Improved clinical outcomes after non-invasive oocyte selection and Day 3 eSET in ICSI patients.

BACKGROUND: Non-invasive oocyte quality scoring, based on cumulus gene expression analysis, in combination with morphology scoring, can increase the clinical pregnancy (CPR) and live birth rates (LBR) in Day 3 eSET (elective single embryo transfer) ICSI patients. This was first investigated in a pilot study and is now confirmed in a large patient cohort of 633 patients. It was investigated whether CPR, LBR and time-to-pregnancy could be improved by analyzing the gene expression profile of three predictive genes in the cumulus cells, compared to patients with morphology-based embryo selection only. METHODS: A large interventional, non-randomized, assessor-blinded cohort study with 633 ICSI patients was conducted in a tertiary fertility center. Non-PCOS patients, 22-39 years old, with good ovarian reserve, were stimulated with HP-hMG using a GnRH antagonist protocol and planned for fresh Day 3 eSET. The cumulus cells from individually denuded oocytes were ranked by a lab-developed cumulus cell test: qRT-PCR for three predictive genes (CAMK1D, EFNB2 and SASH1) and two control genes (UBC, B2M). The embryo selected for transfer was highest ranked from the pool of morphologically transferable Day 3 embryos. Patients in the control (n = 520) and experimental arm (n = 113) were compared for clinical pregnancy and live birth, using a weighted generalized linear model, and time-to-pregnancy using Kaplan-Meier curves. RESULTS: The CPR was 61% in the experimental arm (n = 113) vs 29% in the control arm (n = 520, p < 0.0001). The LBR in the experimental arm (50%) was significantly higher than in the control arm (27%,p < 0.0001). Time-to-pregnancy was significantly shortened by 3 transfer cycles independent of the number of embryos available on Day 3 (Kaplan-Meier, p < 0.0001). Cumulus cell tested patients < 35 years (n = 65) or ≥ 35 years (n = 48) had a CPR of 62 and 60% respectively (ns). For cumulus cell tested patients with 2, 3-4, or > 4 transferable embryos, the CPR was 66, 52, and 67% (ns) respectively, and thus independent of the number of transferable embryos on Day 3. CONCLUSIONS: This study provides further evidence of the clinical usefulness of the non-invasive cumulus cell test over time in a larger patient cohort. TRIAL REGISTRATION: Clinicaltrials.gov, NCT03659786 / NCT02962466 (Registered 6Sep2018/11Nov2016, retrospectively registered.

Significance of premature progesterone rise in IVF.

PURPOSE OF REVIEW: The current review will focus on the cause of premature progesterone and its impact on endometrial receptivity and endometrial gene expression. RECENT FINDINGS: Premature progesterone rise in stimulated IVF cycles seems to have a negative impact on the pregnancy rate. The current knowledge on the cause of a premature progesterone rise is still limited; however, it seems that overstimulating the ovaries during the end of the follicular phase will lead to a premature progesterone rise. The negative impact on the outcome is mainly due to reduced endometrial receptivity as can be seen in the altered endometrial gene expression. SUMMARY: Future studies should focus on how ovarian stimulation can be tailored according to individual requirements of the patients, aiming on preventing a possible progesterone rise during follicular phase of stimulated IVF cycles.

Non-invasive cumulus cell analysis can be applied for oocyte ranking and is useful for countries with legal restrictions on embryo generation or freezing.

RESEARCH QUESTION: Can a strategy for scoring oocyte quality, based on cumulus cell (CC) gene expression, prioritize oocytes with the highest implantation potential, while limiting the number of embryos to be processed in culture and the number of supernumerary embryos to be vitrified? DESIGN: An interventional, blinded, prospective cohort study was retrospectively analyzed. In the original study, patients underwent a fresh Day3 single embryo transfer with embryos ranked based on morphology and CC gene expression (Aurora Test). The additional ranking of the embryos with the Aurora Test resulted in significant higher clinical pregnancy and live birth rates. Now it is investigated if the Aurora Test ranking could be applied to select oocytes. The effect of an Aurora Test based restriction to 2 and 3 2PN or MII oocytes on clinical pregnancy and other outcomes, was analyzed in two subsets of patients with all 2PN (n = 83) or all MII oocytes (n = 45) ranked. RESULTS: Considering only the top three ranked 2PN oocytes, 95% of the patients would have received a fresh SET on Day3 resulting in 65% clinical pregnancies. This was not different from the pregnancy rate obtained in a strategy using all oocytes but significantly reduced the need for vitrification of supernumerary embryos by 3-fold. Considering only top-ranked MII oocytes gave similar results. CONCLUSIONS: In countries with legal restrictions on freezing of embryos, gene expression of CC can be used for the selective processing of oocytes and would thus decrease the twin pregnancy rate and workload, especially for embryo morphology scoring and transfers as the handling and processing of lower competence oocytes is prevented, while improving the ART outcome.

Gene expression profile in the endometrium on the day of oocyte retrieval after ovarian stimulation with low-dose hCG in the follicular phase.

The past decade has seen growing interest in ovarian stimulation protocols with GnRH antagonists in an effort to reduce the incidence of potential complications, such as cyst formation and ovarian hyperstimulation syndrome, and thus improve the clinical experience for patients. Current assisted reproductive technique programmes also increasingly utilize milder protocols for ovarian stimulation. In a recently published randomized controlled trial, we showed that low-dose hCG can be utilized clinically to replace recombinant FSH (rFSH) during the late follicular phase in a GnRH antagonist protocol. This regimen leads to a significant reduction in rFSH consumption, while the ICSI outcome, in terms of oocyte yield and ongoing pregnancy rate, remains comparable with the control regimen of rFSH plus a GnRH antagonist. In the present study, the influence of the administration of low-dose hCG on the endometrium was assessed. A comparison was made between two protocols for ovarian stimulation with GnRH antagonists, namely the classical protocol with rFSH and the protocol with low-dose hCG in the late follicular phase. We analysed the morphological pattern and gene expression profile of human endometrium on the day of oocyte retrieval. No morphological differences were observed and only a minimal set of 65 differentially expressed probe sets between the treatment groups were identified, enabling a similar efficacy to support implantation.

In GnRH antagonist/rec-FSH stimulated cycles, advanced endometrial maturation on the day of oocyte retrieval correlates with altered gene expression.

BACKGROUND: Previously, advanced endometrial maturation on the day of oocyte retrieval in GnRH antagonist and -agonist IVF cycles was observed. In these cycles, endometrial advancement exceeding 3 days between the histological dating and the cycle day never resulted in an ongoing clinical pregnancy. In this study, the gene expression of human endometrium on the day of oocyte retrieval in GnRH antagonist/rec-FSH cycles was analyzed, in correlation with the morphological dating. METHODS: Biopsies were taken on the day of oocyte retrieval in 47 patients with 1 or 2 embryos replaced on Day 3 in the same cycle. Endometrial dating was performed according to Noyes' criteria. Biopsies from 11 patients were analyzed for gene expression with the Affymetrix HG U133 Plus 2 microarray. Data analysis, clustering and pathway analysis were performed with GCOS, GeneSpring 7.3 and Ingenuity, respectively. RESULTS: According to Noyes' criteria, all endometria taken on the day of oocyte retrieval showed an advanced maturation, ranging from +d2 to +d4. The patients with a subsequent clinical pregnancy all showed a histological dating corresponding to +d2 or +d3. When comparing endometria +d2-3 to +d4, the microarray results showed a differential expression of 2550 probe sets. Significantly up-regulated genes were SERPINB6, FOXO3A, SOX17 and CDC42. Down-regulated genes of interest were NRP1, HOXA10 and OSF2. Principal component analysis and hierarchical clustering demonstrated two distinct clusters. CONCLUSIONS: In stimulated cycles, endometrial gene expression on the day of oocyte retrieval discriminates between women with and without histologically advanced endometrial maturation exceeding 3 days and supports histological dating results by Noyes' criteria.

Aberrant endometrial steroid receptor expression in in-vitro maturation cycles despite hormonal luteal support: A pilot study.

Clinical outcomes of fresh embryo transfer in non-hCG triggered in vitro maturation (IVM) cycles are inferior compared to vitrified-warmed embryo transfer. This is a prospective observational pilot study in a consecutive cohort of 31 polycystic ovary syndrome (PCOS) patients and 37 normo-ovulatory egg donors who underwent IVM without fresh embryo transfer between July 2009 and June 2014. All subjects received 150 IU of highly purified menotropin (HP-hMG) daily for three days. On cycle day 6, all patients started transdermal oestradiol (E2) at a daily dose of 9 mg. There was no human chorionic gonadotropin (hCG) trigger before oocyte retrieval (OR). Vaginal micronized progesterone was commenced on the evening after OR, at a daily dose of 600 mg. Additional luteal phase support (LPS) was administered as follows: Group A: no additional LPS; Group B: 1500 IU of hCG administered 4 h after OR and Group C: 5000 IU of hCG administered 4 h after OR + an additional injection of 5000 IU of hCG 1 day before endometrial biopsy. Endometrial biopsy for histology and immunohistochemistry (IHC) was performed on day 5 or 6 after OR. Instead of being downregulated, both PR-B and ERα in endometrial glands and stroma were moderately to strongly expressed in all three protocols, suggesting that the mid-luteal histological signature of endometrial receptivity is deficient in a non-hCG-triggered IVM cycle. Poor clinical outcomes after fresh embryo transfer following IVM are probably related to inappropriate endometrial development which may be linked to the short follicular phase of IVM cycles.

Early luteal phase endocrine profile is affected by the mode of triggering final oocyte maturation and the luteal phase support used in recombinant follicle-stimulating hormone-gonadotropin-releasing hormone antagonist in vitro fertilization cycles.

OBJECTIVE: To assess endocrine differences during early luteal phase according to mode of triggering final oocyte maturation with or without luteal phase support (LPS). DESIGN: A prospective randomized study. SETTING: University center for reproductive medicine. PATIENT(S): Four oocyte donors each underwent four consecutive cycles. INTERVENTION(S): To avoid interpatient variation, each donor underwent the same stimulation regimen. However, different modes of triggering final oocyte maturation and LPS were administered: A) 10,000 IU hCG and standard LPS; B) GnRH agonist (GnRHa; 0.2 mg triptorelin), and 35 hours later 1,500 IU hCG, and standard LPS; C) GnRH agonist (0.2 mg triptorelin) and standard LPS; and D) GnRH agonist (0.2 mg triptorelin) without LPS. MAIN OUTCOME MEASURE(S): Blood sampling was performed on the day of ovulation trigger, ovulation trigger + 1 day, and ovum pick-up + 5 days. Serum E2, FSH, LH, and P were measured. RESULT(S): The early luteal phase steroid levels following GnRHa trigger and modified luteal phase support (B) were similar to those seen after hCG trigger (A). However, significant differences were seen between groups A and B compared with C and D, as well as between groups C and D. CONCLUSION(S): Administration of a single bolus of GnRHa effectively induced LH and FSH surges in oocyte donors stimulated with recombinant FSH and cotreated with a GnRH antagonist. However, gonadotropin and steroid levels differed significantly according to the type of luteal phase support used after GnRHa trigger. EUROPEAN COMMUNITY CLINICAL TRIAL SYSTEM (EUDRACT) NUMBER: 2009-009429-26.

Cyclooxygenase-2 network as predictive molecular marker for clinical pregnancy in in vitro fertilization.

The gene expression of human endometrium on the day of oocyte retrieval of pregnant and nonpregnant patients in a stimulated IVF cycle was compared in two independent groups with different GnRH-antagonist ovarian stimulation protocols. The present data suggest that increased gene expression of cyclooxygenase-2, together with the expression of other molecules in the cyclooxygenase-2 network, on the day of oocyte retrieval in GnRH-antagonist cycles coincides with a lower probability of achieving a clinical pregnancy in this cycle.

Gene expression during successful implantation in a natural cycle.

OBJECTIVE: To determine the human endometrial transcriptome during embryonic implantation. DESIGN: Case report. SETTING: Tertiary fertility center. PATIENT(S): A 24-year-old woman who inadvertently became pregnant during an endometrial biopsy procedure. INTERVENTION(S): An endometrial biopsy was performed with a Pipelle device during the midluteal phase (days 19-21) of the cycle; blood samples for hormonal assessments were collected and a transvaginal ultrasound was performed. MAIN OUTCOME MEASURE(S): Gene expression analysis of the endometrium during the window of implantation (during the implantation of an embryo) in a natural cycle. Localization of selected genes in endometrial tissue with immunohistochemistry. RESULT(S): A total of 394 probe sets were differentially expressed in the pregnant sample when compared with the midsecretory phase nonpregnant endometrial samples. Different gene networks were involved, and selected genes from these signaling pathways were confirmed at the protein level. CONCLUSION(S): Endometrial gene expression of a pregnant patient in a natural cycle is significantly different from nonpregnant patients during the midsecretory phase.