Evrim Unsal

Impairment of BRCA1-Related DNA Double-Strand Break Repair Leads to Ovarian Aging in Mice and Humans

DNA double-strand break repair has a central role in oocyte aging. Preserving Fertility Breeds Flexibility Last month, the U.K. Office for National Statistics reported that, in 2010, ~48% of infants were born to mothers 30 years and older, a level not seen since 1946—just after the end of World War II. Delaying childbearing can allow women flexibility with respect to career options. But unlike many somatic tissues, the female germline ages early, with reproductive capacity beginning to diminish after young adulthood. Attempts to stem oocyte aging and preserve fertility will depend on finely characterizing the molecular mechanisms behind the aging process in the female reproductive system. Now, Titus et al. provide evidence for a new mechanism to explain age-related oocyte dysfunction. The authors showed that double-stranded breaks (DSBs) in DNA—which are essential for normal development—accumulate with age and contribute to reproductive aging in mice and women. In single mouse and human oocytes, the expression of DSB repair genes BRCA1, MRE11, RAD51, and ATM declined with age. Thus DSBs likely collect in the oocyte genome because of age-related missteps in DSB repair, which stimulate apoptosis and diminishes ovarian reserve. Indeed, in Brca1-deficient mice, numbers of primordial follicles—immature primary oocytes—were decreased, DSBs were increased, and reproductive capacity was impaired relative to wild-type mice. Using RNA interference in mouse oocytes, the authors showed that inhibition of Brca1, MRE11, RAD51, and, in turn, ATM expression increased DSBs and reduced oocyte survival. The authors then measured serum concentrations of anti-Müllerian hormone—a measure of fertility—in young women with germline BRCA1 mutations versus controls and showed that ovarian reserve was compromised in the latter group. Together, these findings show that the efficiency of DNA DSB repair is a crucial determinant of oocyte loss. The discovery of therapies that target this pathway might help to enhance the duration of ovarian function. The underlying mechanism behind age-induced wastage of the human ovarian follicle reserve is unknown. We identify impaired ATM (ataxia-telangiectasia mutated)–mediated DNA double-strand break (DSB) repair as a cause of aging in mouse and human oocytes. We show that DSBs accumulate in primordial follicles with age. In parallel, expression of key DNA DSB repair genes BRCA1, MRE11, Rad51, and ATM, but not BRCA2, declines in single mouse and human oocytes. In Brca1-deficient mice, reproductive capacity was impaired, primordial follicle counts were lower, and DSBs were increased in remaining follicles with age relative to wild-type mice. Furthermore, oocyte-specific knockdown of Brca1, MRE11, Rad51, and ATM expression increased DSBs and reduced survival, whereas Brca1 overexpression enhanced both parameters. Likewise, ovarian reserve was impaired in young women with germline BRCA1 mutations compared to controls as determined by serum concentrations of anti-Müllerian hormone. These data implicate DNA DSB repair efficiency as an important determinant of oocyte aging in women.

Relationship between embryo quality and aneuploidies

Many high-grade embryos selected for transfer according to their morphological evaluation were detected to have chromosomal abnormalities after aneuploidy screening for infertility by preimplantation genetic diagnosis (PGD). The aim of this study was to detect if there is any correlation between embryo quality and genetic status. The chromosomal status of the day three embryos was studied by multicolour fluorescence in-situ hybridization for chromosomes 13, 18, 21, X and Y. PGD was performed on 132 patients for 1107 embryos. The correlation between embryo quality and aneuploidy was analysed. The analysis showed that a large proportion of normal embryos (50.7%, n = 280) were grade I. In addition, a considerably high proportion of aneuploid embryos (36.1%, n = 83) were evaluated as grade I. There was a significant relationship between PGD results and embryo grades (P = 0.001). Of the 69 polyploid embryos, 21.7% were grade I and 37.8% were grade II. Of the 83 haploid embryos, 27.8% were grade I and 34.9% were grade II. Euploidy was positively related to morphological grade of embryo (P = 0.001). It was also possible for chromosomally abnormal embryos to have a good developmental potential, and they could be selected for embryo transfer unless the PGD procedure was applied.

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.

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.

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.

The Effect of Piezoelectric Stimulation in Patients with Low Fertilization Potential

Objective: To assess the value of the electrical activation of oocytes in ICSI patients with previous limited fertilization outcomes. Design: Prospective randomized study. Settings: Clinical IVF laboratory. Patient(s): A hundred and seven couples undergoing ICSI with possible low fertilization outcomes. Intervention(s): TESE, TESA, ICSI with Piezoelectric Activation Main Outcome Measure(s): Fertilization, clinical pregnancy rates, embryo grades. Result(s): Patients were subdivided into six study groups. In Group I, testicular elongated spermatids were used and 27.9% fertilization, 2.3% clinical pregnancy rates were evaluated. In testicular immotile spermatozoa injected group (Group II), 56.5% fertilization and 50% clinical pregnancy rates were obtained. 66.7% fertilization and 30%clinical pregnancy rates were achieved in Group III with testicular motile spermatozoa. In Group IV, patients with severe oligozoospermia, 64% fertilization and 28.6% clinical pregnancy rates were achieved. Group V included patients with total immotile spermatozoa and fertilization and clinical pregnancy rates were 50% and 57.1% respectively. In patients with history of low fertilization rate (Group VI), 38.7% fertilization and 19% clinical pregnancy rates were obtained. Conclusion(s): Piezoelectric stimulation can be used for patients with low fertilization rates and total immotile spermatozoa; as we detected an improvement in the fertilization and clinical pregnancy rates of these patients.