Mitochondrial DNA Copy Number in Cleavage Stage Human Embryos-Impact on Infertility Outcome.

A retrospective case control study was undertaken at the molecular biology department of a private center for reproductive medicine in order to determine whether any correlation exists between mitochondrial DNA (mtDNA) content of cleavage-stage preimplantation embryos and their developmental potential. A total of 69 couples underwent IVF treatment (averaged women age: 36.5, SD 4.9) and produced a total of 314 embryos. A single blastomere was biopsied from each embryo at the cleavage stage (day-3 post-fertilization) subjected to low-pass next generation sequencing (NGS), for the purpose of detecting aneuploidy. For each sample, the number of mtDNA reads obtained after analysis using NGS was divided by the number of reads attributable to the nuclear genome. The mtDNA copy number amount was found to be higher in aneuploid embryos than in those that were euploid (mean mtDNA ratio ± SD: 6.3 ± 7.5 versus 7.1 ± 5.8, p < 0.004; U Mann−Whitney test), whereas no statistically significant differences in mtDNA content were seen in relation to embryo morphology (6.6 ± 4.8 vs. 8.5 ± 13.6, p 0.09), sex (6.6 ± 4.1 vs. 6.2 ± 6.8, p 0.16), maternal age (6.9 ± 7.8 vs. 6.7 ± 4.5, p 0.14) or its ability to implant (7.4 ± 6.6 vs. 5.1 ± 4.6, p 0.18). The mtDNA content cannot serve as a useful biomarker at this point in development. However, further studies investigating both quantitative and qualitative aspects of mtDNA are still required to fully evaluate the relationship between mitochondrial DNA and human reproduction.

[QTc duration in the long QT syndrome type 2 - diagnostic dilemma]. / Czas trwania skorygowanego odstepu QT w zespole dlugiego QT typu 2 - problemy diagnostyczne.

We present a case of a 43-year old woman with a long QT syndrome (LQTS). Despite her family history of several sudden deaths in close relatives the patient was misdiagnosed, being treated many years for epilepsy. Finally, after another series of syncopal spells she was diagnosed with LQTS. Although QTc interval was normal on surface ECG, the presence of notched T-waves and QT prolongation up to 543 ms on Holter recording were helpful in establishing the correct diagnosis, which was later confirmed by genotyping revealing G601S mutation in the HERG gene. The patient refused an ICD and despite beta-blocker treatment died suddenly several months later.

Routine use of next-generation sequencing for preimplantation genetic diagnosis of blastomeres obtained from embryos on day 3 in fresh in vitro fertilization cycles.

OBJECTIVE: To determine the usefulness of semiconductor-based next-generation sequencing (NGS) for cleavage-stage preimplantation genetic diagnosis (PGD) of aneuploidy. DESIGN: Prospective case-control study. SETTING: A private center for reproductive medicine. PATIENT(S): A total of 45 patients underwent day-3 embryo biopsy with PGD and fresh cycle transfer. Additionally, 53 patients, matched according to age, anti-Müllerian hormone levels, antral follicles count, and infertility duration were selected as controls. INTERVENTION(S): Choice of embryos for transfer was based on the PGD NGS results. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate (PR) per embryo transfer (ET) was the primary outcome. Secondary outcomes were implantation and miscarriage rates. RESULT(S): The PR per transfer was higher in the NGS group (84.4% vs. 41.5%). The implantation rate (61.5% vs. 34.8%) was higher in the NGS group. The miscarriage rate was similar in the 2 groups (2.8% vs. 4.6%). CONCLUSION(S): We demonstrate the technical feasibility of NGS-based PGD involving cleavage-stage biopsy and fresh ETs. Encouraging data were obtained from a prospective trial using this approach, arguing that cleavage-stage NGS may represent a valuable addition to current aneuploidy screening methods. These findings require further validation in a well-designed randomized controlled trial. CLINICAL TRIAL REGISTRATION NUMBER: ACTRN12614001035617.

Healthy Baby Born to a Robertsonian Translocation Carrier following Next-Generation Sequencing-Based Preimplantation Genetic Diagnosis: A Case Report.

Preimplantation genetic diagnosis (PGD) is well established method for treatment of genetic problems associated with infertility. Moreover, PGD with next-generation sequencing (NGS) provide new possibilities for diagnosis and new parameters for evaluation in, for example, aneuploidy screening. The aim of the study was to report the successful pregnancy outcome following PGD with NGS as the method for 24 chromosome aneuploidy screening in the case of Robertsonian translocation. Day 3 embryos screening for chromosomal aneuploidy was performed in two consecutive in vitro fertilization (IVF) cycles, first with fluorescent in situ hybridization (FISH), and then with NGS-based protocol. In each IVF attempt, three embryos were biopsied. Short duration of procedures enabled fresh embryo transfer without the need for vitrification. First IVF cycle with the embryo selected using PGD analysis with the FISH method ended with pregnancy loss in week 8. The second attempt with NGS-based aneuploidy screening led to exclusion of the following two embryos: one embryo with 22 monosomy and one with multiple aneuploidies. The transfer of the only euploid blastocyst resulted in the successful pregnancy outcome. The identification of the euploid embryo based on the NGS application was the first successful clinical application of NGS-based PGD in the case of the Robertsonian translocation carrier couple.

Prevalence of the most frequent BRCA1 mutations in Polish population.

The purpose of our study was to establish the frequency and distribution of the four most common BRCA1 mutations in Polish general population and in a series of breast cancer patients. Analysis of the population frequency of 5382insC (c.5266dupC), 300T >G (p.181T >G), 185delAG (c.68_69delAG) and 3819del5 (c.3700_3704del5) mutations of the BRCA1 gene were performed on a group of respectively 16,849, 13,462, 12,485 and 3923 anonymous samples collected at birth in seven Polish provinces. The patient group consisted of 1845 consecutive female breast cancer cases. The most frequent BRCA1 mutation in the general population was 5382insC found in 29 out of 16,849 samples (0.17%). 300T >G and 3819del5 mutations were found in respectively 11 of 13,462 (0.08%) and four of 3923 (0.1%) samples. The population prevalence for combined Polish founder 5382insC and 300T >G mutations was 0.25% (1/400). The frequencies of 5382insC and 300T >G carriers among consecutive breast cancer cases were, respectively, 1.9% (35/1845) and 1.2% (18/1486). Comparing these data with the population frequency, we calculated the relative risk of breast cancer for 5382insC mutation at OR = 17 and for 300T >G mutation at OR = 26. Our results, based on large population studies, show high frequencies of founder 5382insC and 300T >G BRCA1 mutations in Polish general population. Carriage of one of these mutations is connected with a very high relative risk of breast cancer.