The GnRH Antagonist Protocol is Associated with a Higher Embryo Aneuploidy Rate Than the GnRH Agonist Long Protocol in Chinese Women

Background: Studies in oocytes have suggested increased aneuploidy rates after ovulation induction in mammals and humans. Conversely, some studies have shown that ovarian stimulation does not significantly increase the embryo aneuploidy rate in humans compared with an unstimulated cycle. In addition, the potential effect of the gonadotropin-releasing hormone (GnRH) antagonist (GnRH-ant) protocol and GnRH agonist (GnRH-a) long protocol on embryo aneuploidy remains unknown.Methods: This is the retrospective cohort study from university-affiliated fertility center. In total, 578 early miscarriage patients who conceived through IVF/intracytoplasmic sperm injection (ICSI) after receiving the gonadotropin-releasing hormone (GnRH) antagonist (GnRH-ant) protocol or the GnRH agonist (GnRH-a) long protocol were analyzed to compare the aneuploidy rates in early aborted tissues. In addition, a total of 466 preimplantation genetic testing for aneuploidy (PGT-A) cycles undergoing GnRH-ant protocol or GnRH-a long protocol were also analyzed to compare the aneuploidy rates in embryo.Results: For early miscarriage patients who conceived through IVF/ICSI, compared to the GnRH-a long protocol group, the GnRH-ant protocol group had a significantly higher rate of aneuploidy in early aborted tissues (48.70% vs. 64.52%), and increased aneuploidy was associated with a significantly higher incidence of trisomy 13, 18, and 21 (p<0.01). Regarding PGT-A cycles, compared to the GnRH-a long protocol group, the rate of embryo aneuploidy was also significantly higher in the GnRH-ant protocol group (48.01% vs. 58%). After stratification and multiple linear regression, the GnRH-ant regimen remained significantly associated with an increased risk of aneuploidy in early aborted tissues and embryos [OR (95% CI) 1.767 (1.174, 2.661), OR (95% CI) 1.465 (1.020, 2.102)]. Furthermore, the embryo aneuploidy rate in the GnRH-ant protocol group was significantly higher than that in the GnRH-a long protocol group but only in young and normal ovarian responders [OR (95% CI) 3.54 (1.48, 8.46)].Conclusions: The GnRH-ant protocol is associated with a higher aneuploidy rate in early aborted tissues and embryos than the GnRH-a long protocol in Chinese women. A multicenter, randomized controlled trial would be the optimal strategy to confirm these results.

Chromosome aneuploidy analysis in embryos derived from in vivo and in vitro matured human oocytes

Background In vitro oocyte maturation (IVM) is being increasingly approached in assisted reproductive technology (ART). This study aimed to evaluate the quality of embryos generated by in-vitro matured immature follicles, as a guideline for further clinical decision-making. Methods A total of 52 couples with normal karyotypes underwent in vitro fertilization, and 162 embryos were donated for genetic screening. Embryos in IVF group were generated by mature follicles retrieved during gonadotrophin-stimulated in vitro fertilization (IVF) cycles. And embryos in IVM group were fertilized from IVM immature oocytes. Results The average age of the women was 30.50 ± 4.55 years (range 21–42 years) with 87 embryos from IVF group and 75 embryos from IVM group. The rate of aneuploid with 28 of the 87 (32.2%) embryos from IVF group and 21 of the 75 (28%) embryos from IVM group, with no significant difference. The frequency of aneuploid embryos was lowest in the youngest age and increased gradually with women’s age, whether in IVF group or IVM group and risen significantly over 35 years old. The embryos with morphological grade 1 have the lowest aneuploidy frequency (16.6%), and increase by the grade, especially in IVF group. In grade 3, embryos in IVM group were more likely to be euploid than IVF group (60% vs 40%, respectively). Conclusions IVM does not affect the quality of embryos and does not increase the aneuploidy rate of embryos. It is clinically recommended that women more than 35 years have a high aneuploidy rate and recommended to test by PGS (strongly recommended to screened by PGS for women more than 40 years). Women aged less than 35 years old for PGS according to their physical and economic conditions. Embryo with poor quality is also recommended to test by PGS, especially for grade III embryos.

P–566 Advanced paternal age can influence aneuploidy rate in egg donation cycles with poor sperm quality

Study question Could advanced paternal age influences the embryos aneuploidy rate in eggs donation cycles with poor sperm quality? Summary answer In case of severe male factors increased paternal age can affect embryos aneuploidy rate in egg donation cycles. What is known already While the impact of advanced maternal age on reproductive is well understood, the effect of paternal age on reproductive function is controversial. Many studies have shown that Advanced Paternal Age (APA) could impact on male fertility potential affecting testicular function and sperm quality. Moreover, APA also has been associated with increased epigenetics changes and DNA mutations. Increased paternal age could be associated with different types of disorders such as autism, schizopherenia and bipolar disorders. Egg donation cycles, controlling female variables, represent the ideal model for the study of the impact of paternal age on reproductive outcomes. Study design, size, duration We retrospectively analyzed 43 egg donation cycles (October 2014-January 2020) with ≥ 50% survival rate of vitrified/warmed oocyte. Only cycles with poor sperm quality were considered. Cycles were divided in two GROUPS: group–1 included male paternal age ≤ 45 while group–2 included male paternal age &gt;45. Data, shown as avarage±SD, were analyzed with Chi square or Student-t test. Participants/materials, setting, methods Group–1 included 20 cycles and 219 oocytes, male age was 40,89 ±6.12; Group–2 included 17 cycles and 173 oocytes, male age was 51±6.06. Respectively, in Group 1 and in Group 2, donor age were 22.4±2.65 and 24.8±3.88 (NS). All oocytes were injected with abnormal sperm samples according to WHO 2010. Embryos were cultured in time-lapse system until blastocyst stage. Trophectoderm biopsy and PGT-A analysis were performed according to standardized laboratory protocols. Main results and the role of chance Oocytes survival rates in Group1 and 2 were 86% (188/219) and 90.7% (157/173) (NS), respectively. Fertilization rates in Group1 and –2 were 71.42 (135/189) and 73.45% (119/162) (NS), respectively. The total number of obtained embryos (transferred + frozen) were 81 and 801 in Group–1 and –2, respectively. The rates of obtained embryos per reiceved occytes were 37% (81/219) and 46.24% (80/173) in Group–1 and –2 (p &lt; 0.7), respectively. The PGT-A analysis showed 38.7% (31/80) and 31.17% (24/77) of euploid (NS) and 25% (20/80)and 42.85% (33/77) of aneuploid embryos (P &lt; 0.05) in Group–1 and –2, respectively. Mosaic embryos were 33.5% (26/80) and 27.27%(21/77), in Group–1 and –2, respectively. (NS). These results indicate that in presence of severe male factor, advanced paternal age could increase embryos aneuploidy rate raising incidence of chromosomal abnormalities. Limitations, reasons for caution Each donor was stimulated with different protocols according to her history and hormones levels. Nothing is known about which type of sperm parameters (semen amount, morphology or motility) have a major impact when focusing on the embryos genetic outcome. Wider implications of the findings: To better known the effect of APA, it could be necessary identify embryos chromosomal abnormalities and the correlation with specific sperm parameters. Further studies should be done to confirm the APA effect in patients with severe male factors and define a cut-off male age where PGT-A should be recommended. Trial registration number Not applicable

P–005 Magnetic-activated cell sorting in couples undergoing preimplantation genetic testing for aneuploidies (PGT-A) using autologous oocytes shows slightly lower aneuploidy rates compared to standard semen processing

Study question Does the selection of non-apoptotic sperm via magnetic-activated cell sorting (MACS) reduce the aneuploidy rate of embryos from couples undergoing ICSI cycles with PGT-A using the patients’ own oocytes? Summary answer It does. The aneuploidy rate in the MACS group was 4.34% lower than the one obtained using semen samples processed according to standard clinical practice. What is known already MACS is a successful tool in eliminating proapoptotic sperm from a semen sample. However, the true effect of this technique on reproductive outcomes and the quality of the resulting embryos are a matter of controversy. Some studies report that its use improves the percentage of good quality blastocysts in women older than 30 years old compared to standard ICSI. Randomized clinical trials that compare MACS to a control sample consider parameters of embryo quality such as morphology at day 3 or day 5, symmetry of the blastomeres, blastocysts’ stage of expansion, but they do not consider embryo ploidy. Study design, size, duration Retrospective, multicentre, observational cohort study. 14,145 patients and 18,710 cycles were evaluated in the reference group. In the MACS group, 615 patients and 974 cycles were considered. Data were exported from cycles performed in Spanish IVIRMA clinics between January 2008 and February 2020. Participants/materials, setting, methods Unselected males in couples undergoing PGT-A cycles, then subdivided into male factor (MF) - total progressive motile sperm count lower than 5 million - and non-male factor (NMF) infertility. Statistical analysis performed using R v.4.0.0. Means were calculated and compared using two-tailed paired t-test, while proportions were compared using Fisher’s exact test and the chi-squared test and the appropriate correction for multiple comparisons. The aneuploidy rates for each group were compared using Fisher’s exact test. Main results and the role of chance In the control group 73,228 biopsied embryos, from which 71,439 were informative in the PGT-A. In the MACS group 3,919 biopsied embryos, from which 3,843 were informative. The aneuploidy rate, computed per informative embryo, was 68.87% (68.40%, 69.34%) in the reference group and 64.53% (62.43%, 66.64%) in the MACS group. Both comparisons were statistically significant (p-value ˂0.00001). According to these results, an embryo in the PGT-A programme using non-apoptotic sperm selected through MACS and autologous oocytes had a 5% less chance of being aneuploid than those embryos fertilised with standardly selected sperm (relative risk of 0.95 (0.91–0.98) p = 0.006769). Embryos conceived from NMF patients whose semen had been processed using MACS had a 4.27% lower aneuploidy rate than the reference (65.52% (63.16%, 67.88%) vs 69.79% (69.20%, 70.37%) respectively). This difference was statistically significant. Those embryos conceived using semen from patients with MF using MACS also showed a lower aneuploidy rate than the reference with MF (0.28% (55.48%, 65.08%) vs (64.94% (63.35%, 66.23%) respectively), although this difference was not statistically significant. Thus, the decrease in aneuploidy rate observed when comparing MACS and reference groups undergoing PGT-A cycles using autologous oocytes remained approximately the same in both MF and NMF semen samples. Limitations, reasons for caution The retrospective nature of the study subjects the data to biases or inaccuracies in their annotation in the clinics’ informatic platform from which they were exported. However, the statistical analysis aimed at controlling these biases as much as possible. Wider implications of the findings: The vast amount of data compiled for this study confirms that the selection of non-apoptotic sperm through MACS slightly decreases the aneuploidy rate of embryos compared to semen samples processed according to the clinics’ standards. This would be interesting for patients who are considering undergoing PGT-A cycles in the future. Trial registration number Not applicable

P–262 Comparison of embryo aneuploidy rate and reproductive outcomes of ART cycles using fresh and vitrified donor oocytes

Study question Is there any difference in blastocyst morphology, embryo aneuploidy rate and ART clinical outcomes when using fresh or vitrified donor oocytes? Summary answer Frequency of good quality blastocyst obtained from fresh oocytes is significantly higher compared to vitrified.No difference in embryo aneuploidy rates nor clinical outcomes were found. What is known already Oocytes vitrification is an efficient method that allows non only fertility preservation but also the creation of donor oocytes banks, optimizing clinical resources for patients undergoing Assisted Reproductive Technology. Although the benefits of donor oocytes vitrification are well known, some studies have shown that this cryopreservation process can induce spindle abnormalities and chromosomal changes, leading to aneuploidy. Comparative studies between fresh and vitrified oocytes to evaluate embryo developmental competence, aneuploidy and clinical pregnancy rate (CPR) are needed. Study design, size, duration This retrospective study includes ICSI cycles with fresh donor oocytes(N = 2795) and vitrified donor oocytes (N = 1225) between January 2019 and September 2020. Pre-implantation Genetic Testing for Aneuploidy (PGT-A) was performed on Day 5 and Day 6 blastocysts. Fertilization rate, blastocyst morphology, aneuploidy status and CPR were analysed and compared between the groups. Recipients were equally distributed in terms of maternal age (40.86 years) and previous history, sperm samples were also similar in profile and origin (fresh-frozen). Participants/materials, setting, methods A total of 266 subfertile couples participated in the study, ICSI was carried out in all cycles. Vitrification and warming protocols were performed with a commercial kit. All embryos were cultured to blastocyst stage in a Time-Lapse incubator and assessed by Gardner’s blastocyst grading scale. PGT-A testing was performed on trophectoderm biopsies by Next Generation Sequencing (NGS). Single/double embryo transfers were performed in all cases. Odd-ratios were calculated,and Chi-square was performed for the statistical analysis. Main results and the role of chance A total of 266 patients underwent 289 donor oocyte cycles yielding an overall of 4557 oocytes. ICSI was performed on 2795 fresh and 1225 vitrified mature oocytes. Similar fertilization rates were achieved with fresh and vitrified oocytes (75.9% (2122/2795) and 75.2% (921/1225), respectively (P = 0.6)) yielding a significant difference in blastocyst rate of 71.7% (1522/2122) and 62.5% (576/921) (OR 1,519; 95% CI 1,290–1,789; p &lt; 0.001). In addition, when blastocysts morphology was analysed, a significant difference was shown in the frequency of good quality embryos that decreases from 56.6% (861/1522) with fresh oocytes to 51% (294/576) with vitrified oocytes (OR 1,249; 95% CI 1,031–1,514; P &lt; 0.02). PGT-A testing of blastocysts revealed not significant differences in euploidy rates (73.6% in fresh oocytes vs 76.8% vitrified oocytes, P = 0.2). With regards to clinical outcomes, similar results were found between the groups. A total of 322 embryo transfers were performed (237 from fresh and 85 vitrified) achieving a CPR of 48.9% (116/237) with fresh oocytes and 54% (46/85) with vitrified (P = 0.7) and a pregnancy loss of 6.7%(16/237) in fresh oocytes and 11.7%(10/85) vitrified oocytes (P = 0.1). Limitations, reasons for caution The study was conducted on a small number of cases. Further studies are needed to confirm our findings. Moreover, although the same stimulation protocol was used, donors from different background were included. Wider implications of the findings: This study supports the use of vitrified oocytes in the laboratory routine without compromising clinical outcomes. Although oocyte vitrification may have an influence on embryo morphology, blastocyst rate, no impact of this cryopreservation process is seen on embryo aneuploidy, developmental competence and CPR. Trial registration number Not applicable

Lapatinib Decreases the Preimplantation Aneuploidy Rate of in vitro Fertilized Mouse Embryos without Affecting Completion of Preimplantation Development

One of the major reasons for implantation failure and spontaneous abortion is a high incidence of preimplantation chromosomal aneuploidy. Lapatinib simultaneously inhibits EGFR and HER2, leading to apoptosis. We hypothesized a higher sensitivity for aneuploid cells in preimplantation embryos to lapatinib based on reports of aneuploid cell lines being sensitive to some anticancer drugs. Late 2-cell mouse embryos were treated with lapatinib after determining a nontoxic dose. Morphologies were recorded 24, 48, and 60 hours later. The effect of lapatinib on the aneuploidy rate was evaluated by studying blastocyst cells using FISH. Although the rate of development to 8-cell and morula stage was higher in the control group (<i>p</i> &#x3c; 0.05), there was no difference in development to the blastocyst stage at the same studied intervals between lapatinib-treated and control groups (<i>p</i> = 0.924). The mean number of cells in morula and blastocyst stages were not different between the groups (<i>p</i> = 0.331 and <i>p</i> = 0.175, respectively). The frequency of aneuploid cells and diploid embryos was, respectively, significantly lower and higher in lapatinib-treated embryos, (<i>p</i> &#x3c; 0.001). Since lapatinib treatment reduced the aneuploidy rate without impact on the development of mouse preimplantation embryos to the blastocyst stage and number of total cells, lapatinib seems useful for prevention of preimplantation aneuploidy in in vitro fertilization.