PREGNANCY OUTCOME OF TRANSFERRED EMBRYOS WITH MOSAICISM AND SEGMENTAL VARIATIONS

sufficient to explain the prevalence of premature loss of centromeric cohesin in oocytes from older females. We are therefore interested in understanding the timing and mechanisms of cohesin depletion during female ageing. Our recent work indicates that cohesin is gradually depleted from oocyte chromosomes during the prolonged arrest at prophase of meiosis I, before oocytes are recruited for growth. Interestingly, the age-related decline in chromosome-associated Rec8 exceeds that of Smc3, which is a subunit of mitotic as well as meiotic cohesin complexes. This suggests that cohesin complexes containing alpha-kleisin subunits other than Rec8 are present on oocyte chromosomes and might be less vulnerable to depletion during female ageing. In addition, we have tested the idea that leaky inhibition of separase during the prolonged arrest at prophase of meiosis I contributes to gradual loss of Rec8. In support of this possibility, we can detect separase in oocytes before they are recruited for growth. We find, however, that deletion of separase specifically in oocytes does not prevent the decline in Rec8 levels observed during female ageing. Together, these findings indicate that cohesin loss occurs gradually during the prolonged prophase arrest and that this occurs by a separase-independent mechanism. doi: 10.1016/j.rbmo.2019.04.027

POLAR BODY ANALYSIS FOR PREIMPLANTATION GENETIC TESTING

Alan H Handyside School of Biosciences, University of Kent, Canterbury, UK

The use of polar bodies for preimplantation genetic testing for both single gene defects (PGT-M) and aneuploidy (PGT-A) was pioneered

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by Verlinsky and colleagues beginning in the late 1980s. For many years, it was applied clinically to diagnose the inheritance of maternal mutations and, using fluorescence in situ hybridisation (FISH) with chromosome specific probes, to detect maternal meiotic segregation errors resulting in aneuploidy. Most clinics now culture to the blastocyst stage and with the advent of vitrification, biopsied blastocysts can be cryopreserved efficiently while the samples are sent to a genetics lab for testing. However, polar body biopsy continues to be used in several European countries with legal or ethical restrictions on the testing of embryos. Although the second polar body can persist to the blastocyst stage, both polar bodies are eventually lost and do not form part of the embryo. Removing both of the polar bodies is therefore minimally invasive. Polar body analysis by NGS-based copy number analysis or SNP genotyping and either karyomapping or meiomapping is highly effective for both aneuploidy and linkage-based testing for maternal mutations. The advantage for PGT-A, in particular, is that only maternal meiotic aneuploidies are identified avoiding the problem of interpreting intermediate copy number changes associated with chromosome mosaicism in multiple cell biopsies. Polar body biopsy therefore remains a valuable alternative strategy for PGT. doi: 10.1016/j.rbmo.2019.04.028

PREGNANCY OUTCOME OF TRANSFERRED EMBRYOS WITH MOSAICISM AND SEGMENTAL VARIATIONS

Francesco Fiorentino GENOMA Group - Molecular Genetics Laboratories, Via di Castel Giubileo, 11, Milan, Rome 00138, Italy Email: [email protected]

Mosaic embryos are characterized by the presence of chromosomally

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different cell lines within the same embryo. Typically, mosaic embryos were not transferred in IVF treatments because, similar to aneuploidy embryos, they were considered abnormal. However, in a recent study we have demonstrated that euploid/diploid mosaic embryos hold the potential to implant and result in the birth of healthy babies. As a consequence, the transfer of these embryos is now offered as an option for women who undergo IVF resulting in mosaic embryos but no euploid embryos. Although the impact of mosaicism on the implantation and developmental potential of embryos is not fully known, it is reasonable to assume that it is likely to influence the clinical outcome of IVF treatments. We also hypothesized that the extent of mosaicism and the type of aneuploidy involved may affect the IVF success rate. However, no definitive conclusion could be drawn because our study was small and the data available were insufficient to test this hypothesis. The aim of this study was to assess whether the extent of mosaicism and the type of the type of aneuploidy may influence the development potential of mosaic embryos. To test this hypothesis we enlarged our previous study, offering the transfer of mosaic embryos, at different aneuploidy percentage and aneuploidy content, to 200 women for which the IVF/PGT cycle resulted in no euploid embryos available for transfer. Comprehensive chromosome testing was performed using high resolution next generation sequencing (NGS) methodology. The clinical outcome obtained after transfer of mosaic embryos with low (<50%) and high (≥50%) aneuploidy percentage or with different types of aneuploidy involved in mosaicism, was compared with that resulting from a control group of 500 euploid blastocysts.

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We found that the reproductive potential of a mosaic euploid/ aneuploid blastocyst is inversely correlated with the abnormalto-normal cells ratio. Mosaic embryos with a high percentage of chromosomally abnormal cells (≥50%) resulted in a statistically significant reduction in clinical pregnancy rate/ET, implantation rate, and live-birth rate compared with mosaic embryos with a lower aneuploidy percentage (<50%). In contrast, embryos with lower aneuploidy percentage (<50%) have a clinical outcome similar to euploid embryos. In addition, we found that the type of aneuploidy involved in mosaicism correlates with the clinical outcome, as embryos with single or double monosomy have significant higher chances in developing into healthy euploid newborns as compared to other types of aneuploidy. The transfer of mosaic blastocysts with complex aneuploidies or segmental rearrangement showed the worst clinical outcome. No statistically significant differences in clinical results were found between mosaic embryos with monosomy and the euploid control group. In conclusion, the results of our study demonstrated that mosaic embryos have poorer clinical outcomes compared with euploid embryos and that their implantation and developmental potential is influenced by the extent of mosaicism and the aneuploidy content. doi: 10.1016/j.rbmo.2019.04.029

IMPACT OF SEVERE MALE INFERTILITY ON CHROMOSOMAL STATUS OF EMBRYOS AND PREGNANCY OUTCOME IN YOUNGER WOMEN

Semra Kahraman, M.D. Istanbul Memorial Hospital, ART&Genetics Center, Turkey

The relationship between severe male infertility and embryo aneuploidy has long been a subject of interest and there have been a limited number

of studies. Our study used next generation sequencing (NGS), to evaluate the chromosomal status of blastocyst stage embryos and evaluated embryo morphokinetics and pregnancy outcomes in young women (<35) according to severe male infertility subgroups.

26.55h, respectively; p=0.0347). There was also a difference between obstructive and non-obstructive azoospermia patients in the time to reach blastocyst, OA patients achieving a blastocyst significantly earlier than NOA (tB= 102.20h vs. tB=104.91h, respectively; p=0.0297).

Severe male factor (SMF) is defined as sperm concentration below 5 million per ml. This covers a very wide range from 5 million down to non-obstructive azoospermia (NOA), in which sperm production is severely impaired. In our study, SMF cases were divided into the following 5 subgroups according to sperm concentration; 1) between five million and one million, 2) between one million and one hundred thousand, 3) less than one hundred thousand (cryptozoospermia), 4) Obstructive Azoospermia (OA) and 5) Nonobstructive Azoospermia (NOA). The control group was composed of males with normal sperm parameters (>39 million and >40% motile sperm in the ejaculate). 228 severe male infertility cases and 67 control cases with normal sperm parameters. In all groups female age was <35 and PGT-A and morphokinetic evaluation were performed.

The cumulative live birth rates of SMF groups were compared. Significantly lower cumulative live birth rates were found in couples with cryptozoospermia (43.7%), in patients with OA (46%) and in patients with NOA (46.1%) compared to other SMF subgroups with five million and one million (58.1%) and between one million and one hundred thousand (71.4%) subgroups.

The PGT-A results showed that the overall aneuploidy rate of patients with SMF was 55.3% and the mosaicism rate was 13.2%. In the control group these figures were 55.8% and 6.7% respectively, a significantly higher mosaicism rate (p=0.001) was observed in the SMF group. Significantly higher chromosomal aneuploidy rates were found in couples with cryptozoospermia, in patients with OA and in patients with NOA compared to other male subgroups. Furthermore, embryo morphokinetic evaluation was carried out using time lapse imaging to investigate embryo development. NOA patients reached the first cleavage significantly later than the control group (27.78h vs.

In conclusion, the chromosomal status of embryos in severe male infertility cases with young female partners (<35) was not significantly different from that of the control groups. However, the mosaicism rate was significantly higher. Higher chromosomal aneuploidy rates were found in the most severe form of male infertility subgroups with cryptozoospermia, OA and NOA. Morphokinetic evaluation showed that NOA patients reached the first cleavage significantly later than the control group indicating that SMF impairs early embryonic competence. Finally, a significantly negative effect on clinical outcomes was observed in the three most severe male infertility (cryptozoospermia, OA and NOA) subgroups. doi: 10.1016/j.rbmo.2019.04.030

GENOME-WIDE PROFILING AND HAPLOTYPING OF CELL-FREE DNA ENABLING COMBINED NON-INVASIVE PRENATAL DIAGNOSIS OF INHERITED MONOGENIC DISEASES AND ANEUPLOIDY

Huiwen Che1, Darine Vileila1, Eftychia Dimitriadou1, Jia Ding1, Thierry Voet2, Joris R. Vermeesch1