An insight into normal and pathological pregnancies using large-scale microarrays: lessons from microarrays.

In the introduction, we briefly recall old but classic evidence that there is no tolerance to paternal alloantigens in a first pregnancy. Therefore, we performed small- and large-scale microarrays in CBA × DBA/2 and CBA × BALB/c combinations, recently described as a murine model for preeclampsia. Our results are in line with other data suggesting a very early deregulation of local immune vascular events rather than a break of immune tolerance. Other data presented at the Tioman 2010 Preeclampsia Workshop supporting this hypothesis are briefly summarised, as well as indications and caveats from a recent human microarray on implantation failure and recurrent pregnancy loss.

Recanalization and particle exclusion after embolization of uterine arteries in sheep: a long-term study.

OBJECTIVE: To compare the long-term evolution of uterine arteries after embolization with the two most commonly used embolic agents for fibroid embolization: nonspherical polyvinyl alcohol (PVA) particles and trisacryl gelatin microspheres (TGMS). DESIGN: Prospective study. SETTING: University-based interventional radiology, pathology, and reproductive physiology units. ANIMAL(S): Two groups of 10 sheep embolized in the uterine artery. INTERVENTION(S): Embolization of the uterine artery with either 600-1000 microm nonspherical polyvinyl alcohol (PVA) particles or with 700-900 microm trisacryl gelatin microspheres (TGMS). Animals were synchronized and naturally inseminated. Animals were killed at 26 months. MAIN OUTCOME MEASURE(S): Uteri were examined pathologically for vessel size, site of occlusion, recanalization rate of vessels, and particle location within the vascular wall. RESULT(S): The PVA particles were more numerous in the vessels' lumen than the TGMS particles (13.3 +/- 20.8 vs. 2.5 +/- 2.7), were located more proximally than TGMS (97% vs. 68% in the trunk and first branches of the uterine artery), and were found almost exclusively in the intima (99.2%). In contrast, 54.4% of the TGMS particles were found in the intima, and 45.6% partially or totally excluded. The rate of recanalization was not statistically significantly different for PVA and TGMS (65.2% vs. 60.6%). CONCLUSION(S): The long-term evolution of uterine arteries was different after uterine artery embolization with PVA and TGMS because PVA particles formed large-sized aggregates that occluded proximal vessels and remained in the vessel intima. Microspheres occluded more distal vessels, and about 50% of them were partially or totally excluded from the vessel.

Fertility after bilateral uterine artery embolization in a sheep model.

OBJECTIVE: To evaluate the impact of bilateral uterine artery embolization (UAE) upon fertility in sheep. DESIGN: Prospective study. SETTING: University-based interventional radiology, pathology, and reproductive physiology units. ANIMAL(S): Nineteen control ewes, 10 ewes embolized with polyvinyl alcohol particles (PVA group), and 10 ewes embolized with Tris-acryl gelatin microspheres (TGMS group). INTERVENTION(S): Bilateral UAE was performed with 600- to 1,000-mum PVA particles or 700- to 900-mum TGMS particles. Animals of three groups were synchronized and naturally inseminated. MAIN OUTCOME MEASURE(S): For each ewe, a hormonal follow-up was performed throughout the gestation. Gestation duration, number and weight of newborns, and fertility and gestation rates were recorded. RESULT(S): Mean number of estrus before insemination and gestation duration were not different between groups. There were 47 living newborns: 26 control, 9 PVA, and 12 TGMS. Overall birth weight of newborns was 3.7 +/- 0.9 kg for controls, 3.6 +/- 1.1 kg for TGMS, and 2.2 +/- 0.7 kg for PVA (which was statistically significant vs. control). In the PVA group, there was a statistically significant decrease of fertility rate and gestation rate vs. controls, but this was not the case in the TGMS group. CONCLUSION(S): Particles of PVA decrease fertility in sheep and lead to intrauterine growth retardation.

Differential receptor subunit affinities of type I interferons govern differential signal activation.

Type I interferons (IFNs) elicit antiviral, antiproliferative and immunmodulatory responses by binding to a shared cell surface receptor comprising the transmembrane proteins ifnar1 and ifnar2. Activation of differential response patterns by IFNs has been observed, suggesting that members of the family play different roles in innate immunity. The molecular basis for differential signaling has not been identified yet. Here, we have investigated the recognition of various IFNs including several human IFNalpha species, human IFNomega and human IFNbeta as well as ovine IFNtau2 by the receptor subunits in detail. Binding to the extracellular domains of ifnar1 (ifnar1-EC) and ifnar2 (ifnar2-EC) was monitored in real time by reflectance interference and total internal reflection fluorescence spectroscopy. For all IFNs investigated, competitive 1:1 interaction not only with ifnar2-EC but also with ifnar1-EC was shown. Furthermore, ternary complex formation was studied with ifnar1-EC and ifnar2-EC tethered onto solid-supported membranes. These analyses confirmed that the signaling complexes recruited by IFNs have very similar architectures. However, differences in rate and affinity constants over several orders of magnitude were observed for both the interactions with ifnar1-EC and ifnar2-EC. These data were correlated with the potencies of ISGF3 activation, antiviral and anti-proliferative activity on 2fTGH cells. The ISGF3 formation and antiviral activity correlated very well with the binding affinity towards ifnar2. In contrast, the affinity towards ifnar1 played a key role for antiproliferative activity. A striking correlation was observed for relative binding affinities towards ifnar1 and ifnar2 with the differential antiproliferative potency. This correlation was confirmed by systematically engineering IFNalpha2 mutants with very high differential antiproliferative potency.

Clinical, biological and hormonal study of mid-pregnancy termination in cats with aglepristone.

In order to evaluate the efficacy, the safety and the variation in plasma concentrations of estrogens, progesterone, PGFM, oxytocin, cortisol and prolactin after mid-pregnancy termination induced by aglepristone, 61 pregnant queens (33.3 + 4.2 days), were injected subcutaneously with 15 [corrected] mg/kg aglepristone, (Alizine) [corrected] repeated once 24 h later. Five queens served as control and received a placebo. The efficacy of aglepristone was 88.5% and termination of pregnancy was achieved in 50% of the queens within 3 days. Brief periods of depression and anorexia were noted in 9.3% of the queens before fetal expulsion (these symptoms were attributed to the phenomenon of fetal expulsions). Not one of the queens that aborted developed uterine disease. There were no changes in plasma concentrations of estrogen, prostaglandin, prolactin or oxytocin following aglepristone administration. However, there were significant increases in plasma concentrations of progesterone and cortisol 60 and 30 h, respectively, after aglepristone administration. Termination of pregnancy occurred with high plasma progesterone concentrations. Fetal expulsion was characterised by an increase in estrogen, PGFM and oxytocin concentrations, whereas prolactin and cortisol levels remained at a basal level.

Involvement of IL-6 in the anti-human immunodeficiency virus activity of IFN-tau in human macrophages.

IFN-tau is a non-cytotoxic type I IFN responsible for maternal recognition of the foetus in ruminants. IFN-tau has been found to inhibit HIV replication more strongly than human IFN-alpha, particularly in human monocyte-derived macrophages, without associated toxicity. Ovine IFN-tau uses the same anti-viral cellular pathways as human IFN-alpha in human macrophages, principally inhibiting the early steps of the biological cycle of HIV, preventing the integration of HIV DNA into the host-cell genome. In this study, we investigated the immunomodulatory properties of IFN-tau in human macrophages. We found that IFN-tau increased the production of IL-10 and IL-6, but not of IL-1beta or tumour necrosis factor alpha, in unstimulated, LPS-stimulated and HIV-1/Ba-L-infected macrophages. We also found that treatment with IL-6 inhibited HIV replication. Moreover, the neutralization of IL-6 activity in the cell culture supernatants of IFN-tau-treated macrophages led to a decrease in the anti-retroviral effects of IFN-tau, suggesting that IL-6 was involved in the anti-viral activity induced by IFN-tau. By focusing on the very early steps of the biological cycle of HIV, we showed that IL-6 co-operated with IFN-tau to decrease intracellular HIV RNA levels 2 h after infection.

Suppressor of cytokine signalling (SOCS) genes are expressed in the endometrium and regulated by conceptus signals during early pregnancy in the ewe.

It is established that the conceptus-endometrium dialogue involves cytokines, growth factors and hormones. Given the crucial functions of the suppressor of cytokine signaling (SOCS) family proteins in cytokine signalling, we analyzed the expression and the regulation of CIS and SOCSs 1-3 transcripts during early pregnancy in the ovine endometrium. An overall stimulation of the SOCS transcripts was described in the pregnant ewes with two specific patterns. Unilaterally pregnant ewes confirmed the conceptus-produced factors as regulators of the SOCSs 1-3 expression at day 16 of pregnancy. Intrauterine injection of recombinant ovine interferon tau (IFNtau) in cyclic ewes stimulated the expression of the SOCS mRNA with various potencies, therefore suggesting that the SOCS could take part in the negative regulation of the IFNtau signalling pathway.

TH1/TH2 paradigm in pregnancy: paradigm lost? Cytokines in pregnancy/early abortion: reexamining the TH1/TH2 paradigm.

In this paper, we briefly survey the history of concepts in reproductive immunology from antibody-mediated tolerance to the "fetal allograft" to the current concept of an embryo "bathing in a sea of cytokines". We then review the paradigm that "allopregnancy is a Th2 phenomenon" and some of the evidence gained in animals and humans supporting it. We continue by discussing the light it sheds on immunologically caused recurrent abortion, and the present status of the concepts. We next show the limits of the Th1/Th2 paradigm by reviewing the role of "inflammatory" cytokines in implantation (as first seen with leukemia inhibitory factor). We go on to discuss recent data showing that interferon-gamma is not solely a "bad guy", e.g. abortifacient as the paradigm would predict, but is needed at low doses for the vascular development and transformation of uterine spiral arteries required for implantation and successful pregnancy. We conclude by discussing the emerging role of NK and IL-12, IL-15, IL-18 tripods and other cytokines in local angiogenesis and tissue remodelling, a series of new data bringing us well beyond the Th1/Th2 paradigm in pregnancy which, in this context, appears now obsolete and an oversimplification, although it has indeed been useful at first. Rather, step-specific events have to be considered and a key role is seen in local tissue remodelling, in which immune cytokines play an important role while not always being secreted by immune cells.

Reproductive immunology 2003: reassessing the Th1/Th2 paradigm?

We briefly review the history of concepts (some of which are still valid) which have lead to the present situation where pregnancy is viewed as being a Th2 phenomenon. We recall some of the early evidence which has been taken as supporting the general validity of this concept in murine and human pregnancy. We then recall some of the recent data dealing with "newer" cytokines and the role of uterine natural killer (NK) cells at the feto-maternal interface which fit neither with a steady-state concept nor with inflammatory cytokines, being solely "bad guys" as the paradigm would predict, nor with the concept of reduction of NK "activity" being required for successful pregnancy. As an example of the newer complexity, we briefly recall some of our recent micro-array studies in mice, and describe briefly our most recent data in human pointing out the importance of the tripod IL-12/IL-18/NK in successful or failed pregnancy in human, perhaps under IL-15 control. We conclude by a repeated warning against the so-called rationales of lymphocyte alloimmunization for therapy of recurrent spontaneous abortion and improvement of implantation rates.

Cytokines, implantation and early abortion: re-examining the Th1/Th2 paradigm leads to question the single pathway, single therapy concept.

PROBLEM: Human in vitro fertilization (IVF) embryo transfer is accompanied by a low implantation rate even after a very successful IVF, and there are a certain number of 'idiopathic sterilities' which are due to repeated implantation failures. In the very same vein, the question of improving implantation rates is of prime importance in agricultural research to improve the management of livestock. Preimplantation prenatal diagnosis cannot be accomplished in individuals who have a high rate of implantation failure, whether women undergoing IVF, or animals, during genetic cloning. Implantation cytokine networks need to be known in such a perspective. METHODS: We review the evolution and theories in reproductive immunology, briefly deal with the complexity of implantation as a step by step developmental event, and then present some of our recent data in mice and human. CONCLUSIONS: We conclude that the T helper cell type 1/2 (Th1/ Th2) paradigm, as useful as it has been to explain pregnancy, is no longer sufficient in view of the emerging complexity of the cytokine network at the materno-fetal interface. This is peculiarly true for implantation, which, as a step by step developmentally regulated process, involving inflammatory molecules, cannot fit into such a scheme.

Anti-human immunodeficiency virus activity of tau interferon in human macrophages: involvement of cellular factors and beta-chemokines.

Tau interferon (IFN-tau) is a noncytotoxic type I IFN responsible for maternal recognition of the fetus in ruminants. IFN-tau inhibits human immunodeficiency virus (HIV) replication more strongly than human IFN-alpha, particularly in human monocyte-derived macrophages. In this study performed in human macrophages, IFN-tau efficiently inhibited the early steps of the biological cycle of HIV, decreasing intracellular HIV RNA and inhibiting the initiation of the reverse transcription of viral RNA into proviral DNA. Two mechanisms induced by IFN-tau treatment in macrophages may account for this inhibition: (i) the synthesis of the cellular antiviral factors such as 2',5'-oligoadenylate synthetase/RNase L and MxA protein and (ii) an increased production of MIP-1alpha, MIP-1beta, and RANTES, which are natural ligands of CCR5, the principal coreceptor of HIV on macrophages. Our results suggest that IFN-tau induces the same antiviral pathways in macrophages as other type I IFNs but without associated toxicity.

Implantation: can immunological parameters of implantation failure be of interest for pre-eclampsia?

We restate briefly why we consider that the Th1/Th2 paradigm, as useful as it has been, is now no longer adequate and is obsolete. We take as an example the role of IL-18, abortifacient at high doses but cardinal for the control of natural killer (NK) cell effects on spiral artery remodelling in mice, and likely also in humans. We then describe briefly our recent studies on cytokine defects and implantation failure in humans, a key feature being the link between uterine cytokine dysregulation and abnormal uterine vascular scores. We draw lessons for preeclampsia, and describe features of a model for its immune aetiology.

Corpus luteum derived copper, zinc-superoxide dismutase serves as a luteinizing hormone-release inhibiting factor in sheep.

In the present study, we report the purification and characterization of a polypeptide from the sheep corpus luteum of pregnancy with a potent luteinizing hormone-release inhibiting factor (LH-RIF) bioactivity that stained as a single band in SDS-PAGE with an apparent molecular mass of 16000 Da. The amino acid sequences obtained after sequence analysis of peptides derived from the trypsin digestion of LH-RIF were subjected to a protein data bank search and were found to be identical with regions of sheep copper, zinc-superoxide dismutase (Cu,Zn-SOD). The measured mass of LH-RIF (15604.2+/-1.9 Da) was found to be similar to the theoretical mass of sheep Cu,Zn-SOD (15603.5 Da), with a disulfide bond and N acetylated alanine at the N-terminus. The inhibitory action of Cu,Zn-SOD on pulsatile LH secretion would suggest that this antioxidant may play an important role, either independently or in concert with some neurotransmitters, in the neuroendocrine regulation of sheep female reproductive function.

Uterine artery embolization in sheep: comparison of acute effects with polyvinyl alcohol particles and calibrated microspheres.

PURPOSE: To compare the effects on the myometrium of polyvinyl alcohol (PVA) particles and calibrated microspheres (MS) in embolization of the uterine arteries in sheep. MATERIALS AND METHODS: Superselective and bilateral embolization of the uterine arteries was performed with PVA particles and calibrated MS within 24 hours after artificial ovulation in 26 adult nonpregnant sheep. PVA particles of four diameters, 150-250, 250-400, 400-600, and 600-1,000 microm, were compared with calibrated MS of similar diameters, 100-300, 300-500, 500-700, and 700-900 microm, in eight groups of sheep. Evaluation was based on histopathologic study of uterus, ovaries, and vascular pedicles after sacrifice 5 days after embolization. The scores of necrosis, the diameter of occluded arteries, and the number of particles were determined. The scores of uterine necrosis were compared by using nonparametric tests (Mann-Whitney U and Kruskal-Wallis). Spearman rank test was used for correlations. RESULTS: PVA particles clumped more readily than did MS. Small particles had a higher score (P =.02) of uterine necrosis than did large particles. PVA particles produced more necrosis than did MS. Size of MS and diameter of occluded arteries showed significant correlation (rho = 0.762, P <.001). Size of PVA particles and diameter of occluded arteries showed no correlation. PVA particles occluded vessels of a wider range of size than did calibrated MS. CONCLUSION: PVA particles are associated with intense uterine necrosis and extensive arterial occlusion regardless of size. Calibrated MS, which are associated with less uterine necrosis, permit a segmental arterial occlusion correlated with size.

A brief review of recent data on some cytokine expressions at the materno-foetal interface which might challenge the classical Th1/Th2 dichotomy.

Focussing attention on cytokines at the materno-foetal interface represents one of the major advances made in the field. This owes much to the visionary views of Tom Wegmann, and to the changes brought about in the field by immunotrophism and Th1/Th2 paradigms. We review these briefly and also point out some emerging problems.However, a certain number of newly discovered cytokines do not fit into the classical Th1/Th2 dichotomy. Yet, by their capacity to activate or downregulate NK cells, by their action on adhesion molecules, and by their regulatory effects on the vascularisation process, they are of possible interest within the materno-foetal relationship. Therefore, as a first step, we have undertaken a systematic study of the expression of IL-11, IL-12, IL-13, IL-15, IL-16, IL-17 and IL-18 in the uterus, the peri-implantation embryo, and later on decidual and placental tissues throughout pregnancy. These cytokines were detected in every case, with, in each case, a precise localisation, which will be detailed, and which indeed suggests important regulatory functions, especially during implantation. In some cases, as will be shown in the peri-implantation uterus, those cells are perfectly expressed by uterine GMG-NK-like cells. Comparative ELISAs and quantitative RT-PCR have been or are being conducted, but already the expression patterns that are observed, and the very precise window of appearance that is observed for some of the GMG NK-like cells, either around or in the implanting embryo, as well as the complexity of the respective distributions, strongly suggest that, as useful as it certainly was for a while, the Th1/Th2 paradigm must now be considered as an over-simplification. Rather, the existing data point to sequential windows and are suggestive of a system where an extreme complexity is allied to very precise timing and tuning. They also suggest that the materno-foetal relationship is not simply maternal tolerance of a foreign tissue, but a series of intricate mutual cytokine interactions governing selective immune regulation and also control of the adhesion and vascularisation processes during this dialogue.

New insights into maternal-fetal interactions at implantation.

The immunotrophic theory was enunciated by Tom Wegmann. Since then, the involvement of cytokines in implantation and materno-fetal tolerance has emerged as a central topic in reproductive immunology. This brief survey covers the historical background leading to the specification of the crucial role of cytokines at the feto-maternal interface, and the present known patterns of their function. Focus is addressed to the most recent concept, e.g. pregnancy as a Th2 phenomenon (the immune response of the mother is biased towards the production of anti inflammatory cytokines, amongst them IL-10 which suppress inflammatory responses). A brief description of the role of inflammatory cytokines in implantation is presented to explain why it does not fit into such a scheme, and other recent data, for example on gamma interferon, also fails to accord with the Th2 phenomenon.