Absence Of Human Chorionic Gonadotropin Research Articles

Severe ovarian hyperstimulation syndrome following sole gonadotropin-releasing hormone (GnRH) agonist trigger: a case series and literature review

Abstract Objective The aim of this study was to report three cases of early severe ovarian hyperstimulation syndrome (OHSS) in patients undergoing a GnRH antagonist protocol triggered with GnRH agonist (GnRH-a), leading to hospitalization and the need for peritoneal drainage. Additionally, a review of the existing literature on this topic is provided. Design This is a retrospective case series and a literature review. Setting This study was conducted at obstetrics and gynecology department of tertiary academic referral hospitals, Israel. Participants This study included three patients presented with severe OHSS symptoms, including abdominal distension, ascites, and hemoconcentration. Main outcome measures The main focus of the treatment was to address the symptoms and prevent any further complications. The outcome was the complete recovery of the patients. Results The presented cases detail instances of severe OHSS following oocyte retrieval, utilizing GnRH-a for triggering. Case 1 involved a 33-year-old patient with a history of polycystic ovary syndrome (PCOS), Case 2 featured a 22-year-old patient with familial adenomatous polyposis (FAP), and Case 3 included a 41-year-old patient with a history of depressive disorder. All patients receiving supportive care, including infusions and medications, exhibited gradual improvement during hospitalization, with complete resolution observed during the 20-day post-hospitalization check-up. Conclusions These three cases highlight the occurrence of severe early OHSS following a GnRH antagonist protocol triggered with GnRH-a in the absence of human chorionic gonadotropin (hCG) administration for trigger or luteal-phase support. Clinicians must be aware that a GnRH-a trigger followed by a freeze-all approach does not guarantee the complete elimination of OHSS in all patients.

Mutation Analysis of the LH Receptor Gene in Leydig Cell Adenoma and Hyperplasia and Functional and Biochemical Studies of Activating Mutations of the LH Receptor Gene

Germline and somatic activating mutations in the LH receptor (LHR) gene have been reported. Our objective was to perform mutation analysis of the LHR gene of patients with Leydig cell adenoma or hyperplasia. Functional studies were conducted to compare the D578H-LHR mutant with the wild-type (WT)-LHR and the D578G-LHR mutant, a classic cause of testotoxicosis. The three main signal transduction pathways in which LHR is involved were studied. We describe eight male patients with gonadotropin-independent precocious puberty due to Leydig cell adenoma or hyperplasia. The D578H-LHR mutation was found in the adenoma or nodule with hyperplasia in all but two patients. D578H-LHR displayed a constitutively increased but noninducible production of cAMP, led to a very high production of inositol phosphates, and induced a slight phosphorylation of p44/42 MAPK in the absence of human chorionic gonadotropin. The D578G-LHR showed a response intermediate between WT-LHR and the D578H-LHR. Subcellular localization studies showed that the WT-LHR was almost exclusively located at the cell membrane, whereas the D578H-LHR showed signs of internalization. D578H-LHR was the only receptor to colocalize with early endosomes in the absence of human chorionic gonadotropin. Although several LHR mutations have been reported in testotoxicosis, the D578H-LHR mutation, which has been found only as a somatic mutation, appears up until now to be specifically responsible for Leydig cell adenomas. This is reflected by the different activation of the signal transduction pathways, when compared with the WT-LHR or D578G-LHR, which may explain the tumorigenesis in the D578H mutant.

Ovulation resumption after medical abortion with mifepristone and misoprostol

Background As an antiprogestin, mifepristone may have an impact on the return to ovulation in a manner that is not only attributable to its abortifacient activity. Our aim was to measure the time-to-ovulation in women who received mifepristone 200 mg orally and misoprostol 800 mcg vaginally for abortion up to 63 days of gestation. Study Design This planned substudy was part of a multicenter randomized trial of mifepristone 200 mg followed immediately or 24 h later by misoprostol 800 mcg vaginally. Women who had successful expulsion of the gestational sac based on ultrasound examination 1 week after mifepristone treatment were enrolled. All subjects used nonhormonal contraception until study completion. Baseline serum progesterone (P) levels were drawn on day 8±1 after mifepristone administration and then twice weekly until the P level was >3 ng/mL, consistent with ovulation. The mean time-to-ovulation was calculated using interval censored regression to address the censoring due to participant discontinuation. Results Fourteen (52%) of 27 enrolled women completed the substudy. The longest period of time that a subject who did not complete the study was followed was 29 days. Ovulation occurred 20.6±5.1 (range 8–36) days after mifepristone administration. Time-to-ovulation was not affected by participant age, gestational age, study arm, body mass index or presence or absence of human chorionic gonadotropin. Conclusions Return to ovulation following medical abortion with mifepristone and misoprostol occurs on average 3 weeks postabortion. Mifepristone 200 mg does not appear to have a lasting effect on ovarian function. Our results should be contextualized by the small sample size, although this is one of the larger studies on return to ovulation after abortion.

Effects of Nonylphenol on the Production of Progesterone by Rat Granulosa Cells.

Nonylphenol (NP) is a metabolite of detergent. The estrogenic effects and acute toxicity of NP causes disturbance in reproductive system. It has been reported that NP inhibits testosterone synthesis in male gonads, while the effects on progesterone release from female ovarian cells are unclear. In the present study, the effects of NP on steroidogenesis of progesterone in rat granulosa cells were examined both in vitro and in vivo. Immature (3-week old) female rats were intraperitoneally injected with pregnant mare serum gonadotropin (PMSG, 20 IU). After 48 hrs, the PMSG-treated rats were sacrificed and granulosa cells were collected from rat ovaries. The granulosa cells were cultured in 24-well plates (105 cells/ 0.5 ml) for 48 hrs, then treated with NP (4.3 µM, 12.8 µM, 43 µM ), in the presence or absence of human chorionic gonadotropin (hCG, 0.05 IU/ml) for 2 to 24 hrs to evaluate the NP effects on progesterone steroidogenesis. The levels of progesterone in medium were measured by radioimmunoassay, and the protein expressions were detected by western blotting. The data indicated that NP stimulated production of progesterone via a dose-dependent manner, especially for 2, 4 and 8 hrs, while treatment of NP for 24 hrs decreased progesterone release. Treatment of NP for 16 and 24 hrs prevented the stimulatory effects of hCG. We also gave the immature female rats gavage of NP with daily dose of 0, 50, 100 and 200 µg per kg body weight of NP for 7 days. The progesterone levels were raised following administration of 100 µg NP. Stimulation with hCG and 8-bromo-adenosine cyclic monophosphate (8-Br-cAMP) showed similar trends. MAPK/ERK kinase (MEK) inhibitor PD98059 did not affect progesterone as compared with vehicle treatment. Expressions of steroidogenic acute regulatory (StAR) protein were enhanced with 100 µg of NP. Moreover, gavage of NP (100 µg /kg/day) during 21-day pregnancy increased progesterone production of the female puppies' granulosa cells compared with those treatment without NP. These data suggested that NP directly stimulated StAR protein expression and affected the synthesis and production of progesterone in rat granulosa cells via activation of cAMP pathway. (poster)

The relationship between the production and the anti-gonadotrophic action of prostaglandin F 2α in luteal cells from the marmoset monkey ( Callithrix jacchus) in the early and mid-luteal phase

To address the potential luteolytic role for prostaglandin F 2α (PGF 2α) in the corpus luteum of the common marmoset monkey ( Callithrix jacchus), the ability of marmoset luteal cells, maintained in monolayer culture, to produce PGF 2α was determined in vitro in the presence and absence of human chorionic gonadotrophin (hCG) and other established pharmacological modulators of PGF 2α synthesis. We also assessed the effects of the PGF 2α analogue, cloprostenol, on progesterone output from luteal cells isolated in the early luteal phase versus the mid-luteal phase (days 3 and 14 post ovulation, respectively). Cloprostenol had no effect on progesterone output from luteal cells isolated on day 3 of the luteal phase, whereas it significantly inhibited both basal and hCG-stimulated progesterone synthesis by day 14 luteal cells during the culture period 48–72 h ( P < 0.001). Intra-luteal PGF 2α concentrations were 5-fold higher in luteal cells isolated in the early luteal phase than in mid-luteal phase cells (16.5 ± 3.5 versus 3.5 ± 0.6 pmol/10 5 cells). While PGF 2α production was unaffected by hCG in vitro, it was decreased by indomethacin (1000 ng/ml) ( P < 0.05) and stimulated by the calcium ionophore A23187 (10 μmol/l) ( P < 0.05) in luteal cells from both stages of the luteal phase. Phospholipase A 2 did not influence PGF 2α production by day 3 luteal cells whereas at 10 IU/ml, it significantly stimulated PGF 2α production by day 14 luteal cells ( P < 0.05). Hence, the timing of luteolysis in the common marmoset monkey appears to involve changes in both the luteal cell response to and production of PGF 2α.

Effects of Nonylphenol and 2,2', 4,6,6'-pentachlorobiphenyl on in vitro Sex Steroid Production in Maturing Oocytes of the Yellowfin Goby, Acanthogobius flavimanus

Several studies have reported that nonylphenol (NP) and 2,2', 4,6,6'-pentachlorobiphenyl (PCB104) exhibit estrogenic activity. To investigate the estrogenic potency of NP and PCB104 during oocyte maturation, fully vitellogenic oocytes (0.76 mm diameter in average) of yellow fin goby, Acanthogobius flavimanus, were exposed in vitro to these chemicals at different concentrations (0.1, 1, 10, 100, and 1,000 ng/mL) with the exogenous precursor <TEX>$17\alpha$</TEX>-hydroxyprogesterone (<TEX>$17{\alpha}OHP$</TEX>) 50 ng/mL in the presence or absence of human chorionic gonadotropin (HCG). The production of testosterone (T), estradiol-<TEX>$17\beta$</TEX> (E2), and <TEX>$17\alpha,20\beta$</TEX>-dihydroxy-4-pregnen-3-one (<TEX>$17\alpha20{\beta}OHP$</TEX>) in response to NP or PCB104 were measured by radioimmunoassay. Steroid levels were also expressed as E2/T and E2/<TEX>$17\alpha20{\beta}OHP$</TEX> ratios. In the absence of HCG, no significant differences in either NP or PCB104 treatment groups were observed. In the presence of HCG, NP treatment did not show significant differences in the production of T, E2, and <TEX>$17\alpha20{\beta}OHP$</TEX> at any concentrations tested, but E2/T ratios were decreased at concentrations of 0.1, 1, 10, and 1,000 ng/mL compared with the control group. PCB104 decreased E2 production at concentrations of 0.1, 10, and 1000 ng/mL, but did not show significant differences in the production of T and <TEX>$17\alpha20{\beta}OHP$</TEX> at any concentration tested. While E2/T ratios were decreased at PCB104 concentrations of 0.1, 1, 10, and 1,000 ng/mL, E2/<TEX>$17\alpha20{\beta}OHP$</TEX> ratios were also decreased at 0.1, 10, and 1,000 ng/mL compared with the control. Results indicate that both NP and PCB104 appeared to have antiestrogenic effects during this phase.

Effects of growth hormone and insulin-like growth factor 1 on progesterone production in human luteinized granulosa cells

To investigate the relationship between growth hormone (GH) and luteal function. In vivo comparative and in vitro culture studies. University hospital. Eighteen women who were interested in becoming pregnant and visited our clinic voluntarily participated in the study for urinary GH measurement. Ten women undergoing in vitro fertilization and embryo transfer (IVF-ET) were enrolled in in vitro culture studies. Ten women received IVF-ET. The GH concentrations in the urine collected for 24 hours, and serum progesterone and estradiol concentrations were measured during the midluteal phase. Effects of GH and insulin-like growth factor 1 (IGF-1) on progesterone and estradiol production were examined in the presence or absence of human chorionic gonadotropin (hCG) in human luteinized granulosa cells. Urinary GH concentrations were statistically significantly correlated with serum progesterone and estradiol concentrations. Progesterone production by luteinized granulosa cells was increased by IGF-1 (1, 10, 100 ng/mL) but not by GH (100 ng/mL) in a dose-dependent manner. Progesterone production stimulated by IGF-1 (100 ng/mL) was further increased in the presence of hCG (1 IU/mL). Estradiol production was increased by IGF-1 and GH. Growth hormone may influence luteal function directly or indirectly via IGF-1.

Effect of Prolactin in the Absence of hCG on Maturation, Fertilization, and Embryonic Development of in Vitro Matured Mouse Oocytes

Oocyte maturation is a complex process involving both the progression of meiotic cycle and the reprogramming of cytoplasmic events. The aim of this study was to investigate the effects of prolactin (PRL) in the in vitro maturation (IVM) of preantral mouse oocytes, in the absence of human chorionic gonadotrophin (hCG). Mouse preantral follicles were collected from female mice without prior hormonal ovarian stimulation and were cultured in the presence of varying concentrations of PRL (20, 100, 200, and 300 ng/mL) for 12 days. A group of in vitro matured oocytes were assessed for polar body (PB) formation, while the rest were fertilized and embryonic development was recorded. The maturation of preantral mouse follicles, as well as their fertilization and cleavage rates, observed when the culture medium was supplemented with middle- and high-range doses of PRL was beneficially affected. This effect was considerably high, although the culture media lacked hCG, a hormone extensively used in modern ovulation induction regiments, as well as in IVM media.

Effects of 1800 MHz GSM-like exposure on the gonadal function and hematological parameters of male mice

The aim of this study was to evaluate the possible effects of in vivo 1800 MHz GSM-like exposure on male reproduction. In five separate experiments, male NMRI mice (35-41 g) were exposed (11-12 mice each) to 1800 MHz GSM-like radiation. The average power density was 100 microW/cm2, the estimated SAR was 0.018-0.023 W/kg. The animals were exposed ten times (over two weeks on workdays) and the duration of exposure was 2 h/day. On the day of the last treatment, mice were anesthetized with i.p. pentobarbital, and blood samples were taken for hematology, serum chemistry and serum testosterone (T) determinations (ELISA). Testicles, epididymes, adrenals, prostates and pituitary glands were removed for histology. One testicle of each animal was used for culture of Leydig cells. The cells were cultured for 48 h in the presence or absence of human chorionic gonadotropin (hCG) to evaluate the in vitro steroidogenic response of Leydig cells. In the exposed animals red blood cell count (RBC: 8.59+/-0.10 T/l, n=37) and volume of packed red cells (VPRC: 42.29+/-0.43%, n=37) were significantly higher (p<0.01) compared with the controls (RBC: 8.12+/-0.08 T/l, n=36; VPRC: 39.76+/-0.36%, n=36). The serum testosterone level of the exposed animals (7.85+/-1.08 ng/ml, n=56) was also significantly elevated (p<0.05) compared to the controls (5.12+/-0.79, n=52), while the in vitro steroidogenic capacity of the Leydig cells was unaltered. No significant differences in the other investigated variables were found between controls and exposed mice. Our results indicate that the applied GSM-like microwave exposure may induce slight, but statistically significant alterations in some hematological and endocrine parameters of male mice within the physiological range. Further investigations are required to establish the biological significance of these phenomena.

Lipoprotein enhancement of ovarian theca-interstitial cell steroidogenesis: relative contribution of scavenger receptor class B (type I) and adenosine 5'-triphosphate- binding cassette (type A1) transporter in high-density lipoprotein-cholesterol transport and androgen synthesis.

The theca-interstitial cells take up plasma high-density lipoprotein (HDL)- and low-density-lipoprotein-derived cholesterol to convert into steroid hormones. The uptake of HDL-derived cholesterol is mediated by the scavenger receptor, class B, type I (SR-BI). In nonsteroidogenic cells, HDL-stimulated efflux of cholesterol has been shown to be mediated by the ATP-binding cassette A1 (ABCA1) transporter. Its expression has not been documented in steroidogenic cells. The goal of the present study was to determine: 1) the role of SR-BI in theca-interstitial cell androgen production; 2) whether theca-interstitial cells express ABCA1 transporter mRNA; and 3) the relative roles of SR-BI and ABCA1 transporter in androgen production. The ABCA1 transporter mRNA expression in rat theca-interstitial cells was shown using RT-PCR and Northern blot analyses. The role of SR-BI and ABCA1 in androstenedione production was also examined by treating cells with anti-SR-BI and 2-hydroxypropyl-beta-cyclodextrin in the presence and absence of human chorionic gonadotropin and/or human HDL(3). The treatment of theca-interstitial cells with anti-SR-BI antibody blocked more than 90% of HDL plus human chorionic gonadotropin-stimulated androstenedione production, and selective HDL-CE uptake. On the other hand, the use of inhibitors of ABCA1 transporter function had no discernible effect on HDL-supported androgen production. These data demonstrate that, although theca-interstitial cells express both SR-BI and ABCA1 transporter mRNA, the SR-BI pathway supplies the majority of the cholesterol required for androgen production. Furthermore, the present study presents evidence for a crucial role for SR-BI in HDL-mediated androgen production.

False-positive serum human chorionic gonadotropin results: Causes, characteristics, and recognition

False-positive serum human chorionic gonadotropin results are estimated to occur in 1 in 103 to 1 in 104 tests. Most of these false-positive results are due to interference by non-human chorionic gonadotropin substances (especially human luteinizing hormone and anti-animal immunoglobulin antibodies) and the detection of pituitary human chorionic gonadotropin. The false-positive human chorionic gonadotropin measurements are characterized by serum levels that are generally <1000 mIU/mL, the absence of human chorionic gonadotropin in the urine, nonparallelism of the human chorionic gonadotropin levels in serial dilutions of the serum with the human chorionic gonadotropin standard, results that are not consistent with the clinical or operative findings, the absence of a substantial change in the serum levels over time or after therapy, and the finding of a negative result when an alternative type of human chorionic gonadotropin assay is used. An algorithm is presented to aid in the recognition of false-positive human chorionic gonadotropin results in patients. (Am J Obstet Gynecol 2002;187:217-24.)

Effects of evodiamine on the secretion of testosterone in rat testicular interstitial cells.

Evodiamine, a bioactive component isolated from the Chinese medicine Wu-chu-yu, exhibits vasodilative and antianoxic action. Although evodiamine indeed has many biological effects, its effects on the endocrine system are not clear. The present study explored the effects of evodiamine on testosterone secretion in vitro. Rat collagenase-dispersed testicular interstitial cells (TICs) were incubated with evodiamine (0 to 10(-4) mol/L) in the presence or absence of human chorionic gonadotropin (hCG), forskolin, 8-bromo-adenosine 3':5'-cyclic monophosphate (8-Br-cAMP), or steroidogenic precursors (including 25-hydroxycholesterol, pregnenolone, progesterone, 17alpha-hydroxyprogesterone, and androstenedione) at 34 degrees C for 1 hour. The testosterone concentration in the media samples was measured by radioimmunoassay. Evodiamine 10(-4) mol/L was effective to reduce both basal and hCG-stimulated testosterone secretion in rat TICs after 1, 2, or 4 hours of incubation. The stimulatory effect of forskolin on testosterone release in TICs was prevented by administration of evodiamine. Evodiamine 10(-4) mol/L also decreased 8-Br-cAMP- and androstenedione-stimulated testosterone secretion. These results suggest that evodiamine reduces testosterone secretion in rat TICs via a mechanism involving reduced activity of cAMP-related pathways and 17beta-hydroxysteroid dehydrogenase (17beta-HSD).

Steroidogenic enzyme expression in human corpora lutea in the presence and absence of exogenous human chorionic gonadotrophin (HCG).

In a human conception cycle, the expected decline in progesterone production by the corpus luteum during the late luteal phase is prevented by human chorionic gonadotrophin (HCG) secreted by the implanting blastocyst. This study investigated the expression of components of the synthetic pathway for progesterone in human corpora lutea in the presence and absence of HCG in vivo. Corpora lutea were obtained from: (i) normally cycling women at the time of hysterectomy and classified on the basis of the urinary luteinizing hormone (LH) surge as early (n = 3), mid- (n = 3), or late luteal (n = 3); or (ii) women who had received daily doubling doses of HCG (n = 3) to 'rescue' the corpus luteum. Expression patterns of steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) were investigated by Northern blotting, in-situ hybridization and immunohistochemistry. Luteal 'rescue' with HCG was associated with the continued expression of these components. In the late luteal phase, in the absence of HCG, expression remained but was more variable. The expression of 3beta-HSD mRNA was significantly reduced during the luteal phase (P<0.01). In conclusion, during luteal 'rescue', HCG acts to maintain the steroidogenic pathway. In the absence of HCG, the decline in progesterone production begins in the presence of the main components of the steroidogenic pathway. While unlikely to initiate this decline, the altered expression levels of these components, particularly that of 3beta-HSD, may contribute to the continued reduction in progesterone production.

Vascular endothelial growth factor production by human luteinized granulosa cells in vitro.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) originating from the follicle or corpus luteum may be a physiological regulator of ovulation and neovascularization of luteinizing tissue, as well as a pathological factor in the development of ovarian hyperstimulation syndrome (OHSS). The objective of this study was to quantify VEGF production by human luteinized granulosa cells in vitro and to determine if gonadotrophin stimulates VEGF production directly and/or indirectly via enhanced synthesis of progesterone. In study 1, luteinized granulosa cells collected from women undergoing ovarian stimulation for in-vitro fertilization were cultured in the presence and absence of human chorionic gonadotrophin (HCG; 100 ng/ml) and/or low density lipoprotein (LDL; 100 microg protein/ml). In study 2, the progesterone synthesis inhibitor trilostane (250 ng/ml) and/or a progesterone receptor antagonist ZK137.316 (3.2 microM) were also added. Medium was harvested on days 1, 3, 5, 7 and 9 of culture and assayed for VEGF and progesterone. Results of study 1 were divided into two categories based on control concentrations of VEGF on day 1: 'low producers' (n = 6; <750 pg VEGF/ml) and 'high producers' (n = 5; >1000 pg VEGF/ml; P < 0.01). VEGF concentrations in cultures of both low and high producers increased (P < 0.01) from day 1 to maximal values on day 3, then steadily declined through to day 9. Chronic exposure to LDL or HCG increased (P < 0.05) VEGF concentrations in cultures of low producers by day 3 and day 5 respectively. In contrast, LDL did not alter VEGF concentrations in cultures of high producers and HCG did not increase VEGF concentrations until day 7. Nevertheless, acute exposure to HCG beginning on day 7 increased (P < 0.05) VEGF concentrations 3-fold in cultures of low or high producers. In study 2, trilostane treatment decreased (P < 0.05) progesterone concentrations by 91% on day 1 of culture but had no effect on VEGF concentrations on any day. ZK137.316 alone or with trilostane did not affect VEGF synthesis. These results suggest that VEGF production by luteinized granulosa cells is enhanced by gonadotrophin (HCG) independent of gonadotrophin-stimulated progesterone synthesis. These data are consistent with the hypothesis that the exacerbation of OHSS in early pregnancy is mediated by the CG stimulation of luteal VEGF production.

Antigonadotrophic effect of Mycobacterium tuberculosis.

The present investigation has been carried out to study if the Mycobacterium tuberculosis has any direct effect on granulosa cell progesterone production. Granulosa cells from pregnant mare serum gonadotropin (PMSG)-treated immature rats were incubated in replicates with and without the ultrasonicated Mycobacterium tuberculosis in the presence and absence of human chorionic gonadotrophin (hCG). Progesterone production and granulosa cell viability were assayed. The mycobacterial lysate significantly inhibited the basal production of progesterone. The lysate completely blocked the stimulatory effect of hCG. Any cytotoxic effect of the lysate was ruled out as none of the treatments decreased the viability of the granulosa cells as compared with the control values.

Gonadotrophin regulation of production of tissue inhibitor of metalloproteinases-1 by luteinized human granulosa cells: a potential mechanism for luteal rescue.

Human granulosa cells were maintained in culture with extracellular matrix in the presence or absence of human chorionic gonadotrophin (HCG) using a defined culture medium. Such cultures are maintained by gonadotrophin in a manner suggesting that features of 'luteal rescue' may be occurring in vitro. Western analysis of culture medium demonstrated that the granulosa cells produced tissue inhibitor of metalloproteinases (TIMP)-1 but not TIMP-2. The presence of TIMP-1 in cultured cells was also detected immunocytochemically. Immunoassay of TIMP-1 output revealed that HCG exposure for 7 days caused a 2-fold increase in TIMP-1 production versus control reaching maximum at approximately 1 ng HCG/ml. The sensitivity of this response to HCG was similar to that observed for stimulation of progesterone production. Delayed addition of HCG, from day 4 of culture, elicited increases in TIMP-1 which were evident within 24 hours, and were not explained by changes in cell replication or survival. Removal of HCG from cultures previously luteinized with HCG for 6 days resulted in a fall in TIMP-1 production. Thus TIMP-1 production by luteinized granulosa cells in culture is gonadotrophin dependent. We speculate that prolonged cellular function associated with 'luteal rescue' may result from increased extracellular matrix stability mediated by up-regulation of TIMP-1 production.

A Possible Mechanism for the Increase in Serum Luteinizing Hormone Levels in Male Rats by Oxolinic Acid

Oxolinic acid (1-ethyl-1,4-dihydro-6,7-methylenedioxy-4-oxo-3-quinolinecarboxylic acid), an antimicrobial agent, raises the serum levels of luteinizing hormone (LH) and increases the incidence of testicular Leydig cell tumors in male rats. In the present study the mechanism by which serum LH levels are raised in male rats receiving oxolinic acid was investigated. Aged Wistar rats were fed a diet containing oxolinic acid at 0 or 3000 ppm for more than 4 weeks. There was no effect of oxolinic acid on either the maximal levels of serum LH after castration nor on the serum levels of LH stimulated with 1 μg/rat of luteinizing hormone-releasing hormone (LHRH). The concentrations of testosterone in serum and testis were not changed by the treatment of oxolinic acid. In the in vitro organ culture, the testes of rats receiving oxolinic acid released testosterone in the same manner as the controls, in the presence or the absence of human chorionic gonadotropin (100 mIU/ml). The oxolinic acid-stimulated serum LH was not increased further by the daily administration of L-dopa (500 mg/kg/day, po, 7 days) and was blocked by the injection of a dopamine antagonist, haloperidol (2 mg/kg, ip). In a microdialysis study, oxolinic acid increased the extracellular concentration of dopamine in the preoptic area of hypothalamus. These findings suggest that a high dietary level of oxolinic acid elevates LH release from the anterior pituitary with an increase in LHRH, in part, by the excitatory input of a dopaminergic system in the preoptic area of rat hypothalamus.

Effects of gossypol on in vitro bovine oocyte maturation and steroidogenesis in bovine granulosa cells

Abstract Recently, we demonstrated an adverse effect of gossypol on steroidogenesis of bovine luteal, porcine granulosa and rat adrenocortical cells as well as on bovine, rat and hamster early embryonic development. The purpose of this study was to evaluate the effects of varying dosages of gossypol on bovine oocytes and granulosa cells from small and large follicles. Oocytes and bovine granulosa cells from small follicles were harvested from 3- to 8-mm follicles, and bovine granulosa cells from larger follicles (>15 mm) were used. Bovine oocytes were cultured in the presence of 10, 50 and 150 μg/ml gossypol for 26 to 28 h. Both bovine granulosa cells and bovine granulosa cells from larger follicles were treated with 0.1, 0.5, 2.5 and 12.5 μg/ml gossypol in the presence or absence of human chorionic gonadotropin (hCG) for 6 h. Cumulus expansion, meiotic progression of oocytes, progesterone production and intracellular cAMP formation in granulosa cells were measured. Gossypol significantly decreased cumulus expansion and inhibited nuclear maturation of oocytes. In bovine granulosa cells, gossypol inhibited cAMP formation only at 12.5 μg/ml. In bovine granulosa cells from larger follicles, hCG significantly increased both progesterone and cAMP levels. Treatment with gossypol significantly decreased hCG-induced cAMP formation in a dose-dependent manner. Basal cAMP level and progesterone secretion were significantly inhibited by only 12.5 μg/ml gossypol. However, inhibition of hCG-induced progesterone secretion was more dramatic. These results indicate that gossypol is capable of inhibiting cumulus expansion and nuclear maturation of bovine oocytes, and progesterone production and cAMP formation in bovine granulosa cells from larger follicles.

The effect of clomiphene citrate isomers on human granulosa-lutein cells in culture.

The effect of racemic clomiphene citrate and its two individual isomeric forms (i.e. en and zu) on corpus luteum function was evaluated. Granulosa-lutein cells were obtained from three normal ovulatory women undergoing oocyte retrieval following ovulation induction with agents other than clomiphene citrate for in vitro fertilization--embryo transfer (IVF-ET). The granulosa cells were cultured in the presence and absence of the three forms of clomiphene citrate, and in the presence and absence of human chorionic gonadotropin (hCG). Patients were recruited from the unit for assisted reproductive technology in a university hospital. The main outcome measured was the production of estradiol and progesterone by cultured human granulosa cells under the various conditions described above. The production of estradiol and progesterone by the cultured granulosa cells was dose-dependently reduced to a similar extent by all three forms of clomiphene citrate. The addition of hCG augmented steroidogenesis in all groups at all concentrations, but this still remained lower in all clomiphene citrate-treated groups compared to controls. The data suggest that all three types of clomiphene citrate (racemic, en, and zu) have inhibitory effects on the production of estradiol and progesterone by cultured human granulosa-lutein cells.

Inhibitory effect of interferon and tumor necrosis factor on human luteal function in vitro

To investigate whether immunological mechanisms may be involved in human luteal function. The effects of the cytokines, interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) on steroidogenesis by human luteal cells were examined in vitro. The dispersed human luteal cells, obtained from a total of 17 women at laparotomy, were cultured separately in the presence or absence of human chorionic gonadotropin (hCG) and IFNs/TNF-alpha with the medium being replaced at 48 hours. The medium was collected at 48 and 96 hours for steroid assays. The IFN-alpha had no significant effect on the production of estradiol or progesterone (P), whereas a dose-related inhibition of basal, as well as hCG-stimulated P formation, was observed after the addition of IFN-gamma (10 to 1,000 U/mL). Progesterone production was inhibited to about 45% of the control at 48 hours and even lower at 96 hours (n = 6, P < 0.001). The combination of IFN-gamma and low doses of TNF-alpha induced a further significant inhibition, whereas there was no effect of TNF-alpha alone. This inhibitory effect of IFN-gamma could be completely neutralized with a monoclonal antibody to IFN-gamma. Incubation with the antibody alone increased the production of P from luteal cells in culture, suggesting a local tonic inhibitory action of endogenous IFN-gamma. Interferon-gamma and TNF-alpha, whose function classically is known as antiviral, also may play a role in human luteal regression by inhibiting luteal P production.