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Preeclampsia, a multi-system hypertensive disorder, is associated with perturbations in the maternal cardiovascular system during early pregnancy. The corpus luteal hormone relaxin, a potent vasodilator, may contribute to physiological circulatory changes especially in early gestation when circulating levels are highest. This study investigated whether first trimester circulating relaxin may be a suitable biomarker for the early prediction of preeclampsia.Relaxin was initially measured in first-trimester samples of women who developed late-onset preeclamptic (LO-PE; delivery ≥ 34 weeks; n = 33) and uncomplicated pregnancies (n = 25) in Pittsburgh, USA. Subsequently, to expand the group numbers, relaxin was measured in women who developed LO-PE (n = 95), early-onset preeclamptic (EO-PE; delivery 34 weeks; n = 57), and uncomplicated pregnancies (n = 469) in Utrecht, the Netherlands.In the Pittsburgh subjects, low relaxin levels (lowest centile:p10) showed an adjusted odds ratio (OR) of 5.29 (95%CI 1.10-25.5) for LO-PE. In the Utrecht population, low relaxin levels (p10) demonstrated adjusted ORs of 1.45 (95%CI 0.54-3.90) and 2.03 (95%CI 1.06-3.88) for EO-PE and LO-PE respectively, the latter increasing to an adjusted OR of 3.18 (95%CI 1.41-7.20) when newborn weight was 10%. Serum relaxin concentrations slightly improved the detection rate of a previously derived prediction model for LO-PE from 42.5% to 45.1% at a fixed 10% false-positive rate.Relaxin shows little improvement in the performance of first trimester prediction models, which does not support its clinical implementation as a biomarker. Although this study was only correlational, the results point to a possible pathophysiologic role for low relaxin levels in pregnancies that later develop LO-PE.

Several different immunoassays have been used in the commercial pharmaceutical development of serelaxin. These assays have been well validated for submission of GLP preclinical and clinical studies to the FDA and EU regulatory bodies. The requirements for these assays exceed that of most research assays commonly developed in academic research but have been and are currently available to academic researchers. Additionally, many human relaxin immunoassays are commercially available from a variety of vendors. Validation procedures for immunoassays are well understood and documented, however validation of these assays is often lacking or completely absent. The data derived from these assays must be questioned if the investigator does not supply information on the validation of the assay used, either from the supplier or through their own efforts. Many recent papers on determination of serum relaxin in clinical settings have recently been published. The assay used for this determination varies but generally is one of two commercially available. These manuscripts and the assay used is discussed. Direct comparisons of assays are lacking but some general conclusions can be drawn by comparing results from similar studies using different assays. There is disagreement among the results of the concentrations of serum relaxin from the use of different assays that raise questions on assay reliability. The differences in the quality of immunoassays used for detection of serum relaxin should be part of the decisions making process in choosing an assay. While the end user bears the ultimate responsibility to demonstrate the assay is valid for the stated claims, reviewers and editors also share responsibility for quality of published results.

Introduction Moving teeth rapidly and avoiding posttreatment relapse are fundamental goals of orthodontic treatment. In-vitro and animal studies suggest that the human hormone relaxin might increase the rate of movement and the stability through its effect on the periodontal ligament. The purpose of this study was to compare relaxin and a placebo with regard to tooth movement and stability in human subjects. Methods A single-center, blinded, placebo-controlled, randomized clinical trial was used to examine the effect of relaxin on tooth movement and stability. Forty subjects were randomized 1:1 and received weekly injections of 50 μg of relaxin or a placebo for 8 weeks. Aligners programmed to move a target tooth 2 mm during treatment were dispensed at weeks 0, 2, 4, and 6. Movement was measured weekly on polyvinyl siloxane impressions that were scanned and digitized. The subjects were followed through week 12 to assess relapse. Results Tooth movement over the 8-week treatment period did not differ by treatment group ( P = 0.995). By using an intent-to-treat analysis, we found that the mean tooth movement for both groups was 0.83 mm (SE, 0.08 for relaxin and 0.09 for the placebo). Relapse from weeks 8 to 12 was the same in both groups ( P = 0.986), and the mean was −0.75 (SE, 0.07 for relaxin and 0.08 for theplacebo). Conclusions No differences in tooth movement over 8 weeks of treatment or relapse at 4 weeks posttreatment were detected when comparing subjects who received weekly injections of relaxin with those who received a placebo. In both groups, an average of less than half of the programmed tooth movement was obtained after 8 weeks of treatment. The local doses of relaxin might have been too low to affect tooth movement or short-term relapse.

Laboratory methods were adapted or developed to analyze approximately 70,000 daily urine samples collected during more than 2,500 menstrual cycles from 448 women working in the semiconductor industry. An immunoenzymometric assay (IEMA) for human chorionic gonadotropin (hCG) was employed for screening cycles in order to optimize laboratory resources and to reduce the number of samples requiring analysis by less efficient methods. The presence of hCG in urine was confirmed by the definitive immunoradiometric assay (IRMA). The screening assay eliminated 78% of cycles from further analysis because there was no evidence of conception. Thirty-eight of 448 cycles identified as having significant levels of hCG with the IEMA were confirmed as hCG positive with the IRMA. HCG-positive cycles were further evaluated by examination of daily diary data and by laboratory assays for ovarian and pituitary hormones. As a result of these evaluations, 17 of the 38 cycles identified by the IRMA as positive for hCG were found to be nonconceptive cycles. These results demonstrate the effectiveness of screening assays for hCG, as well as the importance of using multiple urinary biomarkers for the detection of early fetal loss with daily urine samples.

Objective To characterize the hourly profiles of hCG secretion in blood during conceptive cycles that ended in successful pregnancy. Design Prospective study. Setting University fertility clinic and research laboratories. Patient(s) Healthy spontaneously ovulating women with regular menses, no history of infertility, and either no male partner or an azospermic partner. Intervention(s) Frequent blood samples were collected daily from 11 spontaneously ovulating women during 11 cycles of artifical insemination with donor semen. The concentrations of hCG, LH, and FSH were measured in the blood by immunoassay. Main Outcome Measure(s) The concentration of hCG in the frequent blood samples and the rate that the concentration of hCG changed during the period of frequent sampling. Result(s) For the conceptive cycles resulting in successful pregnancies analyzed, hourly hCG concentrations were observed to increase in a consistent nonpulsatile manner. Conclusion(s) These data provide the first characterization of the hourly secretion profile of hCG in early pregnancy as well as provide further evidence that individual daily blood samples are sufficient for the accurate assessment of pregnancy.

BAS Medical is investigating the use of relaxin to improve ortho-dontic treatments. Relaxin is well known for its effects on remodeling soft tissue, and we believe relaxin will be clinically useful in speeding tooth movement and preventing relapse. We investigated the use of relaxin in preventing relapse in a dog model. Dog maxillary second incisors were orthodontically rotated an average of 42 degrees, and then relaxin was administered by gingival injection to relieve the rotational memory in the connective tissues. Teeth were retained for 30 days to allow fibers to reform. Teeth then were released and relapse was measured by a series of impressions. Animals receiving relaxin gingival injections (n = 8) were compared with placebo-treated animals (n = 8) (exhibiting high relapse) and gingival fiberotomies (n = 8) (low relapse). Gingival fiberotomy is a surgical procedure to cut the gingival connective tissues away from the tooth and has been shown to be effective in preventing relapse clinically and in animal models. There was a significant difference in relapse between the fiberotomy and the placebo control groups, and the relaxin-treated group had an intermediate response between the two groups (nonsignificant). Dose and treatment optimization may improve the response in future studies. To study the underlying mechanisms, we have localized relaxin receptors on gingival and periodontal ligament fibroblasts in tissue slices and cell cultures. Relaxin was found to stimulate collagenase production by relaxin in human gingival fibroblast cultures. Taken together, the data support a role for relaxin therapy to speed tooth movement and prevent relapse in orthodontic practice.

We have developed culture methods for human luteinizing granulosa cells (GLC) that support the timely and dynamic secretion of estrogen (estradiol-17beta; E(2)), progesterone (P(4)), and relaxin (Rlx) in patterns that mimic serum hormone concentrations during the luteal phase of the menstrual cycle. Additional hCG, to simulate rescue of the corpus luteum, prevented the normal decline in GLC hormone production. To test the importance of the P(4) receptor in P(4) production, GLC were treated in vitro with two P(4) receptor antagonists. Human GLC received one of two hCG support protocols: a Baseline group simulating the normal luteal phase or a Rescue group simulating early pregnancy. Baseline and Rescue groups were treated with either RU-486 or HRP2000 either early or late in the cell culture period. The effects of treatments or control on ovarian steroid and peptide hormone production were determined (significant difference was P < 0.05). In the Rescue group, late treatment resulted in an immediate and dramatic decline in E(2), P(4), and Rlx secretion to nearly nondetectable levels within 1 day after treatment, and hormones remained depressed for the remaining 10 days of culture. In contrast, early treatment resulted in a decline in steroid hormone secretion that returned to control levels within 5 days of cessation of treatment, and Rlx secretion was delayed for approximately 5 days more than in controls. The data support the hypothesis that P(4) may be a required autocrine factor, not only for its own production but also for the maintenance of full endocrine function of the corpus luteum.

This study examines the endocrine alterations associated with early fetal loss (EFL) induced by an environmental toxin, TCDD (2,3,7, 8-tetrachlorodibenzo-p-dioxin), in the cynomolgus macaque, a well-documented reproductive/developmental model for humans. Females were administered single doses of 1, 2, and 4 microgram/kg TCDD (n = 4 per dose group) on gestational day (GD) 12. Urinary estrogen metabolites (estrone conjugates) were monitored to establish the day of ovulation, and serum hormones (estradiol, progesterone, chorionic gonadotropin, relaxin) were measured to assess ovarian and placental endocrine status before and after treatment. EFL occurred between GDs 22 and 32 in 10 of the 12 animals treated with TCDD. The primary endocrine alterations associated with TCDD treatment were significant decreases in serum estradiol and bioactive chorionic gonadotropin concentrations (p < 0.02). Less pronounced decreases in serum progesterone (p = 0.10) and relaxin (p < 0.08) also followed TCDD treatment. In contrast, immunoreactive chorionic gonadotropin concentrations were not reduced by TCDD exposure at any level, indicating that TCDD targets specific components of the chorionic gonadotropin synthesis machinery within the trophoblast to alter the functional capacity of the hormone. These data demonstrate the value of endocrine biomarkers in identifying a toxic exposure to primate pregnancy many days before direct signs of reproductive toxicity were apparent. The increased EFL that occurred after exposure to TCDD might reflect a toxic response initially mediated via endocrine imbalance, leading to placental insufficiency, compromised embryonic circulation, and subsequent EFL.

These studies were designed to identify 1) a regimen of a third generation GnRH antagonist that abolishes primate luteal function, and 2) the amount of LH replacement required to maintain the structure and functional life span of the corpus luteum of the menstrual cycle after GnRH antagonist treatment. A single injection of antide at 3 or 5 mg/kg BW on day 6 of the luteal phase suppressed serum progesterone levels within 1 day of treatment, but levels recovered within 4 days. Administration of antide (3 mg/kg) for 3 days (luteal days 6–8) reduced (P < 0.05) serum progesterone below 1 ng/mL and maintained these low levels for the entire sampling period; in subsequent experiments, all monkeys received this antide regimen. Fixed doses (5, 10, or 20 IU) of recombinant human LH administered at 8-h intervals during and after antide treatment stimulated progesterone production in a dose-dependent manner; these monkeys menstruated earlier than controls regardless of treatment group. Replacement with an escalating dose regimen (5–20 IU) of LH resulted in typical serum progesterone and relaxin levels throughout a luteal phase of normal length. Corpora lutea removed on day 10 from monkeys treated with antide alone had decreased wet weight (P < 0.05) and few large luteal cells; coadministration of the escalating dose regimen of LH maintained luteal structure similar to that seen in time-matched controls. Antide-only treatment increased progesterone receptor (PR) messenger ribonucleic acid, but decreased PR immunostaining in luteal tissue; the escalating dose regimen of LH maintained PR messenger ribonucleic acid and immunostaining similar to those in controls. This study indicates that during GnRH antagonist administration, an escalating dose regimen of LH replacement is optimal for maintenance of the structure and functional life span of the primate corpus luteum.

Total concentrations of follicle stimulating hormone (FSH) were evaluated in daily urine samples from conceptive and nonconceptive menstrual cycles by measurement of the FSH beta subunit following treatment of the samples to dissociate the FSH heterodimer. Samples were self-collected by normal subjects during cycles in which daily blood samples also were obtained. Daily blood and urine specimens were collected prospectively from 10 subject in conceptive cycles, which led to normal pregnancies, and from 10 subjects with bilateral tubal ligations to provide control samples form nonconceptive cycles. Mean serum and urinary FSH concentration profiles wer parallel in both groups following ovulation and during he first 9 days of the luteal phase. Mean values for both serum and urinary FSH rose significantly above the postovulatory baseline by 10-12 days following the midcycle luteinizing hormone (LH) peak in nonconceptive cycles, but did not rise at any time following ovulation during conceptive cycles. Following regression analysis of the changing FSH concentration between days 9-14 post-LH surge in conceptive cycles, a slope of

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) caused a significant decrease in estradiol (E2) production when it was administered to human luteinized granulosa cells (hLGCs) in culture. We investigated the involvement of the epidermal growth factor receptor (EGFR) and protein tyrosine kinase (PTK) in this TCDD-induced toxicity. Upregulation in 125I-EGF binding to EGFR was measured after 24 h of TCDD treatment, while downregulation in EGFR binding was measured after 72 h of TCDD treatment. Upregulation of EGFR binding was associated with a significant decrease in postnuclear (7000 x g supernatant) PTK activity, but this activity was stimulated after 72 h of TCDD treatment. TCDD altered the level of tyrosine phosphorylation in proteins with molecular weights 35, 40, 43, 45, 60, and > 205 kDa. TCDD caused a significant increase in postnuclear cAMP-dependent protein kinase (PKA) after 24 h of treatment. The actions of TCDD on protein kinases were partially blocked by the protein synthesis inhibitor, cycloheximide. On the other hand, TCDD increased nuclear PTK and decreased nuclear PKA activity. E2 inhibited the postnuclear and nuclear activity of both PTK and PKA in control samples, but did not affect TCDD actions on either postnuclear or nuclear PTK activity. However, E2 abolished the stimulatory effect of TCDD on PKA activity in postnuclear protein. In the presence of insulin, TCDD did not induce any additional changes in postnuclear or nuclear PTK. Forskolin (FK) alone inhibited postnuclear PTK activity and stimulated its nuclear activity. The addition of TCDD 20 min after FK resulted in an increase in postnuclear PTK, but there was little change in nuclear PTK as compared to the effect of FK alone. The stimulatory effect of TCDD on postnuclear PKA activity was enhanced by insulin and TCDD reversed the negative effect of FK, but there was no effect of either insulin or FK on the inhibition by TCDD of nuclear PKA activity. TCDD decreased the activity of MAP2 kinase and reduced the binding activity of AP-1 DNA when given alone, and also blocked the E2 stimulation of MAP2K. These findings suggest that TCDD may interrupt the endocrine function of hLGCs through the blockage of the mitotic signal directly or indirectly through the interaction of PTK/MAP2K and PKA signaling.

This study examined the changes in cellular glucose uptake, cAMP-dependent protein kinase (PKA), and progesterone production induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in human luteinizing granulosa cells (LGCs) in culture. The role of Ah receptor on TCDD-mediated toxicity in human LGCs was investigated. Treatment of human LGCs with TCDD produced a time- and dose-dependent decrease in the cellular uptake of glucose. The Vmax and the K(m) of glucose transport were decreased by TCDD treatment. Furthermore, cytochalasin B, a specific inhibitor of facilitative glucose transporter proteins, totally abolished the portion of glucose transport activity that is sensitive to TCDD. Pretreatment of the cells with the Ah receptor blockers 4,7-phenanthroline and alpha-naphthoflavone antagonised the effect of TCDD on 3H-Me-glucose uptake. Structure-activity relationship studies with TCDD and three dioxin congeners revealed a rank order for their potency in the inhibition of glucose transport as follows: TCDD > 1,2,3,7,8-PCDD > 1,2,4,7,8-PCDD > 2,7-DCDD. Such a rank order is consistent with the previously determined biological activity of TCDD and the other dioxin congeners. Treatment of cells for 48 h with 10 nM TCDD substantially reduced PKA and progesterone production. The inhibitory effect of TCDD on progesterone production was more pronounced in the presence of insulin (10 micrograms/mL) and D-glucose (13.3 mM). However, cytochalasin B abolished the effect of TCDD on progesterone production. Forskolin (adenylate cyclase activator) abolished the effect of TCDD on glucose uptake and progesterone production but it did not affect the action of TCDD on PKA activity. A relationship between glucose transporting activity and progesterone production in human LGCs treated with TCDD is indicated by several lines of evidence: a) cytochalasin B downregulated glucose transporting activity and progesterone production, b) insulin plus D-glucose downregulated glucose uptake and amplified the negative effect of TCDD on progesterone production, and c) forskolin abolished the negative effect of TCDD on glucose transporting activity and on progesterone production. From the present data we conclude that glucose transporting activity can be used as a sensitive biomarker to detect the very early response to TCDD in human steroid-producing cells and that effect of TCDD on steroid production is mediated through the cAMP-dependent protein kinase.

The factors regulating relaxin production by the primate CL during spontaneous menstrual cycles and in early pregnancy are poorly understood. Since the CL produces steroids, notably progesterone, and expresses progesterone receptors, luteal progesterone may act locally to regulate relaxin production. For the current study, either trilostane (600 mg daily)--a 3 beta-hydroxysteroid dehydrogenase inhibitor--or vehicle was administered to rhesus monkeys during the midluteal phase (Days 6 and 7) of spontaneous menstrual cycles to examine the effects of reduced luteal progesterone synthesis on relaxin secretion. Trilostane treatment reduced serum concentrations of progesterone within 3 h of initial administration and maintained low levels typical of the follicular phase (< 1 ng/ml), causing premature menses without significant alteration in serum bioactive LH levels. Nevertheless, the patterns and levels of circulating relaxin, as measured by homologous macaque ELISA, were not different between trilostane- and vehicle-treated monkeys, with relaxin levels peaking in both groups by Day 13 of the luteal phase. To determine if chorionic gonadotropin (CG) injections simulating early pregnancy could stimulate relaxin production in a progesterone-depleted environment, trilostane or vehicle was administered as described above, followed by injections in increasing dosages of human (h) CG beginning 3 days after initial trilostane administration. Serum progesterone levels in trilostane-treated animals were significantly reduced prior to and during hCG treatment when compared with vehicle-treated animals. However, serum relaxin levels were comparable between these groups; relaxin levels peaked approximately 10-fold above pre-hCG levels in both trilostane- and vehicle-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Objective We determined the ovarian response to human chorionic gonadotrophin (hCG) in terms of relaxin and progesterone secretion during the peri-implantation period of normal and failing pregnancies. We wished to test the hypotheses that relaxin production in failing pregnancies is different from that in normal pregnancies, that relaxin is a reliable, quantitative indicator of the biological activity of endogenous hCG, and that relaxin is a useful predictor of peri-implantation spontaneous abortions. Design Daily blood samples were collected in a prospective longitudinal study from insemination patients. Patients Women undergoing artificial insemination in natural cycles with non-frozen donor semen at a University clinic. Measurements Serum LH, hCG, relaxin and progesterone were measured and the relationship between hCG and the ovarian hormones was evaluated in the peri-implantation period of normal pregnancies and spontaneous abortions. Results Nine of 23 conceptive cycles resulted in a spontaneous abortion between 16 and 70 days after the LH peak. In all normal and failing pregnancies there was a close qualitative relationship between hCG secretion and relaxin production. Six of nine failing pregnancies were associated with abnormally low hCG secretion. Six of the spontaneous abortions were associated with rates of relaxin secretion which were higher than the mean of 14 normal pregnancies. No such alterations in progesterone concentrations were observed. In cases where hCG was extremely low, the quantitative relationship between hCG and relaxin was different from that in cases of normal hCG concentrations. Conclusions There is a close temporal relationship between the secretion of trophoblastic hCG and ovarian secretion of relaxin in the peri-implantation period of normal and failing pregnancies. In failing pregnancies there is substantial variability in the quantitative relationship between relaxin and hCG, indicating that relaxin is not a reliable quantitative indicator of hCG bioactivity. Contrary to previous reports, relaxin concentrations in failing pregnancies tended to be higher than or equal to concentrations in normal pregnancies until the loss was imminent. Because of this relaxin is not a useful predictor of peri-implantation spontaneous abortions.

Circulating relaxin concentrations in the human rise in the late luteal phase and increase further in response to increasing circulating CG concentrations immediately after implantation. Similar events have not been documented in the laboratory macaque because of the lack of sensitivity of heterologous assay systems. A homologous enzyme-linked immunosorbent assay for authentic macaque relaxin was developed and validated. Using this enzyme-linked immunosorbent assay, relaxin concentrations were measured in peripheral and ovarian venous blood collected from cynomolgus and rhesus macaques. Relaxin concentrations rose in the late luteal phase of nonconceptive menstrual cycles in cynomolgus macaques, but it was not detected at other times in the cycle. In conceptive cycles, relaxin concentrations rose rapidly in close association with the appearance of mCG 13-14 days after mating. Pregnant rhesus macaques also had elevated relaxin concentrations in blood samples collected on days 15-17 postbreeding. Relaxin concentrations disappeared immediately after luteectomy or ablation of the trophoblast by either surgery or administration of methotrexate. The rise of relaxin paralleled the rise of mCG until 20-25 days postbreeding, while progesterone concentrations declined during this same time period. The lack of correlation between relaxin and progesterone secretion profiles suggests that either the cellular origins or the intracellular mechanisms promoting the secretion of these hormones are different. The periimplantational profile of serum relaxin in macaques was similar to the profile of relaxin observed during early human pregnancy.

Objective To determine whether preimplantation embryos are targets for relaxin secreted from the corpus luteum of the menstrual cycle. Design Rhesus monkey oocytes obtained from females undergoing controlled ovarian hyperstimulation were inseminated, and the resulting embryos were cultured in medium with or without recombinant human relaxin (20 ng/mL) for 8 days. Setting Research laboratory. Animal(s) Rhesus monkey. Intervention(s) Controlled ovarian stimulation to obtain oocytes for in vitro–produced embryos that were cultured with or without human recombinant relaxin. Main Outcome Measure(s) Rate of blastocyst development, percentage of blastocysts, and inner cell mass/trophectoderm cell ratio were measured on day 8 of culture. The presence of relaxin receptor ( RXFP1 ) messenger RNA in eight-cell embryos was observed by array hybridization. Result(s) RXFP1 receptor expression was localized to the inner cell mass of blastocysts, as shown by immunohistochemistry. The percentage of embryos that developed to blastocyst and the inner cell mass/trophectoderm cell ratio was unchanged with relaxin supplementation; however, the relaxin-treated embryos developed into blastocysts significantly sooner than untreated embryos. Conclusion(s) These results are the first evidence that the preimplantation primate embryo is a target for relaxin and that the addition of relaxin to in vitro culture medium enhances rhesus monkey embryo development.

Relaxin immunological activity has been observed in the plasma of pregnant bitches, and preliminary studies in our laboratory indicated that the highest relaxin concentrations were found in placentas. Therefore, canine placentas were collected at term and also from spay and relaxin was purified by methods developed for equine relaxin. Tissue was prepared by homogenization and purification on a C18 column. The preparation was further purified by stepwise elution ion-exchange chromatography, gel filtration, and gradient elution ion-exchange chromatography. One predominant peak in relaxin immunoactivity was collected. Canine relaxin was found to be larger than either porcine or equine relaxin as determined by SDS-PAGE. It migrated faster under reducing conditions, indicating a subunit structure. Purified canine relaxin was used for tracer and standard in a canine radioimmunoassay (RIA) using an antiporcine relaxin antibody. Concentrations of relaxin immunoactivity using the canine assay were up to 300-fold higher in placental preparations than those measured in the porcine relaxin assay. Sequence analysis of canine relaxin revealed a structure similar to other relaxins in the presence and placement of cystine residues.

Equine relaxin has been previously determined in a small number of pregnant Thoroughbred mares. To better define the normal pregnancy pattern of relaxin, the current study reports on a much larger number of mares. It also was designed to determine if all equids have the same gestational pattern of relaxin secretion. Plasma samples were collected weekly in 24 Standardbred mares, every 7-10 days in 10 pony mares, and daily in late pregnancy from 16 burros. Standardbreds had higher concentrations of relaxin than that reported for Thoroughbreds during most of gestation and did not exhibit the midpregnancy nadir in relaxin concentrations observed in Thoroughbreds. Relaxin concentrations in Standardbreds showed a small but steady decline from Day 150 until delivery. Pony mares had lower relaxin concentrations throughout pregnancy than other mares and had continuously increasing concentrations during gestation. Burros had relaxin concentrations intermediate to ponies and other mares in late gestation. Burros induced to foal with oxytocin showed a sharp increase in relaxin concentrations. No effect of the sex of the offspring was observed in relaxin profiles in Standardbred mares. Each of three Standardbreds with abnormal termination of pregnancy exhibited abnormally low relaxin concentrations at some point in the gestation prior to termination of the pregnancy. Thus, relaxin may be an indicator of placental functioning and used to assess at-risk pregnancies in mares.

Relaxin has previously been tested in rodent wound healing models and been shown to promote angiogenesis and to speed healing. However, pigs have been shown to be a better model for human skin in dermatology studies, so juvenile pigs were selected for a study of scar reduction and cosmetic appearance. Twelve 20- by 6-mm excisional wounds were created on the backs of all animals. Topical formulations of relaxin with 0, 0.5, or 2.5 mg/mL were applied twice daily for weeks 2-3 and then daily for weeks 3-6 in all animals. In addition, some animals received systemic relaxin, which was administered via infusion pumps at a rate of 125 microg/kg of body weight/day. Assessments of healing and cosmetic appearance were made by a dermatologist at weeks 2, 4, and 6. Wound sites were collected at 6 weeks and evaluated histologically for granulation tissue, inflammation, and collagen organization. Wounds in animals receiving systemic relaxin had an improved appearance with less redness, reduced granulation tissue, and lower amounts of inflammation. They showed a more-well-knit collagen structure compared to controls. Wounds treated with topical formulations did not show improvement over controls. The topical formulation used was found to have a short residence time, which likely limited penetration of relaxin. Reformulated relaxin preparations with improved penetration might be useful as a topical treatment for wounds to prevent or reduce scarring.

Recombinant human relaxin was used to attempt cervical ripening in post-delivery date pregnancies. High doses of relaxin were safe but did not advance cervical ripening or induce labor.

Human relaxin was administered to young rats through either minipumps (group P) or subcutaneous injections (group I). Control rats received pump implants containing placebo (group C). Day 0 was the day of orthodontic appliance placement and activation to pull bilateral upper first molars forward, and day 14 was the end time point. Cephalometric radiographs (three repeated exposures) were taken at 0, 3, 5, 7, 10, and 14 days. The total length of three maxillary molar segments and the space between the first and second molars were measured under a dissecting microscope postmortem. Both groups P and I showed rapid tooth movement at day 3. Movement slowed, but it still increased gradually in group P while decreasing in group I after that. Concurrent tooth lateral drifts to the buccal side were smaller in both groups P and I. The length and space were larger in both groups P and I. Thus, administration of human relaxin may accelerate the early stages of orthodontic tooth movement in rats.

Objective To characterize the profiles of human chorionic gonadotropin (hCG) secretion in blood and its subsequent excretion in urine during conceptive cycles that ended in successful pregnancy and in spontaneous abortion. Design A prospective study. Setting University fertility clinic and research laboratories. Patient(s) Healthy, spontaneously ovulating women with regular menses, no history of infertility, and either no male partner or an azoospermic partner. Intervention(s) Blood and urine samples were collected daily from 63 spontaneously ovulating women during 167 cycles of artificial insemination (AI) with donor semen; hCG concentrations were measured in blood and urine, and luteinizing hormone (LH) and follicle-stimulating hormone (FSH) concentrations were measured in blood by immunoassay. Main outcome measure(s) Fecundity, the day of ovulation, the day of hCG detection, and the concentration of hCG on the day of detection in blood and urine. Result(s) In 62 conceptions detected, 14 resulted in clinical spontaneous abortion (CAB) and 8 resulted in early pregnancy loss (EPL). When successful pregnancies and pregnancy losses were compared, no significant differences existed between the days of hCG appearance in serum or in urine, the concentrations of hCG on the day of detection, or the incremental change in hCG concentration on the day of detection. Conclusion(s) These data validate the use of urinary hCG as a biomarker for assessing peri-implantation pregnancy events.

When luteal phase relaxin concentrations were summed to give an integrated measure (pg/mL per cycle), relaxin was found to be significantly lower in those cycles with an out-of-phase endometrial biopsy. In addition, peak relaxin concentrations were lower in out-of-phase cycles compared with normal cycles. These data indicate that relaxin secretion may be related to normal luteal function and suggest that shortening of the luteal phase results in reduced relaxin production. Measurement of circulating relaxin may prove to be useful in making the diagnosis of out-of-phase biopsy and needs to be assessed for its usefulness in diagnosing abnormal luteal function.

We have developed a cell culture system for human luteinizing granulosa cells which supports the timely and dynamic secretion of oestrogen, progesterone and relaxin in patterns that mimic serum concentrations of these hormones during the luteal phase of the menstrual cycle. There was a wide variation in the amount of relaxin secreted by the cultured cells for the 69 patients studied. As relaxin production was generally maximal by day 10 of culture, comparisons were made at this time point. It was observed that most of the conceptions occurred in patients with higher relaxin secretion in vitro. All cycles with relaxin > 800 pg/ml on day 10 had a term pregnancy while only 13% of cycles with relaxin < 200 pg/ml had term pregnancies. A limited number of cycles from donor/recipient cycles did not show similar results. Steroid concentrations were not predictive of conception. These results demonstrated that in-vitro production of relaxin is predictive of implantation success in in-vitro fertilization (IVF)-embryo transfer cycles. This supports the hypothesis that relaxin may be involved in implantation and that lowered relaxin concentrations may be a partial cause of poor pregnancy rates after IVF.

The time of appearance of relaxin in peripheral blood was determined in conceptive and non-conceptive cycles using a sensitive and specific double-antibody enzyme-linked immunoassay for human relaxin. For study of relaxin in early pregnancy, daily plasma samples were collected from women receiving artificial insemination of donor semen. The day of ovulation was determined by daily LH monitoring and ultrasound observation. In three conceptive cycles, relaxin was significantly elevated over baseline 9-10 days following the LH peak. Relaxin concentrations quickly rose over the next 15 days of observation to over 800 pg/ml. Relaxin was observed to increase 1 to 2 days prior to the first detectable increase in plasma hCG as measured by enzyme-linked immunosorbent assay. To compare the relaxin profile in conceptive cycles with normal luteal phase concentrations, relaxin was also measured in daily plasma samples collected from women contracepting with barrier methods, bilateral tubal ligation, or abstinence. A small but consistent rise in relaxin in the late luteal phase was observed in nine of eleven women, which began 6-9 days after the LH peak, averaged approximately 50 pg/ml, and was declining by the next menses. It is concluded that a small but measurable rise in plasma relaxin is associated with the normal luteal phase and that relaxin secretion is accelerated around the time that hCG is first detected in conceptive cycles. This acceleration of relaxin secretion which is associated with the onset of hCG may provide additional evidence for identification of transient early pregnancy.

Previous studies have demonstrated elevations in testosterone and androstenedione initiated within the cycle of conception in pregnant non-human primates, and minimal data in the human support the same picture. In the present study we have investigated a group of patients scheduled for artificial insemination with regular menstrual cycles. For this study all patients provided blood samples at 5 days after the luteinizing hormone (LH) surges and daily through the luteal phase and into early pregnancy (n = 12). Patients who did not become pregnant served as normal controls (n = 9). We have measured 17-hydroxyprogesterone (17-OHP) as a marker of luteal activity not obscured by progesterone within the cycle of conception and testosterone and androstenedione as the major androgens. There were no significant changes in testosterone and androstenedione in the non-pregnant controls, but both testosterone and androstenedione were significantly elevated in the pregnant luteal phase, with the first increases occurring at 15 and 14 days respectively after the LH surge. Three of 12 pregnant patients did not demonstrate a dramatic increase in either testosterone or androstenedione and when examined more carefully a corresponding lack of increase in 17-OHP in those same subjects indicated less than optimal luteal activity, suggesting that these androgens were products of the corpus luteum. In three subjects in which consecutive non-pregnant and pregnant cycles were followed there was a dramatic increase from the non-pregnant luteal phase to the pregnant luteal phase indicating that the more important observation may be the concentrations of androgens in the conceptive luteal phase compared to some baseline, either previous luteal phase or even follicular phase. We have also studied changes in dehydroepiandrosterone sulphate and found that there was no significant contribution to this increase in androgens in early conception. These studies demonstrate a significant increase in both testosterone and androstenedione presumably of ovarian, specifically luteal, origin and that adrenal androgen production is not a factor in these changes.

Human granulosa cells collected from in vitro fertilization have previously been cultured to provide a system to simulate the granulosa lutein cells of the corpus luteum. In most of these systems, the cultures have been relatively short term, and attempts to simulate the normal pattern of hormone production observed during the luteal phase of the cycle have not been reported. Additionally, the hormone relaxin has generally been absent from the endocrine analysis of these systems. In this report, methods were used that supported secretion of ovarian steroids and relaxin that mimics the profiles of these hormones in vivo. This system was used to observe the endocrine responses of the granulosa lutein cells to three different protocols of CG administration designed to mimic the normal luteal phase, early pregnancy, and early pregnancy followed by pregnancy loss. The normal luteal phase was simulated by a constant baseline (0.02 IU/mL) CG model to simulate a nonconceptive cycle (baseline). The second model was baseline CG until day 8 of culture, followed by daily doubling from days 9-17 to simulate an early pregnancy (rescue-plateau). CG concentrations were then held constant from days 17-20 (5.12 IU/mL). A third model (rescue-drop) was used that was identical to the early pregnancy model except that on day 17 CG was returned to baseline concentrations (0.02 IU/mL) to simulate an early pregnancy loss. Baseline CG stimulation resulted in profiles of estrogen, progesterone, and relaxin secretion in culture that were closely related to secretory profiles previously reported in serum during the nonconceptive luteal phase. The timing of appearance of relaxin secretion and later declines in steroid and relaxin secretion paralleled that observed in serum. In the CG rescue protocols, ovarian steroids rose in response to daily doubling of CG and fell when CG either plateaued or fell. Relaxin did not show an increase in response to increasing CG, but its secretion did not drop when CG concentrations plateaued or dropped. This cell culture system model mimics the profile of ovarian steroids and relaxin seen in serum during the nonconceptive luteal phase, although the relative magnitude of the hormones was not the same as seen in vivo. It was also used to investigate responses to luteal rescue protocols designed to simulate early pregnancy and pregnancy loss. This culture system may be useful to study differences in endocrine response in granulosa cells collected from different patients and to provide information of clinical relevance. This culture system provides a model to study luteal function and its response to different protocols of luteal rescue and thus may provide insight into early pregnancy and pregnancy loss.

The present study was undertaken to assess the temporal association between the profiles of serum concentrations of oestradiol-17beta, progesterone, chorionic gonadotrophin (CG) and relaxin in pregnancies established naturally, and after embryo transfer, as well as in failed pregnancies in rhesus monkeys. In naturally mated cycles (group 1) a conception rate of 75% was obtained. In group 1, the mean day of CG detection in serum was 11.5 +/- 1.9 day post-ovulation, and for relaxin, 9.0 +/- 2.5 day post-ovulation. In group 2, embryo transfer to synchronous, non-mated surrogate recipients was performed; seven embryo transfer cycles yielded three pregnancies which were allowed to continue to term and normal infants were delivered. In embryo transfer cycles the mean day of CG detection was 14.8 +/- 1.8 day post-ovulation, and for relaxin, 11.4 +/- 2.6 day post-ovulation. A delay of about 3 days was observed in the appearance in circulation of CG (P < 0.05) and also of relaxin (P < 0.05) between natural mated and embryo transfer conception cycles. Significant differences (P < 0.05 for progesterone and P < 0.03 for oestradiol) were obtained for the areas under the curves for progesterone and oestradiol between days 12 and 16 in conception cycles compared with failed pregnancies. These data provide the first observation of the normal hormonal signals associated with maternal recognition of transferred embryos during the peri-implantation period, and suggest that the use of such an experimental primate embryo transfer model may help to elucidate components of maternal and embryonic signal-response mechanisms during embryo implantation.

CG produced by fetal tissues extends the functional lifespan of the primate corpus luteum during early pregnancy. Previous studies showed that urinary hCG administered to monkeys to simulate the rising CG levels associated with early pregnancy enhanced both progesterone (P) and relaxin (RLX) production by the corpus luteum. The current study was designed: 1) to compare the ability of recombinant (r) and urinary (u) hCG to stimulate luteal function, and 2) to assess the role of P in the regulation of luteal RLX secretion during simulated early pregnancy by concomitant administration of hCG and the 3 beta-hydroxysteroid dehydrogenase inhibitor trilostane to reduce P production. Rhesus monkeys received injections of either r-hCG or u-hCG (Ares Serono) in increasing doses (15-2880 IU/dose, twice daily) for 9 days beginning on day 9 of the luteal phase (n = 5/group). An additional group (n = 4) received r-hCG as described above, with concomitant oral administration of trilostane (500 mg/dose twice daily; Sanofi Winthrop). Daily serum samples were assayed for hCG by immunoradiometric assay, steroid hormones by RIA, and RLX by enzyme-linked immunosorbent assay. Serum hCG levels typically were not different between the r-HCG and u-hCG groups during or after treatment. Concentrations of hCG peaked 1 day after the final injection in monkeys receiving r-hCG (mean +/- SEM. 2759 +/- 120 mIU/mL) and u-hCG (2120 +/- 60 mIU/mL) and dropped below 5 mIU/mL by 10 days after the final treatment in all groups. Both r-hCG and u-hCG stimulated luteal P and RLX production. Progesterone levels rose rapidly after the initiation of hCG treatment and peaked in animals receiving r-hCG (14.4 +/- 2.8 ng/mL) and u-hCG (11.9 +/- 1.4 ng/mL) 4 days after initial administration. RLX levels peaked in the r-hCG (400 +/- pg/mL) and u-hCG (323 +/- 85 pg/mL) groups within 4 days of the final hCG treatment. Trilostane with r-hCG reduced P concentrations to very low levels (0.5 ng/mL; P0.01) within 1 day of administration compared to those in animals receiving r-hCG only and maintained these low levels for the entire treatment interval. Nevertheless, trilostane administration did not alter luteal RLX production, with serum levels peaking at 377 +/- 76 pg/mL. These data indicate that r-hCG and u-hCG were equally efficacious in stimulating the steroidogenic and peptidergic activities of the corpus luteum during simulated early pregnancy. In addition, P deprivation during r-hCG administration did not alter circulating RLX levels, suggesting that P is not a major regulator of RLX production by the primate corpus luteum during early pregnancy.

Jason L Dragoo1, Tiffany N Castillo1, Tatiana A Korotkova1, Ashleigh C Kennedy1, Hyeon Joo Kim1, Dennis R Stewart21Department of Orthopaedic Surgery, Stanford University, Palo Alto, CA, USA; 2Corthera Inc. San Mateo, CA, USAPurpose: This study was designed to investigate the relationship between serum relaxin concentration (SRC) and menstrual history and hormonal contraceptive use among elite collegiate female athletes. Evaluation of SRC in athletes is necessary, because relaxin has been associated with increased knee joint laxity and decreased anterior cruciate ligament (ACL) strength in animal models.Methods: National Collegiate Athletic Association Division I female athletes participating in sports at high risk for ACL tears – basketball, field hockey, gymnastics, lacrosse, soccer, and volleyball – were invited to participate. All participants completed a questionnaire about their menstrual history and hormonal contraceptive use. Venipuncture was performed to obtain samples of serum progesterone and relaxin. Samples were obtained during the mid-luteal phase from ovulating participants, and between the actual or projected cycle days 21 to 24, from anovulatory participants. Serum concentration of relaxin and progesterone was determined by ELISA and the data were analyzed using SPSS statistical software with significance set at P = 0.05.Results: 169 female athletes participated. The mean SRC among all participants was 3.08 ± 6.66 pg/mL). The mean SRC differed significantly between those participants using hormonal contraceptives (1.41 pg/mL) and those not using hormonal contraceptives (3.08 pg/mL, P = 0.002). Mean SRC was lowest among amenorrheic participants (1.02 pg/mL) and highest among oligomenorrheic participants (3.71 pg/mL) and eumenorrheic participants (3.06 pg/mL); these differences were not significant (P = 0.53). Mean serum progesterone concentration (SPC) differed significantly between those participants using hormonal contraceptives (2.80 ng/mL), and those not using hormonal contraceptives (6.99 ng/mL, P < 0.0001).Conclusions: There is a positive correlation between serum progesterone and SRC and an attenuation of SRC with hormonal contraceptive use. Our results underscore the significant role that hormonal contraceptives can play in decreasing relaxin levels, if future investigations establish a link between relaxin levels and ligamentous injury among female athletes.Keywords: hormones, ACL, anterior cruciate ligament, hormonal contraceptives, menstrual cycle

Hormone assays have been developed and applied for monitoring reproductive function using self‐collected urine samples in non‐clinical populations of women. Early pregnancy loss, menstrual dysfunction, reduced fertility as well as the site of toxicity can now be assessed using daily early morning urine samples. The understanding of the specific limitations of individual assay systems is important, however, to make the best use of these systems. The use of multiple end‐points and computer algorithms is suggested to avoid misclassification of adverse reproductive events.

Previous studies have compared ovarian steroid production in the luteal phase of nonconceptive and conceptive cycles. Some investigators reported higher preimplantational levels of progesterone in conceptive cycles vs. nonconceptive cycles, but other studies have found no differences. Many of these results were difficult to interpret because the studies included infertile women and/or women who received exogenous hormones. In this study we have characterized the profiles of gonadotropin secretion and ovarian steroid response during early pregnancy in a population of spontaneously ovulating women and compared them to those in nonconceptive cycles of recently fertile women. Blood samples were collected daily during the luteal phase from 24 women during 51 cycles of artificial insemination with donor semen. Cycles were segregated to those from women who had a successful term pregnancy (normal group) and those having an early spontaneous abortion (SAB group) and were also classified as nonconceptive or conceptive based on measurements of hCG. Serum LH and FSH did not show marked differences between nonconceptive and conceptive cycles in the periimplantation period in either the normal or SAB group. In the normal group, estradiol concentrations were significantly higher in conceptive cycles than in nonconceptive cycles beginning 6 days after the LH peak and continuing through the end of the cycle, while differences in progesterone concentrations bordered on or exceeded significance during the same time period. In the SAB group, preimplantation differences in pituitary gonadotropin and ovarian steroid secretion were not observed, whereas the postimplantation hCG concentrations in the SAB group were significantly lower than those in the normal group. It is reasoned that embryos with defective post-implantation hCG secretion may have had this defect before detection of hCG in serum, thus accounting for the lack of stimulation of steroid secretion in these pregnancies. These findings suggest that the enhanced ovarian steroid secretion in conceptive cycles may be due to a gonadotropic stimulus from the preimplantation embryo.

Relaxin, a polypeptide hormone normally associated with pregnancy, has been purified from many species, and the sequence determined for a growing number. Equine relaxin has been previously purified by acetone extraction, gel filtration, and ion exchange chromatographies. In an attempt to develop a more rapid and efficient method for relaxin purification, the use of affinity chromatography coupled with HPLC was explored. Monoclonal antibodies were raised against highly purified equine relaxin; large quantities of antibody were obtained by ascites production and attached to a solid phase support. An extract of term equine placentas was passed through the affinity column and washed, and relaxin was eluted by a change in pH. The isoforms of equine relaxin were separated by HPLC using a C18 (ODS) reverse phase column and a linear gradient of 25-30% acetonitrile. Four major and several minor isoforms of equine relaxin were obtained. The isoforms share similar mol wt by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), are composed of subunits (SDS-PAGE under reducing conditions), and have similar charges (native PAGE). Five isoforms tested positive for biological activity in the mouse interpubic ligament bioassay. Equine relaxin was sequenced by Edman degradation, and the sequence was confirmed by fast atom bombardment mass spectrometry. The isoforms of equine relaxin were found to be due to heterogeneity of the C-terminus of the B-chain. Equine relaxin appears to be the smallest relaxin sequenced. The A-chain consists of 20 residues, and the B-chain has 28 residues, with a total mol wt of 5253. Equine relaxin shares the greatest sequence homology with porcine relaxin (67% identity).

Objective: We have developed a novel delivery and occlusion method for the purpose of achieving permanent fallopian tube occlusion. This study was designed to determine how the procedure would be tolerated by patients and what early effects there would be on the fallopian tube. Methods: The Adiana procedure consists of applying radio frequency energy to the fallopian tube to ablate the epithelial lining of the tube and depositing a synthetic matrix. Four patients (seven tubes) were treated with the Adiana procedure 12 weeks before an elective hysterectomy. Hysterosalpingography was performed on each patient immediately before the hysterectomy. Additional evaluation of tubal occlusion was performed by applying dye under pressure (up to 150 mm Hg) to the explanted tube. Tissue was then histologically analyzed. Results: All patients tolerated the procedure very well, reporting zero pain on a VAS scale (defined 0 to 100). Patients experienced no pain associated with wearing the implant for the duration of the study. Hysterosalpingography results indicated complete tubal blockage in all treated tubes (seven out of seven). Retrograde dye application determined that six of the seven tubes were resistant to dye passage (150 mm Hg). One of the tubes allowed dye passage after 1 minute of dye application (100 mm Hg), suggesting at least partial occlusion. All seven tubes exhibited host cellular infiltrate into the pores of the matrix. Immunohistologic stains indicated that a variety of host cells made up the occlusive ingrowth. Conclusions: The results of this study indicate that the Adiana procedure is able to promote cellular ingrowth into a matrix and result in occlusion of the fallopian tube for up to 12 weeks.

Plasma relaxin activity was measured by radioimmunoassay (RIA) in the domestic cat utilizing two different antisera developed against highly purified porcine relaxin. One was the 5858 antiserum from our laboratory and the other was the R6 antiserum of Dr. Bernard Steinetz. Relaxin activity could not be detected during the estrous cycle or during pseudopregnancy. Relaxin immunoactivity during early gestation was not detected by either antiserum. Plasma relaxin immunoactivity was first detected by both antisera on about Day 25 of gestation. Relaxin concentrations then increased rapidly, with a plateau reached between Days 30 and 35 that was maintained until 10-15 days prepartum. Relaxin concentrations then declined gradually until parturition. No prepartum increase was observed. Relaxin concentrations were undetectable within 24 h of delivery. Although amounts of immunoactivity measured with the R6 antiserum were consistently higher than measurements with the 5858 antiserum, the patterns of secretions observed were similar for both antisera.

1. In a homologous radioimmunoassay for canine ventricular myosin light chains, the following percentages of cross-reactivities were obtained using the dog as a reference: human, 28%; sheep, 21%; cat, 8%; guinea-pig, 7%; rabbit, 5%; and rat, 4%. 2. In a homologous double diffusion immunoassay using specific gamma G to canine cardiac myosin heavy chains, dog cardiac myosin showed immunological identity with human and sheep cardiac myosin but partial identity with myosins of other species. 3. On a 5-20% polyacrylamide gradient, light chain C1 was electrophoretically distinct in some species; light chain C2 was electrophoretically identical in all species. 4. The K+-activated myosin ATPase of small animals was higher than that of larger animals at an alkaline pH; the same was true for Ca2+-activated myosin when assayed at pH 6.3.

Forty dogs were subjected to coronary ligation by placing snares around the two consecutive diagonal branches of the left anterior descending coronary arteries. Both acute and chronic studies were made of intramyocardial gas tensions using the mass spectrometer. Other studies such as levels of cyclic nucleotides and myosin ATPase activity were made at the chronic periods. Following coronary ligation in dogs there was a significantly elevated tissue P co 2 level in the ischemic region and left ventricular border zone as late as 5 weeks post-ligation. There was a severe decrease in cAMP, whereas cGMP was either the same as normal values or in some cases significantly elevated, in the ischemic area during this post-ligation time. These data were reflected in the border area of the left ventricle, but to a lesser extent. There was a minor, but significant decrease in myosin enzymatic V max values in the border area of the left ventricle during the early post-ligation period when tissue P co 2 levels were significantly elevated.

We have recently reported the secretory profile of relaxin throughout gestation in the cat. Because the appearance of relaxin begins at about Day 20 (Day O = ovulation) and because implantation begins shortly before this at Days 13-14, we hypothesized that relaxin was of feto-placental origin. To test this hypothesis, we used 4 experimental groups: 1) Control (laparotomy-only at Day 23 or 42, n = 4); 2) Early Ovariectomy (Ovx, bilateral ovariectomy between Days 23 and 26, n = 4); 3) Late Ovx (bilateral ovariectomy between Days 40 and 44, n = 4); 4) Tissue Removal (removal of feto-placental units, uterus, and one ovary on Days 16, 21, 28 and 35, n = 1 per day). Pregnancies were maintained in both Ovx groups by progesterone administration. Relaxin secretory patterns in Ovx groups were similar to the Control data. Relaxin was detectable in plasma beginning at about Day 20, with maximum concentrations reached by Day 30. Relaxin concentrations were highest (immunoactivity per mg tissue) in homogenates of placental tissues as compared to luteal, fetal, or uterine tissues. Altogether, these data indicate that the feto-placental unit is the source of relaxin in the cat.

Mild pulmonic stenosis in the dog, where right ventricular peak systolic pressure was increased approximately 150% at the time of sacrifice, induced 100% or more increase in right ventricular free wall weight by 3 weeks postoperative. Accompanying cardiac hypertrophy at these postoperative times, there was a decrease in both tissue PO2 levels and cAMP concentrations in the hemodynamically stressed ventricle, the right ventricle. Myosin ATPase activity was elevated as well as the velocity of contractile element shortening. The hemodynamically nonstressed left ventricle did not hypertrophy at these early postoperative times.

Summary Changes in plasma 15-keto-13, 14-dihydro-prostaglandin F2α were monitored at frequent intervals before, during and after spontaneous deliveries (three mares) and foalings induced by oxytocin (eight mares). No evidence of increased concentrations of the prostaglandin metabolite was observed in the final 10 days of gestation. In spontaneously delivering mares, there was a marked increase from 3 ng/ml at −125 mins to 18 ng/ml at −65 mins to the highest observed value of 182 ng/ml at 20 mins pre-partum. Following delivery, concentrations declined rapidly to around 0.2 ng/ml. Further release of prostaglandins was seen on Days 1 and 3 post partum. In oxytocin induced mares, maximal concentrations of about 100 ng/ml were observed to occur very close to the time of delivery. Large increases were observed as early as 2 mins following oxytocin injection. The significance of these findings is discussed in relation to parturient and post parturient events and changes in levels of the hormone relaxin during the same period.

It has been previously determined that the equine placenta is the sole significant source of relaxin during pregnancy and that relaxin immunoactivity is also present in term placentas. Therefore, placentas obtained at the time of foaling were selected for starting material for purification of equine relaxin. Frozen whole placentas were ground and then extracted with 0.5 N HCl-85% acetone. Relaxin was precipitated by raising the acetone concentration to 97%. Equine relaxin was further purified by stepwise elution ion exchange, gel filtration, and gradient elution ion exchange chromatographies and trichloroacetic acid precipitation. Equine relaxin purified in this manner was shown to be heterogeneous with one major form (R-1) and several minor forms (two of which are identified as R-2 and R-3). About 1.5 mg R-1 were obtained per kg placenta. Purity was assessed by slab and disc gel electrophoresis and dansyl end group analysis. R-1 had a potency of 28 U/mg, as measured in the mouse inter-pubic ligament bioassay and displayed a dose-response curve parallel with porcine relaxin. Amino acid analysis indicated the presence of tyrosine, histidine, and proline, amino acids absent in porcine relaxin. Dansyl end group analysis indicated the blockage of N-terminal groups on R-1 and the presence of Lys on R-3. A lower molecular weight was indicated by the electrophoretic migration of relaxin reduced with 2-mercaptoethanol suggesting that equine relaxin consists of two chains.

Equine relaxin (eRlx) immunoactivity has previously been measured in the mare during pregnancy using the porcine relaxin (pRlx) RIA (pRlx-RIA). This was not the optimal system for measurement of eRlx because the dose-response curve obtained with equine plasma was not parallel to the pRlx standard curve. A homologous eRlx-RIA has been developed and used to measure relaxin immunoactivity during pregnancy and parturition in the mare. Highly purified eRlx was used for the generation of antiserum in rabbits, preparation of tracer, and as assay standards. A double antibody eRlx RIA (eRlx-RIA) was developed which was highly specific and sensitive (0.023 ng relaxin/tube). The dose-response curve obtained with eRlx plasma was parallel to the equine standard curve while there was no parallelism noted between the pRlx and eRlx standard curves. This assay was utilized to measure eRlx in samples collected during pregnancy which were previously measured for relaxin content in the pRlx-RIA. It was found that the two assay systems gave almost identical patterns of secretion throughout pregnancy. The two assays differed in the amount of relaxin measured, with the eRlx-RIA measuring considerably higher amounts during gestation than the pRlx assay. In oxytocin-induced foalings, the differences became greater with the equine assay measuring as much as 10-fold greater concentrations.

Nonclinical studies indicate that the hormone relaxin is a good candidate for a safe cervical ripening agent that does not cause uterine contractions. This Phase II study (conducted November 2, 2005–October 20, 2006) was a randomised, double blind, placebo controlled trial testing 24-h intravenous infusion of serelaxin (recombinant human relaxin) or placebo for cervical ripening in 72 healthy, primiparous women. Eligible subjects had a singleton pregnancy ≥40 weeks, were planned for elective induction, had vertex presentation of the fetus, intact membranes and a Bishop score at screening ≤4. In Part A of the study, safety evaluation of three escalating doses of serelaxin (7.5, 25 or 75 μg/kg/day) or placebo was performed in 22 subjects admitted to the hospital 24 h prior to scheduled induction (n = 7, 4, 4, and 7 subjects, respectively). The highest safe dose from Part A and placebo were then tested in Part B for safety and cervical ripening (n = 25 subjects/arm). Planned randomisation ratio was of 4:2 (serelaxin:placebo) for each dose group in Part A and 1:1 for Part B. For analysis, subjects in Part B were pooled with those receiving the same dose in Part A and all subjects receiving placebo were pooled. The primary efficacy endpoint was change from baseline in Bishop score at 6, 12 and 24 h or end of study drug administration. Maternal safety evaluations included adverse events and vital signs through 4 weeks. Fetal assessments included serial heart rate monitoring and nonstress testing. Neonatal assessments included Apgar scores, NICU admissions, and adverse events through 4 weeks. Overall, 74 subjects were randomized and 72 were treated. There were no significant differences between the groups receiving the highest safe dose of serelaxin (75 μg/kg/day) and placebo in the primary or secondary efficacy endpoints. Changes from baseline in Bishop score at 24 h were 4.19 ± 1.9 and 3.26 ± 2.26 in the pooled placebo and serelaxin groups, respectively (p = 0.2507). Serelaxin was well tolerated and no anti-serelaxin antibodies were detected in either subjects or neonates. Serelaxin infusion at the end of pregnancy was well tolerated but did not advance cervical ripening. Clinicaltrials.gov identifier NCT00259103 (15 November 2005).