Oocyte competence is independent of the ovulation trigger adopted: a large observational study in a setting that entails vitrified-warmed single euploid blastocyst transfer.

PURPOSE: To assess whether the GnRH-agonist or urinary-hCG ovulation triggers affect oocyte competence in a setting entailing vitrified-warmed euploid blastocyst transfer. METHODS: Observational study (April 2013-July 2018) including 2104 patients (1015 and 1089 in the GnRH-a and u-hCG group, respectively) collecting ≥1 cumulus-oocyte-complex (COC) and undergoing ICSI with ejaculated sperm, blastocyst culture, trophectoderm biopsy, comprehensive-chromosome-testing, and vitrified-warmed transfers at a private clinic. The primary outcome measure was the euploid-blastocyst-rate per inseminated oocytes. The secondary outcome measure was the maturation-rate per COCs. Also, the live-birth-rate (LBR) per transfer and the cumulative-live-birth-delivery-rate (CLBdR) among completed cycles were investigated. All data were adjusted for confounders. RESULTS: The generalized-linear-model adjusted for maternal age highlighted no difference in the mean euploid-blastocyst-rate per inseminated oocytes in either group. The LBR per transfer was similar: 44% (n=403/915) and 46% (n=280/608) in GnRH-a and hCG, respectively. On the other hand, a difference was reported regarding the CLBdR per oocyte retrieval among completed cycles, with 42% (n=374/898) and 25% (n=258/1034) in the GnRh-a and u-hCG groups, respectively. Nevertheless, this variance was due to a lower maternal age and higher number of inseminated oocytes in the GnRH-a group, and not imputable to the ovulation trigger itself (multivariate-OR=1.3, 95%CI: 0.9-1.6, adjusted p-value=0.1). CONCLUSION: GnRH-a trigger is a valid alternative to u-hCG in freeze-all cycles, not only for patients at high risk for OHSS. Such strategy might increase the safety and flexibility of controlled-ovarian-stimulation with no impact on oocyte competence and IVF efficacy.

The euploid blastocysts obtained after luteal phase stimulation show the same clinical, obstetric and perinatal outcomes as follicular phase stimulation-derived ones: a multicenter study.

STUDY QUESTION: Are the reproductive outcomes (clinical, obstetric and perinatal) different between follicular phase stimulation (FPS)- and luteal phase stimulation (LPS)-derived euploid blastocysts? SUMMARY ANSWER: No difference was observed between FPS- and LPS-derived euploid blastocysts after vitrified-warmed single embryo transfer (SET). WHAT IS KNOWN ALREADY: Technical improvements in IVF allow the implementation non-conventional controlled ovarian stimulation (COS) protocols for oncologic and poor prognosis patients. One of these protocols begins LPS 5 days after FPS is ended (DuoStim). Although, several studies have reported similar embryological outcomes (e.g. fertilization, blastulation, euploidy) between FPS- and LPS-derived cohort of oocytes, information on the reproductive (clinical, obstetric and perinatal) outcomes of LPS-derived blastocysts is limited to small and retrospective studies. STUDY DESIGN, SIZE, DURATION: Multicenter study conducted between October 2015 and March 2019 including all vitrified-warmed euploid single blastocyst transfers after DuoStim. Only first transfers of good quality blastocysts (≥BB according to Gardner and Schoolcraft's classification) were included. If euploid blastocysts obtained after both FPS and LPS were available the embryo to transfer was chosen blindly. The primary outcome was the live birth rate (LBR) per vitrified-warmed single euploid blastocyst transfer in the two groups. To achieve 80% power (α = 0.05) to rule-out a 15% difference in the LBR, a total of 366 first transfers were required. Every other clinical, as well as obstetric and perinatal outcomes, were recorded. PARTICIPANTS/MATERIALS, SETTING, METHODS: Throughout the study period, 827 patients concluded a DuoStim cycle and among them, 339 did not identify any transferable blastocyst, 145 had an euploid blastocyst after FPS, 186 after LPS and 157 after both FPS and LPS. Fifty transfers of poor quality euploid blastocysts were excluded and 49 patients did not undergo an embryo transfer during the study period. Thus, 389 patients had a vitrified-warmed SET of a good quality euploid blastocyst (182 after FPS and 207 after LPS). For 126 cases (32%) where both FPS- and LPS-derived good quality blastocysts were available, the embryo transferred was chosen blindly with a 'True Random Number Generator' function where '0' stood for FPS-derived euploid blastocysts and '1' for LPS-derived ones (n = 70 and 56, respectively) on the website random.org. All embryos were obtained with the same ovarian stimulation protocol in FPS and LPS (GnRH antagonist protocol with fixed dose of rec-FSH plus rec-LH and GnRH-agonist trigger), culture conditions (continuous culture in a humidified atmosphere with 37°C, 6% CO2 and 5% O2) and laboratory protocols (ICSI, trophectoderm biopsy in Day 5-7 without assisted hatching in Day 3, vitrification and comprehensive chromosome testing). The women whose embryos were included had similar age (FPS: 38.5 ± 3.1 and LPS: 38.5 ± 3.2 years), prevalence of male factor, antral follicle count, basal hormonal characteristics, main cause of infertility and previous reproductive history (i.e. previous live births, miscarriages and implantation failures) whether the embryo came from FPS or LPS. All transfers were conducted after warming in an artificial cycle. The blastocysts transferred after FPS and LPS were similar in terms of day of full-development and morphological quality. MAIN RESULTS AND THE ROLE OF CHANCE: The positive pregnancy test rates for FPS- and LPS-derived euploid blastocysts were 57% and 62%, biochemical pregnancy loss rates were 10% and 8%, miscarriage rates were 15% and 14% and LBRs were 44% (n = 80/182, 95% CI 37-51%) and 49% (n = 102/207, 95% CI 42-56%; P = 0.3), respectively. The overall odds ratio for live birth (LPS vs FPS (reference)) adjusted for day of blastocyst development and quality, was 1.3, 95% CI 0.8-2.0, P = 0.2. Among patients with euploid blastocysts obtained following both FPS and LPS, the LBRs were also similar (53% (n = 37/70, 95% CI 41-65%) and 48% (n = 27/56, 95% CI 35-62%) respectively; P = 0.7). Gestational issues were experienced by 7.5% of pregnant women after FPS- and 10% of women following LPS-derived euploid single blastocyst transfer. Perinatal issues were reported in 5% and 0% of the FPS- and LPS-derived newborns, respectively. The gestational weeks and birthweight were similar in the two groups. A 5% pre-term delivery rate was reported in both groups. A low birthweight was registered in 2.5% and 5% of the newborns, while 4% and 7% showed high birthweight, in FPS- and LPS-derived euploid blastocyst, respectively. Encompassing the 81 FPS-derived newborns, a total of 9% were small and 11% large for gestational age. Among the 102 LPS-derived newborns, 8% were small and 6% large for gestational age. No significant difference was reported for all these comparisons. LIMITATIONS, REASONS FOR CAUTION: The LPS-derived blastocysts were all obtained after FPS in a DuoStim protocol. Therefore, studies are required with LPS-only, late-FPS and random start approaches. The study is powered to assess differences in the LBR per embryo transfer, therefore obstetric and perinatal outcomes should be considered observational. Although prospective, the study was not registered. WIDER IMPLICATIONS OF THE FINDINGS: This study represents a further backing of the safety of non-conventional COS protocols. Therefore, LPS after FPS (DuoStim protocol) is confirmed a feasible and efficient approach also from clinical, obstetric and perinatal perspectives, targeted at patients who need to reach the transfer of an euploid blastocyst in the shortest timeframe possible due to reasons such as cancer, advanced maternal age and/or reduced ovarian reserve and poor ovarian response. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

Reduction of multiple pregnancies in the advanced maternal age population after implementation of an elective single embryo transfer policy coupled with enhanced embryo selection: pre- and post-intervention study.

STUDY QUESTION: Is an elective single-embryo transfer (eSET) policy an efficient approach for women aged >35 years when embryo selection is enhanced via blastocyst culture and preimplantation genetic screening (PGS)? SUMMARY ANSWER: Elective SET coupled with enhanced embryo selection using PGS in women older than 35 years reduced the multiple pregnancy rates while maintaining the cumulative success rate of the IVF programme. WHAT IS KNOWN ALREADY: Multiple pregnancies mean an increased risk of premature birth and perinatal death and occur mainly in older patients when multiple embryos are transferred to increase the chance of pregnancy. A SET policy is usually recommended in cases of good prognosis patients, but no general consensus has been reached for SET application in the advanced maternal age (AMA) population, defined as women older than 35 years. Our objective was to evaluate the results in terms of efficacy, efficiency and safety of an eSET policy coupled with increased application of blastocyst culture and PGS for this population of patients in our IVF programme. STUDY DESIGN, SIZE, DURATION: In January 2013, a multidisciplinary intervention involving optimization of embryo selection procedure and introduction of an eSET policy in an AMA population of women was implemented. This is a retrospective 4-year (January 2010-December 2013) pre- and post-intervention analysis, including 1161 and 499 patients in the pre- and post-intervention period, respectively. The primary outcome measures were the cumulative delivery rate (DR) per oocyte retrieval cycle and multiple DR. PARTICIPANTS/MATERIALS, SETTING, METHODS: Surplus oocytes and/or embryos were vitrified during the entire study period. In the post-intervention period, all couples with good quality embryos and less than two previous implantation failures were offered eSET. Embryo selection was enhanced by blastocyst culture and PGS (blastocyst stage biopsy and 24-chromosomal screening). Elective SET was also applied in cryopreservation cycles. MAIN RESULTS AND THE ROLE OF CHANCE: Patient and cycle characteristics were similar in the pre- and post-intervention groups [mean (SD) female age: 39.6 ± 2.1 and 39.4 ± 2.2 years; range 36-44] as assessed by logistic regression. A total of 1609 versus 574 oocyte retrievals, 937 versus 350 embryo warming and 138 versus 27 oocyte warming cycles were performed in the pre- and post-intervention periods, respectively, resulting in 1854 and 508 embryo transfers, respectively. In the post-intervention period, 289 cycles were blastocyst stage with (n = 182) or without PGS (n = 107). A mean (SD) number of 2.9 ± 1.1 (range 1-4) and 1.4 ± 0.8 (range 1-3) embryos were transferred pre- and post-intervention, respectively (P < 0.01) and similar cumulative clinical pregnancy rates per transfer and per cycle were obtained: 26.8, 30.9% and 29.7, 26.3%, respectively. The total DR per oocyte retrieval cycle (21.0 and 20.4% pre- and post-intervention, respectively) defined as efficacy was not affected by the intervention [odds ratio (OR) = 0.8, 95% confidence interval (CI) = 0.7-1.1; P = 0.23]. However, a significantly increased live birth rate per transferred embryo (defined as efficiency) was observed in the post-intervention group 17.0 versus 10.6% (P < 0.01). Multiple DRs decreased from 21.0 in the preintervention to 6.8% in the post-intervention group (OR = 0.3. 95% CI = 0.1-0.7; P < 0.01). LIMITATIONS, REASONS FOR CAUTION: In this study, the suitability of SET was assessed in individual women on the basis of both clinical and embryological prognostic factors and was not standardized. For the described eSET strategy coupled with an enhanced embryo selection policy, an optimized culture system, cryopreservation and aneuploidy screening programme is necessary. WIDER IMPLICATIONS OF THE FINDINGS: Owing to the increased maternal morbidity and perinatal complications related to multiple pregnancies, it is recommended to extend the eSET policy to the AMA population. As shown in this study, enhanced embryo selection procedures might allow a reduction in the number of embryos transferred and the number of transfers to be performed without affecting the total efficacy of the treatment but increasing efficiency and safety. STUDY FUNDING/COMPETING INTERESTS: None. TRIAL REGISTRATION NUMBER: None.

The worldwide frozen embryo reservoir: methodologies to achieve optimal results.

Cryopreservation of the human embryo has been successfully achieved at the zygote (day 1), cleavage (day 2/3), and blastocyst (day 5) stages; however, each stage presents specific advantages and disadvantages. During the past decades, two major methods have been applied: slow freezing (equilibrium procedure) and vitrification (nonequilibrium procedure). The overwhelming majority of published data prove that the latest vitrification methods induce less cellular trauma and are a more effective cryopreservation technique of human embryos than any other versions of slow freezing. For this reason, fragmented and slow-cleaving embryos that normally would not be recommended may be revaluated for cryopreservation by using the vitrification method. Furthermore, if laser-assisted necrotic blastomere removal is associated with the slow-freezing/thawing procedure, good clinical results can be obtained. Finally, the most proper embryo cleavage stage at which to perform cryopreservation has to be assessed according to clinical indications and laboratory experience.

Metformin effects on ovarian ultrasound appearance and steroidogenic function in normal-weight normoinsulinemic women with polycystic ovary syndrome: a randomized double-blind placebo-controlled clinical trial.

OBJECTIVE: To investigate metformin effects on the endocrine-metabolic parameters and ovarian morphology in normoinsulinemic women with polycystic ovary syndrome (PCOS). DESIGN: Randomized double-blind study. SETTING: Operative Division of Endocrinological Gynecology, Università Cattolica del Sacro Cuore. PATIENT(S): Twenty-eight normal-weight normoinsulinemic PCOS women. INTERVENTION(S): Patients were randomized to receive metformin 500 mg twice a day (group A, 15 subjects) or placebo (group B, 13 subjects) for 6 months. Ultrasonographic pelvic exams, hormonal and lipid features, and oral glucose tolerance test were performed at baseline and after 3 and 6 months of treatment. MAIN OUTCOME MEASURE(S): Hormonal and glycoinsulinemic assessment, ovarian ultrasound appearance. RESULT(S): Glycoinsulinemic assessment remained unvaried in both groups. About 70% of patients in group A experienced a restoration of menstrual cyclicity. Metformin significantly decreased testosterone levels at 3 and 6 months) and 17-hydroxyprogesterone levels at 6 months, and improved hirsutism score at 6 months. No clinical or hormonal modifications occurred in group B. Metformin, but not placebo, reduced ovarian volume and stromal/total area ratio at 3 and 6 months. CONCLUSION(S): Metformin seems to improve the menstrual pattern and ultrasonographic ovarian features in normoinsulinemic PCOS women. These effects seem to be, at least in part, independent of the insulin-lowering properties of the drug.

Pioglitazone reduces the adrenal androgen response to corticotropin-releasing factor without changes in ACTH release in hyperinsulinemic women with polycystic ovary syndrome.

OBJECTIVE: The hypothalamic-pituitary-adrenal (HPA) axis seems to hyperfunction at both central and peripheral levels in polycystic ovary syndrome (PCOS). Hyperinsulinemia is involved in the adrenal hyper-responsiveness to ACTH. The present study was performed to investigate the role of insulin in the derangement of the hypothalamic-pituitary compartment of the HPA axis in PCOS. DESIGN: Prospective clinical study. SETTING: Academic research center. PATIENT(S): Fifteen hyperinsulinemic PCOS women. INTERVENTION(S): Hormonal and lipid assays, oral glucose tolerance test, and corticotropin-releasing factor (1 microg/kg CRF) test before and after 4 months of treatment with the insulin sensitizer pioglitazone (30 mg/day). MAIN OUTCOME MEASURE(S): Glycemic and insulinemic response to glucose load; pituitary and adrenal response to CRF. RESULT(S): We observed a significant reduction in insulin secretion after therapy. Pioglitazone administration did not modify ACTH and cortisol response to CRF. A significant reduction in the adrenal CRF-induced secretion of androstenedione (A) (area under the curve [AUC] 202.76 +/- 78.68 ng/mL x 90 minutes to 147.05 +/- 52.06 ng/mL x 90 minutes) and 17OH-progesterone (AUC 152.92 +/- 59.56 ng/mL x 90 minutes to 117.10 +/- 63.25 ng/mL x 90 minutes') occurred after treatment. A trace response to CRF was observed for DHEAS and testosterone both before and after pioglitazone. CONCLUSION(S): In PCOS subjects, insulin may enhance adrenal steroidogenesis by acting directly on the peripheral gland, with no significant effects on the pituitary response to CRF stimulation.

Administration of exogenous ghrelin in obese patients with polycystic ovary syndrome: effects on plasma levels of growth hormone, glucose, and insulin.

OBJECTIVE: To assess the effects of the administration of exogenous ghrelin, a peptide with potent GH-releasing activity and glucose-enhancing and insulin-lowering properties, in obese patients with polycystic ovary syndrome (PCOS). DESIGN: Prospective, controlled study. SETTING: Academic research environment. PATIENT(S): Twenty obese women with PCOS, and 15 obese controls. INTERVENTION(S): Oral glucose tolerance test (OGTT) and ghrelin test (1 microg/kg i.v. bolus). MAIN OUTCOME MEASURE(S): Basal hormonal assays, including ghrelin, were performed. Glucose, insulin, and C-peptide were assessed in a fasting condition and during the OGTT. Growth hormone, insulin, and glucose were measured basally and every 15 minutes for 90 minutes after the injection of ghrelin. RESULT(S): Both groups showed an insulin response to the glucose load above the normal range. Significantly lower levels of ghrelin were detected in patients with PCOS compared to controls (108.96 +/- 27.65 Fmol/mL versus 162.47 +/- 42.23 Fmol/mL). Administration of ghrelin markedly enhanced GH levels in both groups (1,888.59 +/- 1,209.53 ng/mL and 1,639.95 +/- 631.79 ng/mL per 90 minutes as GH area under the curve, respectively), with a peak occurring 30 minutes after injection. Ghrelin also induced a trend toward an increase in plasma glucose levels, and a significant decrease in insulin concentrations in both groups. CONCLUSION(S): The injection of ghrelin seems to override the GH secretion defect in obese women with PCOS, and to induce glucoinsulinemic changes in both controls and obese patients with PCOS.

Effect of metformin on the growth hormone response to growth hormone-releasing hormone in obese women with polycystic ovary syndrome.

OBJECTIVE: Obese women with polycystic ovary syndrome (PCOS) show a marked growth hormone (GH) hyporesponsiveness to several stimuli. We aimed to evaluate the impact of insulin metabolism on the GH secretion impairment in these subjects in relation to food ingestion. DESIGN: Prospective clinical study. SETTING: Academic research center. PATIENT(S): Nine obese women with PCOS. INTERVENTION(S): Metformin (1,500 mg/daily) was administered for three months. The study protocol, which was performed before and after therapy, included hormonal and lipid assays, oral glucose tolerance test (75 g), euglycemic hyperinsulinemic clamp, and growth hormone-releasing hormone (GHRH) test (50 microg/ev), both on fasting and after a standard meal. MAIN OUTCOME MEASURE(S): Growth hormone response to GHRH (expressed as the area under the curve) in different experimental conditions. RESULT(S): The preprandial GH response to GHRH was not modified by the therapy, whereas a significant increase (P<.05) occurred in the postprandial GH secretion, thus resembling the response of obese normal persons. This change was accompanied by a trend towards improvement, though not statistically significant, of all the evaluated glycoinsulinemic parameters. A significant reduction in cholesterol (P<.01) and androstenedione (P<.05) and an increase in sex hormone-binding globulin (P<.05) were also achieved. CONCLUSION(S): These data suggest that metformin is able to affect GH secretion in obese women with PCOS, even with minimal metabolic modifications.