ABSTRACT
STUDY QUESTION: Is embryo culture in a closed time-lapse system associated with any differences in perinatal and maternal outcomes in comparison to conventional culture and spontaneous conception? SUMMARY ANSWER: There were no significant differences between time-lapse and conventional embryo culture in preterm birth (PTB, <37 weeks), low birth weight (LBW, >2500 g) and hypertensive disorders of pregnancy for singleton deliveries, the primary outcomes of this study. WHAT IS KNOWN ALREADY: Evidence from prospective trials evaluating the safety of time-lapse incubation for clinical use show similar embryo development rates, implantation rates, and ongoing pregnancy and live birth rates when compared to conventional incubation. Few studies have investigated if uninterrupted culture can alter risks of adverse perinatal outcomes presently associated with IVF when compared to conventional culture and spontaneous conceptions. STUDY DESIGN, SIZE, DURATION: This study is a Swedish population-based retrospective registry study, including 7379 singleton deliveries after fresh embryo transfer between 2013 and 2018 from selected IVF clinics. Perinatal outcomes of singletons born from time-lapse-cultured embryos were compared to singletons from embryos cultured in conventional incubators and 71 300 singletons from spontaneous conceptions. Main perinatal outcomes included PTB and LBW. Main maternal outcomes included hypertensive disorders of pregnancy (pregnancy hypertension and preeclampsia). PARTICIPANTS/MATERIALS, SETTING, METHODS: From nine IVF clinics, 2683 singletons born after fresh embryo transfer in a time-lapse system were compared to 4696 singletons born after culture in a conventional incubator and 71 300 singletons born after spontaneous conception matched for year of birth, parity, and maternal age. Patient and treatment characteristics from IVF deliveries were cross-linked with the Swedish Medical Birth Register, Register of Birth Defects, National Patient Register and Statistics Sweden. Children born after sperm and oocyte donation cycles and after Preimplantation Genetic testing cycles were excluded. Odds ratio (OR) and adjusted OR were calculated, adjusting for relevant confounders. MAIN RESULTS AND THE ROLE OF CHANCE: In the adjusted analyses, no significant differences were found for risk of PTB (adjusted OR 1.11, 95% CI 0.87-1.41) and LBW (adjusted OR 0.86, 95% CI 0.66-1.14) or hypertensive disorders of pregnancy; preeclampsia and hypertension (adjusted OR 0.99, 95% CI 0.67-1.45 and adjusted OR 0.98, 95% CI 0.62-1.53, respectively) between time-lapse and conventional incubation systems. A significantly increased risk of PTB (adjusted OR 1.31, 95% CI 1.08-1.60) and LBW (adjusted OR 1.36, 95% CI 1.08-1.72) was found for singletons born after time-lapse incubation compared to singletons born after spontaneous conceptions. In addition, a lower risk for pregnancy hypertension (adjusted OR 0.72 95% CI 0.53-0.99) but no significant difference for preeclampsia (adjusted OR 0.87, 95% CI 0.68-1.12) was found compared to spontaneous conceptions. Subgroup analyses showed that some risks were related to the day of embryo transfer, with more adverse outcomes after blastocyst transfer in comparison to cleavage stage transfer. LIMITATIONS, REASONS FOR CAUTION: This study is retrospective in design and different clinical strategies may have been used to select specific patient groups for time-lapse versus conventional incubation. The number of patients is limited and larger datasets are required to obtain more precise estimates and adjust for possible effect of additional embryo culture variables. WIDER IMPLICATIONS OF THE FINDINGS: Embryo culture in time-lapse systems is not associated with major differences in perinatal and maternal outcomes, compared to conventional embryo culture, suggesting that this technology is an acceptable alternative for embryo incubation. STUDY FUNDING/COMPETING INTEREST(S): The study was financed by a research grant from Gedeon Richter. There are no conflicts of interest for all authors to declare. TRIAL REGISTRATION NUMBER: N/A.
Subject(s)
Hypertension, Pregnancy-Induced , Pre-Eclampsia , Premature Birth , Pregnancy , Female , Child , Infant, Newborn , Humans , Male , Retrospective Studies , Premature Birth/epidemiology , Premature Birth/etiology , Hypertension, Pregnancy-Induced/etiology , Prospective Studies , Time-Lapse Imaging , Semen , Fertilization in Vitro/adverse effectsABSTRACT
BACKGROUND: MCM5 is a protein involved in DNA replication, facilitating cell proliferation. In normal epithelium MCM5 expression is restricted to the cells in the basal proliferative compartments, however in the presence of a tumour MCM5 positive cells are present at the surface epithelium and are shed into bodily fluids. The aim of this study was to determine the sensitivity of MCM5 as a biomarker for the detection of endometrial and ovarian cancer. METHODS: Patients with known ovarian or endometrial cancers, or known benign gynaecological conditions, were enrolled. Informed consent was obtained prior to the collection of full void urine, and either a vaginal tampon (worn for 6-8 h), or a vaginal swab. Vaginal secretions were extracted from the tampon or swab, centrifuged and lysed. Urine samples were centrifuged and lysed. MCM5 levels were determined by MCM5-ELISA (Arquer Diagnostics Ltd). RESULTS: 125 patients completed the study protocol, 41 patients had endometrial cancer, 26 ovarian cancer, and 58 benign controls. All patients provided a urine sample and either a tampon or vaginal swab sample. Urine MCM5 levels were higher in cancer patients than controls (p < 0.0001), there was no significant difference in levels between tampon samples or vaginal swab samples in cancer patients when compared to controls. Performance of MCM5 to discriminate cancer from benign disease was high with an area under the ROC curve of 0.83 for endometrial cancer and 0.68 for ovarian cancer. Using a cut off of 12 pg/mL, overall sensitivity for endometrial cancer was 87.8, and 61.5% for ovarian cancer with a specificity of 75.9%. CONCLUSIONS: MCM5 is a novel sensitive and specific biomarker for the detection of ovarian and endometrial tumours in urine samples, which is likely to have clinical utility as a diagnostic aid.
Subject(s)
Biomarkers, Tumor/metabolism , Cell Cycle Proteins/metabolism , Endometrial Neoplasms/diagnosis , Ovarian Neoplasms/diagnosis , Aged , Early Detection of Cancer , Female , Humans , Middle AgedABSTRACT
OBJECTIVES: The findings of a previous multigene study indicated that the expression of a panel of seven specific genes had strong differential power regarding inflammatory bowel disease (IBD) versus non-IBD, as well as ulcerative colitis (UC) versus Crohn's disease (CD). This prospective confirmatory study based on an independent patient cohort from a national Danish IBD centre was conducted in an attempt to verify these earlier observations. DESIGN, SETTING AND PARTICIPANTS: A total of 119 patients were included in the study (CD, UC and controls). Three mucosal biopsies were retrieved from the left side of the colon of each patient. RNA was extracted, and RT-PCR was performed to retain expression profiles from the seven selected genes. Expression data from the training set (18 CD, 20 UC and 20 controls) were used to build a classification model, using quadratic discriminant analysis, and data from the test set (20 CD, 21 UC and 20 controls) were used to test the validity of the model. RESULTS: The present investigation did not confirm the previous observation that a panel of seven specific genes is able to distinguish between patients with CD and UC, whereas the discriminative power for IBD versus control subjects was substantiated. CONCLUSION: Our results fail to demonstrate that the previously identified seven-gene classification model is able to discriminate between CD and UC but suggest that the gene panel merely discriminates between inflamed and noninflamed colonic tissue. Thus, a reliable and simple diagnostic tool is still warranted for optimal diagnosis and treatment of patients with IBD, especially the subgroup with unclassified disease.
Subject(s)
Colitis, Ulcerative/diagnosis , Crohn Disease/diagnosis , Adult , Amidohydrolases/genetics , Amino Acid Transport Systems , Amino Acid Transport Systems, Neutral/genetics , Anion Transport Proteins/genetics , Area Under Curve , Chemokine CXCL1/genetics , Colitis, Ulcerative/genetics , Crohn Disease/genetics , Female , GPI-Linked Proteins/genetics , Gene Expression/genetics , Genetic Markers/genetics , Humans , Lectins, C-Type/genetics , Male , Matrix Metalloproteinase 7/genetics , Membrane Proteins/genetics , Pancreatitis-Associated Proteins , Prospective Studies , Real-Time Polymerase Chain Reaction , Sulfate TransportersABSTRACT
Although controversial, chronic uric acid nephropathy is a tubulointerstitial disease capable of developing renal function loss. On the other hand, potassium citrate (KCi) administration has demonstrated to be effective in calcium as well as uric acid nephrolithiasis therapy. Therefore, the aim of the present study was to evaluate the possible benefit of KCi treatment in the prevention or amelioration of renal interstitial damage in uric acid nephropathy. Two-month-old male Sprague-Dawley rats were divided into 3 groups: G1 hyperuricemic (HU), G2 hyperuricemic + KCi (HU+KCi), and G3 KCi. G1 and G2 were fed on oxonic acid (inhibitor of rat liver uricase), and a uric acid supplement, during 4 weeks. G2 and G3 were given 2% KCi in drinking water, and G1 regular tap water and standard rat chow. At the end of the study, renal tissue was processed for light and electron microscopy and immunostaining by alpha-smooth muscle actin (SMA). Tubulointerstitial lesions and the amount of alpha-SMA immunostaining in renal tissue were evaluated by histomorphometric quantitation. Rats belonging to the hyperuricemic groups treated with KCi (G2) showed fewer tubulointerstitial lesions as follows: % tubular atrophy: 1.7 +/- 0.3 versus 7.2 +/- 1.2, p < 0.05; inflammatory cells infiltrate (number of cells/area): 0.6 +/- 0.1 versus 2.4 +/- 0.2, p < 0.01; % interstitial fibrosis (cortex): 3.3 +/- 0.3 versus 9.3 +/- 0.5, p < 0.05; % interstitial fibrosis (medulla): 5.2 +/- 0.3 versus 21.9 +/- 1.2, p < 0.01, lower albuminuria (32.8 +/- 11.2 mg/day versus 128.5 +/- 10.4, p < 0.01), higher creatinine clearance ( 1.36 +/- 0.02 ml/min versus 0.74 +/- 0.01, p < 0.01 ) and less percentage of alpha-SMA in renal tissue (1.8 +/- 0.1 versus 10.5 +/- 1.4, p < 0.05), when compared with the hyperuricemic group not treated with KCi (G1). These data suggest that KCi administration could provide a substantial benefit in the regard to tubulointerstitial lesion and progressive renal damage.
Subject(s)
Kidney Diseases/drug therapy , Potassium Citrate/therapeutic use , Uric Acid/metabolism , Animals , Kidney/chemistry , Kidney/pathology , Kidney Diseases/metabolism , Kidney Diseases/pathology , Male , Rats , Rats, Sprague-DawleyABSTRACT
El objetivo de este trabajo fue evaluar las diferentes alteraciones renales morfológicas y funcionales secundarias a la pérdida de la masa renal por nefrectomía subtotal (Nx5/6) e investigar los mecanismos patogénicos involucrados en el daño TI en este modelo.Se utilizaron machos Sprague-Dawley (280-290 g) divididos en dos grupos. GI, (Nx5/6) N=15 y G2, (Sham) n= 15, los que fueron sometidos a una operación simulada "sham operation". La duración del experimento fue 24 semanas obteniéndose en el período basal y cada 4 semanas hasta la finalización del estudio, registros de presión arterial (PA) y muestras de sangre y orina. Los resultados indicaron que este modelo de nefrectomía subtotal, produce en 24 semanas un DRC con ascenso de la PA correlacionándose significativamente con el ascenso de la creatininemia
Subject(s)
Animals , Rats , Kidney/physiopathology , NephrectomyABSTRACT
El objetivo de este trabajo fue evaluar las diferentes alteraciones renales morfológicas y funcionales secundarias a la pérdida de la masa renal por nefrectomía subtotal (Nx5/6) e investigar los mecanismos patogénicos involucrados en el daño TI en este modelo.Se utilizaron machos Sprague-Dawley (280-290 g) divididos en dos grupos. GI, (Nx5/6) N=15 y G2, (Sham) n= 15, los que fueron sometidos a una operación simulada "sham operation". La duración del experimento fue 24 semanas obteniéndose en el período basal y cada 4 semanas hasta la finalización del estudio, registros de presión arterial (PA) y muestras de sangre y orina. Los resultados indicaron que este modelo de nefrectomía subtotal, produce en 24 semanas un DRC con ascenso de la PA correlacionándose significativamente con el ascenso de la creatininemia
Subject(s)
Animals , Rats , Nephrectomy , Kidney/physiopathologyABSTRACT
Previously we have shown that release of gamma-aminobutyric acid (GABA) in the stalk-median eminence (S-ME) is high in prepubertal monkeys and that a decrease in GABA release triggers the onset of puberty. However, it is still unclear how disinhibition of the luteinizing hormone releasing hormone (LHRH) neuronal system from GABA input is followed (or accompanied) by an increase in stimulatory signals, such as glutamatergic input to LHRH neurons. To clarify the temporal relationship between the reduction of the GABAergic inhibitory signal and the enhancement of the glutamatergic stimulatory signal in the control of LHRH release at the onset of puberty, we conducted two experiments using a push-pull perfusion method. In the first experiment, we measured developmental changes in release of LHRH, GABA, and glutamate in the S-ME. LHRH levels were very low in prepubertal monkeys, increased to higher levels in early pubertal monkeys, with the highest LHRH levels occurring in mid-pubertal monkeys. As we previously observed, GABA levels were high in prepubertal monkeys and then decreased in early- and mid-pubertal monkeys. In contrast, glutamate levels were very low in prepubertal monkeys, increased dramatically in early pubertal monkeys, and then slightly decreased in mid-pubertal monkeys, although mid-pubertal levels remained much higher than prepubertal levels. In the second experiment, we measured GABA, glutamate and LHRH in the same samples obtained from prepubertal monkeys which were infused with an antisense oligodeoxynucleotide (AS) for glutamic acid decarboxylase (GAD) 67 mRNA into the S-ME. GAD67 is a catalytic enzyme for GABA synthesis from glutamate, and AS GAD67 mRNA interferes with GAD67 synthesis. Infusion of the AS GAD67 induced a decrease in GABA release, which subsequently resulted in an increase in LHRH release. Surprisingly, glutamate release also increased several hours after the decrease in GABA release, and the increased LHRH release continued. These data are interpreted to mean that a decrease in GABA synthesis by interference with GAD67 synthesis and the reduction of GABA release in the S-ME trigger an increase in LHRH release, but that a subsequent increase in glutamate release in the S-ME further contributes to the pubertal increase in LHRH release at the onset of puberty. The data further support our hypothesis that GAD plays an important role in the mechanism of the onset of puberty.
Subject(s)
Glutamates/metabolism , Gonadotropin-Releasing Hormone/metabolism , Sexual Maturation , gamma-Aminobutyric Acid/metabolism , Animals , Female , Glutamate Decarboxylase/genetics , Macaca mulatta , Median Eminence/metabolism , RNA, Antisense/pharmacologyABSTRACT
Previously we have shown that gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter restricting the pubertal increase in LHRH release in juvenile monkeys, and that interfering with GABA synthesis with an antisense oligodeoxynucleotide (AS) for glutamic acid decarboxylase (GAD67) mRNA results in an increase in LHRH release in prepubertal monkeys. GAD67 is a catalytic enzyme that synthesizes GABA from glutamate. To further clarify the role of GABA in puberty, we examined whether the inhibition of LHRH release by GABA continues after the onset of puberty and whether input from glutamatergic neurons plays any role in the onset of puberty when GABA inhibition declines, using a push-pull perfusion method. In Study I, the effects of the AS GAD67 mRNA on LHRH release in pubertal monkeys (34.3 +/- 1.5 months of age, n = 8) were examined, and the results were compared with those in prepubertal monkeys (18.5 +/- 0.4 months, n = 12). Direct infusion of AS GAD67 (1 microM) into the stalk-median eminence (S-ME) for 5 h stimulated LHRH release in both prepubertal and pubertal monkeys. However, the increase in LHRH release in pubertal monkeys was significantly (P < 0.01) smaller than that in prepubertal monkeys. Infusion of a scrambled oligo as a control was without effect in either group. In Study II, to examine the possibility that an increase in glutamate tone after the reduction of an inhibitory GABA tone contributes to the AS GAD67-induced LHRH increase, the effects of the NMDA receptor blocker MK801 (5 microM) on LHRH release were tested in monkeys treated with AS GAD67. MK801 infusion into the S-ME during the treatment of AS GAD67 (1 microM) suppressed the AS GAD67-induced LHRH release in both age groups. MK801 alone did not cause any significant effect in either group. The data are interpreted to mean that GABA continues to suppress LHRH release after the onset of puberty, although the degree of suppression is weakened considerably after the onset of puberty, and that the increased LHRH release after AS GAD67 treatment may be partly due to an increase in glutamate tone mediated by NMDA receptors, as well as due to the decrease in GABA release following the decrease in GAD synthesis. Taken together, the present results suggest that GAD may play an important role in the onset and progress of puberty in nonhuman primates.
Subject(s)
Glutamic Acid/physiology , gamma-Aminobutyric Acid/physiology , Animals , Dizocilpine Maleate/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Female , Glutamate Decarboxylase/genetics , Gonadotropin-Releasing Hormone/metabolism , Injections , Macaca mulatta , Median Eminence/metabolism , Oligonucleotides, Antisense/pharmacology , RNA, Messenger/geneticsABSTRACT
In several species, including humans, circulating insulin-like growth factor I (IGF-I) levels increase during the onset of puberty, suggesting that this peptide contributes to attaining sexual maturity. Because IGF-I elicits LHRH release from the median eminence (ME) of immature female rats in vitro, we hypothesized that it may represent one of the peripheral signals suspected to link somatic development to the LHRH-releasing system at puberty. We now present evidence in support of this concept. Quantitation of IGF-I messenger RNA (mRNA) levels by ribonuclease protection assay revealed that expression of the IGF-I gene did not change in the medial basal hypothalamus or preoptic area of female rats during peripubertal development. In contrast, the contents of both IGF-Ia and IGF-Ib mRNA, the two alternatively spliced forms of the IGF-I gene, increased significantly in the liver during the early proestrous phase of puberty. This change was followed by an elevation in serum IGF-I levels during the late proestrous phase of puberty along with a concomitant increase is serum gonadotropin levels. The proestrous change in serum IGF-I levels was accompanied by a selective increase in IGF-I receptor (IGF-IR) mRNA in the ME. Small doses of IGF-I (2-200 ng), administered intraventricularly, effectively induced LH release in both juvenile and peripubertal female rats, an increase prevented by prior immunoneutralization of LHRH actions. Importantly, intraventricular injections of IGF-I (20 ng), administered twice daily in the afternoon to immature animals, significantly advanced puberty. Thus, these results suggest that IGF-I of peripheral origin contributes to the initiation of female puberty by stimulating LHRH release from the hypothalamus, an effect that appears to be amplified by the increased synthesis of IGF-I receptors in the ME during first proestrus.
Subject(s)
Brain/physiology , Insulin-Like Growth Factor I/physiology , Sex Characteristics , Animals , Female , Hormones/blood , Injections, Intraventricular , Insulin-Like Growth Factor I/genetics , Luteinizing Hormone/metabolism , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Somatomedin/genetics , Reproduction/physiologyABSTRACT
For several years, it has been well accepted that insulin-like growth factor 1 (IGF-1) plays a critical role in peripubertal growth. Recently, we have provided evidence to suggest that this peptide may also be involved in the sexual maturation process, via an action to stimulate hypothalamic luteinizing hormone releasing hormone release. Because ethanol (ETOH) delays puberty, an event that is associated with depressed growth rates and decreased growth hormone and luteinizing hormone (LH) secretion via actions at the hypothalamic level, we investigated whether this drug is capable of altering the expression of genes encoding IGF-1 in liver and brain, as well as the expression of the type 1 IGF receptor (IGF-1R) within the median eminence (ME). Also, we wanted to determine if any regional changes in the expression of these genes were associated with concomitant alterations in the serum levels of IGF-1 and LH. Rats were implanted with gastric cannulae on day 24 and began receiving specific control or ETOH diets on day 29. Rats were killed on day 34, determined to be in the late juvenile stage of development, and their tissues and blood were collected. Results indicate that the ETOH-fed rats showed a decrease (p < 0.01) in the expression of hepatic IGF-1 mRNA when compared with the controls, and this paralleled depressions in both serum IGF-1 (p < 0.01) and LH (p < 0.01). In contrast, no changes were detected in IGF-1 mRNA expression in the preoptic area and hypothalamus, as well as in IGF-1R mRNA expression within the ME. These results suggest that the well-known detrimental effects of ETOH on growth rates and the progression of the female pubertal process in the rat may be associated with the drug's ability to depress the hepatic synthesis of IGF-1 and the subsequent prepubertal circulating levels of the protein.
Subject(s)
Alcoholism/blood , Ethanol/toxicity , Insulin-Like Growth Factor I/genetics , Receptor, IGF Type 1/genetics , Sexual Maturation/drug effects , Alcoholism/genetics , Animals , Body Weight/drug effects , Dose-Response Relationship, Drug , Female , Gene Expression Regulation/drug effects , Median Eminence/drug effects , Preoptic Area/drug effects , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Sexual Maturation/geneticsABSTRACT
This research was designed to determine 1) whether changes occur in the levels of N-methyl-D-aspartic acid (NMDA) receptor (NMDA-R) messenger RNA (mRNA) in the reproductive hypothalamus of female rats as they approach puberty, 2) whether NMDA-R stimulation would promote differential LH responses during the specific stages of peripubertal development, and 3) whether ethanol (ETOH), which is known to affect the NMDA-R in other brain systems, can alter NMDA-R-activated LH secretion at puberty. In the first experiment, female rats were killed at 15, 20, 25, and 34-36 days of age to determine the levels of mRNA that code for the NMDA-R, specifically NMDA-R1, in the arcuate nucleus-median eminence (AN-ME) and preoptic area (POA) during pre- and peripubertal development by a ribonuclease protection assay. Results indicate that in juvenile animals, NMDA-R mRNA levels in the AN-ME increased at 25 days (P < 0.01). In the POA, the levels increased at 20 days (P < 0.05), but were unchanged at 25 days. During the peripubertal period, NMDA-R gene expression in the AN-ME did not change; however, gene expression in the POA increased (P < 0.05) during first proestrus, then declined during first estrus. In the second experiment, NMDA-R stimulation with N-methyl-D,L-aspartic acid (NMA; 2.5 mg/kg) produced differential stimulatory effects on LH release depending upon the stage of pubertal development. In this regard, significant post-NMA percent increases in LH released over pre-NMA (basal) levels occurred during anestrus (46%; P < 0.01) and first proestrus (95%; P < 0.01), with nonsignificant increases of 18% and 28% during first estrus and diestrus, respectively. Finally, a 3 g/kg dose of ETOH given intragastrically 90 min before the NMA challenge blocked (P < 0.05) NMA-induced LH release during first proestrus. In conclusion, these findings demonstrate regional differences in the timing of NMDA-R gene expression in the reproductive hypothalamus during pubertal development, show differential responses of LH to NMDA-R activation during the peripubertal period, and continue to demonstrate the vulnerability of the hypothalamic-pituitary axis to the detrimental effects of ETOH at this critical time of development.
Subject(s)
Ethanol/pharmacology , Luteinizing Hormone/metabolism , RNA, Messenger/metabolism , Receptors, N-Methyl-D-Aspartate/genetics , Sexual Maturation/physiology , Animals , Arcuate Nucleus of Hypothalamus/metabolism , Female , Median Eminence/metabolism , N-Methylaspartate/pharmacology , Preoptic Area/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
Nitric oxide (NO) synthase, the enzyme which converts arginine into citrulline plus NO, a highly active free radical, has been found in many neurons in the brain, including neurons in the hypothalamus. Our previous experiments showed that norepinephrine-induced prostaglandin E2 release from hypothalamic explants incubated in vitro is mediated by NO. Since the release of luteinizing hormone-releasing hormone (LHRH) is also driven by norepinephrine and prostaglandin E2, we hypothesized that NO might also control pulsatile release of LHRH in vivo, resulting in turn in pulsatile release of luteinizing hormone (LH). To ascertain the role of NO in control of pulsatile LH release in vivo, an inhibitor of NO synthase, NG-monomethyl-L-arginine (NMMA), was microinjected into the third cerebral ventricle (1 mg/5 microliters) of conscious castrate male rats at time 0 and 60 min later; blood samples were taken every 10 min during this period. NMMA blocked pulsatile LH release within 20 min, and plasma LH concentration declined further without pulses after the injection at 60 min. Pulsatile release of LH was not altered in diluent-injected controls. NMMA did not alter pulsatile release of follicle-stimulating hormone, which suggests that its release does not require NO. Incubation of medial basal hypothalami with norepinephrine (10 microM) induced an increase in LHRH release that was inhibited by NMMA (300 microM). NMMA alone did not alter basal LHRH release, whereas it was augmented by sodium nitroprusside (100 microM), which releases NO spontaneously. This augmentation was prevented by hemoglobin (2 micrograms/ml), which binds the NO released by nitroprusside. Our previous experiments showed that norepinephrine-induced release of prostaglandin E2 is mediated by NO. Nitric oxidergic neurons were visualized in the median eminence adjacent to the LHRH terminals. The combined in vivo and in vitro results indicate that the pulsatile release of LHRH induced by norepinephrine is brought about by alpha 1-adrenergic activation of NO synthase. NO then induces prostaglandin E2 release that activates exocytosis of LHRH secretory granules into the portal vessels to induce pulsatile LH release.
Subject(s)
Arginine/analogs & derivatives , Cerebral Ventricles/physiology , Gonadotropin-Releasing Hormone/metabolism , Hypothalamus, Middle/metabolism , Luteinizing Hormone/metabolism , Neurons/physiology , Nitric Oxide/physiology , Amino Acid Oxidoreductases/metabolism , Animals , Arginine/administration & dosage , Arginine/pharmacology , Cerebral Ventricles/drug effects , Hypothalamus, Middle/drug effects , Injections, Intraventricular , Interneurons/physiology , Kinetics , Male , Microinjections , Models, Neurological , Neurons/drug effects , Nitric Oxide Synthase , Nitroprusside/pharmacology , Norepinephrine/pharmacology , Orchiectomy , Rats , Rats, Sprague-Dawley , Time Factors , omega-N-MethylarginineABSTRACT
In the present study, we have evaluated the effects of ethanol (ETOH) on luteinizing hormone releasing hormone (LHRH) secretion induced by N-methyl-DL-aspartic acid (NMA) in vitro and the ability of NMA to induce precocious puberty in vivo. For the in vitro experiments, the basal and NMA-stimulated release of LHRH from arcuate nucleus-median eminence (AN-ME) fragments obtained from immature female rats was measured by RIA following static incubation in medium consisting of Krebs-Ringer bicarbonate glucose buffer. Control vials contained medium only and the test vials contained medium plus ETOH doses of 30, 50, and 70 mM. These data demonstrate that ETOH did not alter basal LHRH release, but dose-dependently blocked (p < 0.01) the NMA-induced release of the peptide during anestrus, as well as first proestrus and estrus. For the in vivo experiment, 26-day-old females began receiving saline or saline-ETOH (3 g/kg) solution by gastric gavage daily at 12.30 p.m. Each day at 2.00 and 4.00 p.m., each of the animals received subcutaneous injections of a 40-mg/kg solution of NMA in saline. Other control animals received saline gastrically, as well as subcutaneously. The timing of puberty was assessed in all animals by monitoring vaginal opening (VO) and first diestrus (D1). The mean (+/- SEM) age at VO for animals in the non-ETOH group, which received NMA, was 31.7 +/- 0.40 days. Vaginal smears revealed that D1 occurred at a mean (+/- SEM) age of 33.0 +/- 0.42 days.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Ethanol/pharmacology , Gonadotropin-Releasing Hormone/metabolism , N-Methylaspartate/analogs & derivatives , Sexual Maturation/drug effects , Anestrus , Animals , Arcuate Nucleus of Hypothalamus/metabolism , Diestrus , Female , In Vitro Techniques , Median Eminence/metabolism , N-Methylaspartate/pharmacology , Radioimmunoassay , Rats , Rats, Sprague-Dawley , Time Factors , Vagina/physiologyABSTRACT
Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to alter testicular function. However, its effect on the efficiency of spermatogenesis or on Leydig cell volume has not been determined in adult rats. In two replicas, adult male rats received a single intraperitoneal injection of TCDD at a rate of 0, 12.5, 25.0, or 50.0 micrograms/kg body weight. Rats were sacrificed 4 weeks after treatment. The cytosolic Ah receptor in the testis was estimated at 10.3 +/- 1.2 fmol/mg protein in these adult rats. The presence of the Ah receptor at this concentration in the testis reveals that the testis is a possible target organ for TCDD-induced responses. Left testes were homogenized and testicular spermatids were counted by phase contrast cytometry to determine daily sperm production. Right testes were vascularly perfused with glutaraldehyde, embedded in Epon 812, sectioned at 0.5 micron, stained with toluidine blue and evaluated by stereology for germ cells or Leydig cells. Body weight was reduced (P < 0.01) in a dose-dependent fashion. Testicular weight and daily sperm production per testis were not significantly reduced by TCDD. Androgen receptor concentrations in the testis and prostate were not affected. Weights of two androgen-sensitive organs (seminal vesicles and epididymis) were reduced (P < 0.01) in a dose-dependent fashion and serum concentrations of testosterone were reduced in a dose-dependent fashion in Replica 2. Due to low numbers of animals in Replica 1, the reduced Leydig cell volume was not significant after TCDD treatment; however, in Replica 2 there was a dose-dependent reduction (P < 0.01) in volume per testis of Leydig cell cytoplasm, nuclei, or total Leydig cell volume. Production of testosterone was sufficient to maintain spermatogenesis quantitatively; however, TCDD caused a dose-dependent reduction in Leydig cell function and Leydig cell volume per testis. This study showed for the first time that TCDD-induced androgen deficiency of male rats may be explained by the loss of total volume of Leydig cell cytoplasm. This study also further illustrates the reserve capacity of Leydig cell function to maintain spermatogenesis when the volume of these cells is significantly reduced.
Subject(s)
Leydig Cells/drug effects , Polychlorinated Dibenzodioxins/toxicity , Receptors, Androgen/drug effects , Spermatogenesis/drug effects , Testis/drug effects , Animals , Body Weight/drug effects , Cell Count/drug effects , Dose-Response Relationship, Drug , Epididymis/drug effects , Leydig Cells/physiology , Leydig Cells/ultrastructure , Male , Microscopy, Electron , Organ Size/drug effects , Prostate/drug effects , Rats , Rats, Sprague-Dawley , Seminal Vesicles/drug effects , Spermatozoa/drug effects , Spermatozoa/physiology , Testosterone/bloodABSTRACT
In a randomized double-blind study we evaluated the effects on cervical ripening and labor induction of 0.5 mg PGE2 in gel given intracervically and 2.0 mg PGE2 given as a vaginal suppository. All patients were at term with unfavorable cervical scores. The indications for induction were toxemia, diabetes mellitus, Rh-immunization, or intrauterine growth retardation. Significantly better results for both cervical priming and labor induction were obtained after intracervical PGE2-gel application than after treatment with placebo or vaginal suppositories. Eleven out of 19 patients (58%) were delivered within 24 h after intracervical PGE2-gel compared to two out of 19 patients given placebo (p less than 0.01). In patients not delivered 24 h after the start of treatment, the mean cervical score had changed from 3.7 to 6.0 (p less than 0.05) after PGE2-gel application compared to a change from 3.9 to 4.3 after placebo treatment (n.s.). The outcome after treatment with PGE2 suppositories did not differ significantly from that with placebo treatment. In a subsequent study 25 patients were given 0.5 mg PGE2-gel intracervically. The results were consistent with those obtained in patients receiving PGE2-gel intracervically in the double-blind study. Few side effects were noted. No patient complained of gastro-intestinal discomfort but increased myometrial activity was observed in two patients; one after placebo and the other after active intracervical PGE2-gel treatment. The hyperactivity was readily countered with the beta 2-agonist, terbutaline. All infants were born in good condition with Apgar scores of 7 or more within 5 min. At pediatric examinations at 1 week and at 6 months of age all children seemed healthy.