Eleuterio R. Hernandez

Human intraovarian interleukin-1 (IL-1) system: highly compartmentalized and hormonally dependent regulation of the genes encoding IL-1, its receptor, and its receptor antagonist.

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.

Ovarian transforming growth factor-β (TGFβ): cellular site(s), and mechanism(s) of action☆

It is the objective of the experiments reported herein to examine the possible relevance of transforming growth factor-beta (TGF beta) to theca-interstitial cell function, and to further characterize the established interaction of TGF beta with the granulosa cell. In examining the interaction of TGF beta (10 ng/ml) with murine theca-interstitial cells, no significant effect was observed on either basal or human chorionic gonadotropin (hCG)-stimulated androsterone accumulation. In contrast, given murine granulosa cells, TGF beta (10 ng/ml) produced dose- and time-dependent augmentation of follicle-stimulating hormone (FSH)-supported aromatase activity with a minimal and median effective doses of 20 +/- 3 and 123 +/- 24 pg/ml, respectively and a minimal time requirement of less than or equal to 48 h. The ability of TGF beta to augment FSH hormonal action could not be accounted for by alteration(s) of specific FSH binding (13965 +/- 298 and 12614 +/- 694 cpm/4 X 10(5) cells for FSH and FSH + TGF beta). However, TGF beta proved capable of exerting a direct upregulatory effect on stimulatable adenylate cyclase activity, further enhancement occurring at site(s) distal to cAMP generation (dibutyryl cyclic AMP (Bt2cAMP) = 1.4 +/- 0.2 ng/culture; Bt2cAMP + TGF beta = 4.1 +/- 0.6 ng/culture). Taken together, our findings are in keeping with the notion that TGF beta, possibly of intraovarian origin, comprises the central signal of autocrine or paracrine loop(s) capable of amplifying gonadotropin action at the level of the granulosa, but not theca-interstitial cell.

Granulosa cell-derived insulin-like growth factor (IGF) binding proteins are inhibitory to IGF-I hormonal action. Evidence derived from the use of a truncated IGF-I analogue.

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.

Triggering ovulation with gonadotropin-releasing hormone agonist in in vitro fertilization patients with polycystic ovaries does not cause ovarian hyperstimulation syndrome despite very high estradiol levels

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.

No room for cancellation, coasting, or ovarian hyperstimulation syndrome in oocyte donation cycles

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.

Ovarian granulosa cell-derived insulin-like growth factor (IGF) binding proteins: release of low molecular weight, high-affinity IGF-selective species.

The ovarian granulosa cell has previously been shown to be a site of insulin-like growth factor (IGF) I production, reception, and action. It is the objective of this study to characterize in greater detail the soluble IGF binding activity released by this cell type. To this end, use was made of granulosa cells from immature diethylstilbestrol-treated rats. Serum-free media conditioned for 72 h by cultured untreated cells acquired polyethylene glycol (PEG)-precipitable [125I]IGF-I binding activity. The latter proved cell density-dependent, displaying a minimal inoculum requirement of less than or equal to 3 x 10(5) cells/culture. The daily elaboration of IGF-I binding activity appeared constant throughout the 72 h experimental period, the overall time-dependent accumulation of binding activity (over the same time period) proving virtually additive. Scatchard analysis of detailed competition studies with IGF-I suggests that the latter ligand binds to granulosa cell-derived IGF binding protein(s) (IGFBPs) with an apparent affinity of 3 x 10(-10) M. Qualitatively similar results were obtained when using [125I]IGF-II suggesting that the IGFBPs in question are not IGF-I-selective. In fact, specificity studies using either [125I]IGF-I or [125I]IGF-II revealed a rank order of competitive potencies compatible with that observed in other tissues so studied (IGF-II greater than IGF-I much greater than insulin). The proteinacious nature of the acid-stable IGF binding activity under study was indicated by its sensitivity to relatively low concentrations of cycloheximide, its apparent deactivation following repeated cycles of freezing and thawing, and its virtual elimination when subjected to boiling or trypsin treatment. Cycloheximide-induced blockade of protein biosynthesis also revealed that the IGF binding activity is subject to measurable turnover thereby suggesting that its accumulation represents the balance struck between synthetic and degradative processes. Western ligand blotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated media revealed a non-glycosylated major band doublet of 28-29 kDa. A single minor IGFBP species represented by a 23 kDa band was also appreciated in some but not all experiments. Taken together, these findings document the ability of ovarian granulosa cells to secrete a heterogenous mix of low molecular weight, high-affinity IGF-selective binding species. As such, these observations are in keeping with the concept of a complete intraovarian IGF system replete with ligands, receptors, and soluble binding activity.

What next for preimplantation genetic screening? Beyond aneuploidy

Many published papers suggest a favourable impact of preimplantation genetic screening (PGS) on implantation and pregnancy rates, but more recent randomized studies have not confirmed, or could not conclude, that PGS actually improved implantation rates. Team inexperience in embryo screening has been mentioned as the origin of the discrepancies; thus some clinicians allege a need for more powerful, well-designed, randomized studies performed by specialized teams. However, what if all the contradictory results about the benefits of PGS and implantation were not due to technical problems or team specialization but were biological in origin? The developmental programme of an eight-cell embryo relies on signals of maternal origin retrieved from the cytoplasm to initiate a new transcriptional network that will eventually serve as a filter (checkpoints and apoptosis) for aneuploidy. Thus, the use of PGS with the objective of improving the likelihood of a successful pregnancy based only on nuclear abnormalities (aneuploidies) in an early cleavage stage embryo could be invalid since the information (diagnosis) obtained at the moment of biopsy could be overturned by the transcriptional machinery of the new zygote genome.

The Ovarian and Testicular IGF-I System: A Comparative Analysis

As the significance of putative intraovarian regulators becomes increasingly recognized, much of the attention centers on insulin-like growth factors (IGFs). Indeed, a large body of evidence now suggests the existence of an intraovarian IGF system complete with ligands, re-ceptors, and binding proteins (Adashi et al. 1985c). More importantly, IGFs have been shown to exert a variety of significant effects in murine (Adashi et al. 1985a,b,d; 1986b; Bicsak et al. 1986; Cara and Rosenfeld 1988; Davoren et al. 1985; Magoffin et al. 1990; Zhiwen et al. 1987), porcine (Baranao et al. 1984; Maruo et al. 1988; May et al. 1988; Veldhuis and Demers 1985; Veldhuis and Furlanetto 1985, Veldhuis et al. 1986a, b; Veldhuis et al. 1987; Veldhuis and Rodgers, 1987; Veldhuis and Gwynne 1989), and human (Erickson et al. 1989, 1990; Steinkampf et al. 1988) somatic ovarian cells, thereby raising the possibility of a meaningful in vivo role.

Rodent Studies on the Potential Relevance of Insulin-Like Growth Factor (IGF-I) to Ovarian Physiology

The recurring process of ovarian follicular growth is an exponential rather than a linear process characterized by substantial dramatic proliferation and differentiation of the granulosa cell. Although the pivotal role(s) of gonadotropins and of gonadal steroids in this explosive agenda is well recognized, the variable fate of follicles subject to comparable gonadotropic support suggests the existence of additional intraovarian regulatory mechanism(s). Among potential novel intraovarian regulators, insulin-like growth factor I (IGF-I) has been receiving increasingly intense scrutiny (1–2). Taken together these studies strongly suggest the existence of an intraovarian autocrine control mechanism, wherein IGF-I may serve as the central signal, and the granulosa cell its site of production, reception, and action. Viewed in this light, IGF-I may promote the growth and/or differentiation of the granulosa cell, acting for the most part as an amplifier of gonadotropin action. Granulosa cell-derived IGF-I may also provide paracrine input to the nearby theca-interstitial cell compartment in the interest of coordinated follicular function.

Intraovarian IGF-I System

As the significance of putative intraovarian regulators becomes increasingly recognized, much of the attention centers on insulin-like growth factors (IGFs). Indeed, a large body of evidence now suggests the existence of an intraovarian IGF system complete with ligands, receptors, and binding proteins. More importantly, IGFs have been shown to exert a variety of significant effects at the level of the somatic ovarian cell, raising the possibility of a meaningful in vivo role. The above notwithstanding, there is at this time no compelling evidence to indicate that IGFs (or for that matter any other putative intraovarian regulator) are indispensable to ovarian function. However, a large body of a somewhat indirect nature strongly suggests such a possibility. It is the purpose of this communication to review and summarize key developments in this area.