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A large body of information now supports the existence of a complete intraovarian insulin-like growth factor (IGF) system replete with ligands, receptors, and binding proteins. Although much remains to be learned, the emerging consensus would suggest that the intraovarian IGF system is concerned largely with the amplification of gonadotrophin hormonal action for the facilitation of follicular growth and development. Future studies are likely to address the central issue of indispensability and the documentation of a meaningful in vivo role for this and related putative intraovarian regulators. [ABSTRACT FROM AUTHOR]

Study Question: Can artificial intelligence (AI) algorithms developed to assist embryologists in evaluating embryo morphokinetics be enriched with multi-centric clinical data to better predict clinical pregnancy outcome?, Summary Answer: Training algorithms on multi-centric clinical data significantly increased AUC compared to algorithms that only analyzed the time-lapse system (TLS) videos., What Is Known Already: Several AI-based algorithms have been developed to predict pregnancy, most of them based only on analysis of the time-lapse recording of embryo development. It remains unclear, however, whether considering numerous clinical features can improve the predictive performances of time-lapse based embryo evaluation., Study Design, Size, Duration: A dataset of 9986 embryos (95.60% known clinical pregnancy outcome, 32.47% frozen transfers) from 5226 patients from 14 European fertility centers (in two countries) recorded with three different TLS was used to train and validate the algorithms. A total of 31 clinical factors were collected. A separate test set (447 videos) was used to compare performances between embryologists and the algorithm., Participants/materials, Setting, Methods: Clinical pregnancy (defined as a pregnancy leading to a fetal heartbeat) outcome was first predicted using a 3D convolutional neural network that analyzed videos of the embryonic development up to 2 or 3 days of development (33% of the database) or up to 5 or 6 days of development (67% of the database). The output video score was then fed as input alongside clinical features to a gradient boosting algorithm that generated a second score corresponding to the hybrid model. AUC was computed across 7-fold of the validation dataset for both models. These predictions were compared to those of 13 senior embryologists made on the test dataset., Main Results and the Role of Chance: The average AUC of the hybrid model across all 7-fold was significantly higher than that of the video model (0.727 versus 0.684, respectively, P = 0.015; Wilcoxon test). A SHapley Additive exPlanations (SHAP) analysis of the hybrid model showed that the six first most important features to predict pregnancy were morphokinetics of the embryo (video score), oocyte age, total gonadotrophin dose intake, number of embryos generated, number of oocytes retrieved, and endometrium thickness. The hybrid model was shown to be superior to embryologists with respect to different metrics, including the balanced accuracy (P ≤ 0.003; Wilcoxon test). The likelihood of pregnancy was linearly linked to the hybrid score, with increasing odds ratio (maximum P-value = 0.001), demonstrating the ranking capacity of the model. Training individual hybrid models did not improve predictive performance. A clinic hold-out experiment was conducted and resulted in AUCs ranging between 0.63 and 0.73. Performance of the hybrid model did not vary between TLS or between subgroups of embryos transferred at different days of embryonic development. The hybrid model did fare better for patients older than 35 years (P < 0.001; Mann-Whitney test), and for fresh transfers (P < 0.001; Mann-Whitney test)., Limitations, Reasons for Caution: Participant centers were located in two countries, thus limiting the generalization of our conclusion to wider subpopulations of patients. Not all clinical features were available for all embryos, thus limiting the performances of the hybrid model in some instances., Wider Implications of the Findings: Our study suggests that considering clinical data improves pregnancy predictive performances and that there is no need to retrain algorithms at the clinic level unless they follow strikingly different practices. This study characterizes a versatile AI algorithm with similar performance on different time-lapse microscopes and on embryos transferred at different development stages. It can also help with patients of different ages and protocols used but with varying performances, presumably because the task of predicting fetal heartbeat becomes more or less hard depending on the clinical context. This AI model can be made widely available and can help embryologists in a wide range of clinical scenarios to standardize their practices., Study Funding/competing Interest(s): Funding for the study was provided by ImVitro with grant funding received in part from BPIFrance (Bourse French Tech Emergence (DOS0106572/00), Paris Innovation Amorçage (DOS0132841/00), and Aide au Développement DeepTech (DOS0152872/00)). A.B.-C. is a co-owner of, and holds stocks in, ImVitro SAS. A.B.-C. and F.D.M. hold a patent for 'Devices and processes for machine learning prediction of in vitro fertilization' (EP20305914.2). A.D., N.D., M.M.F., and F.D.M. are or have been employees of ImVitro and have been granted stock options. X.P.-V. has been paid as a consultant to ImVitro and has been granted stocks options of ImVitro. L.C.-D. and C.G.-S. have undertaken paid consultancy for ImVitro SAS. The remaining authors have no conflicts to declare., Trial Registration Number: N/A., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Objective: To determine whether inducing ovulation with a GnRH agonist in patients with polycystic ovaries (PCO) would permit oocyte retrieval without the burden or risk of cancellation, coasting, or ovarian hyperstimulation syndrome (OHSS), thus maintaining pregnancy rates by allowing embryo cryopreservation for transfer in a subsequent cycle., Design: Retrospective observational study., Setting: Private institution., Patient(s): Forty-two women who had previously experienced a controlled ovarian hyperstimulation (COH)/IVF cycle that had to be cancelled because of an elevated risk of OHSS., Intervention(s): Forty-two PCO patients with a previous cancelled IVF cycle were assigned to a second controlled ovarian stimulation with recombinant FSH (75-150 IU/day) + GnRH antagonist (0.25 mg/day). Embryos were cryopreserved and transferred in a later cycle., Main Outcome Measure(s): OHSS, oocyte retrieval, and pregnancy rates., Result(s): In the first COH, the cycle had to be cancelled to avoid OHSS because E(2) serum levels were above safety levels (4809.6 +/- 2947.7). However, in the second cycle (ovulation triggered with a GnRH agonist) and independent of E(2) serum levels (4518.5 +/- 2118.85), all PCO patients eventually completed oocyte retrieval and frozen ET. With regard to pregnancy rates, 33% of patients receiving a transfer of a previously frozen embryo were successful. No patient developed OHSS., Conclusion(s): Triggering ovulation with a GnRH agonist followed by embryo cryopreservation allows PCO patients to complete a COH/IVF cycle with no cycle cancellation, coasting, or OHSS and, finally, to attain good pregnancy rates., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

We retrospectively studied 429 IVF donor cycles in which ovulation was triggered with either hCG (175 cycles) or GnRH agonist (254 cycles). Of the donors in whom ovulation was triggered with hCG, 3.2% developed symptoms of moderate (2.2%) or severe (1%) ovarian hyperstimulation syndrome, while none of the IVF donor cycles that were triggered with the GnRH agonist presented ovarian hyperstimulation syndrome, needed coasting, or were cancelled.

Objective: To determine whether including a GnRH antagonist in controlled ovarian stimulation-intrauterine insemination cycles would increase pregnancy rates., Design: Prospective randomized study., Setting: Private reproductive medicine clinic in Spain., Patient(s): Three hundred sixty-seven women with primary or secondary infertility., Intervention(s): Patients were randomly assigned to controlled ovarian stimulation with recombinant FSH (75-150 IU/d) alone (controls, n = 183) or with recombinant FSH (75-150 IU/d) + the GnRH antagonist (0.25 mg/d), initiated when the recruited follicles were >or=16 mm (n = 184). A single insemination was performed, 36-38 hours after hCG (5,000 IU, IM), in both groups., Main Outcome Measure(s): Follicular recruitment, pregnancy rates., Result(s): Numbers of mature follicles (2.4 +/- 1.3 vs. 1.3 +/- 1.09) and clinical pregnancy rates (23% vs. 11%) were statistically significantly higher in patients who were treated with GnRH antagonist than in those who were in the control group. The pregnancy rate was only higher in the antagonist group if more than one follicle sized >or=18 mm was present on the day that the hCG was given. A similar number of twin pregnancies occurred in both groups: two in the antagonist group and three in the control group. The antagonist group also had one triplet gestation., Conclusion(s): Adding GnRH antagonist to controlled ovarian stimulation-intrauterine insemination cycles significantly increases pregnancy rates in multifollicular, but not monofollicular, cycles.

Objective: To evaluate the implant capacity of embryos derived from oocytes matured with a bolus of GnRH agonist., Design: Donors were randomly assigned to a protocol using either GnRH agonist or recombinant (r) hCG to trigger ovulation. Analysis of variance, Student t test, and Fisher exact test were used where appropriate., Setting: Private clinical setting., Patient(s): Young voluntary donors receiving GnRH agonist (n = 30) or rhCG (n = 30). Eighty-nine patients received oocytes., Intervention(s): Controlled ovarian stimulation was carried out with GnRH antagonist and FSH/LH in a step-down protocol. Donors received a single bolus of GnRH agonist (0.2 mg) or rhCG (250 microg). The endometrial tissue of recipient patients was prepared with oral E(2) and P., Main Outcome Measure(s): Pregnancy and implantation rates and ovarian hyperstimulation syndrome (OHSS) in an IVF donor program., Result(s): No significant differences in the number of retrieved oocytes (327 vs. 288), MII oocytes (70% vs. 76%), fertilization (80% vs. 65%,), pregnancy/transfer (55% vs. 59%), and implantation rates (29% vs. 32%) were found between recipients whose embryos originated from donors in whom final oocyte maturation was triggered with GnRH agonist and those whose donors received hCG. Significant differences in luteal phase length (4.16 + 0.70 days vs. 13.63 + 2.12 days) and in OHSS (0/30 vs. 5/30) were seen between donors ovulated with the agonist and the donors in whom ovulation was triggered with hCG., Conclusion(s): In controlled ovarian stimulation IVF donor cycles, GnRH agonists trigger ovulation and induce luteolysis but do not compromise embryo implantation capacity.

Objective: To determine whether LH supplementation improved pregnancy and implantation rates in GnRH antagonist donor cycles., Design: Donors were randomly assigned to a protocol using GnRH antagonist (GnRH-a) alone or GnRH-a + recombinant LH. Analysis of variance, Student's t-test and Fisher's exact test were used where appropriate., Setting: Private clinical setting., Patient(s): Young voluntary donors with antagonist (n = 20) and antagonist + LH (n = 22). Fifty-five patients received oocytes., Intervention(s): Donors received the GnRH-a (Cetrorelix, 0.25 mg/day) alone or in combination with recombinant LH (75 IU/day). Ovulation induction was carried out with recombinant FSH in a step-down protocol. The endometrial tissue of recipient patients was prepared with oral E(2) and P., Main Outcome Measure(s): Pregnancy and implantation rates in a donor program., Result(s): A significant increase in MII oocyte (80% vs. 71%), fertilization rates (83% vs. 71%), G1 embryos (17% vs. 3%), and implantation rates (35% vs. 15%), were found in recipients whose embryos originated from donors receiving GnRH-a + recombinant LH as compared to donors receiving GnRH-a alone. Estradiol levels, pregnancy/transfer and clinical pregnancies were lower (not significant) in donors treated with the GnRH-a alone vs. those receiving the recombinant LH-supplemented GnRH-a., Conclusion(s): The LH supplementation improved the possibilities of gestation for recipients whose embryos originated from GnRH-a-treated donors.

Objective: To report two cases of monozygotic twinning after IVF-blastocyst transfer., Design: Case report., Setting: Private practice in an assisted reproductive technology clinic., Patient(s): Two women treated with IVF-ET at the blastocyst stage., Intervention(s): Pituitary down-regulation with luteal leuprolide acetate, ovulation induction with gonadotropins, IVF, sequential culture, blastocyst transfer, and P for luteal support., Main Outcome Measure(s): Levels of hCG, pelvic ultrasound examination, amniocentesis, obstetric follow-up, and cesarean section., Result(s): Two intrauterine monozygotic twin pregnancies occurred after IVF and blastocyst transfer. One of them was complicated by fetus-to-fetus transfusion syndrome and was delivered preterm by cesarean section; the other woman had a normal pregnancy and vaginal delivery., Conclusion(s): Monozygotic multiple gestations may be increased in IVF blastocyst transfers. The potential obstetric complications of this type of pregnancy should be discussed with patients.

Objective: To assess the in vivo regulation of ovarian insulin-like growth factor binding protein-4 (IGFBP-4) mRNA expression by gonadotropins and estrogen., Methods: Whole ovarian RNA, obtained from two models of follicular development, was extracted and analyzed by Northern blotting. Immature rats were treated with pregnant more serum gonadotropin (PMSG) followed 48 hours later with hCG, or alternatively were hypophysectomized and treated with FSH and/or diethylstilbestrol (DES). Localization of IGFBP-4 expression was assessed in the former study by in situ hybridization. Finally, the ability of human IGFBP-4 to antagonize FSH-stimulated progesterone accumulation was assessed in vitro., Results: The ovarian content of IGFBP-4 transcripts increased threefold (P < .05) at 12 hours after PMSG but was near baseline at 24 and 48 hours. The abundance of IGFBP-4 mRNA increased (P < .05) again at 6 and 24 hours after hCG. The expression of IGFBP-4 was localized to granulosa cells of preantral (untreated) and small antral (12 hours after PMSG) follicles. No IGFBP-4 expression was noted in large (gonadotropin-primed) antral follicles. Hypophysectomy increased (P < .05) the ovarian content of IGFBP-4 mRNA by 1.5-fold, an effect further enhanced (1.8-fold; P < .05) by the provision of FSH and DES. In vitro studies revealed the ability of increasing concentrations (0.01-1 microgram/mL) of recombinant human IGFBP-4 to inhibit the FSH-supported accumulation of progesterone., Conclusion: Increased expression after administration of PMSG, hCG, and FSH/DES suggests that IGFBP-4 is a dynamic and hormonally responsive component of the ovarian cycle. The lack of expression in preovulatory follicles and its antigonadotropic actions in vitro imply that the attenuated expression of IGFBP-4 may constitute a requirement for successful follicular maturation.

We evaluated the effects of gestational weight gain on neonatal birthweight women in whom gestational diabetes mellitus (GDM) was diagnosed after the 32nd week of gestation. The prevalence of macrosomia, the birthweight differences from 50th percentile value of a reference population, and the relationships among plasma glucose values during oral glucose tolerance test and neonatal birthweight were evaluated in 60 newborns from mothers with gestational diabetes mellitus divided according to pregravid body-mass index. Serving as controls were 132 newborns of mothers with normal glucose tolerance. The prevalence of macrosomia was higher in the GDM group; the neonatal birthweight difference above 50th percentile value was higher in newborns of mothers with GDM; and a strong relationship between maternal gestational weight gain and neonatal birth weight was present in all pregnant women. In conclusion, (1) the gestational weight gain is a good predictor of neonatal birth weight in all pregnant women; (2) GDM enhances the increase in neonatal size induced by excessive gestational weight gain alone, and (3) a weight gain of more than 9 kg makes the relative risk of macrosomia twofold higher in GDM than in control mothers.

Most but not all of the established insulin-like growth factor-binding proteins (IGFBPs) are expressed in the rat ovary. To continue the process of characterizing these ovarian IGFBPs, a solution hybridization/RNase protection assay was used to study IGFBP-6 gene expression, cellular localization, and hormonal regulation in the immature rat ovary. Total RNA was hybridized with a 458-base long 32P-labeled rat IGFBP-6 cRNA. A single protected fragment (380 bases long) corresponding to IGFBP-6 transcripts was identified in RNA from ovary and lung, but not kidney or liver. The amount of IGFBP-6 transcripts was higher in ovaries from immature rats (25-28 days old) than in ovaries from adult rats and was higher in theca-interstitial than in granulosa cell preparations. Hypophysectomy resulted in a significant (P < 0.05) 2.3 +/- 0.7-fold (mean +/- SD) increase in whole ovarian IGFBP-6 transcripts. This suggests that ovarian IGFBP-6 gene expression is subject to inhibition by one or more pituitary hormones. Therefore, the effect of replacement of FSH, GH, diethylstilbestrol (DES), or combinations thereof was evaluated. Treatment with FSH resulted in a 2.4-fold decrease (P < 0.05) in the abundance of ovarian IGFBP-6 transcripts relative to that in hypophysectomized controls. Provision of DES yielded comparable results. Moreover, the combined provision of FSH and DES resulted in a synergistic decrease (6.0-fold) in ovarian IGFBP-6 transcripts. In contrast, treatment of hypophysectomized rats with GH proved without effect. However, treatment with FSH or DES in addition to GH resulted in a decrease in ovarian IGFBP-6 transcripts (3.9- and 2.7-fold, respectively). To assess the role of ovarian IGFBP-6, its influence on gonadotropin action in primary cultures of rat granulosa cells was also studied. Increasing concentrations (0.01-1 micrograms/ml) of recombinantly expressed human IGFBP-6 did not inhibit the FSH-supported accumulation of either progesterone or estradiol. These findings 1) establish the theca-interstitial compartment of the immature rat ovary as a prominent site of IGFBP-6 gene expression, 2) implicate FSH and DES as inhibitors of IGFBP-6 transcript levels in the whole ovary, 3) and disclose the limited antigonadotropic action of IGBP-6F on the rat granulosa cell.

Objective: To assess possible interfacing between the somatotrophic and reproductive axes., Design: Literature review., Main Outcome Measures: Ovarian growth hormone reception and action., Results: The available literature strongly supports a permissive role for the somatotrophic axis in the reproductive process., Conclusions: Although a role for growth hormone in reproductive biology appears highly likely, its relevance to the process of puberty and to the normal workings of the menstrual cycle, as well as its possible application in reproductive pathology must await further investigation.

Endothelin (ET)-1, is a 21 amino acid vasoactive peptide subject to regulation by cellular oxygen tension. However, an increasing body of information now suggests that ET-1 is a multifunctional peptidergic regulator the actions of which are not limited to the vascular system. Although ET-1 has been shown to inhibit the gonadotropin-supported accumulation of progesterone by cultured granulosa cells, the precise cellular mechanism(s) involved remain unknown. It was therefore the objective of this study to examine in greater detail the effects of ET-1 on progestin economy in cultured granulosa cells from immature rats. Treatment with ET-1 was inhibitory to the FSH-supported accumulation of progesterone in a dose-dependent manner, an action characterized by a median inhibitory dose of 2 x 10(-11) M and a maximal inhibitory effect of 90%. This inhibitory action of ET-1 was reversible following extensive washing and could not be accounted for by a decrease in the viable cell mass. Evaluation of the activities of progesterone-forming enzymes revealed ET-1 to be a potent (P < 0.01) inhibitor of cholesterol side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase (HSD)/isomerase (76.1 +/- 1.2% and 47.3 +/- 8.6% inhibition, respectively). Cellular radiolabeling with [3H]pregnenolone confirmed an ET-1-induced inhibition of the FSH-supported accumulation of radiolabeled progesterone. However, this effect was concomitant with enhancement of the accumulation of more distal metabolites, i.e. 20 alpha-dihydroprogesterone, 5 alpha-pregnane-3 alpha, 20 alpha-diol, and 5 alpha-pregnane-3 alpha-ol-20-one. Analysis of the FSH-supported activities of the progesterone-degrading enzymes revealed ET-1 as a potent (P < 0.05) stimulator of 20 alpha-HSD and 5 alpha-reductase (3.6 +/- 1.0 and 1.7 +/- 0.3-fold, stimulation respectively). In contrast, no significant changes were observed in 3 alpha-HSD activity. Taken together, our findings demonstrate that the ET-1 induced inhibition of gonadotropin-supported progesterone accumulation constitutes a complex phenomenon wherein ET-1 inhibits the activities of steroidogenic enzymes concerned with progesterone formation while enhancing the activities of enzymes concerned with progesterone degradation. We speculate that ET-1, possibly of intraovarian origin, acts as a luteinization-inhibitor to suppress premature luteinization at a time when continued preovulatory expression of ET-1 (in the intact but not ruptured follicle) may be contingent upon relative intrafollicular hypoxia.

An increasing body of information now suggests that insulin-like growth factor (IGF) binding proteins (BPs) may serve as antigonadotropins at the level of the ovary. It is the objective of the present communication to evaluate the functional role of endogenous (granulosa cell-derived) IGFBPs by exploiting the unique properties of des(1-3)IGF-I, a naturally occurring IGF-I analogue characterized as a weak ligand of IGFBPs but not of type I IGF receptors. Given IGFBP-replete circumstances, des(1-3)IGF-I proved more potent (10-fold) than its intact counterpart in promoting the follicle stimulating hormone (FSH)-stimulated accumulation of progesterone by cultured rat granulosa cells. In contrast, des(1-3)IGF-I proved virtually equipotent to the unmodified principle under IGFBP-deplete circumstances. Taken together, these findings are in keeping with the notion and that the apparently enhanced potency of des(1-3)IGF-I (under IGFBP-replete conditions) is due to its diminished affinity for endogenously generated IGFBPs and that rat granulosa cell-derived IGFBPs are inhibitory to IGF (and thus inevitably to gonadotropin) hormonal action. Accordingly, the reported ability of gonadotropins to attenuate IGFBP release by granulosa cells may be designed to enhance the bioavailability of endogenously generated IGFs in the best interest of ovarian steroidogenesis.

To delineate the scope of the human intraovarian IL-1 system we used a solution hybridization/RNase protection assay to test for expression of the genes encoding IL-1, its type I receptor (IL-1R), and its receptor antagonist (IL-1RA). IL-1 transcripts were not detected in whole ovarian material from days 4 or 12 of an unstimulated menstrual cycle but transcripts (IL-1 beta much greater than IL-11 alpha) were detected in preovulatory follicular aspirates from gonadotropin-stimulated cycles. Concurrently obtained peripheral monocytes did not contain IL-1 beta transcripts but macrophage-depleted follicular aspirates did, thus implicating the granulosa cells as the site of IL-1 expression. IL-1R transcripts were detected in RNA from whole ovaries and follicular aspirates but not in RNA from peripheral monocytes. IL-1RA transcripts were detected in whole ovarian material as well as in macrophage-free follicular aspirates. Cultured human granulosa and theca cells did not contain mRNA for IL-1 beta or IL-1RA but did contain mRNA for IL-1R. Treatment of cell cultures with forskolin (25 microM) induced IL-1 beta transcripts in granulosa but not theca cells. Forskolin also increased the basal levels of IL-1R transcripts in both granulosa and theca cells but did not induce IL-RA transcripts in either cell type. Taken together, these findings reveal the existence of a complete, highly compartmentalized, hormonally dependent intraovarian IL-1 system replete with ligands, receptor, and receptor antagonist.

To further the identification and characterization of insulin-like growth factor binding proteins at the level of the immature rat ovary, we have set out to study the ovarian expression, cellular localization, and hormonal regulation of the insulin-like growth factor binding protein (IGFBP)-3. To this end, use was made of a solution hybridization/RNAse protection assay wherein ovarian total RNA from immature (21-23 days old) female rats was hybridized with a 343 bases-long [32P]-labeled rat IGFBP-3 riboprobe. As in liver, a single protected fragment (315 bases-long) corresponding to IGFBP-3 transcripts was identified in whole ovarian material. Cellular localization studies revealed the IGFBP-3 gene to be exclusively expressed in the theca-interstitial rather than the granulosa cell compartment. To confirm presence and cellular distribution of the IGFBP-3 protein, media conditioned by cultured granulosa cells, theca-interstitial cells, and whole ovarian dispersates were subjected to Western Ligand Blotting. Importantly, media conditioned by cultured theca-interstitial (but not granulosa) cells displayed an IGFBP the size of rat IGFBP-3 (46kDa) as determined by comigration with a rat serum standard. A similarly-sized band was apparent in media conditioned by cultured whole ovarian dispersates reflecting in all likelihood the contribution of the theca-interstitial cell component. Significantly, deglycosylation of media conditioned by cultured theca-interstitial cells revealed the glycosylated nature of the 46kDa IGFBP species as judged by the apparent reduction in its molecular size to 35kDa. Similar alterations were noted in corresponding rat serum samples. Hypophysectomy of immature rats resulted in a modest but statistically insignificant decrease in the relative (densitometrically-quantified) abundance of ovarian IGFBP-3 transcripts, an effect further augmented by the systemic provision of either FSH or diethylstilbestrol (DES). In contrast, systemic treatment of hypophysectomized rats with GH produced a marked (3.2-fold) increase (P less than 0.05) in the steady state levels of ovarian (as well as hepatic) IGFBP-3 transcripts. However, the concurrent provision of either FSH or DES resulted in substantial (P less than 0.05) attenuation (78 and 57% inhibition, respectively) of the upregulatory GH effect. These findings document the highly compartmentalized expression of the IGFBP-3 gene at the level of the immature rat ovary, implicate the theca-interstitial cell as the sole source of its generation, reveal its pituitary dependence, and disclose its diametrically-opposed (indeed antagonistic) regulation by FSH (or estrogens) and GH.(ABSTRACT TRUNCATED AT 400 WORDS)

An increasing body of information now suggests the existence of a complete intraovarian IL-1 system replete with ligands, receptor, and receptor antagonist. Since IL-1 is an established mediator of inflammation and since ovulation may constitute an inflammatory-like reaction, consideration may be given to the possibility that IL-1 may play an intermediary role in the ovulatory process. To further assess the above hypothesis, we have set out to determine whether IL-1 is capable of promoting ovarian prostaglandin biosynthesis, an established component of the ovulatory cascade. Cultured whole ovarian dispersates from immature (25 day old) rats constitutively elaborated major prostaglandin species (PGE2 greater than PGF2 alpha) in a cell density-dependent fashion. Treatment with IL-1 produced dose-dependent increments in prostaglandin (PGE2 greater than PGF2 alpha) accumulation as compared with untreated controls. Comparable cellular densities of untreated or IL-1 beta-treated whole ovarian dispersates elaborated substantially more PGE2 as compared with isolated granulosa or theca-interstitial cell preparations suggesting a requirement for cell-cell interaction. Indeed, cell contact-dependent reconstitution experiments involving isolated granulosa and theca-interstitial cells at a projected physiologic ratio of 4:1 revealed synergistic interactions in the elaboration of PGE2 under both basal and IL-1 beta-treated circumstances. Identical results were obtained for cell contact-independent heterologous (but not homologous) coculture experiments. Taken together, our present findings reveal optimal basal and IL-1-stimulated ovarian prostaglandin (PGE2 greater than PGF2 alpha) biosynthesis to require heterologous, contact-independent, presumably humorally-mediated, cell-cell interaction. These observations along with the demonstration of the gonadotropin-dependent preovulatory induction of ovarian IL-1 beta gene expression provide strong support for the view that IL-1 may be the centerpiece of an intraovarian regulatory loop concerned with the promotion of the preovulatory cascade.

The intraovarian insulin-like growth factor (IGF) system constitutes a triad composed of ligands, receptors, and binding proteins. Although conventional radioligand receptor assays have documented the presence of specific receptors for insulin and insulin-like peptides in some rat somatic ovarian cell types, the exact cellular localization and hormonal regulation of the receptors in question remain matters of inquiry. To reevaluate the very presence, cellular localization, and hormonal regulation of the IGF receptor gene family in the rat ovary, solution hybridization/RNase protection assays were used wherein ovarian total RNA (20 micrograms) from immature (21-23 days old) rats was hybridized with 32P-labeled type I IGF receptor, type II IGF/mannose-6-phosphate receptor, and insulin receptor riboprobes. Single protected fragments 261 (type I IGF receptor), 500 (type II IGF/mannose-6-phosphate receptor), and 478 (insulin receptor) bases long were evident in whole ovary, granulosa, and theca-interstitial cells. Hypophysectomy of immature rats led to significant (P less than 0.05) albeit variable decrements in the relative (densitometrically quantified) ovarian abundance of transcripts corresponding to the type I IGF (but not insulin or type II IGF/mannose-6-phosphate) receptor. Treatment of immature hypophysectomized rats with FSH (10 micrograms/rat.day x 2.5 days) resulted in a significant (P less than 0.05) increase (4-fold) in transcripts corresponding to the type I IGF receptor in both whole ovarian material and freshly isolated granulosa cells. Similar (3.7-fold) increments (P less than 0.05) were noted after treatment with a diethylstilbestrol-containing sc silastic implant applied for a total of 5 days.(ABSTRACT TRUNCATED AT 250 WORDS)

We, and others have recently demonstrated the ovary to be a site of interleukin-1 (IL-1) reception and action. Since IL-1 is an established mediator of inflammation and since ovulation may constitute an inflammatory-like reaction, consideration was given to the possibility that IL-1 may play an intermediary role in the ovulatory process. To begin to evaluate the above hypothesis, we have set out to evaluate rat ovarian IL-1 beta gene expression, to determine its cellular localization, and to study its modulation by key endocrine and autocrine regulatory signals. To this end, use was made of a solution hybridization/RNase protection assay in which rat ovarian total RNA (20 micrograms) was hybridized with a [32P]-labeled 272 base rat IL-1 beta antisense riboprobe. To assess rat ovarian IL-1 beta gene expression under in vivo circumstances, use was made of an established experimental model capable of simulating naturally-occurring follicular maturation, ovulation, and corpus luteum formation. Specifically, a single subcutaneous injection of PMSG (15IU/rat) was followed (48h) later by an ovulatory dose (15IU) of human chorionic gonadotropin (hCG). A faint protected fragment 222 bases long corresponding to the IL-1 beta message was detectable in whole ovarian material prior to gonadotropic stimulation. Treatment with PMSG for 48h resulted in a modest, albeit measurable increase in the densitometrically-quantified steady state levels of the ovarian IL-1 beta message. Most striking, however, were the increments noted in the relative abundance of ovarian IL-1 beta transcripts following a 6h exposure to hCG producing a 4 to 5-fold increase (P less than 0.05) over the untreated state at a time point approximately 6h prior to projected follicular rupture. Subsequent evaluation of ovarian IL-1 beta transcripts, 24 and 48h following hCG administration, revealed significant (P less than 0.05) decrements (relative to the 6h peak) to a level comparable to that seen at the conclusion of 48h of treatment with PMSG. Cellular localization studies revealed the gonadotropin-dependent IL-1 beta mRNA to be theca-interstitial cell-exclusive. To assess rat ovarian IL-1 beta gene expression under in vitro circumstances, we have set out to determine whether IL-1 itself may influence the relative level of its own message. Treatment of whole ovarian dispersates with rhIL-1 beta (10ng/ml) for 4 and 24h resulted in a marked (P less than 0.05) time-dependent increase (up to 12-fold) in the relative abundance of IL-1 beta transcripts when compared with untreated controls.(ABSTRACT TRUNCATED AT 400 WORDS)

To begin the process of identification and charactization of rat ovarian insulin-like growth factor binding proteins, we have undertaken to explore the ovarian expression, cellular localization, and hormonal regulation of the insulin-like growth factor binding protein-2 (IGFBP-2) gene for which an antigonadotropic potential has recently been demonstrated. To this end, a solution hybridization/RNase protection assay was employed wherein total ovarian RNA (20 micrograms) from immature (21-23 days old) female rats was hybridized with a [32P]-labeled IGFBP-2 riboprobe. As in liver, a single protected fragment (550 bases long) corresponding to IGFBP-2 transcripts was identified in whole ovarian material. Cellular localization studies revealed the IGFBP-2 gene to be exclusively expressed in the theca-interstitial rather than the granulosa cell compartment. To confirm the cellular distribution of the IGFBP-2 protein, media conditioned by cultured granulosa or theca-interstitial cells were subjected to immunoprecipitation using two IGFBP-2-directed polyclonal antisera. Expectedly, both antibodies (but not non-immune rabbit serum) readily immunoprecipitated the 28 kDa rat IGFBP-2 species generated by hepatic BRL-3A cells. Similarly, both antibodies effectively immunoprecipitated an IGFBP the size of rat IGFBP-2 elaborated by theca-interstitial cells. In contrast, neither antibody immunoprecipitated the 28-29 kDa IGFBP species elaborated by granulosa cells otherwise readily apparent in conventional Western ligand blots. Hypophysectomy resulted in a 3-fold decrease (P less than 0.05) in the relative (densitometrically-quantified) abundance of ovarian IGFBP-2 transcripts, a diametrically opposed effect (P less than 0.05) being noted at the level of the liver. In contrast, treatment of immature hypophysectomized rats with a diethylstilbestrol-containing subcutaneous silastic implant for a total of 5 days resulted in a concordant 3-fold increase (P less than 0.05) in the relative abundance of IGFBP-2 transcripts in both ovary and liver when compared with untreated hypophysectomized controls. Taken together, these findings document rat ovarian IGFPB-2 gene expression to be theca-interstitial (rather than granulosa) cell-selective, and subject to upregulatory control by pituitary principle(s) and/or by estrogens. Although equally estrogen-dependent, hepatic IGFBP-2 gene expression proved constitutive in nature and subject to (diametrically opposed) inhibitory control by (potentially distinct) pituitary principle(s).

Recent observations disclosed the multiplicity of granulosa cell-derived high affinity insulin-like growth factor binding proteins (IGFBPs) and revealed the striking ability of high doses of FSH to suppress their constitutive release under both in vitro and in vivo circumstances. It is the objective of this communication to characterize in greater detail the modulatory effect(s) exerted by FSH on the elaboration of IGFBPs by granulosa cells from immature, estrogen-primed rats. The ability of FSH to regulate the elaboration of granulosa cell-derived IGFBPs proved dose-dependent but biphasic in nature. Specifically, FSH concentrations in the range of 1-3 ng/ml applied for 72 h produced a significant (P less than 0.05) increase in polyethylene glycol-precipitable [125I]IGF-I binding activity corresponding to all IGFBP species detectable by ligand blotting. In contrast, treatment with higher concentrations (greater than or equal to 10 ng/ml) of FSH resulted in progressive dose-dependent inhibition of the constitutive release of IGF-I binding activity (80% inhibition at the 100 ng/ml dose level) and the virtual elimination of all detectable IGFBP species. Time-course studies disclosed a significant (P less than 0.05) initial (apparent at the 24-h time point) stimulatory effect of a high dose of FSH (100 ng/ml) corresponding mostly (but not solely) to the single minor (23K) IGFBP band. In contrast, more prolonged exposure to FSH (greater than or equal to 48 h) produced progressive time-dependent decrements in IGF-I binding activity, an effect associated with a decrease in the relative representation of the major band doublet (28-29K), the 23K species being all but eliminated under these experimental circumstances. Hypophysectomy produced significant (P less than 0.05) inhibition of the subsequent in vitro release of granulosa cell-derived precipitable IGF-I binding activity strongly suggesting that (presumptively stimulatory) pituitary principles other than FSH are likely involved in the regulation of granulosa cell IGFBP release and that the trophic influence of these putative agents appears to outweight the potential disinhibition of FSH hormonal action. Taken together, these findings indicate that the pituitary regulation of granulosa cell-derived IGFBPs is complex and is not FSH-exclusive. Our findings further indicate that the ability of FSH to regulate granulosa cell-derived IGFBPs is dose- and time-dependent but biphasic in nature, an effect characterized by an early low-dose stimulatory effect and a late high-dose inhibitory effect.(ABSTRACT TRUNCATED AT 400 WORDS)