Dominique Royère

The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting

This paper reports the proceedings of an international consensus meeting on oocyte and embryo morphology assessment. Following background presentations about current practice, the expert panel developed a set of consensus points to define the minimum criteria for oocyte and embryo morphology assessment. It is expected that the definition of common terminology and standardization of laboratory practice related to embryo morphology assessment will result in more effective comparisons of treatment outcomes. This document is intended to be referenced as a global consensus to allow standardized reporting of the minimum dataset required for the accurate description of embryo development. This paper reports the proceedings and outcomes of an international consensus meeting on human oocyte and embryo morphology assessment. An expert panel developed a series of consensus points to define the minimum criteria for such assessments. The definition of common terminology, and standardization of laboratory practices related to these morphological assessments, will permit more effective comparisons of treatment outcomes around the world. This report is intended to be referenced as a global consensus to allow standardized reporting of the minimum descriptive criteria required for routine clinical evaluations of human embryo development in vitro.

Adiponectin increases insulin-like growth factor I-induced progesterone and estradiol secretion in human granulosa cells

OBJECTIVE To identify adiponectin and its receptors (AdipoR1 and AdipoR2) in human granulosa cells (GC) and to study the effects of recombinant human adiponectin on P and E(2) secretion from these cells. DESIGN The effects of recombinant human adiponectin on the secretion of P and E(2) by cultured human GCs were investigated. SETTING Academic institutions. PATIENT(S) Seventeen infertile and healthy women undergoing IVF. INTERVENTION(S) Primary human GC cultures stimulated with human recombinant adiponectin (5 microg/mL). MAIN OUTCOME MEASURE(S) Determination of messenger RNA (mRNA) and protein expression of adiponectin and its receptors AdipoR1 and AdipoR2 in fresh human GCs by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot, respectively. Measurement of P and E(2) levels in the conditioned media by RIA and determination of cell proliferation by tritied thymidine incorporation. RESULT(S) Human GCs express adiponectin receptors AdipoR1 and AdipoR2 but not adiponectin. In primary human GCs, adiponectin increases P and E(2) secretion in response to insulin-like growth factor I (IGF-I). This was associated with an increase in the p450 aromatase protein level but not those of p450scc, 3 beta HSD, or StAR. Adiponectin treatment does not affect IGF-1-induced cell proliferation and basal steroidogenesis (no IGF-1 or FSH stimulation). Adiponectin rapidly stimulates MAPK ERK1/2 and p38 phosphorylation in primary human GCs. CONCLUSION(S) Adiponectin receptors AdipoR1 and AdipoR2, but not adiponectin, are present in human GCs. Adiponectin increases IGF-1-induced P and E(2) secretion in primary human GCs.

Chemerin inhibits IGF-1-induced progesterone and estradiol secretion in human granulosa cells

BACKGROUND Chemerin is a novel adipokine involved in the regulation of adipocyte development, inflammation and metabolic functions. To date, no role of this adipokine in reproductive functions has been described. In the present study, we identified chemerin and its receptor, CMKLR1 (chemokine-like receptor 1), in primary human granulosa cells (hGCs) and in a human ovarian granulosa-like tumour cell line (KGN). We also investigated the effects of recombinant human chemerin (rhChem) on steroid production and on various signalling pathways. METHODS AND RESULTS By RT-PCR immunoblotting and immunohistochemistry, we showed that chemerin and CMKLR1 are expressed in hGCs and KGN cells. By ELISA, we also found chemerin in human follicular fluid and we observed that in 8 of 10 women the chemerin level was at least 2-fold higher in follicular fluid than in plasma. rhChem (10 or 100 ng/ml) significantly decreased insulin-like growth factor-1 (IGF-1) (10(-8) M)-induced secretion of progesterone and estradiol (as determined by radioimmunoassay) but did not affect basal-or FSH (10(-8) M)-induced steroid secretion in hGCs and KGN cells. In parallel, it also decreased IGF-1-induced p450 aromatase protein levels without affecting the protein levels of other factors involved in steroidogenesis (steroidogenic acute regulatory protein, 3-beta-hydroxysteroid dehydrogenase and p450 side-chain cleavage enzyme) in hGCs cells. All these changes were associated with a decrease in the IGF-1-induced tyrosine phosphorylation of IGF-1 receptor beta subunit and phosphorylation of mitogen-activated protein kinase extracellular signal-regulated kinases 1/2 (MAPK ERK1/2) and Akt. In hGCs and KGN cells, rhChem also decreased IGF-1-induced thymidine incorporation. Finally, we showed that rhChem rapidly activates MAPK ERK1/2, MAPK P38 and Akt phosphorylation and more slowly AMP-activated protein kinase phosphorylation under basal conditions (no IGF-1 or FSH) in primary hGC cells. CONCLUSIONS Taken together, chemerin and its receptor (CMKLR1) are present and active in hGCs. Chemerin reduces IGF-1-induced steroidogenesis and cell proliferation through a decrease in the activation of IGF-1R signalling pathways in primary hGCs.

A randomized prospective study comparing pregnancy rates after clomiphene citrate and human menopausal gonadotropin before intrauterine insemination

OBJECTIVE To determine whether hMG offers an advantage over clomiphene citrate (CC) in achieving pregnancy after IUI with husbands sperm. DESIGN Randomized prospective trial. SETTING Infertility patients in a university teaching hospital. PATIENT(S) Fifty-eight women under 39 years old undergoing ovulation induction before IUI. INTERVENTION(S) The women were assigned randomly to one of two treatment groups. Patients in group I (CCHH) received CC for the first two cycles and hMG for the last two cycles. Patients in group II (HHCC) received hMG for the first two cycles and CC for the last two cycles. MAIN OUTCOME MEASURE(S) Cycle fecundity rates for the two treatment modalities were compared statistically with use of life-table analysis. RESULT(S) Of the 174 cycles studied, overall cycle fecundity rate was 11.11 (9 of 81 cycles) in the CCHH group and 10.75 (10 of 93 cycles) in the HHCC group. The difference was not statistically significant. The cycle fecundity rate was 14.44% (13 of 90 cycles) for cycles with CC and 7.14% (6 of 84) with hMG. The difference was not statistically significant. CONCLUSION(S) These data suggest that CC is an effective alternative to hMG in the population examined.

Visfatin is expressed in human granulosa cells: regulation by metformin through AMPK/SIRT1 pathways and its role in steroidogenesis

Visfatin is a cytokine hormone and an enzyme involved in metabolic (obesity, type II diabetes) and immune disorders. Some data suggest a role of visfatin in ovarian function. Here, we identified visfatin in human follicles and investigated the molecular mechanisms involved in the regulation of its expression in response to insulin sensitizers, metformin (MetF) and rosiglitazone, in primary human granulosa cells (hGCs) and in a human ovarian granulosa-like tumour cell line (KGN). We also studied the effects of human recombinant visfatin (RhVisf) on steroid production and on the activation of various signalling pathways. By RT-PCR, immunoblotting and immunohistochemistry, we showed that visfatin is expressed not only in hGCs and KGN cells, but also in human cumulus cells and oocytes. In hGCs and KGN cells, MetF increased visfatin mRNA in a dose-dependent manner (0.1, 1 and 10 mM), and rosiglitazone increased visfatin mRNA expression (only at 10 μM) after treatments for 24 h, whereas both reduced it after 48 h of incubation. This regulation was confirmed at the protein level for the MetF treatment only. Using the compound C and Aicar, inhibitor and activator of AMP-activated protein kinase (AMPK), respectively, and Sirtinol, an inhibitor of sirtuin-1 (SIRT1), we observed that these MetF effects on visfatin expression were mediated through the AMPK/SIRT1 signalling pathways. RhVisf (10 ng/ml) significantly increased insulin-like growth factor-1 (IGF-1) (10 nM)- but not FSH (10 nM)-induced secretion of progesterone and estradiol as determined by radioimmunoassay and IGF-1-induced thymidine incorporation in hGCs and KGN cells. Finally, rhVisf rapidly activates the mitogen-activated protein kinase pathway via ERK1/2, P38 and Akt phosphorylation under basal conditions in primary hGC cells. In conclusion, visfatin is present in ovarian human follicles, and in hGCs and KGN cells, visfatin increases IGF-1-induced steroidogenesis and cell proliferation and MetF regulates visfatin expression through the AMPK/SIRT1 signalling pathway.

Resistin decreases insulin-like growth factor I–induced steroid production and insulin-like growth factor I receptor signaling in human granulosa cells

OBJECTIVE To identify resistin in human ovarian follicles and investigate the effect and the molecular mechanisms associated with resistin on steroidogenesis in human granulosa cells (GCs). DESIGN The effects of recombinant human resistin on the secretion of progesterone (P) and estradiol (E2) by cultured human GCs were investigated. SETTING Academic institutions. PATIENT(S) Twenty infertile and healthy women undergoing IVF. INTERVENTION(S) Primary human GC cultures stimulated with recombinant human resistin (10 ng/mL). MAIN OUTCOME MEASURE(S) Determination of messenger RNA (mRNA) and protein expression of resistin in fresh human GCs by reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblot and immunohistochemistry, respectively; measurement of P and E2 levels in the conditioned media by radioimmunoassay; determination of cell proliferation by tritiated thymidine incorporation; and analysis of signaling pathways activation by immunoblot analysis. RESULT(S) Human GCs and theca cells express resistin. In primary human GCs, resistin decreases P and E2 secretion in response to insulin-like growth factor I (IGF-I). This was associated with a reduction in the P450 aromatase and P450scc (cholesterol side-chain cleavage cytochromes P450) (P450scc) protein levels but not those of 3β-hydroxysteroid dehydrogenase (3β-HSD) or steroidogenic acute regulatory protein (StAR) and with a decrease in IGF-I-induced IGF-I receptor and mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Resistin treatment does not affect IGF-I-induced cell proliferation and basal steroidogenesis (there is no IGF-I or follicle-stimulating hormone stimulation). In the basal state, resistin rapidly stimulates Akt and MAPK ERK1/2 and p38 phosphorylation in primary human GCs. CONCLUSION(S) Resistin is present in human GCs and theca cells. It decreases P and E2 secretion, P450scc and P450 aromatase protein levels, and IGF-IR signaling in response to IGF-I in primary human GCs.

Treating women under 36 years old without top-quality embryos on day 2: a prospective study comparing double embryo transfer with single blastocyst transfer

BACKGROUND Embryologists currently face a challenge when counselling patients regarding the stage and the number of embryos to transfer when no top-quality embryos (TQE) are available. METHODS The aim of this study was to evaluate the efficacy of single blastocyst transfer (SBT) in comparison with the transfer of two cleavage-stage embryos in women under 36 years old. A total of 450 women under 36 years undergoing their first or second IVF treatment who had no TQE on Day 2 were included in this prospective study. Couples were assigned to either a SBT or a double cleavage-stage embryo transfer (DET). The clinical end-points monitored were rates of implantation, delivery and multiple deliveries. RESULTS The rate of transfer was significantly lower for couples assigned to the SBT group compared with the DET group (88 versus 100%, respectively, P < 0.001) while the delivery rate per oocyte retrieval was similar in both groups (26.7%). By contrast, the rate of multiple deliveries was significantly lower in the SBT group compared with the DET group (3.3 versus 23.3%, respectively, P < 0.01). Blastocyst cryopreservation was twice as high in the SBT group compared with the DET group (39 versus 18%, respectively, P < 0.001). CONCLUSIONS These findings show the value of extended embryo culture for couples without TQE. In such situations, delaying embryo transfer in order to select a single blastocyst with the highest potential for implantation can reduce the number of multiple pregnancies. Furthermore, our results demonstrate that extended culture allows blastocyst cryopreservation from embryos not available for Day 2 cryopreservation.