Diurnal variation of serum progesterone, but not relaxin, prolactin, or estradiol-17 beta in the pregnant bitch.

The concentrations of progesterone (P), relaxin (Rlx), estradiol (E2) and PRL were measured by specific RIAs in serum samples collected twice daily at 8:00 am and 3:00 pm at weekly intervals after mating and until whelping in five Labrador Retriever bitches. From weeks 3 to 6 of pregnancy, P exhibited a conspicuous and statistically significant diurnal pattern (P less than 0.05), its serum concentration being approximately twice as high at 8:00 am as at 3:00 pm. A similar but nonsignificant trend was observed weeks 2, 7, and 8, and the overall ratio of the am/pm P concentrations was 2.4 +/- 0.28 (P less than 0.001). Rlx, E2, and PRL did not show a diurnal pattern at any time during pregnancy. The glandular sources and regulation of secretion of Rlx were further investigated. Rlx bioactivity was detected in canine placentas and ovaries, providing supportive evidence that these organs may be a dual source of the hormone in canine pregnancy. Injection of ovine PRL in three pseudopregnant bitches significantly increased serum P concentration as compared with seven untreated pseudopregnant controls, but Rlx was not detectable in serum before, during or after PRL treatment. The data support the view that Rlx and P are independently regulated in the bitch; PRL may be a regulator of P, but not of Rlx secretion. However, as PRL showed no diurnal changes in concentration, it does not appear to be directly responsible for the diurnal pattern exhibited by P.

Demonstration of a relaxin receptor and relaxin-stimulated tyrosine phosphorylation in human lower uterine segment fibroblasts.

To elucidate the mechanism of relaxin action, we studied the binding characteristics of human relaxin and its effects on intracellular concentrations of cAMP and tyrosine phosphorylation of cellular proteins in a model system of human cervix, human lower uterine segment fibroblasts. Human relaxin labeled with 125I bound specifically to a single class of high-affinity relaxin binding sites, distinct from insulin receptors, with a mean (+/-SEM) dissociation constant (Kd) of 4.36 +/- 1.7 x 10(-9) M and a mean of 3220 +/- 557 binding sites per cell in human lower uterine segment fibroblasts. Relaxin, in quantities that were shown previously to stimulate intracellular levels of cAMP in other cell types, had no effect on intracellular levels of cAMP in human lower uterine segment fibroblasts even in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). Incubation of the cells with relaxin caused a significant increase in tyrosine phosphorylation of a protein with an apparent Mr of approximately 220 kDa in these cells. In concert with results of recent studies that demonstrated that the Mr of the relaxin receptor is approximately 220 kDa, our data suggest that the phosphorylated protein is likely to be the relaxin receptor.

Placental control of ovarian immunoreactive relaxin secretion in the pregnant rat.

To investigate the control mechanisms for the secretion of relaxin in pregnant rats, the effects of the fetus, placenta, and uterus were studied. Plasma immunoreactive relaxin and progesterone were measured in pregnant rats, from days 8--1 post partum. On day 16 of pregnancy, groups of animals were subjected to removal of the fetuses, conceptuses (fetuses and placentae), or uteri. To determine whether there are extraovarian sources of circulating relaxin, a group of pregnant rats was ovariectomized on day 16. Immunoreactive relaxin was undetectable in the plasma of pregnant rats before day 10, and increased to a mean concentration of 0.52 +/- 0.01 (SEM) ng/ml on day 13. In control animals, immunoreactive relaxin levels remained at about this concentration throughout the remainder of pregnancy and declined rapidly post partum. The pattern of secretion of relaxin in fetectomized animals was similar to that in controls. In contrast, a significant decline in immunoreactive relaxin was seen, within 24 h after surgery, in those animals in which removal of the conceptuses or hysterectomy was performed. In these animals, immunoreactive relaxin was undetectable within 48 h after surgery and remained undetectable throughout the experimental period. In animals that were ovariectomized, immunoreactive relaxin was undetectable 24 h after surgery. Progesterone secretion in animals that had fetectomy or removal of the conceptuses performed was similar to that in controls. These groups showed a significant decline in progesterone on day 17 of pregnancy, and progesterone continued to decline until day 1 post partum. Progesterone in hysterectomized animals declined more abruptly than in either controls or other experimental groups. Ovariectomy resulted in a prompt fall in plasma progesterone. These results indicate that in the rat, the fetus is not needed for the maintenance of relaxin secretion throughout pregnancy, the placenta controls the ovarian secretion of relaxin. The uterus does not exert a tropic effect upon relaxin secretion, no extraovarian sources of circulating relaxin exist in the rat, and there is a divergence between progesterone and relaxin secretion during rat pregnancy.

Relaxin positively regulates matrix metalloproteinase expression in human lower uterine segment fibroblasts using a tyrosine kinase signaling pathway.

Despite the importance of relaxin to normal parturition in various species and its potential as an etiological agent in preterm delivery in women, knowledge regarding the mechanisms by which relaxin alters cervical connective tissue is extremely limited. An established in vitro model for human pregnancy cervix, human lower uterine segment fibroblasts, was used to determine the effects of relaxin as well as those of progesterone on the expression of matrix metalloproteinases and tissue inhibitor of metalloproteinase-1. The results demonstrate that relaxin is a positive regulator of matrix metalloproteinase expression, as it stimulates the expression of procollagenase protein and mRNA levels, stimulates prostromelysin-1 protein and mRNA levels, and inhibits tissue inhibitor of metalloproteinase-1 protein expression. Stimulation of procollagenase and prostromelysin-1 expression by relaxin does not involve phorbol-12-myristate-13-acetate- sensitive PKCs. Relaxin-stimulated tyrosine phosphorylation of the putative receptor and inhibition by a receptor tyrosine kinase inhibitor suggest that the relaxin receptor is probably a tyrosine kinase receptor. Inhibition of c-Raf protein expression using an antisense oligonucleotide inhibits relaxin regulation of matrix metalloproteinase and tissue inhibitor of metalloproteinase-1, suggesting that a signaling pathway involving c-Raf kinase mediates relaxin action.

The source of relaxin in pregnant Syrian hamsters.

Serum immunoreactive relaxin (IR) was measured on days 8, 10, and 14 of gestation in intact and ovariectomized (day 8 of pregnancy) hamsters. In intact hamsters, IR increased from 3-4 ng/ml on day 8 to 20 ng/ml by day 14 of pregnancy. After ovariectomy on day 8, pregnancy failed, and IR decreased rapidly to 0.29 ng/ml on day 14. However, when pregnancy was maintained in ovariectomized hamsters by daily injections of 0.1 microgram 17 beta-estradiol and 4 mg progesterone, serum IR rose to levels similar to those in intact hamsters on days 10 and 14 of pregnancy (i.e. 15 and 20 ng/ml, respectively). Placentas were obtained from other groups of hamsters on days 11, 14, and 15 of pregnancy and homogenized for bioassay by the classical guinea pig pubic symphysis palpation bioassay. Homogenates of placentas obtained on days 14 and 15 contained, respectively, 4 and 10 micrograms eq porcine relaxin/serum relaxin/g fresh tissue. The placenta, rather than the ovary, appears to be the source of during pregnancy in the hamster.

Human seminal relaxin is a product of the same gene as human luteal relaxin.

Unlike that of other species, which have only one gene encoding relaxin, the human genome contains two nonallelic genes for relaxin, designated H1 and H2, which encode markedly different relaxin peptides. Whereas human relaxin gene H2 is selectively expressed in the ovary, no ovarian expression of gene H1 has been detected. Since relaxin is actively produced in the human male, it is possible to postulate divergent gene expression of relaxin in the male and female. We examined this question directly through the structural determination of human seminal relaxin and its comparison with the structure of human luteal relaxin. Partially purified relaxin, prepared from pooled human seminal plasma which had been delipidated by extraction with acid acetone and hexane, subjected to two cycles of HPLC and an additional purification step by ion-exchange chromatography, was further purified by immunoaffinity chromatography, using a monoclonal antibody to the H2 relaxin A chain which cross-reacts with synthetic H1 relaxin, followed by an additional HPLC step performed on a C4 reverse-phase column. The recovered, purified relaxin was then analyzed by N-terminal gas-phase sequencing and fast atom bombardment mass spectroscopy for determination of the amino acid sequence and molecular ions of the A and B chains, respectively. The results demonstrate that the structure of the predominant relaxin in human semen plasma is derived from the product of the H2 gene, consisting of a N-terminal pyroglutamic acid A-24 A chain and a mixture of B-26 and B-27 B chains. With the exception of degradation of the seminal relaxin B chain C-terminus, this structure is identical to the structure of human luteal relaxin. Therefore, both human seminal and luteal relaxin are products of the H2 gene.

Hormone secretion by monolayer cultures of human luteal cells.

To be able to study the control mechanisms for human luteal function, a system was designed to maintain human luteal cells in culture. Collagenase dispersed cells of human corpora lutea of the menstrual cycle and pregnancy were maintained as monolayer cultures for 26 days. Progesterone (P) and relaxin (R) in culture media were measured by radioimmunoassay. Both menstrual cycle and pregnancy luteal cells secreted P for 26 days. hCG increased P secretion by menstrual cycle luteal cells, but not by pregnancy luteal cells. R was not detected in media of menstrual cycle luteal cell controls, nor in media of cells incubated with hCG. R was detected in media of pregnancy luteal cells for 6 days. Addition of hCG caused a significant increase in media R levels only on day 2 of culture. These studies show that human luteal cells can be maintained as viable monolayer cultures for at least 26 days and these cultures can be used to study control of human luteal function.

The role of prolactin in the regulation of the primate corpus luteum.

The role of PRL in the control of corpus luteum (CL) function was examined in hyperprolactinemic and euprolactinemic female rhesus monkeys with hypothalamic lesions in which ovulatory menstrual cycles were induced by the pulsatile administration of GnRH, in intact postpartum monkeys nursing their infants, as well as in animals treated with bromocriptine. In the lesioned monkeys, neither markedly elevated nor undetectable (bromocriptine-suppressed) PRL levels influenced the time courses of postovulatory plasma progesterone concentrations compared to those in monkeys with normal PRL concentrations. Plasma progesterone concentrations in the hyperprolactinemic monkeys, however, did not decline to undetectable levels at the end of the luteal phase, as is the case in normal animals, but remained slightly elevated for extended periods, a pattern characteristic of lactating animals. In both lesioned and postpartum animals, bromocriptine administration or removal of suckling infants from the latter resulted in complete functional luteolysis. These observations suggest that PRL does not play a significant role in the control of the CL of the menstrual cycle, but that in high concentrations, this hormone can partially maintain previously established CL.

Human corpus luteum secretion of relaxin, oxytocin, and progesterone.

To determine whether the human corpus luteum is a source of relaxin and oxytocin, we measured the concentrations of these peptides in plasma obtained from the ovarian veins of ovaries with and without a corpus luteum and compared these to peripheral plasma levels. Peripheral and ovarian venous blood samples were obtained from 34 nonpregnant women, 13 during the luteal phase and 21 during the follicular phase of their cycles, and from a 6-week pregnant woman. Plasma relaxin, oxytocin, and progesterone concentrations were determined by sensitive and specific RIAs. Plasma relaxin levels were not detectable (less than 0.16 microgram/L) in peripheral or ovarian venous plasma not draining a corpus luteum. The mean relaxin concentration in plasma draining an ovary with a corpus luteum was 0.41 +/- 0.09 (+/- SE) microgram/L. Oxytocin levels also were significantly higher in plasma draining an ovary with a corpus luteum (6.70 +/- 1.86 pmol/L) than in that draining the ovary with no corpus luteum (1.58 +/- 0.09 pmol/L; P less than 0.01) or in peripheral plasma (1.58 +/- 0.09 pmol/L; P less than 0.025). The mean progesterone concentration also was highest in plasma draining an ovary with a corpus luteum (210.2 +/- 50.5 nmol/L) compared with those in plasma from the contralateral ovarian vein (40.3 +/- 16.5 nmol/L P less than 0.005) and peripheral plasma (30.2 +/- 5.7 nmol/L; P less than 0.005) during the luteal phase. In a woman who was 6 weeks pregnant, plasma draining the ovary with a corpus luteum had 1.9 micrograms relaxin/L, but only 0.49 pmol/L oxytocin; the latter was similar to concentrations in noncorpus luteum-bearing ovarian venous plasma. These findings indicate that the human corpus luteum secretes relaxin, oxytocin, and progesterone. Both ovarian oxytocin and relaxin may function as paracrine or autocrine modulators of luteal function.

Ovarian control of pituitary hormone secretion in early human pregnancy.

To determine the influence of ovarian relaxin on the secretion of pituitary GH and PRL in vivo, we evaluated circulating serum hormone levels in 17 pregnant patients with functional corpora lutea (group I) and compared them to levels in 10 patients with premature ovarian failure (POF; group II) who became pregnant with egg donation and did not have corpora lutea. Group II patients had exogenous hormonal support. Serum relaxin (RLX), GH, PRL, estradiol (E2), and progesterone levels were measured weekly by RIA from weeks 4-8 of pregnancy. Analysis of variance and covariance were used to determine hormonal relationships. Serum RLX was present in the natural pregnancy group, with a mean of 1.94 micrograms/L over the study period. Serum RLX was undetectable in the POF patients (less than 0.16 micrograms/L). No significant difference in PRL or progesterone levels between the two groups was noted. E2 levels showed an upward trend in both groups with time and were significantly higher in patients of the POF group than in group I women (P = 0.001). GH levels were significantly higher in the natural cycle patients (P = 0.02) despite lower E2 levels. These data provide additional support for the concept that RLX production in early pregnancy originates from the corpus luteum. They suggest that a luteal product, probably RLX, stimulates GH secretion in early pregnancy. This is a previously undescribed role for RLX in pituitary physiology during human pregnancy.

Luteal progesterone relates to histological endometrial maturation in fertile women.

To examine the relationship between endometrial histological maturation and reproductive hormones, we studied 11 fertile women, aged 18-37 yr. All participants had had at least 1 previous pregnancy and cycled regularly, every 25-35 days. Women collected daily, first morning voided urine for measurement of estradiol and progesterone metabolite excretion, estrone conjugates (E1c), and pregnanediol glucuronide (Pdg), respectively, throughout the cycle of study. Hormones were normalized for creatinine. Between 7-9 days after home detection of a LH surge (Sure Step), participants underwent an endometrial biopsy using a small bore (Pipelle) catheter. Tissue was prepared for histological and biochemical analyses. The histological analysis is reported herein. Endometrium was dated by 3 authors (N.S., D.H., and S.P.), all of whom were blinded to the participant's identity or timing of biopsy within her cycle. Final dating was agreed upon based upon the method of Noyes et al. E1c and Pdg were integrated throughout the cycle using the trapezoidal rule, and correlations were sought between deviation from expected histology (based upon urinary hormones and LH surge) and integrated hormone values. E1c varied over a 2-fold range in these normal women, from 1196-2040 ng/cycle. Pdg excretion was much more variable, ranging from 22-119 microg/cycle. No relationship could be found between histological lagging of endometrial maturation and lower excretion of E1c. A moderate correlation was observed (Spearman's r = 0.6; P < 0.05) between degree of histological maturation and integrated Pdg. Of two women with evidence of a disparity between gland and stromal development (glands lagging behind stroma by >2 days), one excreted 24 microg Pdg/cycle, the next to lowest value. We conclude that normal fertile women experience a wide range of hormone concentrations in the face of normal endometrial maturation. Progesterone appears to exert a dose-related effect on endometrial maturation, and the techniques we used, although relatively crude clinical measures, appeared to be sufficient to detect this relationship.

Relaxin and its role in pregnancy.

Relaxin is a 6000-d polypeptide, structurally related to insulin and the insulin-like growth factors. Unlike insulin, the structure of which is remarkably well conserved among the vertebrates, relaxin sequences can vary by more than 50% between different species. Despite these large sequence variations, relaxins (with few exceptions) have very similar biologic activities in animal test systems. The reason for this has recently come to light: the receptor binding region of the B chain, in contrast to the rest of the molecule, is highly conserved between species. Relaxin is measured by bioassays employing interpubic ligament formation in mice and guinea pigs, and by inhibition of uterine motility. A more sensitive and efficient bioassay is urgently needed. In women, the target organs for relaxin are the uterine cervix, myometrium, endometrium, and decidua. Other presumptive but unproven targets are the pubic symphysis and sacroiliac joints, mammary glands, and pituitary gland. Circulating relaxin is secreted by the corpus luteum. The placenta, decidua, or both also produce relaxin, which does not enter the circulation but may act in an autocrine or paracrine fashion. hCG is a stimulus to luteal relaxin secretion. Other regulatory factors are poorly defined. Aluteal women are hyporelaxinemic, and yet are capable of normal vaginal delivery of their infants. Local effects of placental or decidual relaxin cannot be discounted in such subjects. Hyperrelaxinemia may occur in women with multiple gestations and ovarian stimulation, and may be associated with increased premature births. Serum relaxin also is elevated in pregnant diabetics, but its role in this condition has not been defined. Clearly, further investigations are needed to delineate the precise role of relaxin in human pregnancy.

Detection of Ki-ras proto-oncogene protein by a specific monoclonal antibody.

We have used a commercially available mouse monoclonal antibody and shown it to bind specifically to cellular Ki-ras p21 proteins and not to cellular N- and Ha-ras p21 proteins. In conjunction with electrophoresis and Western blotting, this antibody can be used, with further detailing, to assess levels of the cellular Ki-ras p21 against a background of total p21s.

Elevated first-trimester serum relaxin concentrations in pregnant women following ovarian stimulation predict prematurity risk and preterm delivery.

OBJECTIVE: To determine whether ovarian stimulation would result in higher circulating relaxin concentrations and whether this hyperrelaxinemia would be associated with prematurity. METHODS: Two groups of women were studied: 1) women achieving pregnancy after ovarian stimulation (n = 114) and 2) women achieving pregnancy without treatment (n = 37). Serum was obtained at 6-12 weeks' gestational age; fetal number was determined by transvaginal ultrasound. Prematurity risk or preterm delivery was determined from the obstetric record. A specific human relaxin enzyme-linked immunosorbent assay was used to measure serum relaxin concentrations. Hyperrelaxinemia was defined as levels greater than 3 standard deviations above the weighted mean of levels in normal unstimulated singleton pregnancies at 6-12 weeks' gestation. RESULTS: An association was found between prematurity risk or premature delivery and peripheral relaxin concentrations during weeks 6-12 of pregnancy in women having ovarian stimulation and in women having multiple gestations. Circulating relaxin concentrations greater than 16 ng/mL in women having ovarian stimulation and levels greater than 7 ng/mL in women who had multiple gestations predicted prematurity risk or premature delivery in 50% of the women. CONCLUSIONS: These data demonstrate that after ovarian stimulation, some women have highly elevated circulating first-trimester relaxin concentrations. First-trimester hyperrelaxinemia identifies a group of women at risk for prematurity who can be monitored aggressively.

The effect of multifetal pregnancy reduction on serum relaxin.

OBJECTIVE: To determine the effect of multifetal pregnancy reduction on circulating relaxin levels. METHODS: Patients with multifetal pregnancies had relaxin levels determined on the day of multifetal pregnancy reduction, after the procedure, and late in pregnancy. RESULTS: Forty-eight women (26 presenting with three fetuses and 22 with four or more) were studied. All pregnancies followed some form of ovulation induction. All pregnancies (except for one, which was reduced to a singleton) were reduced to twins. Pre-procedure, post-procedure and late-pregnancy relaxin levels were significantly higher in the in vitro fertilization (IVF)-gamete intrafallopian transfer (GIFT) group compared with the human menopausal gonadotropin (hMG)-alone group (P < .05). The initial number of fetuses had no significant effect on relaxin levels. Although post-procedure relaxin levels were significantly lower than pre-procedure levels (P = .002), relaxin levels continued to decrease throughout pregnancy, as evidenced by even lower levels later on (P = .0001). CONCLUSIONS: Serum relaxin levels were significantly higher in the IVF-GIFT group than in the hMG-alone group, which probably reflects more aggressive ovulation induction in the former. Because relaxin levels continued to decrease throughout pregnancy, the difference observed between pre- and post-procedure levels are not considered to be due to the procedure itself.

Secretion of progesterone and relaxin by the humancorpus luteum at midpregnancy and at term.

PIP: The secretion of progesterone (P) and relaxin (R):by the human corpus luteum at midpregnancy and at term was studied in 19 women, 6 of whom had their pregnancies terminated by hysterotomy at 14-18 weeks and 13 whose pregnancies went to term and were delivered by Caesarean section. Luteectomies were performed on 6 of the women at the time of Caesarean section. P and R levels were measured. R section correlated well with luteal P secretion. After Caesarean section at term the decline in serum P was parallel to that observed after hysterotomya at midtrimester. Absolute levels of P are higher at term. P decline after luteectomy was precipitous compared with Caesarean section. Results indicate that the corpus luteum in the human remains active throughout pregnancy.^ieng

Human chorionic gonadotropin and relaxin concentrations in early ectopic and normal pregnancies.

The present study was performed to investigate whether determination of serum relaxin concentrations would allow assessment of the gestation further than that provided by determination of hCG. Serum relaxin concentrations were quantified in women with resorbing ectopic gestations (as documented by declining titers of beta-hCG). The control group consisted of individuals with intrauterine pregnancies. As an additional control, we studied pregnancies conceived through ovulation induction, which usually have an increased volume of relaxin-secreting luteal tissue. On days 39-70 of gestation, the mean serum relaxin concentrations were significantly lower in ten resorbing ectopic gestations (P less than .001, permutation test) than in the normal control group of 13 intrauterine pregnancies. The median serum relaxin concentrations in patients who had ovulation induction with Pergonal were substantially higher than the median for all normal controls; values in clomiphene citrate-treated patients were within the normal range. These data suggest that relaxin secretion correlates with luteal function in both normal and abnormal gestations and reflects the status of the pregnancy. Thus, relaxin may serve as a useful clinical marker.

Elevated maternal serum relaxin concentrations throughout pregnancy in singleton gestations after superovulation.

OBJECTIVE: To test the hypothesis that superovulation results in elevated maternal circulating relaxin concentrations throughout the second and third trimesters of pregnancy, independent of the pattern of hCG secretion. METHODS: Two groups of women with singleton gestations were studied: a group of nine women who achieved pregnancy after stimulation with human menopausal gonadotropin and a group of six women who achieved pregnancy without prior stimulation. Peripheral blood samples were drawn approximately every 5 weeks throughout the second and third trimesters. Serum relaxin concentrations were measured using a human relaxin-specific enzyme-linked immunosorbent assay; hCG was measured by an immunofluorometric assay. RESULTS: The stimulated group had significantly higher relaxin levels throughout pregnancy (P=.007, multivariate analysis of variance) than did nonstimulated controls. The mean relaxin level in stimulated patients was 1.78 ng/mL (95% confidence interval [CI] 1.5, 2.17) and in nonstimulated subjects the level was 0.73 ng/mL (95% CI 0.59, 1.25). Spline fits demonstrated that stimulated patients had higher relaxin levels throughout the second and third trimesters. There was no significant difference in hCG concentrations between the two groups (P=.61). CONCLUSION: In singleton gestations after superovulation, maternal serum relaxin concentrations are significantly higher throughout the second and third trimesters of pregnancy. These differences are independent of the pattern of hCG secretion. It appears that luteal relaxin secretion is controlled by factors in addition to hCG.

Gonadotropin-releasing hormone agonist and human menopausal gonadotropin (hMG) short protocol, but not hMG alone, rescues the corpus luteum from the preceding cycle.

OBJECTIVE: To determine whether ovulation induction regimens, particularly the short protocol, has an effect on the corpus luteum (CL) from the previous cycle. DESIGN: Infertility patients were followed in an academic research environment. Patients were treated with either the short protocol (GnRH agonist [GnRH-a] and hMG) or hMG alone in a controlled ovarian hyperstimulation cycle. SETTING: Infertility center in academic setting. PATIENTS: Patients requiring ovulation induction. INTERVENTIONS: The blood samples were drawn on day 2 or 3 as a baseline before initiation of any treatments, generally on day 5 and usually every other day thereafter until ovulation. Serum P, E2, and relaxin were determined. MAIN OUTCOME MEASURES: Serum P, E2 and relaxin. RESULTS: Patients receiving hMG alone showed no change in serum P levels in the first few days of treatment whereas most short-protocol patients (18/30; 60%) showed an increase of P within 3 days of the initiation of treatment. The increase in P almost always was associated with an increase in relaxin as a marker of the luteal production of this P. CONCLUSION: The short protocol with its flare of gonadotropins is able to stimulate the CL from the previous cycle, resulting in an early increase in P that comes from the CL as indicated by its association with an increased relaxin in the same subjects.

Human chorionic gonadotropin stimulation of relaxin secretion by luteinized human granulosa cells.

OBJECTIVE: To determine the effect of human chorionic gonadotropin (hCG) on relaxin secretion by long-term cultures of luteinized human granulosa cells (GC). DESIGN: Luteinized human GC were collected from 10 women undergoing in vitro fertilization (IVF) cycles. Luteinized human GC from each woman were plated in replicate wells at 1 x 10(5) cells/well and exposed to medium 199 (GIBCO, Grand Island, NY), medium 199 with 1 IU/mL hCG, and/or medium 199 with 100 IU hCG/mL. Luteinized human GC were maintained for up to 40 days in culture. Spent media were changed every 2 days and assayed for relaxin and progesterone (P) at the conclusion of each experiment. SETTING: Tertiary care center. PATIENTS, PARTICIPANTS: Luteinized human GC were obtained from women undergoing controlled ovarian hyperstimulation for IVF with one of the following regimens: (1) clomiphene citrate with human menopausal gonadotropins (hMG); (2) hMG alone; or (3) hMG with leuprolide acetate. All women were less than 40 years of age, in good health, and were not taking medications other than those used in the ovulation-induction regimen. MAIN OUTCOME MEASURES: Levels of P and relaxin in spent media. RESULTS: Relaxin secretion by luteinized human GC was dependent on hCG stimulation and was detected only after a time lag in culture. After relaxin secretion was detected, it was maintained throughout the culture period (10 to 22 days). Luteinized human GC produced P immediately under both basal and stimulated conditions. Progesterone production continued throughout the culture period with hCG-stimulated cells producing significantly greater P after 4 to 8 days in culture. CONCLUSIONS: Luteinized human GC obtained at the time of oocyte retrieval secrete relaxin in response to hCG stimulation and secrete P under both basal and hCG-stimulated conditions, thereby serving as a model to explore luteal function and control.