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The oil overlay in microdrop culture systems prevents medium evaporation, helps to maintain appropriate pH and osmotic conditions and protects from microbial contamination. In the present study, we prospectively compared covering by Ovoil™, a paraffin oil, and LiteOil®, a mineral oil, on the in vitro development of human embryos and their suitability for transfer/freezing at day 3 and live birth rate. One hundred and one patients undergoing in vitro fertilization (IVF) treatment by  intracytoplasmic sperm injection (ICSI) were enrolled in our study. After ICSI, 1237 oocytes were 1:1 randomly allocated into 2 groups according to the type of overlaying oil: Ovoil™ (616 oocytes) or LiteOil® (621 oocytes). Fertilization rate was assessed around 18 hours post-insemination (hpi) and embryos were checked for early cleavage at 25 hpi. Embryo morphology was recorded on days 2 and 3. A total of 437 (Ovoil™) and 438 day 3 embryos (LiteOil®) were analyzed. There were no differences between the two groups in terms of fertilization rate and occurrence of early cleavage. The proportion of top quality embryos (41.7% vs. 41.2%) and the final utilization rates (92.2% vs. 92.0%) were similar in Ovoil and LiteOil groups, respectively, at day 3. Live birth rate per transfer was essentially the same with Ovoil™ overlay (26.9%) when compared to LiteOil® (26.2%). Live birth rate in patients who simultaneously received  embryos from both overlay types was 17.2%. Despite the different characteristics of these two oils regarding hydrocarbon saturation, packing and temperature storage, Ovoil™ and LiteOil® can be used in parallel in the same IVF protocol. Abbreviations: ART: assisted reproductive technologies; hpi: hours post-insemination; hSA: human serum albumin; HTF: human tubal fluid; ICSI: intracytoplasmic sperm injection; IVF: in vitro fertilization; MII: metaphase II; MEA: mouse embryo assay; RT: room temperature. [ABSTRACT FROM AUTHOR]

Embryo implantation requires extensive angiogenesis at the maternal-fetal interface. Hyperglycosylated human chorionic gonadotropin (hCG-H), a trophoblast invasive signal produced by extravillous cytotrophoblasts and by choriocarcinoma, was evaluated for its angiogenic role. hCG-H was purified by HPLC from choriocarcinoma supernatant, and the glycosylation pattern was determined by 2D gel analysis. Angiogenesis models used were aortic ring assay with wild-type and LHCGR-knockout mice, endothelial and mural cell proliferation, and migration assays. The TGF-β signaling pathway was studied by coimmunoprecipitation, competitive binding, TGF-β reporter gene assays, and Smad immunoblotting. hCG-H displayed a potent angiogenic effect [3.2-fold increase of number of vessel intersections in wild-type aortic rings (11.406 to 36.964)]. hCG-H-induced angiostimulation was independent of the classic hCG signaling pathway since it persisted in LHCGR-knockout mice [4.73-fold increase of number of vessel intersections (10.826 to 51.288)]. Using TGF-β signaling inhibitors, Tβ-RII was identified as the hCG-H receptor responsible for its angiogenic switch. hCG-H exposure enhanced phosphorylation of Smad 2 in endothelial and mural cells and genomic activation of Smad-responsive elements. Interaction between hCG-H and Tβ-RII was demonstrated by coimmunoprecipitation and binding competition with 125I-TGF-β. This new paracrine interaction between trophoblast and endothelial cells through the hCG-H and the TGF-β receptor complex plays a key role in angiogenesis associated with placental development and tumorigenesis. [ABSTRACT FROM AUTHOR]

Introduction: Pregnancy is an immune paradox. While the immune system is required for embryo implantation, placental development and progression of gestation, excessive inflammation is associated with pregnancy failure. Similarly, the cytokine IL-17A plays an important role in defence against extracellular pathogens, but its dysregulation can lead to pathogenic inflammation and tissue damage. Although expression of IL-17 has been reported during pregnancy, the cellular source of this cytokine and its relevance to gestation are not clear., Objectives: Here we define the kinetics and cellular source of IL-17A in the uterus during healthy and abortion-prone murine pregnancy., Methods: The CBA/J x DBA/2J abortion-prone mating was used and compared to CBA/J x BALB/c control mating., Results: We demonstrate that, irrespective of gestational health, the number of IL-17-producing cells peaks during midterm pregnancy and is largely derived from the γδ T-cell lineage. We identify γδ T, Th17, CD8 T and NKT cells as the cellular source of IL-17A in pregnant mice. Furthermore, we positively identify the Vγ6 + subset of uterine γδ T cells as the main producer of IL-17A during both healthy pregnancy and abortive pregnancy., Conclusions: To conclude, the accumulation of uterine IL-17 + innate-like T cells appears not to adversely impact the developing foetus. Collectively, our results show that IL-17 + γδ T cells are present in the uterus throughout the course of normal gestation and therefore may play an important role in healthy pregnancy.

Acceptance of the fetal semi-allograft by the mother's immune system has become the focus of intensive research. CD4+ T cells are important actors in the establishment of pregnancy. Th1/Th2 paradigm has been expanded to include CD4+ regulatory T (Treg) and T helper 17 (Th17) cells. Pregnancy hormones exert very significant modulatory properties on the maternal immune system. In this review, we describe mechanisms by which the endocrine milieu modulates CD4 T cell polarization during pregnancy. We first focused on Treg and Th17 cells and on their importance for pregnancy. Secondly, we review the effects of pregnancy hormones [progesterone (P4) and estradiol (E2)] on immune cells previously described, with a particular attention to human chorionic gonadotropin (hCG). The importance of Treg cells for pregnancy is evidenced. They are recruited before implantation and are essential for pregnancy maintenance. Decreased number or less efficient Treg cells are implicated in fertility disorders. As for Th17 cells, the few available studies suggest that they have a negative impact on fertility. Th17 frequency is increased in infertile patients. With the combination of its pro-effects on Th2 and Treg cells and anti-effects on Th1 and Th17 cells, P4 contributes to establishment of a favorable environment for pregnancy. E2 effects are more dependent on the context but it seems that E2 promotes Treg and Th2 cells while it inhibits Th1 cells. hCG positively influences activities of Treg and uterine natural killer cells. This embryo signal is an essential actor for the success of pregnancy, both as the endocrine factor regulating P4 secretion by the ovarian corpus luteum, but also as a paracrine agent during implantation as well as an angiogenic and immunologic mediator during the course of gestation. Luteinizing hormone (LH) immune properties begin to be studied but its positive impact on Treg cells suggests that LH could be a considerable immunomodulator in the mouse.

Follicular fluid (FF) contains various cytokines that are involved with folliculogenesis, some of which have been shown to be associated with oocyte quality and the implantation potential of a resulting embryo. Several IL-1 family members have previously been identified in FF. This study investigates a newly identified member of the family, IL-33, and its receptor ST2, comparing values to those of FF Granulocyte-Colony Stimulating Factor (G-CSF)--a known predictor of Assisted Reproductive Technology (ART) success. FF was collected from patients undergoing in vitro fertilisation/intra-cytoplasmic sperm injection (IVF/ICSI) at oocyte retrieval to analyse IL-33 and sST2 expression in human follicles. sST2, but not IL-33, is highly increased in the FF compared to plasma levels (up to 7.9-fold), with higher levels in larger follicles (p<0.05). Furthermore, we identify that human luteinised granulosa cells are one possible source of the FF sST2, as these cells express and secrete sST2 when cultured ex vivo. FF associated with oocytes which when fertilised develop into good quality embryos have higher sST2 levels than those which are graded average (p<0.01). These embryos were transferred to the patient and levels of FF sST2 compared between successful and unsuccessful ICSI cycles. However unlike G-CSF, sST2 levels cannot be used to predict cycle outcome.

Background: Cryopreservation of cortex ovarian tissue before anticancer therapy is a promising technique for fertility preservation mainly in children and young women. Ischemia in the early stage after ovarian graft causes massive follicle loss by apoptosis. VEGF111 is a recently described vascular endothelial growth factor (VEGF) isoform that does not bind to the extracellular matrix, diffuses extensively, and is resistant to proteolysis. These properties confer a significantly higher angiogenic potential to VEGF111 in comparison with the other VEGF isoforms., Methods: We evaluated the morphology of cryopreserved sheep ovarian cortex grafted in the presence or absence of VEGF111. Ovarian cortex biopsies were embedded in type I collagen with or without VEGF111 addition before transplantation to severe combined immunodeficient mice ovaries. Transplants were retrieved 3 days or 3 weeks later. Follicular density, vasculature network, hemoglobin content, and cell proliferation were analyzed., Results: Addition of VEGF111 increased density of functional capillaries (P=0.01) 3 days after grafting. By double immunostaining of Ki-67 and von Willebrand factor, we demonstrated that proliferating endothelial cells were found in 83% of the VEGF111 group compared with 33% in the control group (P=0.001). This angiostimulation was associated with a significant enhancement of hemoglobin content (P=0.03). Three weeks after transplantation, the number of primary follicles was significantly higher in VEGF111 grafts (P=0.02)., Conclusion: VEGF111 accelerates blood vessel recruitment and functional angiogenesis and improves the viability of ovarian cortex by limiting ischemia and ovarian cortex damage.

G-CSF in individual follicular fluids correlates with the potential of the corresponding embryo to result in a live birth after transfer in IVF. To evaluate the requirements for routine follicular fluid G-CSF quantification, we compared follicular fluid G-CSF measurements made with two multiplexed microbead assays purchased from Bio-Rad Laboratories and R&D Systems, and a commercial G-CSF ELISA (R&D Systems). Individual follicular fluids (n=139) associated with transferred embryos were analysed to determine cytokine profile and the fate of each transferred embryo was recorded. The effect of multiplexing as well as comparison of the respective performances of the microbead assay with a flow cytometry assay was explored. Multivariable logistic regression analysis was performed and receiver operating characteristic (ROC) analysis was used to determine the performance and sensitivity/specificity of each method for individual follicular fluids. Covariate factors known to influence IVF outcome such as age, serum oestradiol and embryo score were systematically integrated in each analysis. The quantification of follicular fluid G-CSF using microbead assay methodologies, but not ELISA, yielded results showing the utility of follicular fluid G-CSF as a biomarker predictive of a successful delivery (Au(roc): 0.77 [0.68-0.84] (p=0.003) and 0.75 [0.66-0.82] (p=0.004) for Bio-Rad and R&D Systems microbead assays respectively), whereas follicular fluid G-CSF values quantified by ELISA were not predictive (Au(roc):0.61 [0.52-0.70] p=0.84). Microbead assay and flow cytometry appeared similarly efficient for quantifying follicular fluid G-CSF and multiplex versus single-plex assays did not influence the reliability of quantification., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

The success of implantation depends on a receptive endometrium, a normal blastocyst and synchronized cross-talk at the maternal-fetal interface. The progression of pregnancy then requires immunological tolerance which allows conceptus survival. A cascade of cytokines mediates this dialogue and is crucial in the cross-talk between the immune and endocrine systems. The first known human embryo-derived signal is chorionic gonadotropin (hCG) by which the embryo profoundly influences immunological tolerance and angiogenesis at the maternal-fetal interface. hCG levels coincide with the development of trophoblast tolerance. Indeed, it increases the number of uterine natural killer cells that play a key role in the establishment of pregnancy. hCG also intervenes in the development of local immune tolerance through the cellular system of apoptosis via Fas/Fas-Ligand. It modulates the Th1/Th2 balance and acts on complement C3 and C4A/B factors modulating decidual immunity. The transient tolerance evident during gestation is at least partially achieved via the presence of regulatory T cells which are attracted by hCG at the fetal-maternal interface. Finally, hCG treatment of activated dendritic cells results in an up-regulation of MHC class II, IL-10 and IDO expression, reducing the ability to stimulate T cell proliferation. Successful implantation requires an extensive endometrial angiogenesis in the implantation site. Recent data demonstrate angiogenic effects of hCG via its interaction with endometrial and endothelial LH/hCG receptors. Our review focuses on these functions of hCG, giving new insight into the endocrine-immune dialogue that exists between the conceptus and immune cells within the receptive endometrium at the time of implantation., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Background: Oral contraceptives (OCs) with estrogens and progestins may affect oxidative stress (OS) status., Study Design: A group of 32 women using oral contraceptives (OCU) containing 0.03 mg ethinylestradiol and 3 mg drospirenone have been compared to a matched control group of 30 noncontraception users (NCU). Blood levels of antioxidants, trace elements and markers of lipid peroxidation were assessed by biochemical methods. A microarray analysis of whole blood mRNA levels of 200 genes involved in OS-dependant pathway was also performed., Results: Levels of zinc, vitamin E and antibodies to oxidized low-density lipoproteins (LDLs) were not significantly different between the two groups. On the other hand, significant increases in the mean levels of lipid peroxides (+176%, p<.001), oxidized LDLs (+145%, p<.002), copper (+103%, p<.001), Cu/Zn ratio (+100%, p<.001) and a significant decrease in the mean level of beta-carotene (-41%, p<.01) were observed in the OCU compared to NCU. There was a highly significant positive correlation between the lipid peroxide levels and the copper-to-zinc ratio. From the 200 genes tested by microarray, one coding for HSP70 was significantly up-regulated (log(2) fold change=+ 0.45, p<.02) and one coding for inducible nitric oxide synthase significantly down-regulated (log(2) fold change=-0.24, p<.05) in the OCU compared to the NCU., Conclusions: The recently introduced combination of ethinylestradiol and drospirenone induced the heightening of lipid peroxidation correlated with high levels of copper, a situation that could be associated with increased cardiovascular risk.

Several reports have described an association between the presence of soluble human leukocyte antigen G (sHLA-G) in human embryo culture supernatants (ES) and implantation success. However, not all studies agree with these findings. To further document this debate, a multicentre blinded study was performed to investigate, on a large number of IVF ES and ICSI ES, whether sHLA-G is a useful criterion for embryo selection before transfer. A total of 1405 ES from 355 patients were collected from three assisted reproductive technique (ART) centres and evaluated for their sHLA-G content in a single laboratory, using a chemiluminescence enzyme-linked immunosorbent assay. In only one centre was a significant association between sHLA-G-positive ES and successful implantation established (P = 0.0379), whereas no such association was observed in the other centres. It was found that the percentages and concentrations of sHLA-G-positive ES varied between centres, depending on culture media and ART conditions. The percentage of sHLA-G-positive ES was significantly higher in IVF ES than ICSI ES (P < 0.001 and P < 0.01 for two centres). These data demonstrate that substantial variations of sHLA-G content in ES occur between different ART centres, highlighting the influence of several technical parameters that differ from one centre to another.

Objective(s): The implantation process is closely linked to the fundamental question of the tolerance of the maternal immune system. The main objective of this study was to investigate whether different members of the transforming growth factor-beta (TGF-beta) superfamily could intervene in the first steps of embryo implantation by modulating the secretion of proimplantatory leukemia inhibitory factor (LIF) and in the tolerance of the fetal graft by regulating proinflammatory interleukin (IL)-6 secretion by human endometrial epithelium (EEC) in vitro., Methods: EEC were isolated from biopsies collected from 16 informed and consenting fertile women and were cultured for 72 h. Cytokine measurements (LIF and IL-6) were realized by ELISA., Results: TGF-beta(1) (from 10(-12) to 10(-8)M), -beta(2), -beta(3) and activin A (10(-10) and 10(-8)M) increased LIF secretion by EEC cultures. Inhibin B (10(-10) and 10(-8)M) did not stimulate LIF production by human EEC. Contrastingly, TGF-beta(1) (from 10(-12) to 10(-8)M), -beta(2), -beta(3) and activin A (10(-10) and 10(-8)M) reduced IL-6 release by the same cells. Activin A at 10(-8) M also significantly reduced the stimulating effect of IL-1beta (10(-9)M) which is known to stimulate LIF production by EEC. Only the highest concentration of inhibin B (10(-8)M) reduced IL-6 secretion by EEC, but did not modulate IL-1beta-induced stimulation of IL-6 secretion., Conclusion(s): Besides their role in the control of the process of implantation and in the induction of embryonic mesoderm, different members of the TGF-beta superfamily may also contribute in the reproductive process by enhancing endometrial proimplantatory LIF secretion and reducing proinflammatory IL-6 release by EEC., (Copyright (c) 2005 S. Karger AG, Basel.)