Cumulus-corona gene expression analysis combined with morphological embryo scoring in single embryo transfer cycles increases live birth after fresh transfer and decreases time to pregnancy.

PURPOSE: Clinical pregnancy rate after IVF with eSET stagnates between 30 and 40%. In order to increase pregnancy and live birth rates, multiple embryo transfer is still common practice. Providing additional non-invasive tools to choose the competent embryo for transfer could avoid multiple pregnancy and improve time to pregnancy. Cumulus mRNA analysis with quantitative PCR (QPCR) is a non-invasive approach. However, so far, no gene sets have been validated in prospective interventional studies. METHODS: A prospective interventional single-center pilot study with two matched controls (day-3 and day-5 eSET) was performed in 96 patients consenting to the analysis of the cumulus-corona of their oocytes. All patients were super-ovulated for ICSI and eSET at day 3. All oocytes were denuded individually and cumulus was analyzed by quantitative PCR using three predictive genes (EFNB2, SASH1, CAMK1D) and two housekeeping genes (UBC and ß2M). Patients (n = 62) with 2 or more day-3 embryos (good or excellent morphology) had their embryo chosen following the normalized expression of the genes. RESULTS: Corona testing significantly increased the clinical pregnancy and live births rates (63% and 55%) compared to single embryo transfer (eSET) on day 3 (27% and 23%: p < 0.001) and day 5 (43% and 39%: p = 0.022 and p = 0.050) fresh transfer cycle controls with morphology-only selection. Time-to-pregnancy was significantly reduced, regardless of the number of good-quality embryos available on day 3. CONCLUSION: Combining standard morphology scoring and cumulus/corona gene expression analysis increases day-3 eSET results and significantly reduces the time to pregnancy. TRIAL REGISTRATION NUMBER: This is not an RCT study and was only registered by the ethical committee of the University Hospital UZBRUSSEL of the Vrije Universiteit Brussel VUB (BUN: 143201318000).

Impact of clomiphene citrate during ovarian stimulation on the luteal phase after GnRH agonist trigger.

The use of a gonadotrophin-releasing hormone (GnRH) agonist to trigger final oocyte maturation in a GnRH antagonist protocol has been associated with poorer clinical outcomes due to an increased luteal-phase defect. It has been shown that LH activity is crucial in a normal luteal phase. Studies assessing the LH concentrations after clomiphene citrate co-treatment have observed increased luteal-phase LH concentrations. The purpose of this prospective cohort study was to analyse the effect of clomiphene citrate on the endocrine profile in the luteal phase when using GnRH agonist trigger. This was evaluated in eight oocyte donors undergoing ovarian stimulation using clomiphene citrate in combination with recombinant FSH compared with a control group of five donors treated with recombinant FSH only. The endocrine profile was comparable in both groups, except for serum LH concentrations on the day after trigger (121.3±53.0IU/l versus 52.9±21.5IU/l, respectively, P=0.022). No significant differences in LH concentrations were found on the day of trigger or 5days after oocyte retrieval. In conclusion, a luteal-phase defect was observed despite treatment with clomiphene citrate during ovarian stimulation. The use of gonadotrophin-releasing hormone (GnRH) agonist to trigger ovulation in IVF has been associated with poorer pregnancy outcomes due to an increased luteal-phase defect. The luteal phase is the last phase of the menstrual cycle and is defined as the period between ovulation and the beginning of pregnancy or menses. It has been shown the activity of LH is crucial in a normal luteal phase. Studies assessing the LH concentrations after clomiphene citrate, an oestrogen receptor inhibitor, co-treatment have observed increased luteal-phase LH concentrations. The purpose of this prospective cohort study was to analyse the effect of clomiphene citrate on menstrual cycle day 2-6 on the hormone profile in the luteal phase when using GnRH agonist trigger. This was evaluated was in eight oocyte donors undergoing ovarian stimulation using recombinant FSH compared with a control cohort of donors treated with recombinant FSH only. The current prospective cohort study reports higher LH concentrations on the day after GnRH agonist trigger, but not 5days after oocyte retrieval (i.e. in the luteal phase). In conclusion, a luteal-phase defect was observed despite the administration of clomiphene citrate during ovarian stimulation. Additional treatment with clomiphene citrate in the follicular phase is therefore not a valid alternative to prevent luteal-phase defect after GnRH agonist trigger.

Endometrial gene expression in the early luteal phase is impacted by mode of triggering final oocyte maturation in recFSH stimulated and GnRH antagonist co-treated IVF cycles.

STUDY QUESTION: Do differences in endometrial gene expression exist after ovarian stimulation with four different regimens of triggering final oocyte maturation and luteal phase support in the same patient? SUMMARY ANSWER: Significant differences in the expression of genes involved in receptivity and early implantation were seen between the four protocols. WHAT IS KNOWN ALREADY: GnRH agonist triggering is an alternative to hCG triggering in GnRH antagonist co-treated cycles, resulting in an elimination of early ovarian hyperstimulation syndrome. Whereas previous studies have revealed a low ongoing clinical pregnancy rate after GnRH agonist trigger due to a high early pregnancy loss rate, despite supplementation with the standard luteal phase support, more recent studies, employing a 'modified' luteal phase support including a bolus of 1500 IU hCG on the day of oocyte aspiration, have reported ongoing pregnancy rates similar to those seen after hCG triggering. STUDY DESIGN, SIZE DURATION: A prospective randomized study was performed in four oocyte donors recruited from an oocyte donation program during the period 2010-2011. PARTICIPANTS, MATERIALS, SETTING, METHODS: Four oocyte donors in a university IVF center each prospectively underwent four consecutive stimulation protocols, with different modes of triggering final oocyte maturation and a different luteal phase support, followed by endometrial biopsy on Day 5 after oocyte retrieval. The following protocols were used: (A) 10 000 IU hCG and standard luteal phase support, (B) GnRH agonist (triptorelin 0.2 mg), followed by 1500 IU hCG 35 h after triggering final oocyte maturation, and standard luteal phase support, (C) GnRH agonist (triptorelin 0.2 mg) and standard luteal phase support and (D) GnRH agonist (triptorelin 0.2 mg) without luteal phase support. Microarray data analysis was performed with GeneSpring GX 11.5 (RMA algorithm). Pathway and network analysis was performed with the gene ontology software Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com, Redwood City, CA, USA). Samples were grouped and background intensity values were removed (cutoff at the lowest 20th percentile). A one-way ANOVA test (P< 0.05) was performed with Benjamini-Hochberg multiple testing correction. MAIN RESULTS: Significant differences were seen in endometrial gene expression, related to the type of ovulation trigger and luteal phase support. However, the endometrial gene expression after the GnRH agonist trigger and a modified luteal phase support (B) was similar to the pattern seen after the hCG trigger (A). LIMITATIONS, REASONS FOR CAUTION: The study was performed in four oocyte donors only; however, it is a strength of the study that the same donor underwent four consecutive stimulation protocols within 1 year to avoid inter-individual variations. WIDER IMPLICATIONS OF THE FINDINGS: These endometrial gene-expression findings support the clinical reports of a non-significant difference in live birth rates between the GnRH agonist trigger and the hCG trigger, when the GnRH agonist trigger is followed by a bolus of 1500 IU hCG at 35 h post trigger in addition to the standard luteal phase support. STUDY FUNDING/ COMPETING INTERESTS: This study was supported by an un-restricted research grant by MSD Belgium. TRIAL REGISTRATION NUMBER: EudraCT number 2009-009429-26, protocol number 997 (P06034).

Elevated progesterone during ovarian stimulation for IVF.

There is an ongoing debate regarding the impact of premature progesterone rise on the IVF outcome. The objective of this review is to assess evidence of poorer ongoing pregnancy rate in IVF cycles with elevated serum progesterone at the end of follicular phase in ovarian stimulation. It also explores the origin of the progesterone rise, potential modifying factors and possible methods to prevent its rise during ovarian stimulation. This review draws on information already published from monitoring progesterone concentrations at the end of follicular phase in ovarian stimulation. The databases of Medline and PubMed were searched to identify relevant publications. Good-quality evidence supports the negative impact on endometrial receptivity of elevated progesterone concentrations at the end of the follicular phase in ovarian stimulation. Future trials should document the cause and origin of premature progesterone in stimulated IVF cycles. There is an ongoing debate regarding the impact of premature progesterone rise on the IVF outcome. The objective of this review is to assess evidence of poorer ongoing pregnancy rate in IVF cycles with elevated serum progesterone at the end of follicular phase in ovarian stimulation. It also explores the origin of the progesterone rise, potential modifying factors and possible methods to prevent its rise during ovarian stimulation. This review draws on information already published from monitoring progesterone concentrations at the end of follicular phase in ovarian stimulation. The databases of Medline and PubMed were searched to identify relevant publications. Good-quality evidence supports the negative impact on endometrial receptivity of elevated progesterone concentrations at the end of follicular phase in ovarian stimulation. Future trials should document the cause and origin of premature progesterone in stimulated IVF cycles.

Progesterone rise on HCG day in GnRH antagonist/rFSH stimulated cycles affects endometrial gene expression.

Premature progesterone rise during gonadotrophin-releasing hormone (GnRH) antagonist cycles for IVF is a frequent phenomenon and has been associated with lower pregnancy and implantation rates. This study evaluated endometrial gene expression on the day of oocyte retrieval according to the concentration of serum progesterone on the day of human chorionic gonadotrophin (HCG) administration in GnRH-antagonist/recombinant FSH IVF cycles with fresh embryo transfer. Endometrial biopsies (n=14) were analysed with Affymetrix HG U133 Plus 2.0 Arrays. Patients were divided into three groups according to their progesterone serum concentration on the day of HCG administration: ≤ 0.9 ng/ml (group A), 1-1.5 ng/ml (group B) and >1.5 ng/ml (group C). Gene expression analysis showed a small number of significantly differentially expressed probe sets between groups A and B (five up/23 down in B) and a large difference between groups B and C (607 up/212 down; P ≤ 0.05, fold change ≥ 1.4). Validation was performed with quantitative real-time PCR on selected genes. As far as is known, this is the first study to demonstrate a distinct difference in endometrial gene expression profile between patients with a progesterone serum concentration above and below the threshold of 1.5 ng/ml on the day of HCG administration.