P–042 Impact of semen parameters, sperm DNA fragmentation and sperm aneuploidy in male infertility

Study question Should sperm aneuploidies and sperm DNA fragmentation (sDNAfrag) be included as valid tests in the routine investigation of male infertility? Summary answer Sperm DNA fragmentation was associated with male age, oligozoospermia (OZ), oligoteratozoospermia (OT), astenoteratozoospermia (AT) and oligoastenoteratozoospermia (OAT). Sperm aneuploidies were associated with OT and OAT. What is known already Semen parameters assist male infertility diagnosis and treatment, but sDNAfrag and aneuploidy analysis could add useful information, as abnormal values compromise fertility. To include these tests in the routine diagnosis it should be determined if behave as informative parameter and add information regarding the fertility status. For that, further studies comparing these tests to semen parameters are needed, since previous results are not consensual. Additionally, standardization of a sDNAfrag cut-off is needed, as different sample sizes and techniques originate distinct results. Also, until a standardization of the protocol is missing, a cut-off value should be defined for each laboratory. Study design, size, duration A retrospective and prospective investigation was performed, within a 12 years period (April 2007-December 2019). A total of 835 infertile males with a normal karyotype (46,XY) were included. Karyotyping and evaluation of sDNAfrag and sperm aneuploidies were made at a public Genetic unit. All normozoospermic (NZ) patients with a born child and patients whose infertility treatments were done due to female factors were selected from our database and used as controls (60 individuals). Participants/materials, setting, methods Semen analysis followed WHO–2010 guidelines. sDNAfrag was evaluated using the TUNEL assay. Sperm aneuploidies were detected using FISH (chromosomes 13, 18, 21, X, Y). Several tests were applied: correlations for linear associations between numerical variables, ANOVA for comparisons between means, Dunn-test for post-hoc comparisons. To determine the sDNAfrag cut-off value, the area under the ROC curve, sensitivity and specificity, were calculated, with the Youden-Index used to find a threshold that maximizes both sensitivity and specificity. Main results and the role of chance Regarding male age, it was observed a positive correlation with sperm concentration, a negative correlation with sperm vitality (VT) and hypoosmolality, and a positive correlation with sDNAfrag. Regarding sDNAfrag, it was observed negative correlation with sperm concentration, total progressive motility (TPM), morphology, VT and hypoosmolality. Regarding sperm aneuploidies, both total sperm aneuploidy and total sperm disomy exhibited a negative association with sperm concentration, TPM and morphology. It was also investigated whose groups of individuals could be indicated for sDNAfrag or sperm aneuploidy testing. The NZ group evidenced significant lower sDNAfrag, total sperm aneuploidy and total sperm disomy in relation to the non-NZ group. In the NZ group, sDNAfrag was significantly lower in relation to the OZ, OT, AT and OAT groups. The NZ group presented significant lower percentages of sperm aneuploidy in relation to the OT and OAT groups, and significant lower percentages of sperm disomy in relation to the OAT group. Additionally, sDNAfrag was positively correlated with total sperm aneuploidy and total sperm disomy. From the present large population, ROC curve analysis allowed estimating a cut-off value of 18.8% for the TUNEL-assay (sDNAfrag), with 0.658 of area under the curve, 53.9% sensitivity and 76.7% specificity. Limitations, reasons for caution Although presenting a high number of cases and strict controls, the present study was unable to include as controls healthy men with proven fertility. Additionally, the present study did not take into account life-style factors and male associated pathologies besides infertility. Wider implications of the findings: Semen parameters were shown to be negatively correlated with sDNAfrag and sperm aneuploidies. As sDNAfrag testing and sperm aneuploidy testing were associated with semen abnormalities and male age, it is suggested their inclusion in the routine evaluation of infertile men, thus adding important complementary information about the fertility status. Trial registration number Not Appliable

O-009 Data from the ESHRE PGT consortium – year 2019

text Study question Which are the trends shown in data collection XXI of the European Society of Human Reproduction and Embryology (ESHRE) PGT Consortium compared with previous years? Summary answer Data collection XXI, year 2019, represents valuable data on PGT activity in (mainly) Europe and reports on the main trends observed, being the further expansion of comprehensive testing technology in PGT-SR and PGT-A. What is known already The ESHRE PGT Consortium was set up in 1997 and from that time has been collecting data on PGT and PGT-A. The PGT database comprises the world’s largest collection of PGT / PGT-A data providing a valuable resource for data mining and for following trends in PGT practice. So far, up to the year 2015, data collections were carried out in a retrospective data way, from 2016 onwards a prospective data collection was in place. Study design, size, duration As the nature of PGT/ PGT-A treatments has changed significantly over the last years and IVF cycle management and genetic analysis techniques are getting more complex, ESHRE uses an online data collection system in which data are collected prospectively from oocyte retrieval to analysis, embryo transfer and pregnancy / live birth. Data are collected cycle by cycle on a voluntary basis. Participants/materials, settings, method For the 2019 data, individual centres (31) from 19 countries directly entered the data into the PGT database through software developed by ESHRE. Data were analysed at ESHRE headquarters and include all aspects of PGT/PGT-A cycles. Main results and the role of chance The Consortium has analysed the PGT analyses (n = 2735) performed in 2019. The indications for PGT included inherited chromosomal abnormalities (n = 253 analyses), monogenic disorders (n = 1105 analyses), aneuploidy testing for infertility (n = 1111 analyses) or combinations of the above (n = 266 analyses). In addition, 662 clinical pregnancies and 216 deliveries have been analysed in detail. The methods used for biopsy were polar body (2%), cleavage stage biopsy (35%) and blastocyst biopsy (61%; comparable with data from 2018). The methodology used for diagnosis is what is evolving most over the last years, with data set XXI (2019) showing around 7% of FISH, 37% of PCR and 55% of WGA. Within WGA 90.6% of the analysis were done using NGS, in 4.4% cases SNP arrays were used and in 2.4% array-CGH was used. The overall clinical pregnancy rate is about 24% per analysis. The baby data show that it is difficult for most centres to have a detailed follow-up. Limitations, reasons for caution The findings apply to the 31 participating centres and may not represent worldwide trends in PGT. Data were collected prospectively, but details of the follow-up on PGT pregnancies and babies born were limited. Wider implications of the findings The ESHRE PGD Consortium continues its activities as an important forum for PGT practitioners to share data and exchange experiences. The information extracted from the data collections helps to monitor quality issues in PGT and survey the introduction and effectiveness of new PGT technologies and methods.

P–560 Comparative analysis of non-invasive preimplantation genetic testing of aneuploidies (niPGT-A), PGT-A and IVF cycles without aneuploidy testing: preliminary results

Study question Can non-invasive preimplantation genetic testing of aneuploidies (niPGT-A) improve the clinical outcome in IVF patients after proper validation? Summary answer We demonstrate the usefulness of the embryonic cell-free DNA (cfDNA) in the blastocyst culture medium to select more objectively the blastocysts with higher implantation potential. What is known already One of the greatest challenges in IVF is accurately selecting viable embryos that are more likely to achieve healthy livebirths following embryo transfer. Trophectoderm (TE) biopsy and PGT-A provide a direct assessment of chromosome status and improve implantation and clinical pregnancy rates per transfer. A non-invasive alternative is to analyse embryonic cfDNA in the blastocyst culture medium. Previous studies have shown that cfDNA testing in culture medium of blastocysts on day 6 of development allows aneuploidy detection with high concordance rates compared to TE biopsy and inner cell mass (Rubio et al., 2020). Study design, size, duration Observational study of the clinical application of niPGT-A (July 2020-December 2020). The clinical application consisted in a first validation phase, comparing TE biopsies with cfDNA in the media of 28 blastocysts. And, in a second phase, niPGT-A was applied and the outcome of 13 single embryo transfers (SETs) compared to 13 PGT-A SETs and 130 IVF/ICSI SETs performed in a period of six months. In the three groups, women and donors age was ≤38 years. Participants/materials, setting, methods Embryos were cultured in a Geri incubator (Merck) up to day 4, and then individually cultured in 10µl drops of CCSS (Fujifilm) until day 6 in a bench-top K-system. At day 6, blastocysts were vitrified, and media collected in sterile PCR tubes after at least 40 hours in culture. After collection, media were immediately frozen and analyzed by NGS analysis in our reference laboratory (Igenomix, Spain). Deferred transfer was performed according to media results. Main results and the role of chance Before the first clinical cases, a validation of the protocol comparing the results of cfDNA with the TE biopsies of the same day–6 blastocyst was performed, and ploidy concordance rates were 87.5%. Similar results were found for niPGT-A and PGT-A in terms of aneuploidy results and in clinical outcomes. The percentages of informative results were 95% and 97% and the aneuploidy rates were 44% and 46%, for niPGT-A and PGT-A, respectively. Clinical pregnancy rates were in both groups of aneuploidy testing, 69.2%, with 8 ongoing pregnancies (61.5%) and 4 tested by prenatal screaning NACE. For untested embryos clinical pregnancy (57.7%) and ongoing pregnancy rates (48.5%) were lower than in the two groups of tested embryos (niPGT-A and PGT-A). In the niPGT-A cycles embryo transfer was performed according to media results and morphology. We did a secondary analysis of which blastocyst we would transfer, if only morphology is considered. We observed that if we only select the embryos by morphology, in 61.5% of the cases we would choose the same embryo than with niPGT-A, and in 30.4% of the cases we would transfer a blastocyst with an aneuploid medium. Limitations, reasons for caution Our results are encouraging but should be interpreted with caution due to the small sample size. Larger and randomized controlled trials are needed to verify and extend our findings in each group. Wider implications of the findings: We observed consistent results for niPGT-A compared to TE biopsies in our internal validation. These results endorse the clinical application of niPGT-A in the routine of the laboratory and can avoid the embryo manipulation also reducing the subjectivity when embryos are selected only by morphology. Trial registration number Sa–16552/19-EC:428

Virtual partition digital PCR for high precision chromosomal counting applications

Digital PCR (dPCR) is the gold standard analytical platform for rapid high precision quantification of genomic fragments. However, current dPCR assays are generally limited to monitoring 1-2 analytes per sample, thereby limiting the platform’s ability to address some clinical applications that require the simultaneous monitoring of 20 – 50 analytes per sample. Here we present Virtual Partition dPCR (VPdPCR), a novel analysis methodology enabling the detection of 10 or more target regions per color channel using conventional dPCR hardware and workflow. Furthermore, VPdPCR enables dPCR instruments to overcome upper quantitation limits caused by partitioning error. While traditional dPCR analysis establishes a single threshold to separate negative and positive partitions, VPdPCR establishes multiple thresholds to identify the number of unique targets present in each positive droplet based on fluorescent intensity. Each physical partition is then divided into a series of virtual partitions, and the resulting increase in partition count substantially decreases partitioning error. We present both a theoretical analysis of the advantages of VPdPCR and an experimental demonstration in the form of a 20-plex assay for non-invasive fetal aneuploidy testing. This demonstration assay – tested on 432 samples contrived from sheared cell-line DNA at multiple input concentrations and simulated fractions of euploid or trisomy-21 “fetal” DNA – is analyzed using both traditional dPCR thresholding and VPdPCR. VPdPCR analysis significantly lowers variance of chromosome ratio across replicates and increases the accuracy of trisomy identification when compared to traditional dPCR, yielding >98% single-well sensitivity and specificity. VPdPCR has substantial promise for increasing the utility of dPCR in applications requiring ultra-high-precision quantitation.

Modern Methods for Assessment of the Implantation Potential of Embryos in Assisted Reproductive Programs

Objective of the Review: To analyse the modern methods for assessment of the implantation potential of embryos in assisted reproductive programs. Key Points. We present the study results for selection of a most optimal embryo for transfer, using visual assessment of embryo quality, preimplantation genetic aneuploidy testing, analysis of metabolomic, proteomic, transcriptomic profiles of culture media and embryo blastocele. We have paid special attention to assessment of small non-coding RNA (sncRNA) in embryo culture medium. Conclusion. Due to the high sensitivity, objectivity and biomarker resistance to degradation, the most promising non-invasive method to assess the implantation potential of an embryo is analysis of the sncRNA profile in embryo culture media. Keywords: aneuploidy, pre-implantation genetic testing, small non-coding RNAs, proteomic analysis, metabolomic analysis.