An Incidental Detection of a Cryptic Complex Chromosome Rearrangement Found During NGS Based PGT-SR: A Case Report.

Background: Complex chromosome rearrangements (CCRs) involve more than 2 chromosomal breakpoints and cause the exchanges of chromosomal segments between two or more chromosomes. The carriers of CCRs have normal phenotypes, but they have a higher risk of reproductive failure. Case Presentation: This paper presents a couple with a history of two affected children, one spontaneous abortion, three in vitro fertilization (IVF) failures, and one healthy boy who were referred to our laboratory for preimplantation genetic testing (PGT). The wife had been evaluated as a carrier of 46,XX,t (2;6)(p21;p25); therefore, four IVF treatment cycles supported with PGT for this translocation had been performed in different IVF centers until the couple consulted our laboratory. Only one of these four IVF attempts had resulted in a healthy boy and this IVF study had been performed with fluorescence in situ hybridization (FISH)-based preimplantation genetic testing for structural chromosomal rearrangements (PGT-SR). The fifth IVF study with next-generation sequencing (NGS)-based PGT was performed by our laboratory and no healthy embryo was found in evaluated 6 embryos. During our NGS-based PGT, the cryptic involvement of 12p was firstly detected. FISH with chromosome 2,6, and 12 specific probes revealed that the mother was a carrier of a balanced 3-way translocation of 46,XX,t(2;6;12)(p21;p25;p13). Conclusion: NGS based PGT-SR method is an accurate method for detecting the copy number variations and is helpful to find out the cryptic CCRs.

Effects of pentoxifylline and platelet activating factor on sperm DNA damage.

OBJECTIVE: Pentoxifylline and platelet-activating factor (PAF) have been used to increase sperm motility in embryology laboratories. In the present study, we aimed to investigate whether these agents pose sperm DNA damage using DNA sperm chromatin dispersion (SCD) assay. STUDY DESIGN: Following application of pentoxifylline and PAF, sperm samples of 50 individuals with different sperm parameters were compared to baseline in terms of DNA damage using SCD assay. Furthermore, the relationship between DNA damage and sperm parameters in predicting DNA damage was assessed. RESULTS AND CONCLUSIONS: Significant increase in DNA damage was observed following application of PAF and pentoxifylline. Furthermore, DNA damage was significantly increased with application of pentoxifylline compared to PAF. Sperm motility was observed to be a statistically significant indicator in predicting alterations in DNA damage in baseline and subsequent to application of PAF and pentoxifylline independent of sperm concentration and morphology. Increased DNA damage was observed in both groups following application of pentoxifylline and PAF. Furthermore, the increase in DNA damage was higher in samples treated with pentoxifylline compared to samples treated with PAF. Thus, PAF seems to be more innocent in choosing viable sperm cells and in achieving sperm motility in the in vitro fertilization laboratory.

Oogonial Precursor Cell-Derived Autologous Mitochondria Injection to Improve Outcomes in Women With Multiple IVF Failures Due to Low Oocyte Quality: A Clinical Translation.

BACKGROUND: Mitochondrial dysfunction has been suggested as a major cause of age-induced decline in oocyte quality. In the past, donor oocyte cytoplasmic transfer showed some success but was abandoned due to the concerns with heteroplasmy. Recent studies indicated presence of oogonial precursor cells (OPCs) in the human ovary, which could be an autologous source of "healthy mitochondria." We sought to investigate the clinical efficacy of OPC-derived autologous mitochondrial injection (AMI) to improve oocyte quality in women with multiple in vitro fertilization (IVF) failures. METHODS: The OPCs were isolated from laparoscopically obtained ovarian cortical pieces by cell sorting using a monoclonal anti-vasa homolog (anti-DDX) antibody. They were then disrupted and mitochondria were isolated. Reconstituted mitochondria were injected into each oocyte during intracytoplasmic sperm injection. Paired comparisons were made between the first failed cycles and the post-AMI cycles. RESULTS: Of the 15 women undergoing ovarian stimulation, 2 were canceled and 3 decided to pool oocytes for later AMI. In remaining 10 (mean age 34.7 ± 4.1), AMI significantly improved fertilization rates (49.7 ± 31.3 vs 78.3 ± 18.9; P = .03) with a trend for better embryo grades (2.3 ± 0.3 vs 3.1 ± 0.7; P = .08). Four of 10 women conceived after single frozen embryo transfer and 3 after confirmation of diploidy via array comparative genomic hybridization (aCGH) (clinical pregnancy/embryo transfer = 4/10). CONCLUSION: These data show encouraging results for AMI in comparison to previous failed IVF cycles.

Effect of storage solutions on microhardness of crown enamel and dentin.

OBJECTIVE: The aim of this study was to determine alterations in microhardness of crown dentin and enamel, after 2 and 12-month storage in de-ionized water, 0.2% glutaraldehyde, Hanks' Balanced Salt Solution (HBSS), 0.1% sodium hypochlorite (NaOCl) or 0.1% thymol. MATERIALS AND METHODS: Freshly extracted, nonsterile 60 intact human premolars were distributed to five groups. Six teeth from each group were evaluated after two, and other six teeth were evaluated after 12 months storage. After grinding and polishing of teeth, Vickers hardness was evaluated with making indentations on enamel and dentin, using a pyramid diamond indenter tip exerting 100 g load for 15 s. RESULTS: After 2 months storage in solutions, range of the hardness values (HV) of enamel and dentin were in between 315-357 and 64-67, respectively. However, 12 months storage of the teeth resulted in a statistically significant decrease in microhardness when compared to microhardness of teeth stored for 2 months (P = 0.001). Although the differences were not significant regarding solutions, all solutions decreased the microhardness both in enamel and dentin (P > 0.05). However, decrease in microhardness was relatively less in de-ionized water and thymol solutions while glutaraldehyde decreased microhardness the most: 63% for enamel and 53% for dentin. CONCLUSIONS: Microhardness of enamel and dentin was in an acceptable range when teeth were stored for 2 months in de-ionized water, glutaraldehyde, HBSS, NaOCl or in thymol; thus, teeth kept up to 2 months in these solutions can be used for mechanical in vitro tests. However, 12 months storage significantly decreased the microhardness of enamel and dentin.

The effectiveness of intracytoplasmic sperm injection combined with piezoelectric stimulation in infertile couples with total fertilization failure.

OBJECTIVE: To assess the effectiveness of intracytoplasmic sperm injection (ICSI) combined with piezoelectric stimulation in infertile couples with a history of total fertilization failure (TFF). DESIGN: Prospective controlled trial. SETTING: Clinical IVF laboratory. PATIENT(S): Seventy-one couples undergoing ICSI on sibling oocytes having at least one previous ICSI attempt with TFF. INTERVENTION(S): ICSI or ICSI with piezoelectric activation. MAIN OUTCOME MEASURE(S): Fertilization rate. RESULT(S): The patients were allocated to two groups: group I included 21 patients with only one previous TFF and group II included 50 patients with more than one previous TFF. Collectively, a total of 823 metaphase II (MII) oocytes were retrieved in 78 oocyte retrievals. In Group I, combined ICSI with piezoelectric stimulation was applied to 123/211 (58.2%) of MII oocytes (group IA), whereas standard ICSI procedure was applied to 88/211 (41.8%) of MII oocytes (group IB). The fertilization rate was 62% and 12% in group IA and group IB respectively. In group II, piezoelectric activation was applied in all 612 MII oocytes, of which 296 (48.3%) were fertilized. The rates for implantation and pregnancy/embryo transfer were obtained as 30.6% and 44.1%, respectively. CONCLUSION(S): Piezoelectric activation seems to improve IVF outcome in patients with previous TFF history.

Successful application of preimplantation genetic diagnosis for Leigh syndrome.

OBJECTIVE: To perform preimplantation genetic diagnosis (PGD) for a SURF1 gene mutation of the Leigh syndrome to transfer unaffected or carrier embryo/embryos. DESIGN: Case report. SETTING: Clinical IVF laboratory. PATIENT(S): A couple carrying an nt769 G/A mutation that is associated with Leigh syndrome. INTERVENTION(S): Oocytes were fertilized by means of intracytoplasmic sperm injection. The resulting embryos were biopsied 3 days after fertilization. One blastomere was taken and whole-genome amplification was performed. Amplification of the mutation site was achieved by polymerase chain reaction (PCR) and restriction digestion was completed. Gel Imager was used to measure the digests of normal and mutant load. MAIN OUTCOME MEASURE(S): Embryo testing by means of PGD-PCR and pregnancy. Successful preimplantation genetic diagnosis for a SURF1 gene mutation and transfer of healthy or carrier embryos. RESULT(S): Successful singleton pregnancy resulting in the delivery of healthy baby girl. CONCLUSION(S): We report the first case of successful PGD for Leigh syndrome resulting in delivery of a healthy newborn.