P–080 Higher sperm DNA fragmentation reduces the proportion of good quality embryos at day 5 on IVF and ICSI cycles from unselected males

Study question Does sperm DNA fragmentation (SDF) reduce the ratio of good-quality embryos in day 3 (D3) and day 5 (D5) of embryonic development? Summary answer High sperm DNA fragmentation (SDF >15%) is associated with poor embryo quality at blastocyst-stage per cycle in unselected patients undergoing IVF and ICSI. What is known already It has been shown that the proportion of spermatozoa with DNA fragmentation is higher in infertile men than in semen from fertile men. However, the controversy regarding the impact that sperm genome damage can have on IVF or ICSI treatments is evident in the published literature. The effects of SDF would become evident after activation of the embryonic genome at 8-cell stage, compromising not only the quality of the embryos obtained, but also the reproductive outcomes, as reduced implantation rates, higher miscarriages rates and thus, a decreased chance of pregnancy. Study design, size, duration This multicentric observational retrospective study included 1339 couples who underwent 2759 IVF-ICSI cycles using autologous oocytes from January 2000 to March 2019. All men have an SDF test in their ejaculated spermatozoa by TUNEL assay (Terminal deoxynucleotidyl transferase dUTP nick end labeling). The subjects were divided into two groups according to their sperm DNA integrity: low (≤15%) (n = 2287 cycles) or high (>15%) (n = 472) SDF. Participants/materials, setting, methods Embryo quality was assessed complying morphological standards at cleavage-stage on D3 and at blastocyst-stage on D5 (inner cell mass (ICM) and trophectoderm (TE) grade (A, B, C or D)) in according to ASEBIR’s embryo selection criteria, being embryos of good quality those categorized as A+B. The outcomes were calculated in relation to the total number of zygotes obtained. The results were compared by Student t test; p value <0.05 was considered significant. Main results and the role of chance The SDF average of the low group was 5.8% (95% CI 5.6–5.9) whereas in high group was 23.7% (95% CI 23.0–24.4). The female age was equal, 37.1 years (95%CI 37.0–37.2) and 37.1 years (95% CI 36.8–37.4) respectively. A total of 9796 embryos were evaluated. The optimal cleavage-stage embryo rate per cycle was 25.0% (95% CI 21.7–28.3) (8.0 average cells number, 1.5 embryo fragmentation average, symmetry 1, mononucleated cells) versus 26.7% (95%CI 19.1–34.2) (7.9 average cells number, 1.8 embryo fragmentation average, symmetry 1, mononucleated cells) when comparing between groups (p < 0.001). Blastocyst-stage arrival rate (number of embryos at D5) per cycle was 55.8% (95% CI 54.3–57.2) in ≤ 15% SDF group (embryo quality score was ICM A:12.1%, B:69.5%, C:8.8%, D:4.5%; TE A: 7.5%, B:42.2%, C:42.2%, D:8.1%) and 55.9% (95% CI 52.8–59.1) in the >15% SDF group (ICM A:12.0%, B:68.7%, C:10.6%, D: 5.2%; TE A:9.1%, B:44.8%, C:37.8%, D:8.3%) (p < 0.001). The good quality blastocyst rate per cycle was significantly higher in the group with SDF ≤15%, 27.7% (95%CI 26.5–29.0) versus SDF >15% (27.4% (95%CI 24.6–30.2)). Of the total number of blastocysts, the proportion of A+B blastocyst was 60.5% (95% CI 58.3–62.7) and 64.2% (95% CI 59.2–69.2) (p < 0.001), respectively. Limitations, reasons for caution The retrospective and multicenter nature of this study leads to uncontrolled biases derived from the clinical practice. Although the results were not adjusted for female’s age, it was not statistically different between groups. Embryo morphology evaluation was performed by senior embryologists, it still remains a subjective evaluation, though. Wider implications of the findings: In this study, a higher amount of data was compiled so that a large number of embryos were analyzed. The DNA integrity of the sperm may be an important consideration when poor quality embryos were obtained in the previous cycle when deciding on the next clinical strategy to apply. Trial registration number NA

P–030 A sperm chromatin damage >15% negatively impacts on the quality of embryos obtained from ovum donation ICSI cycles of unselected couples

Study question Is embryo quality downgraded in couples with elevated sperm DNA fragmentation (SDF) in the ejaculated semen of male partner using donated eggs? Summary answer The rate of good quality embryos at day 3 and blastocyst-stage is statistically inferior in males with SDF>15% undergoing ICSI cycles with donated oocytes. What is known already The effect of a damaged paternal chromatin will be shown from the 8-cell stage of embryo development, a time which the genome of the embryo is transcriptionally active. Fertilization with a spermatozoon with fragmented DNA may impair the quality of the embryos obtained per cycle, and therefore reduce the chances of pregnancy. The use of donated oocytes is an ideal model to evaluate the real effect of SDF on embryo quality by standardizing the female factor. In addition, we have a large cohort of ovum donation cases in our history, which allows a more proper evaluation of the effect. Study design, size, duration Retrospective multicentric study including the clinical data of 864 couples of ovum donation program who underwent 1903 ICSI cycles between January 2000 and March 2019. The DNA fragmentation of their ejaculated spermatozoa was measured by TUNEL assay (Terminal deoxynucleotidyl transferase dUTP nick end labeling). Two study groups were created according to the SDF level: ≤15% (low) (n = 1626) or > 15% (high) (n = 277). Participants/materials, setting, methods Embryos were evaluated throughout embryonic development according to classical morphological parameters at day 3 (D3), on cleavage-stage, and at day 5 (D5), on blastocyst-stage (trophectoderm (TE) and inner cell mass (ICM)), following ASEBIR guidelines, categorized from A to D. Embryos scored as A and B were considered to be good quality. The proportion of embryos was calculated according to the total number of correctly fertilized oocytes or zygotes. A p < 0.05 was considered significant. Main results and the role of chance A total of 6130 embryos were evaluated. The SDF average of ≤ 15% group was 5.9% (95%CI 5.7–6.1) and 24.3% (95%CI 23.2–25.3) in the >15% group. The cycle-related characteristics and the seminal parameters were comparable. The proportion of optimal cleavage-stage embryo (number of A+B embryos at D3) per cycle was 21.7% (95%CI 19.0–24.5) (8.1 average cells number, 0.8 embryo fragmentation average, symmetry 1, mononucleated cells) in ≤ 15% SDF group versus 21.1% (95%CI 13.9–28.3) (8.2 cells number average, 1.3 embryo fragmentation average, symmetry 1, mononucleated cells) (p < 0.001). The blastocyst-stage arrival rate (number of embryos at D5) per cycle was higher in the >15% SDF group (p < 0.001), 53.4% (95%CI 48.8–58.1) (TE quality A:20.5%, B:42.5%, C:22.7%, D:14.8%, and the ICM quality A:26.1%, B:52.1%, C:13.2%, D:6.2%) versus 49.9% (95%CI 48.1–51.6) (TE quality A:21.1%, B:42.8%, C:21.85, D:14.1% and ICM A:26.6%, B:55.5%, C:11.1%, D:4.7%) in the low SDF group. The rate of good quality blastocyst (number of quality A+B embryos in D5) per cycle was significantly higher in the couples with low SDF (24.8% (95%CI 23.6–25.9)) than in those with elevated SDF (23.5% (95%CI20.9–26.2)) (p < 0.001). Accordingly, the A+B blastocyst rate divided by the total number of blastocysts was 59.1% (95%CI 56.7–61.4) versus 55.9% (95%CI 49.9–62.0) (p < 0.001), respectively. Limitations, reasons for caution The main limitation is that retrospective design of the study may not eliminate the potential unaccounted-for bias derived from the clinical practice of multiple centers even though both groups were statistically comparable. Also, the assessment of embryo quality is still remaining highly subjective to embryologists. Wider implications of the findings: Although the effect size is small, it may be useful in clinical practice when an ICSI cycle yields no good-quality embryos, as one of the underlying causes of that fact. Knowing the SDF level can be a helpful tool in making subsequent clinical decisions aimed at improving outcomes for couples. Trial registration number Not applicable

Expression patterns of mitochondrial OXPHOS components, mitofusin 1 and dynamin-related protein 1 are associated with human embryo fragmentation

Developmental dysfunction in embryos, such as a lethal level of fragmentation, is assumed to be mitochondrial in origin. This study investigated the molecular basis of mitochondrial impairment in embryo fragmentation. Transcription patterns of factors that determine mitochondrial functionality: (i) components of the oxidative phosphorylation (OXPHOS) – complex I, cytochrome b, complex IV and ATP synthase; (ii) mitochondrial membrane potential (MMP); (iii) mitochondrial DNA (mtDNA) content and (iv) proteins involved in mitochondrial dynamics, mitofusin 1 (Mfn1) and dynamin related protein 1 (Drp1) were examined in six-cells Day 3 non-fragmented (control), low-fragmented (LF) and high-fragmented (HF) human embryos. Gene expression of mitochondria-encoded components of complex I and IV, cytochrome b and mtDNA were increased in HF embryos compared with control and LF embryos. In LF embryos, expression of these molecules was decreased compared with control and HF embryos. Both classes of fragmented embryos had decreased MMP compared with control. LF embryos had increased gene expression of Mfn1 accompanied by decreased expression of Drp1, while HF embryos had decreased Mfn1 expression but increased Drp1 expression. The study revealed that each improper transcriptional (in)activation of mitochondria-encoded components of the OXPHOS during early in vitro embryo development is associated with a decrease in MMP and with embryo fragmentation. The results also showed the importance of mitochondrial dynamics in fragmentation, at least in the extent of this process.