Clinical performance of a specific granulocyte colony stimulating factor ELISA to determine its concentration in follicular fluid as a predictor of implantation success during in vitro fertilization.

This study aimed to demonstrate the clinical performance of an ultra-sensitive follicular fluid (FF) granulocyte colony stimulating factor (G-CSF) immunoassay to confirm previous work, indicating a correlation between FF G-CSF concentration and live birth potential of the corresponding embryo after in vitro fertilization. This study was a noninterventional, prospective, diagnostic clinical multicentric study conducted between August 2012 and January 2014 with 396 single embryo transfers (SETs) from 278 subjects. During oocyte retrieval, FF was individually collected. Embryo morphology and implantation success were evaluated. The implantation success rate in the high G-CSF group (32.3%) was higher than the overall rate (27.5%). Similarly, for embryos with optimal morphology, implantation success rates were highest among those in the high G-CSF concentration category (34.5%) compared with low (19.6%) and intermediate (29.8%) G-CSF concentration categories. Significant differences in mean G-CSF concentrations were observed between the study sites. To minimize bias, analyses were repeated using data from the center with the largest number of SETs. In alignment with the overall analysis, this center demonstrated a 43% greater probability of implantation for optimal embryos with high G-CSF compared to the general implantation rate among optimal embryos and a 327% increase compared with the implantation rate of optimal embryos with low G-CSF.

The history of Belgian assisted reproduction technology cycle registration and control: a case study in reducing the incidence of multiple pregnancy.

STUDY QUESTION: What is the effect of a legal limitation of the number of embryos that can be transferred in an assisted reproductive technology (ART) cycle on the multiple delivery rate? SUMMARY ANSWER: The Belgian national register shows that the introduction of reimbursement of ART laboratory costs in July 2003, and the imposition of a legal limitation of the number of embryos transferred in the same year, were associated with a >50% reduction of the multiple pregnancy rate from 27 to 11% between 2003 and the last assessment in 2010, without any reduction of the pregnancy rate per cycle. WHAT IS KNOWN ALREADY: Individual Belgian IVF centres have published their results since the implementation of the law, and these show a decrease in the multiple pregnancy rate on a centre by centre basis. However, the overall national picture remains unpublished. STUDY DESIGN, SIZE, DURATION: Cohort study from 1990 to 2010 of all ART cycles in Belgium (2685 cycles in 1990 evolving to 19 110 cycles in 2010), with a retrospective analysis from 1990 to 2000 and prospective online data collection since 2001. PARTICIPANTS/MATERIALS, SETTING, METHODS: Registration evolved from paper written reports per centre to a compulsory online registration of all ART cycles. From 2001 up to mid-2009, data were collected from Excel spread sheets or MS Access files into an MS Access database. Since mid-2009, data collection is done via a remote and secured web-based system (www.belrap.be) where centres can upload their data and get immediate feedback about missing data, errors and inconsistencies. MAIN RESULTS AND THE ROLE OF CHANCE: National Belgian registration data show that reimbursement of IVF laboratory costs in July 2003, coupled to a legal limitation in the number of embryos transferred in utero, were associated with a 50% reduction of the multiple pregnancy rate from 27 to 11% without reduction of the pregnancy rate per cycle, and with an increase in the number of fresh and frozen ART cycles due to improved access to treatment. LIMITATIONS, REASONS FOR CAUTION: There is potential underreporting of complications of ART treatment, pregnancy outcome and neonatal health. WIDER IMPLICATIONS OF THE FINDINGS: Over the 20 years of registration, the pregnancy rate has remained constant, despite the reduction in the number of embryos transferred, optimization of laboratory procedures and stimulation protocols, introduction of quality systems and implementation of the EU Tissue Directive over the period 2004-2010. STUDY FUNDING/COMPETING INTEREST(S): No external funding was sought for this study. None of the authors has any conflict of interest to declare.

Impact of follicular G-CSF quantification on subsequent embryo transfer decisions: a proof of concept study.

BACKGROUND: Previous experiments have shown that granulocyte colony-stimulating factor (G-CSF), quantified in the follicular fluid (FF) of individual oocytes, correlates with the potential for an ongoing pregnancy of the corresponding fertilized oocytes among selected transferred embryos. Here we present a proof of concept study aimed at evaluating the impact of including FF G-CSF quantification in the embryo transfer decisions. METHODS: FF G-CSF was quantified with the Luminex XMap technology in 523 individual FF samples corresponding to 116 fresh transferred embryos, 275 frozen embryos and 131 destroyed embryos from 78 patients undergoing ICSI. RESULTS: Follicular G-CSF was highly predictive of subsequent implantation. The receiving operator characteristics curve methodology showed its higher discriminatory power to predict ongoing pregnancy in multivariate logistic regression analysis for FF G-CSF compared with embryo morphology [0.77 (0.69-0.83), P < 0.001 versus 0.66 (0.58-0.73), P = 0.01)]. Embryos were classified by their FF G-CSF concentration: Class I over 30 pg/ml (a highest positive predictive value for implantation), Class II from 30 to 18.4 pg/ml and Class III <18.4 pg/ml (a highest negative predictive value). Embryos derived from Class I follicles had a significantly higher implantation rate (IR) than those from Class II and III follicles (36 versus 16.6 and 6%, P < 0.001). Embryos derived from Class I follicles with an optimal morphology reached an IR of 54%. Frozen-thawed embryos transfer derived from Class I follicles had an IR of 37% significantly higher than those from Class II and III follicles, respectively, of 8 and 5% (P < 0.001). Thirty-five per cent of the frozen embryos but also 10% of the destroyed embryos were derived from G-CSF Class I follicles. Non-optimal embryos appear to have been transferred in 28% (22/78) of the women, and their pregnancy rate was significantly lower than that of women who received at least one optimal embryo (18 versus 36%, P = 0.04). CONCLUSIONS: Monitoring FF G-CSF for the selection of embryos with a better potential for pregnancy might improve the effectiveness of IVF by reducing the time and cost required for obtaining a pregnancy.

New pre-conception immune biomarkers for clinical practice: interleukin-18, interleukin-15 and TWEAK on the endometrial side, G-CSF on the follicular side.

Identification of biomarkers of optimal uterine receptivity to the implanting embryo as well as biomarkers of oocyte competence would undoubtedly improve the efficiency of assisted reproductive technology (ART). Expression of IL-15 and IL-18 has been shown to be different in patients with failed implantation after IVF/ICSI compared with fertile controls and both correlate with local uNK (CD56+) recruitment and angiogenesis. Tumor necrosis factor weak inducer of apoptosis (TWEAK) has been described in mice as a potent early immune regulator able to protect the conceptus. The results of our studies in human suggest that TWEAK modulates the IL-18 related cytotoxicity of uNK cells. Quantification of IL-18, TWEAK and IL-15 mRNA expression by real-time PCR in endometrial tissue collected in mid-luteal phase of non-conception cycles allowed documentation of physiological events that occur at the time of uterine receptivity. Such information may be useful for the physician especially in patients where embryos fail to implant. Cytokine quantification may assist in understanding the mechanisms leading to repeated IVF/ICSI failure: either depletion of cytokines necessary for the apposition-adhesion, or an excess of cytokines leading to local cytotoxicity, may impair the implantation of the embryo. Other new data suggest that a pre-conception dialogue mediated by the oocyte and the follicular fluid and the oocyte may contribute to later implantation success. Follicular concentration of G-CSF appears as a useful biomarker of oocyte competence before fertilization. Moreover both in human and animal models, evidence of a role of the endometrium as a biosensor of the embryo is emerging.

Dialogue between blastocyst hCG and endometrial LH/hCG receptor: which role in implantation?

The specific interaction of blastocyst-derived human chorionic gonadotropin (hCG) and endometrial LH/hCG-R constitutes a fundamental component of the molecular dialogue at the materno-fetal interface. From our observations and studies from other groups, hCG was indeed shown to play a significant role in implantation and tolerance of the embryo, decidual differentiation and remodeling, as well as in placentation. The profile pattern of LH/hCG-R expression by endometrial epithelium correlates with the theoretical timing of the implantation window. Studies are currently being conducted in assisted medical procreation and in an animal model of implantation to establish the index of LH/hCG-R expression as a new biomarker of uterine receptivity for embryo implantation.

[What's new at the maternal-foetal interface: role of the hCG/LH-hCG receptor couple during embryo implantation]. / Du nouveau à l'interface materno-foetale: rô1e du couple hCG/récepteur LH-hCG dans l'implantation embryonnaire.

Implantation of the embryo into the maternal endometrium represents a unique biological process, combining an immunological (tolerance of an allograft) and biological (adhesion of two epitheliums) paradox. The success of implantation depends on a receptive endometrium, a functionally normal blastocyst and a synchronized cross-talk between embryonic and maternal tissues. Though sexual steroids control the process, a cascade of growth factors or cytokines are the prime paracrine mediators of the dialogue at the maternal-embryonic interface. HCG is one of the molecules most precociously produced by the embryo and is the most specific marker of its presence. HCG is a luteotropic factor which relays the inadequate support provided by the reduced rates of LH, but also influences the pregnancy on a paracrine mode by a local action on implantation process, probably by interacting with its receptor, the LH/hCG-R that we have evidenced on endometrial epithelium. We demonstrate that embryo actively participate into its implantation, tolerance and placentation.

Human chorionic gonadotropin and growth factors at the embryonic-endometrial interface control leukemia inhibitory factor (LIF) and interleukin 6 (IL-6) secretion by human endometrial epithelium.

BACKGROUND: The elucidation of the molecular mechanisms by which the embryo contributes to its implantation is an area of extensive research. The main objective of this study was to investigate the pattern of leukemia inhibitory factor (LIF) and interleukin-6 (IL-6) secretion by human endometrial epithelium, and their regulation by human chorionic gonadotropin (hCG) and other growth factors present at the embryonic-endometrial interface. METHODS: Endometrial epithelial cells (EEC) were isolated from biopsies collected at both proliferative and secretory phases of fertile women. RESULTS: HCG (1-50 IU/ml) increased LIF secretion by EEC cultures derived from follicular phase (up to 285+/-75%) or from secretory phase (up to 212+/-16%). In contrast, hCG reduced IL-6 secretion by EEC in both phases. The hCG/LH receptor gene was transcribed by EEC as evidenced by RT-PCR. Insulin-like growth factors 1 and 2 increased LIF secretion by EEC. Transforming growth factor beta1 stimulated LIF and reduced IL-6 secretion. CONCLUSIONS: Through hCG, the blastocyst may be involved in the control of its implantation (via an increase of proimplantatory LIF) and tolerance (via an inhibition of proinflammatory IL-6). Other growth factors present at the embryonic-endometrial interface are also involved in the control of LIF and IL-6 endometrial secretion.

[Implantation: the first maternal-embryo crosstalk]. / L'implantation: premier dialogue entre la mère et l'embryon.

Despite progress in assisted reproduction technologies, the lack of control of implantation remains a major obstacle to obtain successful pregnancies. It is of prime importance to determine the characteristic features of a receptive endometrium. Embryo implantation is a complex event involving apposition followed by the adhesion of the blastocyst to the maternal endometrium, and finally the invasion of this endometrium. Though implantation could occur in any human tissue, the endometrium is the only tissue where embryo implantation cannot occur except during a restricted period called the "implantation window". During this window, the endometrium is highly receptive to the embryo. Maternal-embryo crosstalk is favored by the implantation window. The molecular basis of the implantation window remains to be defined In the present study, we investigated the role of hCG, TGFbeta1, IGF1 and IGF2 in the secretion of leukemia inhibitory factor (LIF) and interleukin-6 (IL-6) by human epithelial endometrial cells in culture. Epithelial endometrial cells were obtained from endometrial biopsy in 28 fertile women. HCG and TGFbeta1 added to primary cultures of human epithelial endometrial cells showed a stimulatory effect on LIF secretion and a reducing effect on IL-6 secretion. All these findings suggest a role for hCG and TGFbeta1 in human embryo implantation.

[The implantation window]. / La fenêtre implantatoire.

BACKGROUND: Embryo implantation is a complex event involving apposition followed by adhesion of the blastocyst to the maternal endometrium, and finally invasion of this endometrium. Though implantation could occur in any human tissue, the endometrium is the only tissue where embryo implantation cannot occur except during a restricted period called the implantation window. During this window, the endometrium is highly receptive to the embryo. MATERIAL: and methods. We reviewed the literature concerning the different factors involved in improved endometrial receptivity and implantation. RESULTS: Maternal - embryo crosstalk is favored by the implantation window. Endometrial receptivity results from the acquisition of ligands or receptors facilitating apposition, then adhesion of the embryo, or from the loss of components preventing it. The molecular basis of the implantation window remains to be defined. CONCLUSION: Despite progress in assisted reproduction technologies, the lack of control of implantation remains a major obstacle to successful pregnancy. It is of prime importance to determine the characteristic features of a receptive endometrium and, among the many markers proposed by in vitro studies, to analyze in humans those demonstrated by knock-out experiments to play a crucial role in mice.

Human endometrial epithelial cells modulate the activation of gelatinase a by stromal cells.

Metalloproteinases (MMPs) are central effectors in endometrial physiology. Their production is tightly regulated by ovarian steroids and cytokines. Using zymography, we investigated MMP-2 production by human endometrial cells treated with estradiol-17beta + progesterone (E(2)+P) and by various key cytokines in endometrial physiology (IL-1beta, LIF, TGF-beta, and TNF-alpha). No gelatinase activity was detected in the culture media of epithelial cells. In basal conditions, stromal cells produced the pro form of MMP-2. MMP-2 production/activation was not directly affected by cytokine treatment. Interestingly, activated MMP-2 was only detected after treatment of stromal cells with culture medium from epithelial cells. Cytokine treatment of epithelial cells increased the capacity of conditioned medium to stimulate stromal cells to activate MMP-2. As the tissue inhibitor of MMP-2 (TIMP-2) is a regulator of gelatinase A activity, its concentration was measured by ELISA. TIMP-2 production by stromal cells was not affected by cytokines or by epithelial cell-conditioned medium. These results strongly suggest that regulation of stromal MMP-2 activation involves soluble factor(s) derived from the epithelial compartment.