ObjectiveThe aim of this study was to investigate the effect of sperm DNA fragmentation on fertilization rate, embryo development (blastulation rate), and pregnancy outcomes for ICSI cycles performed in a cohort of couples using donor eggs and to assess the remaining embryos that were not transferred or frozen for apoptotic markers.MethodsEighty-two women (egg recipients) were included in the study (2016) were included in the study. The recipients' mean age was 41.8±5.1 y/o (36-49), while the egg donors' mean age was 30.8±2.1 y/o (27-33). Even though donor egg cycles with frozen sperm samples are performed regularly in our center, 35 cycles were done using fresh sperm samples. The mean age of the males involved in the procedure was 40.1±5.2 y/o. Fertilization, blastulation, and pregnancy rates were assessed. The patients were divided into two groups, TUNEL <15% and ≥15%. In arrested embryos, ICC was performed to detect cleaved caspase-3, survivin, TUNEL, and DNA. The Student's t-test was used in between-group comparisons. The Mann-Whitney U-test was used to assess homogeneity. Pearson's correlation coefficient was also calculated. p<0.05 was considered statistically significant.Results This study showed that there is a negative correlation (R=-0.5) between DNA fragmentation and blastulation rate. High levels of DNA fragmentation were associated with low blastulation and pregnancy rates (per transfer); however, fertilization rate was not affected. Samples with higher levels of DNA fragmentation were associated with higher levels of DNA fragmentation in blastomeres without activating the apoptotic pathway (9.1% vs. 15.9%) (p<0.05). Blastomeres from samples with high DNA fragmentation activated the apoptotic pathway in higher levels than samples with TUNEL <15% (16.4% vs. 21.9%) (p<0.05).ConclusionSperm DNA fragmentation was negatively correlated with blastulation and pregnancy rates even in good quality oocytes. High levels of DNA damage promote embryo arrest and induce the activation of the apoptotic pathway.

Objective: To present the development of the first custom gene panel for the diagnosis of male and female infertility in Latin America.Methods: We developed a next-generation sequencing (NGS) panel that assesses genes associated with infertility. The panel targeted exons and their flanking regions. Selected introns in the CFTR gene were also included. The FMR1 gene and Y chromosome microdeletions were analyzed with other recommended methodologies. An inhouse developed bioinformatic pipeline was applied for the interpretation of the results. Clear infertility phenotypes, idiopathic infertility, and samples with known pathogenic variants were evaluated.Results: A total of 75 genes were selected based on female (primary ovarian insufficiency, risk of ovarian hyperstimulation syndrome, recurrent pregnancy loss, oocyte maturation defects, and embryo development arrest) and male conditions (azoospermia, severe oligospermia, asthenozoospermia, and teratozoospermia). The panel designed was used to assess 25 DNA samples. Two of the variants found were classified as pathogenic and enable the diagnosis of a woman with secondary amenorrhea and a man with oligoasthenoteratozoospermia. Targeted NGS assay metrics resulted in a mean of 180X coverage, with more than 98% of the bases covered ≥20X. Conclusion:Our custom gene sequencing panel designed for the diagnosis of male and female infertility caused by genetic defects revealed the underlying genetic cause of some cases of infertility. The panel will allow us to develop more precise approaches in assisted reproduction.

51%) were greater (p<0.001) than Day 6 (35%) and 7 (33%), but Day 6 euploid rates were not different from Day 7. Failed amplification rates were similar at 2%, 2%, and 3% on Day 5, 6, and 7, respectively. Euploid rates correlated with blastocyst quality on Day 5 (GOOD ¼ 55%; FAIR ¼ 47%, POOR ¼ 35%) and Day 6 (GOOD ¼ 49%; FAIR ¼ 32%, POOR ¼ 25%). but not on Day 7 (GOOD ¼ 25%; FAIR ¼ 43%, POOR ¼ 30%).Single euploid blastocyst FET pregnancy rates as detailed in Table 1 on Day 5, 6, and 7 were 75%, 63%, 35%, respectively (p<0.01). Furthermore, pregnancy rates were positively correlated to blastocyst quality.CONCLUSIONS: Although Day 7 blastocysts represented only 11% of all the blastocysts biopsied in these PGT-A cycles, the euploid rate was similar to those seen in Day 6 blastocysts, but markedly lower than Day 5 blastocysts. Furthermore, although the blastocyst quality is low in Day 7 biopsied blastocysts with only 7% GOOD quality, 35% of our patients achieved pregnancy from Day 7 single euploid FETs. These results support the continued practice of extended blastocyst culture to Day 7 to increase PGT-A pregnancy rates per retrieval cycle.

hybridization, next-generation sequencing, or single-nucleotide polymorphism microarray. Patients who utilized monogenic preimplantation genetic testing were not included in this study. Mosaic (0.14%) and inconclusive (1.04%) results were excluded from the data set. Statistical comparisons were made by Chi-Square analysis. RESULTS: The average age of autologous patients was 36.7. An average of 3.9 embryos were biopsied per cycle. Good quality embryos that were biopsied on day 5 showed the highest euploid rate of 63% (857/1370). Good quality embryos biopsied on day 6 had a euploid rate of 56% (588/1048). Fair quality embryos were significantly less likely to yield euploid results, with embryos biopsied on day 5 returning a euploid rate of 48% (187/393) and embryos biopsied on day 6 returning a euploid rate of 39% (271/691). When stratified by age, this trend held significance (p< .01). CONCLUSIONS: Establishing a hierarchy of embryo quality and rate of development can help guide embryo selection for frozen embryo transfers for patients who did not utilize PGT. These data suggest that in the absence of PGT, embryo quality is the single most important factor in selecting a euploid embryo for transfer, while rate of development to the blastocyst stage should be considered secondarily. Further studies will be necessary to identify other laboratory and clinical factors that contribute to the success of an FET cycle.

Most of human breast cancers are aneuploid because of chromosome missegregation occurring during mitosis. Deregulation in the control of cell cycle progression is frequently found in mammary carcinomas and is associated with mitotic spindle checkpoint defects and aneuploidy. The Laboratory of Hormonal Carcinogenesis has developed an experimental model of mammary carcinomas by the administration of medroxyprogesterone acetate (MPA) to BALB/c mice. These tumors are metastatic luminal ductal carcinomas, which express estrogen and progesterone receptors (PR) and show different responses to antiprogestin treatment. An association between PR isoform expression and endocrine therapy resistance was found in these mammary carcinomas. Tumors with higher levels of isoform A of PR (PRA) than isoform B (PRB) are inhibited by antiprogestin treatment while those with the opposite ratio are antiprogestin resistant tumors. The cytogenetic analysis of these MPA-induced mammary carcinomas showed that all the diploid mammary tumors respond to antiprogestin treatment, whereas aneuploid mammary tumors can be responsive or resistant to the endocrine therapy. Taking into account that Cyclin A has been related to chromosome instability and has also been involved in PR activation, in this study we evaluated its role in aneuploidy and hormone resistance using the MPA breast cancer model. We analyzed the Cyclin A expression by Western blot (WB) and immunohistochemistry (IHC) in the hormone dependent and the independent (HI) derived variants with different antiprogestin responsiveness and ploidy levels. WB studies revealed for Cyclin A two bands of estimated molecular weights of 52 kDa and 48 kDa in all the mammary tumors analyzed. The 52 kDa form of Cyclin A was overexpressed in the aneuploid unresponsive C4-2-HI tumor (10 fold; p&lt;0.05), whereas the 48 kDa Cyclin A form was overexpressed in the resistant C4-HIR tumor (6 fold; p&lt;0.05), compared to the responsive tumors in the C4 tumor family. On the other hand, both forms of Cyclin A were overexpressed in the resistant C7-2-HIR tumor (52 kDa: 6 fold; 48 kDa: 1.7, p&lt;0.05), while the unresponsive C7-HI tumor expressed higher levels of the 48 kDa form (1.5 fold), as compared to the diploid responsive tumor. No differences were observed in the expression of the 48 kDa form of the cyclin in the mammary tumors of 59 tumor family, although the 52 kDa band was increased in one of the responsive HI tumors. These results were confirmed by IHC analysis of the paraffin sections of these tumors. A higher percentage of Cyclin A positive nuclei was observed in the resistant C4-HIR and C7-HI tumors, compared to the responsive tumors studied. These results suggest that in addition to its role in cell cycle control and its participation in aneuploidy, Cyclin A might be involved in the onset of antiprogestin resistance in the MPA breast cancer model. Citation Format: Melina Bilinski, Claudia Lanari, Victoria T. Fabris. Cyclin A expression and endocrine resistance in an experimental model of murine mammary carcinomas. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A11.