New protocol based on massive parallel sequencing for aneuploidy screening of preimplantation human embryos.

Novel next-generation sequencing procedures have rapidly emerged into the preimplantation genetic screening framework. This work presents the design and validation of a new low-coverage whole-genome sequencing assay for aneuploidy detection in single blastomeres and trophectodermal samples from preimplantation embryos. The validation ensures analytical sensitivity, specificity, robustness, precision, limit of detection, resolution, and reproducibility. Specific parameters to measure the performance are defined, and the results are compared with a standardized array-based method to stablish the concordance. From the single cell genomics point of view, the main novelties are the length of reads of the libraries (150 nucleotides) together with a paired-end strategy and the design of an original algorithm and copy number viewer. A total of 129 samples were included in six experimental runs using a MiSeq Illumina platform. Samples included: single amniocytes, single blastomeres (cleavage-stage embryos), trophectoderm samples (blastocyst), and diluted DNA. Sensitivity and specificity were calculated per chromosome yielding 96% and 99%, respectively. The percentage of concordant samples was 98.2% and all of the aneuploid samples were confirmed. In conclusion, the validation yields highly reliable and reproducible results, representing an accurate and cost-effective strategy for the routine detection of aneuploidy in human embryos.

Tiempos de equidad de género: descripción de las desigualdades entre comunidades autónomas, España 2006-2014 / Times of gender equity: a description of the inequalities between autonomous communities, Spain 2006–2014

Objetivo: La equidad de género es un determinante estructural de las desigualdades en salud. Por ello, se pretende visibilizar su evolución en las comunidades autónomas (CC.AA.) desde 2006, previamente a la promulgación de la Ley de Igualdad (2007) y la crisis económica (2008), hasta 2014. Método: Estudio ecológico sobre la equidad de género en las 17 CC.AA. en 2006-2011-2014. Cálculo de: 1) índice de equidad de género modificado (IEGM) de las CC.AA. (0=equidad, ±1=inequidad); 2) convergencia interregional y temporal en equidad de género. Resultados: El IEGM de las CC.AA.2014 toma valores negativos próximos a 0 (inequidad desfavorable a las mujeres). No hay convergencia interregional en la equidad de género, pues aumenta la dispersión (2006: 0,1503; 2011: 0,2280; 2014: 0,4964). Tampoco existe convergencia temporal, al no evolucionar mejor las CC.AA. menos equitativas. La brecha de género en actividad económica sigue desfavorable a las mujeres. En 2006-2011 disminuye en todas las CC.AA., y en 2014 aumenta en seis CCAA. La brecha de género en educación tiene valores positivos próximos a 0 (desfavorable a los hombres) en 2006-2011-2014, y en empoderamiento es desfavorable a las mujeres, siendo la dimensión que más pesa en la equidad de género. Se mantiene la dispersión entre CC.AA. en 2006-2014 en actividad económica y educación, y aumenta en empoderamiento. Conclusiones: El contexto de equidad de género alcanzado en las CC.AA. españolas en 2006 se ha perdido durante la crisis económica, al aumentar la desigualdad en la equidad de género entre CC.AA. en 2014. La inequidad de género sigue siendo desfavorable a las mujeres (AU)

Objective: Gender equity (GE) is a structural determinant of health inequalities. In this light, our objective is to show the evolution of gender equity in the Spanish autonomous communities since 2006, prior to the enactment of the Equality Act (2007) and the economic crisis (2008), until 2014. Method: Ecological study of gender equity in the 17 Spanish autonomous communities from 2006-2011-2014. We have calculated: 1) modified gender equity index (MGEI) for the autonomous communities (0=equity, ±1=inequity); 2) interregional and temporal convergences in gender equity. Results: The MGEI in the autonomous communities in 2014 has negative values close to 0 (inequity towards women). There is no interregional convergence due to the dispersion increase (2006: 0.1503; 2011: 0.2280; 2014: 0.4964), and no temporal convergence due to the lack of progress of the autonomous communities with poor gender equity. The gender gap in economic activity continues to be unfavourable to women, decreasing in all communities between 2006 and 2011 but increasing in six communities in 2014. The gender gap in education from 2006-2011-2014 has positive values close to 0 (unfavourable to men), while the gender gap in empowerment is unfavourable to women, representing the most significant gender equity disparity. Inter-community dispersion of economic activity and education did not change between 2006 and 2014, while inter-community dispersion of empowerment increased. Conclusions: The level of gender equity achieved in the Spanish autonomous communities in 2006 was lost during the economic crisis, as gender equity disparities between the communities had increased by 2014. Gender inequity continues to be unfavourable to women (AU)

[Times of gender equity: a description of the inequalities between autonomous communities, Spain 2006-2014]. / Tiempos de equidad de género: descripción de las desigualdades entre comunidades autónomas, España 2006-2014.

OBJECTIVE: Gender equity (GE) is a structural determinant of health inequalities. In this light, our objective is to show the evolution of gender equity in the Spanish autonomous communities since 2006, prior to the enactment of the Equality Act (2007) and the economic crisis (2008), until 2014. METHOD: Ecological study of gender equity in the 17 Spanish autonomous communities from 2006-2011-2014. We have calculated: 1) modified gender equity index (MGEI) for the autonomous communities (0=equity, ±1=inequity); 2) interregional and temporal convergences in gender equity. RESULTS: The MGEI in the autonomous communities in 2014 has negative values close to 0 (inequity towards women). There is no interregional convergence due to the dispersion increase (2006: 0.1503; 2011: 0.2280; 2014: 0.4964), and no temporal convergence due to the lack of progress of the autonomous communities with poor gender equity. The gender gap in economic activity continues to be unfavourable to women, decreasing in all communities between 2006 and 2011 but increasing in six communities in 2014. The gender gap in education from 2006-2011-2014 has positive values close to 0 (unfavourable to men), while the gender gap in empowerment is unfavourable to women, representing the most significant gender equity disparity. Inter-community dispersion of economic activity and education did not change between 2006 and 2014, while inter-community dispersion of empowerment increased. CONCLUSIONS: The level of gender equity achieved in the Spanish autonomous communities in 2006 was lost during the economic crisis, as gender equity disparities between the communities had increased by 2014. Gender inequity continues to be unfavourable to women.

Correlation between aneuploidy, apoptotic markers and DNA fragmentation in spermatozoa from normozoospermic patients.

Genetic and biochemical sperm integrity is essential to ensure the reproductive competence. However, spermatogenesis involves physiological changes that could endanger sperm integrity. DNA protamination and apoptosis have been studied extensively. Furthermore, elevated rates of aneuploidy and DNA injury correlate with reproductive failures. Consequently, this study applied the conventional spermiogram method in combination with molecular tests to assess genetic integrity in ejaculate from normozoospermic patients with implantation failure by retrospectively analysing aneuploidy (chromosomes 18, X, Y), DNA fragmentation, externalization of phosphatidylserine and mitochondrial membrane potential status before and after magnetic activated cell sorting (MACS). Aneuploid, apoptotic and DNA-injured spermatozoa decreased significantly after MACS. A positive correlation was detected between reduction of aneuploidy and decreased DNA damage, but no correlation was determined with apoptotic markers. The interactions between apoptotic markers, DNA integrity and aneuploidy, and the effect of MACS on these parameters, remain unknown. In conclusion, use of MACS reduced aneuploidy, DNA fragmentation and apoptosis. A postulated mechanism relating aneuploidy and DNA injury is discussed; on the contrary, cell death markers could not be related. An 'apoptotic-like' route could explain this situation.

Case report: birth of healthy twins after preimplantation genetic diagnosis of propionic acidemia.

PURPOSE: Development of an ad hoc protocol for the preimplantion genetic diagnosis of propionic acidemia in a couple carrying the mutations c.737G>T (G246V) and c.1218del14ins12 (ins/del) in the PCCB gene. Propionic acidemia is an autosomal recessive metabolic disorder where the body is unable to process certain parts of proteins and lipids. Symptoms manifest few days after birth and sometimes progress to more serious medical problems, including heart abnormalities, coma and death. METHODS: Four short tandem repeat markers closely linked to the PCCB gene were tested, in order to support the direct mutation detection diagnosis. Multiplex fluorescent heminested polymerase chain reaction followed by fragment analysis and minisequencing was used. RESULTS: Fourteen single blastomeres from nine embryos were tested and two carrier embryos were transferred, resulting in the birth of two healthy boys. CONCLUSIONS: Preimplantation genetic diagnosis represents a valid reproductive option for couples affected of propionic acidemia, in order to avoid transmission to offspring.

Successful application of preimplantation genetic diagnosis for hypokalaemic periodic paralysis.

Hypokalaemic periodic paralysis is a rare dominant inherited disease where a person suffers sudden falls of circulating potassium concentrations, producing muscle weakness and sometimes severe paralysis. Attacks can occur as frequently as several times a day or once in a year. The age of onset is usually adolescence but symptoms can appear as early as 10 years of age. Muscle weakness can compromise vital functions such as breathing or swallowing and heart arrhythmias are also frequent during attacks. Preimplantation genetic diagnosis, an early form of prenatal diagnosis for couples at risk of transmitting inherited diseases, was used to prevent the transmission of this disease. Six polymorphic short tandem repeat or microsatellite markers (STR) closely linked to the CACNA1S gene were tested. Three fully informative markers were chosen to establish the disease-bearing haplotype in the family and to determine the genetic status of five embryos by multiplex fluorescent heminested PCR. Four of the five embryos tested were diagnosed as non-affected and one as affected. Two embryos were transferred resulting in a singleton pregnancy and the birth of a healthy girl.

Could the addition of hp-hMG and GnRH antagonists modulate the response in IVF-ICSI cycles?

OBJECTIVE: To assess if the luteinizing hormone/human chorionic gonadotropin present in some gonadotropin formulations may be of benefit in protocols with GnRH antagonists. METHODS: Open, quasi-experimental, multicenter, prospective, parallel-controlled study compared 136 women undergoing in vitro fertilization--intracytoplasmic sperm injection after stimulation with highly purified human menopausal gonadotropin (hp-hMG) (n = 44), recombinant-follicle stimulating hormone (r-FSH) (n = 46), or a combination of both (r FSH + hp-hMG) (n = 46) following an antagonist protocol. Blood determinations were made on day 6 of stimulation and on the day of ovulation induction, with centralized analysis. RESULTS: No differences were found in the ongoing pregnancy rates between groups [37.0% versus 29.5% (hp-hMG) and 23.9% (r-FSH); p = 0.688]. However, the ratio top-quality embryos/retrieved oocytes (TQE/RO) was higher in the combined therapy group (19.6%)--reaching significance versus the r-FSH group (6.5%) (p = 0.008), but not versus hp-hMG (12.3%) (p = 0.137). CONCLUSIONS: An improved TQE/RO ratio was obtained together with a greater percentage of frozen embryos in the patients that incorporated hp-hMG to their stimulation protocol. Despite good results of adding hp-hMG, non statistical differences were found in terms of ongoing pregnancy rate.

Association of genetic markers within the BMP15 gene with anovulation and infertility in women with polycystic ovary syndrome.

We analyzed two polymorphisms (-9C>G and IVS1+905A>G) within the BMP15 gene in women from Spain with polycystic ovary syndrome (PCOS). In this study, the BMP15 gene does not seem to be associated with PCOS. Nonetheless, we observed in both markers a genetic association with anovulation or infertility in these patients.