Oocytes with smooth endoplasmic reticulum aggregates do not impact blastocyst euploidy rate

Objective: To investigate whether the euploidy rate of blastocysts derived from smooth endoplasmic reticulum (SERa) positive cycles and oocytes are impacted.Design: Retrospective cohort study.Setting: A tertiary hospital-based reproductive medicine center.Patient(s): A total of 601 preimplantation genetic testing (PGT) cycles with obtained oocytes in our center between April 2017 and May 2021 were included in the study. Intervention(s): Women＞35 years and PGT cycles with chromosomal structural rearrangements (PGT-SR) were excluded. Embryological and blastocyst ploidy outcomes were compared between SERa+ oocyte, sibling SERa- oocytes and oocytes in SERa- cycles.Main Outcome Measure(s): Embryological outcomes and blastocyst euploidy rate.Results: No significant difference was observed in the normal fertilization rate (82.1 % vs. 77.8 % vs. 83.1 %, respectively, P=0.061), blastocyst formation rate (71.0 % vs. 72.5 % vs. 68.4 %, respectively, P=0.393), good quality blastocyst formation rate (46.4 % vs. 48.3 % vs. 42.6 %, respectively, P=0.198) between the SERa+ oocyte group, sibling SERa- oocyte group and SERa- oocyte group. No significant difference was observed in the euploidy rate (50.0 % vs. 62.5 % vs. 63.3 %, respectively, P=0.324), mosaic rate (12.5 % vs. 9.7 % vs. 13.4 %, respectively, P=0.506) and aneuploidy rate (37.5 % vs. 27.8% vs. 23.2 %, respectively, P=0.137) between the three groups.Conclusion: Our results suggest that the euploidy rate of blastocysts derived from SERa+ cycles and oocytes are not impacted.

P-631 Embryo euploidy rates following follicular or luteal start ovarian stimulation. A prospective study with repeated ovarian stimulation ovarian stimulation cycles

Study question Is there any difference in embryo euploidy rates following luteal phase phase (LS) and follicular phase (FS) start ovarian stimulation. Summary answer The number of euploid blastocysts and embryo euploidy rate are comparable when comparing FS and LS. What is known already Random start ovarian stimulation (starting at any time of the cycle) has been traditionally used in women undergoing urgent fertility preservation for medical reason. Although there is accumulating evidence that in infertile women, LS can result in equivalent number of oocytes and embryos as compared with FS, no study has evaluated the effect of luteal phase start ovarian stimulation on embryo euploidy rates. The current study is the first prospective study designed to evaluate embryo euploidy rates in donors undergoing two identical consecutive ovarian stimulation protocols within a period of 6 months starting either in the (FS), or (LS). Study design, size, duration In a prospective study, conducted between May 2018 and January 2020, 40 oocyte donors underwent two consecutive ovarian stimulation protocols within a period of 6 months with an identical fixed GnRH antagonist protocol starting either in the early follicular (FS), or and luteal menstrual cycle phase (LS). Participants/materials, setting, methods All participants underwent two identical consecutive ovarian stimulation cycles with 150μg corifollitropin alfa followed by 200 IU rFSH in a fixed GnRH antagonist protocol either in the FS or LS. Six MII oocytes from the same oocyte donor, from each stimulation cycle, were allocated to the recipients and were inseminated with the same sperm sample (recipients partner sperm or donor sperm). Embryos were cultivated to blastocyst stage followed by preimplantation genetic testing for aneuploidies (PGT-A). Main results and the role of chance When comparing FP with LP, the duration of ovarian stimulation was significantly shorter (9.68± 2.09 vs 10.93± 1.55 days), 95% CI [-1.95; -0.55] and a higher total additional dose of daily recFSH was significantly lower (526.14± 338.94 IU vs 726.14± 366.27), 95% CI [-315,12; -84,88] when CPT was administered in the luteal phase. . There were no differences in the hormone values on the triggering day (Estradiol 2137.61±1198.25 pg/ml vs 2362.96±1472.89); 95% CI [-1160.45;709.76]. Overall no differences were observed in the number of oocytes (24.84± 11.200 vs 24.27± 9.08); 95% CI[-2,61; 3.75] and MII oocytes (21.41±10.19 vs 21.59± 8.81), 95%CI [-2.72; 2.35] retrieved between FP and LP cycles in the oocytes donors. Following oocyte allocation and fertilization to the recipients, a total of 245 blastocysts were biopsied (blastocyst formation rate 245/408, 60.05%), 117 in FP group and 128 in LP group. The overall blastocyst euploidy rate was 59.18% . There were no differences in the number of euploid embryos between FS (1.59±1.32) and LS (1.70±1.29), mean difference 0.11, 95%CI [-0.65; 0.46]. Finally, there were no differences in the percentage of euploid embryos per oocytes inseminated between FS [70/287 (24.4%)] and LP [75/278 (24.7%), mean difference -0.027, 95%CI [-0.11; 0.06]. Limitations, reasons for caution The study was performed in oocyte derived from potentially fertile young oocyte donors thus caution is needed when extrapolating the results in oocytes derived from infertile women of older age. Wider implications of the findings Luteal phase stimulation does not alter embryo euploidy status as compared with follicular phase stimulation and thus it appears that it can be safely used not only in cases of urgent medical fertility preservation but also in patients undergoing ovarian stimulation for IVF/ICSI. Trial registration number Clinical Trials Gov (NCT03555942).

P–519 Investigation of embryo chromosomal constitution and live birth rate after vitrified-warmed euploid single blastocyst transfer across ranges of maternal body-mass-index

Study question Does maternal body-mass-index (BMI) associate with blastocysts’ chromosomal constitution and clinical outcomes in infertile patients undergoing preimplantation genetic testing for aneuploidies (PGT-A)? Summary answer A higher euploidy rate per biopsied blastocyst was reported among underweight women. Overweight women were instead subject to higher miscarriage (MR) and lower live-birth-rates (LBR). What is known already Different studies in the literature revealed an association between BMI and infertility, suggesting a J-shaped relationship: both underweight and overweight women can suffer from infertility issues. Even if IVF might increase the success rate in both these categories of patients, it seems insufficient per se to overcome the complex and multifactorial fertility impairment derived from unbalanced nutritional intakes. Miscarriage, in particular, is common in both underweight and overweight women. However, most of the literature is based on chromosomally-untested embryos. Study design, size, duration: Retrospective observational study. Only the first IVF cycle with ≥1 biopsied blastocyst from each woman was included. The primary outcome was the association between maternal BMI (underweight, BMI<18.5, n = 160; normal-weight, BMI=18–25, N = 1392; overweight, BMI>25, N = 259) and the mean euploidy rate per cohort of biopsied blastocysts (m-ER). The secondary outcomes were the association between maternal BMI with clinical (mainly MR and LBR), gestational and perinatal outcomes after first vitrified-warmed single euploid blastocyst transfers. Participants/materials, setting, methods We included 1811 women undergoing PGT-A at a private IVF center between April–2013 and March–2020. The secondary outcomes were investigated on 1125 first vitrified-warmed single euploid blastocyst transfers from all patients obtaining ≥1 transferable blastocyst. Only ICSI with ejaculated sperm and continuous culture in standard incubators were performed. Logistic regressions were conducted to identify putative confounders and adjust the results accordingly. Main results and the role of chance Except for a lower maternal age among underweight women (38.3±3.1 versus 38.9±3.4 yr, p < 0.01) and higher among overweight ones (39.3±3.6 yr, p = 0.04), no difference was reported with respect to normal-weight women in terms of duration of infertility, hormonal levels, main cause of infertility, sperm quality, and reproductive history. The mean number of biopsied blastocysts was ∼3 in all groups. The m-ER shows a decreasing trend as the maternal BMI increases between 17 and 22–23, to then plateau. In fact, a significant difference was reported between underweight (50.8%±36.4%) and normal-weight women (41.4%±37.5%, p < 0.01). A linear regression adjusted for maternal age confirmed this moderate association between increasing BMI and m-ER (unstandardized-coefficient-B –0.6%, 95%CI:–1.1% to –0.1%, p = 0.02). Morphological quality and day of full-blastulation among transferred euploid blastocysts was similar in the three groups. Overweight women showed higher MR per pregnancy (N = 20/75, 26.7%, 95%CI:17.4%–38.3% versus N = 67/461, 14.5%, 95%CI:11.5%–18.2%; OR 2.0, 95%CI:1.1–3.6, p = 0.01) and lower LBR per transfer (N = 55/154, 35.7%, 95%CI:28.3%–43.8% versus N = 388/859, 45.2%, 95%CI:41.8%–48.6%; OR adjusted for euploid blastocysts’ features 0.67, 95%CI:0.46–0.96, p = 0.03). Clinical outcomes were instead similar among underweight and normal-weight women. All gestational and perinatal outcomes were comparable in the tree groups. Limitations, reasons for caution Our study is limited by its retrospective nature, and the fact that maternal BMI was measured only before oocyte retrieval and not before embryo transfer. Moreover, the reduced sample size did not allow for further relevant sub-analyses among solely obese women. Wider implications of the findings: When possible nutritional/lifestyle modifications should be encouraged to adjust maternal BMI before IVF. Overweight patients should be especially informed of their higher risk for miscarriage. Yet, BMI is just a gross marker, future studies based on body fat localization and percentage (e.g. by bioelectrical impedance analyses) are desirable. Trial registration number None

P–601 Anovulatory patients with PCOS have lower euploidy rates compared to those with hypothalamic amenorrhea and to normo-ovulatory patients

Study question How do euploidy rates differ in anovulatory women with polycystic ovarian syndrome (PCOS) and hypothalamic hypogonadism (HH) compared to normo-ovulatory women undergoing IVF/ICSI? Summary answer Patients with PCOS have a significantly lower euploidy rate compared to patients with HH and patients with tubal factor infertility. What is known already Previous studies have demonstrated similar blastocyst conversion rates in women with PCOS and tubal factor infertility. Reported aneuploidy rates in preimplantation genetic testing cycles are similar in women with PCOS and tubal infertility. There are no data on blastocyst conversion or aneuploidy rates in women with HH. While PCOS and HH are different physiologic processes, patients with these disorders are reported together to SART and to the CDC National ART Surveillance System under the diagnosis of “ovulatory dysfunction”. Study design, size, duration: Retrospective cohort study of all autologous IVF and ICSI cycles for patients with oligo-anovulation (PCOS, n = 552 and HH, n = 48) and normo-ovulation (tubal factor infertility, n = 423) from 1/1/2012 to 6/30/2019. A total of 1023 cycles from 720 patients were analyzed. Participants/materials, setting, methods Cycle outcomes, including number of oocytes, mature oocytes, blastocysts and euploid blastocysts were assessed for each diagnosis. Adjusted relative risks (aRR) and 95% confidence intervals (CI) were calculated adjusting for age, BMI, AMH, and stimulation protocol. Poisson regression was used for counts and with an offset for ratios. Patients contributing multiple cycles were accounted for using general estimating equations. Main results and the role of chance PCOS patients were given a lower starting dose of gonadotropins and received less total gonadotropins compared to patients with tubal factor infertility or HH, but had similar stimulation durations as tubal-factor patients. Patients with HH received higher total doses of gonadotropins and had longer stimulation durations. PCOS patients had significantly more oocytes retrieved and a higher number of blastocysts than patients with tubal factor infertility (18.9 vs. 13.6 aRR 1.16 95% CI: 1.05–1.28 and 6.6 vs. 3.7 aRR 1.32 95% CI 1.10–1.57, respectively). Patients with HH had a similar number of oocytes retrieved and number of blastocysts compared to tubal factor patients. The blastocyst conversion rate was higher for PCOS than tubal (59.4% vs. 49.7%), but not significantly different (aRR 1.04 95% CI: 0.94–1.15). Blastocyst conversion and euploidy rates were similar for HH and tubal factor patients (51.9% vs. 49.7% and 39.1% vs. 44.9%, respectively, aRR 1.01 95% CI: 0.81–1.26 and aRR 1.05 95% CI: 0.85–1.31, respectively). In the adjusted model, patients with PCOS had a significantly lower euploidy rate than patients with tubal infertility (aRR 0.75 95% CI: 0.58–0.96). Patients with HH also had a significantly higher euploidy rate compared to women with PCOS (aRR 1.41 95% CI: 1.05–1.89). Limitations, reasons for caution This study is limited by its retrospective nature and the small sample size of women with hypothalamic hypogonadism. Additionally, these data represent outcomes from a single academic center, so generalizability of our findings may be limited. Wider implications of the findings: Cycle outcomes differ for ovulatory dysfunction patients with PCOS as compared to those with HH. HH patients require higher total doses of gonadotropins and longer stimulations to achieve similar cycle outcomes as normo-ovulatory patients. While PCOS patients have more embryos, the percent of euploid blastocysts is lower. Trial registration number Not applicable

P–615 The effect of late-follicular phase progesterone rise on embryo ploidy, embryo quality and cumulative live birth rates following a freeze-only strategy

Study question Is late-follicular phase progesterone elevation (PE) associated with a deleterious effect on embryo euploidy, embryo blastulation and cumulative live birth rates (CLBRs)? Summary answer Late-follicular phase PE has no impact on impact on embryo euploidy rate, embryo blastulation rate nor on the CLBR. What is known already The effect of PE in ART outcomes has been extensively studied, yielding so far conflicting results. While some authors claim it is only detrimental to endometrial receptivity, others have suggested that it may also impair oocyte/embryo quality. Moreover, little is known regarding the potential effect PE may have on embryo ploidy and, consequently, CLBR. Study design, size, duration A multicenter retrospective cross-sectional study was performed between August 2017 and December 2019. A total of 1495 ICSI cycles coupled with preimplantation genetic diagnosis for aneuploidies (PGT-A) and deferred frozen embryo transfer (FET) were analyzed. Participants/materials, setting, methods All patients underwent ovarian stimulation with GnRH antagonist protocol and performed a serum progesterone measurement at one of the participating private fertility clinics on the day of trigger. The sample was stratified according to the progesterone levels: normal (≤1.50 ng/ml) and high (>1.50 ng/ml). The primary outcome was the embryo euploidy rate. Secondary outcomes were the number of euploid blastocysts, the blastulation rate and CLBR. Main results and the role of chance Late-follicular phase PE was associated with higher late-follicular estradiol levels (2847.56±1091.10 pg/ml vs. 2240.94± 996.37 pg/ml, p < 0.001) and more oocytes retrieved (17.67±8.86 vs. 12.70±7.00, p < 0.001). The number of euploid embryos was higher in the PE group (2.32±1.74 vs. 1.86±1.42, p < 0.001), whereas the embryo euploidy rate (48.3% [44.9%–51.7%] vs. 49.1% [47.7%–50.6%] and blastulation rate (47.1% [43.7%–50.5%] vs. 51.0% [49.7%–52.4%]) were comparable between the two groups. Likewise, no significant differences were found regarding the live birth rate (LBR) after the first FET (34.1% vs. 31.1%, p = 0.427) nor the CLBRs (38.9% vs. 37.0%, p = 0.637). Mixed-model analysis was performed in order to account for the clustering of cycles in the same patient. Adjusting for patients’ age, PE and BMI, PE failed to demonstrate any effect on the embryo euploidy rate (OR 1.03 [95% CI 0.89–1.20]). Mixed-model analysis for the number of euploid embryos was also performed. After adjusting for PE, age, BMI and ovarian response, PE did not affect the number of euploid embryos (0.02 [95%CI –0.21;0.25]. Multivariate logistic regression adjusted for PE, age, BMI and ovarian response revealed that PE was not associated with the CLBR (adjOR 0.96 [95% CI 0.66–1.38]). Limitations, reasons for caution Limitations of the study include its retrospective nature. Moreover, including only GnRH antagonist protocol and ICSI does not allow the extrapolation of these results to other populations. Wider implications of the findings: Our findings question results from previous studies claiming a detrimental effect of PE on embryo implantation potential. According to our results, PE has no impact on embryo euploidy rate, blastulation rate nor on CLBRs. Trial registration number Not applicable

P–631 Embryo euploidy rates following follicular or luteal start ovarian stimulation. A prospective study with repeated ovarian stimulation ovarian stimulation cycles

Study question Is there any difference in embryo euploidy rates following luteal phase phase (LS) and follicular phase (FS) start ovarian stimulation. Summary answer: The number of euploid blastocysts and embryo euploidy rate are comparable when comparing FS and LS. What is known already Random start ovarian stimulation (starting at any time of the cycle) has been traditionally used in women undergoing urgent fertility preservation for medical reason. Although there is accumulating evidence that in infertile women, LS can result in equivalent number of oocytes and embryos as compared with FS, no study has evaluated the effect of luteal phase start ovarian stimulation on embryo euploidy rates. The current study is the first prospective study designed to evaluate embryo euploidy rates in donors undergoing two identical consecutive ovarian stimulation protocols within a period of 6 months starting either in the (FS), or (LS). Study design, size, duration In a prospective study, conducted between May 2018 and January 2020, 40 oocyte donors underwent two consecutive ovarian stimulation protocols within a period of 6 months with an identical fixed GnRH antagonist protocol starting either in the early follicular (FS), or and luteal menstrual cycle phase (LS). Participants/materials, setting, methods All participants underwent two identical consecutive ovarian stimulation cycles with 150μg corifollitropin alfa followed by 200 IU rFSH in a fixed GnRH antagonist protocol either in the FS or LS. Six MII oocytes from the same oocyte donor, from each stimulation cycle, were allocated to the recipients and were inseminated with the same sperm sample (recipients partner sperm or donor sperm). Embryos were cultivated to blastocyst stage followed by preimplantation genetic testing for aneuploidies (PGT-A). Main results and the role of chance When comparing FP with LP, the duration of ovarian stimulation was significantly shorter (9.68± 2.09 vs 10.93± 1.55 days), 95% CI [–1.95; –0.55] and a higher total additional dose of daily recFSH was significantly lower (526.14± 338.94 IU vs 726.14± 366.27), 95% CI [–315,12; –84,88] when CPT was administered in the luteal phase. . There were no differences in the hormone values on the triggering day (Estradiol 2137.61±1198.25 pg/ml vs 2362.96±1472.89); 95% CI [–1160.45;709.76]. Overall no differences were observed in the number of oocytes (24.84± 11.200 vs 24.27± 9.08); 95% CI[–2,61; 3.75] and MII oocytes (21.41±10.19 vs 21.59± 8.81), 95%CI [–2.72; 2.35] retrieved between FP and LP cycles in the oocytes donors. Following oocyte allocation and fertilization to the recipients, a total of 245 blastocysts were biopsied (blastocyst formation rate 245/408, 60.05%), 117 in FP group and 128 in LP group. The overall blastocyst euploidy rate was 59.18% . There were no differences in the number of euploid embryos between FS (1.59±1.32) and LS (1.70±1.29), mean difference 0.11, 95%CI [–0.65; 0.46]. Finally, there were no differences in the percentage of euploid embryos per oocytes inseminated between FS [70/287 (24.4%)] and LP [75/278 (24.7%), mean difference –0.027, 95%CI [–0.11; 0.06]. Limitations, reasons for caution The study was performed in oocyte derived from potentially fertile young oocyte donors thus caution is needed when extrapolating the results in oocytes derived from infertile women of older age. Wider implications of the findings: Luteal phase stimulation does not alter embryo euploidy status as compared with follicular phase stimulation and thus it appears that it can be safely used not only in cases of urgent medical fertility preservation but also in patients undergoing ovarian stimulation for IVF/ICSI. Trial registration number Clinical Trials Gov (NCT03555942).

P–224 Multinucleated and non-multinucleated embryos in PGT-A cycles: Is there any difference?

Study question Is multinucleation during cleavage stage correlated with the ploidy status of embryos and how does it affect the clinical outcome? Summary answer The presence of multinucleated embryos does not affect clinical outcome, although the risk of aneuploidy is higher in multinucleated embryos. What is known already Multinucleated blastomeres (ΜΝ) of cleavage stage embryos has been reported widely in scientific literature. Multinucleation has been associated with diminished embryo developmental competency and clinical outcomes such as lower implantation. Although this is an intriguing subject of research, it is not clear yet whether multinucleation is related to aneuploidy. Morphological irregularities such as multinucleation in blastomeres became a constant finding only after the perpetually evolving technology of time-lapse culture of embryos which in combination with PGT-A analysis, creates new research paths which aim to develop a new tool for selection or deselection of embryos for transfer. Study design, size, duration This retrospective study, included 97 PGT-A cycles, performed at Embryolab fertility clinic from May 2017 to December 2020, all cultured in time-lapse incubator (EmbryoScope). Two study groups were formed; the MN Group consisted of PGT-A cycles with at least one multinucleated embryo (n = 56) and the Control Group in which all PGT-A cycles had no multinucleated embryos (n = 38). Euploidy rate, type of chromosomal abnormality, cumulative pregnancy and live birth rates were compared between the two groups. Participants/materials, setting, methods Embryos were annotated for the existence of multinucleated blastomeres on Day 2 of their development. Biopsy was performed on Days 5/6 and embryos were genetically tested. One or two euploid embryos were transferred. Euploidy rate and clinical outcomes between the two groups were compared. Within the MN group, euploidy rate between multinucleated and non-multinucleated embryos was compared. For abnormal embryos, association of multinucleation with the type of abnormality was tested. SAS statistical analysis was performed. Main results and the role of chance Mean female age was 35.93 years in the MN group and 38.39 years in the control group. Blastocyst formation rate (expressed per fertilised oocytes) was similar between MN and Control group (74% vs 76%, p = 0.6303). In the MN group, 56 cases resulted in 44 embryo transfers while in the control group 38 cases resulted in 23 embryo transfers. Pregnancy rates (59.09% vs 65.21%, p = 0.6255) and clinical pregnancy rates (45.45% vs 39.13%, p = 0.4245) were not significantly different between MN and Control group. Initially, cumulative live birth rate was found to be significantly higher in the MN group compared to the Control group (62.96% vs 33.34%, p = 0.0417). However, when logistic and poisson regression was applied, it became obvious that this difference was not affected by multinucleation but from other factors such as female age. When comparing multinucleated and non-multinucleated embryos within the MN group, it was found that the mean number of euploid embryos was significantly higher in the non-multinucleated subgroup of embryos (p = 0.0021). No correlation was found between multinucleation and the type of chromosomal abnormality. Limitations, reasons for caution The sample size is the main limitation of the present study. More research with bigger sample size is needed in order to confirm the finding of the present study. Wider implications of the findings: The present study suggests that multinucleated blastomeres during embryo development is not an indication for diminished blastocyst formation and does not affect the clinical outcomes. However, within a sibling embryo population, non-multinucleated embryos tend to be euploid and this finding can be used to advance embryo selection efficiency. Trial registration number Not applicable