going to bed at night)., Results: Reduction in pain after a treatment session was significantly (P < .001) greater in the magnet-on group (46%) compared to the magnet-off group (8%)., Conclusion: Low-amplitude, extremely low frequency magnetic fields are safe and effective for treating patients with chronic knee pain due to osteoarthritis.

We describe a patient with onset of puberty at the age of 5 yr. characterized by accelerated growth, enlargement of genitalia, pubarche, and serum hormone levels compatible with noncentral precocious puberty. Exon 11 of the LH receptor gene was amplified from genomic DNA by PCR and directly sequenced. We identified a heterozygous C to T base change at nucleotide position 1126, exchanging codon 373 from Ala to Val in the first transmembrane domain. The LH receptor sequence of the parents was normal. The mutated receptor displayed an up to 7.5-fold increase in basal cAMP production compared to that of the wild-type receptor in transiently transfected COS-7 cells. Treatment of the patient with ketoconazole resulted in inconsistent suppression of serum testosterone levels. At the age of 9.1 yr, central activation of the hypothalamic-pituitary-gonadal axis occurred. Additional treatment with a GnRH agonist led to complete suppression of testosterone secretion. This is the first description of constitutive activation of the LH receptor in the first transmembrane segment. It suggests the involvement of the first transmembrane helix in signal transduction and provides further insight into the structural organization of the seven transmembrane domains of the glycoprotein hormone receptor proteins.

Safety, contraceptive efficacy and biodegradability of subdermal norethindrone (NET)-cholesterol implants (Anuelle) were investigated in 19 healthy, sexually active women. Mean serum NET levels in women with four and five pellets, containing 174 mg and 266.5 mg NET, showed a "burst-effect" of 3.17 and 3.71 ng/ml, respectively, within 24 h post implantation. Subsequently, for the four- and five-pellet groups, respectively, the levels declined from 0.75 to 0.40 ng/ml and 1.05 to 0.61 ng/ml during the months 12-15, from 0.40 to 0.11 ng/ml and 0.61 to 0.25 ng/ml up to month 36. The serum NET levels were undetectable at 36 months and beyond, indicating complete biodegradability of NET pellets. Serum E2 levels remained within normal limits (> 50 pg/ml), whereas serum P indicated anovulatory cycles in 78% of the four-pellet group and 99% of the five-pellet group. FSH and LH levels were subnormal and acyclic. Plasma lipids showed reduced total and LDL cholesterol and triglycerides. HDL cholesterol remained unchanged. Drug-related adverse effects were essentially limited to irregular bleeding. There were no pregnancies in either group.

Objective: To evaluate the contraceptive efficacy, pharmacokinetics, and safety of two formulations of Annuelle (Endocon, Inc., South Walpole, MA) biodegradable norethindrone (NET) SC pellet implants., Design: Prospective observational study., Setting: Two clinical sites in the United States., Patient(s): Thirty-nine healthy, fertile, sexually active women., Intervention(s): Nineteen women received a four-pellet system containing 174 mg NET; 20 women received a five-pellet system containing 266.5 mg NET., Main Outcome Measure(s): Contraceptive efficacy, median serum NET levels, adverse events., Result(s): No pregnancies were observed in 293 woman-months in the four-pellet group or in 375 woman-months in the five-pellet group. An initial burst in median serum NET levels occurred in the first 24 hours postinsertion followed by a steady decline over the next 3 years. Norethindrone levels varied considerably among women. The main side effect was bleeding abnormalities, which persisted in half the participants for up to 2 years. No serious adverse events were reported that were related to the pellets. Pellet insertion and removal generally were uncomplicated., Conclusion(s): Annuelle shows potential as an effective, safe contraceptive with distinct advantages over other long-acting agents, because it is biodegradable but can be removed if problems arise or if fertility is desired.

Objective: The primary objective of this study was to compare the safety, contraceptive efficacy, and menstrual cycle patterns in women using triphasic oral contraceptive pills, namely CTR-05, containing 50/100/150 micrograms desogestrel and 35/30/30 micrograms ethinylestradiol, and Orthonovum777 containing 500/750/1000 micrograms norethindrone and 35/35/35 micrograms ethinylestradiol., Method: Forty-six female volunteers, satisfying the selection criteria, were evaluated for six cycles, in an open-label, randomized study. Volunteers using CTR-05 were studied for 13 additional cycles for efficacy and safety., Results: No serious adverse effects were observed in either group. The incidences of other drug-related adverse effects, such as headache and nausea, were transient in both groups. CTR-05 did not lower levels of high density lipoprotein (HDL) cholesterol. This may be attributed to the lower androgenicity of its progestin component, desogestrel. No pregnancies were reported in either group. Clinical and laboratory parameters remained within normal limits in both groups. In the CTR-05 group, the lower dose of ethinylestradiol did not affect the safety, efficacy and acceptability of the product., Conclusion: Desogestrel, with little estrogenic activity and only minimal androgenic activity, leads to lipoprotein changes, resulting in a favorable cardiovascular profile, as well as minimal androgen-related effects, such as hirsutism and acne.

A 7000 g supernatant, obtained during the purification of luteinizing hormone (LH) receptor from bovine corpora lutea homogenate, was concentrated by ultrafiltration. The filtrate, containing < 50,000 molecular weight material, exhibited LH receptor binding inhibitor (LH-RBI) activity. The filtrate was ultrafiltered sequentially through Amicon PM-10, PM-30 and UM-2 filters to yield a LH-RBI-containing fraction in the higher molecular weight range of 30,000-10,000 and a LH-RBI-containing fraction in the lower molecular weight range of 10,000-1000. The higher molecular weight LH-RBI fraction was purified on Sephadex G-25 and the lower molecular weight LH-RBI fraction was purified on Sephadex G-50. Both the high- and the low-molecular-weight LH-RBI species inhibited the binding of 125I-labeled human chorionic gonadotropin (hCG) to bovine corpora lutea and to rat Leydig cell membrane receptors. Similarly, the production of testosterone by hCG-stimulated rat Leydig cells was inhibited in a dose-response manner by both the high- and the low-molecular-weight LH-RBI species. The LH-RBI activity in the low-molecular-weight species was stable at 4 degrees C for up to 6 months and at temperatures up to 90 degrees C for 15 mins, whereas the LH-RBI activity of the high-molecular-weight species was stable at 4 degrees C for 15 months and unstable at 60 degrees C after 15 min. The 7000 g supernatant provided a much-needed source to obtain larger than previously reported quantities of LH-RBI for isolation as well as for structure and function studies.

Three New Zealand white adult female rabbits, designated as A, B, and C, were immunized to produce lutropin receptor antibodies. Antisera inhibited the binding of 125I-hCG to the lutropin receptor and the production of testosterone by hCG stimulated rat Leydig cells. During the study period of, approximately 10 months after the last immunization, rabbits did not ovulate in response to an injection of 75 IU of human chorionic gonadotropin or mating as revealed by laparotomy. As the antibody titers declined, induction of ovulation and laparotomy revealed restoration of ovulation and corpus luteum formation. However, no pregnancy occurred when rabbits A and B were mated and artificially inseminated. These observations indicate that lutropin receptor antibodies can cause infertility in female rabbits.

Monoclonal antibodies (Mabs) were produced against bovine Lutropin receptor (LH-R). Antibodies were detected by an enzyme linked immunosorbant assay (ELISA). Hybridomas were subcloned to achieve monoclonality. Ascites were developed in Balb/c mice. Hybridoma supernatants were purified by ammonium sulfate precipitation and chromatography on hydroxylapatite columns. LH-R antibodies showed upto 50% inhibition of 125I-labeled hCG binding to bovine luteal cell membranes and up to 80% inhibition of testosterone production by hCG stimulated mouse Leydig cells. LH-R antibodies were predominantly IgM isotype. Purified antibodies showed a 78-kDa band, in SDS-PAGE, as the heavy chain of the immunoglobulin. LH-R antibodies were localized specifically in the thecal and luteal cells of the rat ovaries as well as in the Leydig cell of mouse testes. Injections of the LH-R antibody caused a constant estrus in normal rats. One month after the cessation of the injections the animals returned to normal estrus cycle and fertility. Pregnant mice injected with LH-R antibodies produced only 3 viable pregnancies and 10 pups, as compared to 8 pregnancies and 45 pups born to normal controls. LH-R antibodies also caused, approximately, a 50% reduction in testosterone production in normal male rats. These observations indicate a high degree of specificity of the Mab to LH-R and their potential in studies on gonadal function.

Four baboons (Papio anubis) were actively immunized with bovine LH receptor for periods of 6-22 months. Serum antibody levels were measured by an enzyme immunoabsorbant assay (ELISA). Antibodies against the receptor were detected 2 weeks after the first injection. Antisera caused an inhibition in the binding of human chorionic gonadotropin (hCG) to its receptor as well as inhibited the production of hCG induced testosterone by rat Leydig cells in culture. Serum estradiol and progesterone levels were determined by radioimmunoassay (RIA). Progesterone levels were suppressed during the post immunization period. Two baboons experienced periods of anovulation. Serum estradiol levels were cyclic and appeared elevated. Baboons were mated with males of proven fertility; none of the immunized females conceived over eight cycles of observation. Fertility parameters returned to normal, when antibodies against LH-receptor were undetectable in the serum.

Follicle-stimulating hormone of a high state of physicochemical and biological purity was isolated from acetone-preserved human pituitary glands. The follicle-stimulating hormone was dissociated into alpha and beta subunits by treatment with 8 M urea and the subunits were separated by ion exchange chromatography on DEAE-Sephadex A-25. The subunits were freed of undissociated or reassociated follicle-stimulating hormone by gel filtration on Sephadex G-100. For the establishment of the primary amino acid sequence, the alpha subunit was reduced and either carboxyamidomethylated or S-aminoethylated prior to a thermolytic or a tryptic digestion. Each digest was gel filtered on a column of Sephadex G-50 to separate the glycopeptides from the peptides. The glycopeptides and the peptides were purified further by sequential gel filtration on Sephadex G-25, G-15, and Bio-Gel-P-2 and were isolated by high voltage electrophoresis at pH 6, 3.5, and 2. The purity of the isolated peptides was ascertained further by amino acid analysis. The amino acid sequences of the peptides were determined by Edman degradation followed by subtractive amino acid analysis. COOH-terminal sequences were established by digestion with carboxypeptidases A and B. The primary amino acid sequence of human follicle-stimulating hormone-alpha is identical to that of human chorionic gonadotropin-alpha and differs from that of human luteinizing hormone-alpha in having the tripeptide Ala-Pro-Asx- at the NH2-terminal end.

An aqueous solution of 66 per cent ethanol and 6.6 per cent ammonium acetate has been used in the development of a relatively simpler and rapid as well as highly reproducible method of separation of antibody-bound and free hormones in the radioimmunoassay of human growth hormone (HGH). The present assay is sensitive to detect 20 pg. of hormone with a precision of plus or minus 5 per cent, hence, as little as 20 mul of plasma is required to perform the radioimmunoassay. Serum levels of HGH have been found to be approximately 70 per cent of the plasma concentrations in the same subjects.

Human follicle-stimulating hormone (FSH) was digested with subtilisin, thermolysin, cyanogen gromide, pronase and trypsin to isolate the cystine-containing peptides. These peptides were purified by gel filtration through Sephadex G-50 column and by high-voltage paper electrophoresis at pH 6, 3.5 and/or 2. The location of the cystine-containing peptides in human FSH alpha- and beta-subunits was established by amino acid composition, end-group analysis and determination of the amino acid sequence by Edman degradation. The results indicate that the disulfide bonds are present between half-cystine residues located between positions 7 and 10, 28 and 87 and 82 and 84 in the alpha-subunit, and between positions 3 and 28, 17 and 51 and 32 and 104 in the beta-subunit of human FSH.

One thousand radioreceptorassays for human chorionic gonadotropin (hCG) for the diagnosis of pregnancy were analyzed under eight categories. The high sensitivity (50 pg/ml) and specificity, coupled with performance time of the assay (1 hour), provided a new modality for the early diagnosis and management of normal and abnormal pregnancy in the first trimester. The radioreceptorassay becomes positive 1 week after conception. In the normal pregnancy this assay has been 100% correct at the time of the first missed menses. All ectopic pregnancies demonstrated positive tests, and no false negatives occurred. On the other hand, a negative assay excluded ectopic pregnancy.

Based on the regeneration of the hormonal activity following recombination, the alpha and beta subunits of human follicle-stimulating hormone have been designated as 'functional' or 'nonfunctional'. Chemical modifications of the tryptophan, methionine, tyrosine and arginine residues of human follicle-stimulating hormone, luteinizing hormone, and the 'functional' human follicle-stimulating hormone alpha and beta subunits have indicated that the tryptophan in human follicle-stimulating hormone-beta and human luteinizing hormone-beta is essential for the biological activity. The iodination of human follicle-stimulating hormone-alpha did not interfere with the hormonal activity. The modification of arginine abolishes the biological activity of the hormones. The accessibility of tyrosine and methionine in human follicle-stimulating hormone-alpha, of arginine in both native hormones and subunits, and the non-availability of the tryptophan residues to 2-hydroxy 5-nitrobenzyl bromide suggest that the alpha subunit lies on the surface of the native molecule.

A rapid, sensitive, and specific radioreceptor assay for the determination of human chorionic gonadotropin and luteinizing hormone in plasma is described. Plasma membranes of bovine corpora lutea of early pregnancy, which bind biologically active labeled human chorionic gonadotropin, have been used as receptor. Pregnancy could be detected by assaying the gonadotropin in plasma samples obtained from day 6 to 8 after conception.

Plasma follicle stimulating hormone (FSH) and luteinizing hormone (LH), prolactin (PRL), estradiol (E2), progesterone (P), testosterone (T), and dihydrotestosterone (DHT) were measured by radioimmunoassay in 7 patients who had ovulation induction with clomiphene citrate and human chorionic gonadotropin. Six of the 7 patients ovulated. There was higher increase in FSH than LH in 3 patients who also showed a midcycle FSH and LH peak. Plasma PRL was initially elevated in 2 patients, remained normal (less than 25 ng/ml) throughout the cycle in one patient, and showed an increase in the late luteal phase in 3 patients. Plasma E2 showed a good response at the end of clomiphene intake, with a subsequent preovulatory surge. Plasma P increased to more than 25 ng/ml in patients who ovulated and reached higher levels than in normal ovulatory cycles. Plasma T increased and decreased in a similar pattern to plasma E2 and was higher with clomiphene therapy than in the normal menstrual cycle. Plasma DHT remained similar to concentrations found in normal menstrual cycles. The findings are discussed in relation to the effect of clomiphene and these hormones on the pituitary-ovarian axis, ovulation rate, and conception rate.

An improved method is described for the purification of prolactin from the same batch of acetone-dried pituitary glands which is used for the isolation of other anterior pituitary hormones. The residue obtained after the extraction of the glycoprotein fraction from the pituitary acetone powder was extracted for growth hormone. The supernatant after the precipitation of growth hormone at 50% ethanol concentration was adjusted to 85% in ethanol. To the supernatant obtained from that step an equal volume of acetone was added to precipitate the 'PRL fraction.' This fraction was further purified by gel filtration on Sephadex G-100, ion-exchange chromatography on DEAE-cellulose and isoelectric focussing. A yield of 23 mg prolactin, 37 U/mg, was obtained per 1000 g pituitary acetone powder.

A batch of 24 mg of luteinizing hormone-human chorionic gonadotropin (LH-hCG) receptor was isolated from bovine corpora lutea. The LH-hCG receptor showed specific binding with hCG. The receptor-hCG complex activated the regulatory Ns protein isolated from rabbit liver, which in turn stimulated adenylate cyclase to convert ATP into cAMP in vitro, attesting to the biological activity of the purified LH-hCG receptor. The LH-hCG receptor was treated with 2% sodium dodecyl sulfate (SDS) to prepare the molecular weight (Mr) 280K dimer and with 50 mM dithiothreitol (DTT) to prepare the Mr 120K monomer and subunits of Mr 85K and 38K. Oligomers of various molecular weights were recovered from gel filtration columns due to the reassociation of disulfide bonds between monomers and subunits. Hence, the receptor monomer was also dissociated into subunits of Mr 85K and 38K by reduction of -S-S-bonds with 50 mM DTT in 2% SDS and alkylation of sulfhydryl groups in the presence of 100 mM N-ethyl-maleimide. The subunits were separated by gel filtration through columns of Ultrogel AcA-44 and Sephadex G-75. The yields of the purified alkylated subunits of Mr 85K and 38K were 1.8 and 1.5 mg, respectively. Each subunit migrated as a single entity in SDS-polyacrylamide gel electrophoresis. The monomer of the receptor of Mr 120K showed specific binding with 125I-hCG, suggesting it to be the minimum molecular weight functional unit of the receptor. The Mr 85K and 38K subunits bound 125I-hCG, which could not be displaced with unlabeled hCG.(ABSTRACT TRUNCATED AT 250 WORDS)

A tentative amino acid sequence of the beta-subunit of equine follicle-stimulating hormone (FSH) was derived from the sequences of tryptic, thermolytic as well as peptic, subtilisin, and chymotryptic peptides. Equine FSH-beta is analogous to human FSH-beta except six amino acid substitutions at positions 12, 16, 21, 62, 108, and 114. The amino acid sequence suggests that the hormone-specific beta-subunits of FSH are similar at the interspecies level, whereas the amino acid sequences of the hormone nonspecific alpha-subunits show variations.

Twelve pregnancies, in which the initial serum hCG titer as measured by RRA was subnormal, were studied to determine if a hormonal profile would serve as a prognostic index of spontaneous abortion. Three of the 12 patients delivered term babies. The hCG titers remained subnormal throughout the pregnancy, however, the serum E2, P, 17alpha-OHP, and PRL remained normal. One of the 12 patients aborted in week 25 of gestation. The serum hCG and PRL were subnormal; however, the serum E2, P, and 17alpha-OHP were normal. Eight of the 12 patients aborted prior to 100 days' gestation. The serum hCG, E2, and PRL were subnormal whereas the P and 17alpha-OHP were normal. Therefore, in patients who have subnormal serum hCG titers, serum E2 and PRL may provide a prognostic index of spontaneous abortion.

The use of specific receptors has provided a novel method to prepare LH-free serum. LH-free serum was used in RRA to measure basal LH levels in serum of men and women during menstrual cycle as well as during post menopause.

The contraceptive efficacy of norethindrone (NET) fused pellets was evaluated over 12 months in a Phase II clinical study with three and four pellets, each pellet containing 35 mg of NET. Volunteers were healthy, fertile, sexually active women. The release rate of NET from three and four pellets, respectively, was 150.3 +/- 7.2 micrograms and 212.5 +/- 8.6 micrograms NET/day. Following the implantation of NET pellets, serum NET levels did not show any 'burst effect' and were sustained at levels between 0.4 and 0.6 ng NET/ml serum with three pellets and 0.6-0.7 ng NET/ml serum with four pellets. With three and four pellets, respectively, 40% and 27% of the women had normal menstrual cycles; 20% and 14% were amenorrheic; 27% and 37% had mid-menstrual spotting or bleeding; and 13% and 22% had prolonged episodes of bleeding. Cardiovascular, hepatic, and renal functions were normal throughout the study. Ovulation was inhibited in 85% and 92% of the cycles with three and four pellets, respectively. In women using three pellets, two pregnancies occurred, one at the 6th cycle and another at the 12th cycle. In women using four pellets, no pregnancies occurred during the entire period of study.

Maternal and umbillical arterial and venous plasma and amniotic fluid testosterone (T) and dihydrotestosterone (DHT) were measured by radioimmunoassay. Maternal plasma T was 690 +/- 80 pg. per milliliter (mean +/- S.E.) in early pregnancy (less than 20 weeks) and increased significantly (p = 0.0002) to 1,095 +/- 177 pg. per milliliter in late pregnancy (greater than 20 weeks). DHT was 113.0 +/- 18.8 pg. per milliliter in early pregnancy and 179.8 +/- 30.5 pg. per milliliter in late pregnancy. Both umbilical arterial (UA) and umbilical venous (UV) plasma T were significantly higher in 11 male infants (UA T = 135.6 +/- 16.5 pg. per milliliter; UV T = 227.5 +/- 40.8 pg. per milliliter) than in 12 female infants (UA T = 92.1 +/- 9.7 pg. per milliliter; UV T = 89.6 +/- 12.6 pg. per milliliter) (p = less than 0.05 and less than 0.005, respectively). UV DHT and UA DHT showed no significant difference between male and female neonates. In midtrimester pregnancy, amniotic fluid T (AFT) was 165.2 +/- 15.4 pg. per millitier in pregnancies with a male fetus and was significantly higher (p = less than 0.001) than in pregnancies with a female fetus (mean +/- S.E. = 27.6 +/- 2.6 pg. per millitier). In late pregnancy, AFT levels were similar to those of early pregnancy, but a considerable overlap in AFT between fetuses of both sexes was observed. DHT was not detectable in amniotic fluid. The results suggest the potential value of AFT for determining fetal sex in midtrimester pregnancy and confirm that maternal T and DHT increase during pregnancy and that cord T levels reflect fetal gonadal androgen synthesis.

Blastocyst fluid was aspirated from Day 6 1/2--7 rabbit blastocysts and was added to cultures of granulosa cells obtained from preovulatory follicles of untreated rhesus monkeys or from follicles of monkeys or from follicles of monkeys treated with PMSG. The stimulation of progesterone secretion was measured and equated with that produced by hCG. The hCG-like activity was also measured in a radioreceptor assay using 125I-labelled hCG and porcine granulosa cells. In 8 out of 10 experiments with cultured cells from untreated monkeys, addition of 20% blastocyst fluid from Days 6--9 of culture stimulated progesterone secretion by 2- to 6-fold. Similar findings were obtained in 5 experiments with cultures from PMSG-treated monkeys except that the blastocyst fluid was added from Days 0 to 6 of culture. The granulosa cells in such cultures underwent morphological luteinization. Compared to a standard of purified hCG the blastocyst fluid contained about 0.76--2.5 ng hCG-like activity/ml which was non-dialysable. The radioreceptor assay indicated the presence of 0.5--2.5 ng hCG-like material/ml.

This study was designed to test the feasibility of a patient-monitored glucose determination program to establish and maintain normal blood glucose levels. Ten pregnant women, who were insulin-dependent diabetics prior to becoming pregnant and who were in their eighth week or less of pregnancy, were offered the program. All 10 accepted and continued the program for the duration of their pregnancy. Normal plasma glucose (60 to 140; mean = 80 mg/dl) levels were achieved after one week of the program and were maintained throughout the pregnancy as documented by 5 to 8 blood glucose determinations a day. The hemoglobin A1c level, which was elevated in all 10 patients at the start (9.4 +/- 1.6 per cent) of the program, fell into the normal range (2 to 5.0 per cent) five weeks after glucose values became normal. Serum estradiol (0.8 +/- 0.6 ng/ml), serum prolactin (10 +/- 9 ng/ml) and serum human chorionic gonadotropin (5,500 +/- 1,700 ng/ml), although all abnormal at the start of the program, became normal after glucose control was achieved (program weeks 4, 5 and 6, respectively). The infants showed no signs of macrosomnia (2,988 +/- 959 g), hypoglycemia, hyperbilirubinemia, hypocalcemia, erythremia or respiratory distress. Therefore, a program to maintain normal blood glucose levels during a diabetic patient's pregnancy is not only possible but may also improve the pregnancy and the outcome.

A role of prolactin (PRL) in ovarian function has been suggested in several species, but not unequivocally established except in the rat. We, therefore, examined the presence of specific receptor for PRL in ovaries of rat, cow, and human. Human PRL (hPRL) labelled with 125I by the lactoperoxidase method was shown to be capable of specific binding to rat mammary tissue homogenate. Human, cow, and rat ovarian homogenates and/or partially purified plasma membranes were also shown to specifically bind 125I-hPRL. Binding was a saturable phenomenon and was dependent on receptor protein concentration. Optimal binding was observed at pH 7.0 and 37 degrees C. Binding was reversibly inhibited by exposure of membranes to pH 10.0 and irreversibly destroyed by exposure to pH 3.0. Bound 125I-hPRL was displaceable by unlabelled human, ovine, and bovine PRL but not by FSH or LH. However, human chorionic somatomammotrophin (hCS) and hGH showed some competition with 125I-hPRL. Number of binding sites/mg protein was lowest (0.8 x 10(-12)M) during metoestrus and increased during dioestrus (11 x 10(-12)M) reaching the maximum number at pro-oestrus (24.6 x 10(-12)M). These results demonstrate that presence of specific PRL receptor in the ovaries and are consistent with a role of PRL at the ovarian level.

In the receptor-immunoassay, the binding of anti-HCG-beta sera to 125I-HCG and 125I-HLH is measured in the presence of bovine corpus luteum "receptor". Anti-sera specific for HCG show little binding with 125I-HLH. The receptor-immunoassay permits an evaluation of specificity, affinity and the ability of anti-HCG-beta sera to compete with the "receptor" for binding to 125I-HCG.

Contraceptive efficacy of injectable norethindrone (NET) microspheres of 90 day duration was evaluated for 6 months: nine women received two injections of 65 mg each and eight received two injections of 100 mg each with 90 days between each injection. Average serum NET levels were 5 to 9 ng/mL on the day after injection and subsequently remained at 1 to 3 ng/mL. With 65 and 100 mg doses, respectively, 36% and 25% of cycles had normal bleeding patterns, 36% and 48% were amenorrheic, and the remaining 28% and 27% had prolonged bleeding. Also, 56% and 40% of cycles showed increased spotting for the 65 mg and 100 mg dose, respectively. There was no incidence of pregnancy or serious side effects. The mean serum NET concentration returned to RIA nonspecific basal levels 100 days after the second injection. Women returned to ovulatory cycles between days 100 to 115 after the second injection.

Receptors specific for hCG were found in human corpora lutea and follicles. hCG and LH were found to bind at a similar receptor site. The dissociation constant for hCG ranged from 10-minus 10 to 10-minus 11 mol/1 in human corpora lutea. The number of binding sites for 125-I-hCG ranged from 10-minus 14 to 10-minus 15 moles/mg protein in human corpora lutea. The binding of 125-I-hCG to ovary was found to vary at different stages of the menstrual cycle. The binding of 125-I-hCG to human ovaries increased on days 13-15 of the cycle, then declined slightly, and increased again on days 22-23. Following day 23, there was a slow decline until day 27 when binding activity could no longer be measured. No binding could be measured by the corpus luteum after the onset of menstruation or in corpora albicans.

The presence of a gonadotropin-like substance in preimplanted rabbit blastocyst fluid was determined bu radioreceptorassay of human chorionic gonadotropin (hCG)/luteinizing hormone (LH), using receptors prepared from bovine corpus luteum, rat testis, and rat ovaries. An average of 16.6 microliter of fluid containing 0.83 ng of luteotropic material was recovered from each blastocyst. An intense fluorescence was exhibited by the trophoblastic cells of the blastocysts treated with fluorescein-conjugated gamma-globulin isolated from antiserum raised against human chorionic gonadotropin. Serum concentrations of LH as well as estradiol, progesterone, and 17 alpha-hydroxyprogesterone up to day 6 after mating were determined in pregnant rabbits and compared with those in pseudopregnant and normal rabbits. In pregnant rabbits after mating, a surge of 62 +/- 15 ng of luteinizing hormone and 73 +/- 22 ng of progesterone/ml of serum occurred which returned to basal levels at 6 hours and on day 1, respectively. Secondary increases in serum luteinizing hormone of 26 +/- 12 to 36 +/- 16 ng/ml on days 3 and 5 and in serum progesterone of 16 +/- 4 and 14 +/- 5 ng/ml on days 5 and 6, respectively, were observed in pregnant rabbits.

Highly sensitive and specific radioreceptorassay and radioimmunoassay of human chorionic gonadtropin (hCG) have been used in the detection of hCG in random serum samples during the luteal phase of the menstrual cycles of 200 women and in daily serum samples obtained a few days prior to expected ovulation through the luteal phase in 3 women with regular bleeding patterns and using a copper intrauterine device (IUD). Twelve to nineteen per cent of IUD users showed hCG in serum during the luteal phase, indicating that the presence of the IUD, while permitting fertilization, probably causes interference through degeneration of the blastocyst and consequent lack of implantation.