The effectiveness of flow cytometric sorting of human sperm (MicroSort®) for influencing a child's sex.

BACKGROUND: Flow cytometric sorting can be used to separate sperm based on sex chromosome content. Differential fluorescence emitted by stained X- vs. Y-chromosome-bearing sperm enables sorting and collection of samples enriched in either X- or Y-bearing sperm for use to influence the likelihood that the offspring will be a particular sex. Herein we report the effectiveness of flow cytometric sorting of human sperm and its use in human ART procedures. METHODS: This prospective, observational cohort study of the series of subjects treated with flow cytometrically sorted human sperm was conducted at investigational sites at two private reproductive centers. After meeting inclusion criteria, married couples (n = 4993) enrolled to reduce the likelihood of sex-linked or sex-limited disease in future children (n = 383) or to balance the sex ratio of their children (n = 4610). Fresh or frozen-thawed semen was processed and recovered sperm were stained with Hoechst 33342 and sorted by flow cytometry (n = 7718) to increase the percentage of X-bearing sperm (n = 5635) or Y-bearing sperm (n = 2083) in the sorted specimen. Sorted sperm were used for IUI (n = 4448) and IVF/ICSI (n = 2957). Measures of effectiveness were the percentage of X- and Y-bearing sperm in sorted samples, determined by fluorescence in situ hybridization, sex of babies born, IVF/ICSI fertilization- and cleavage rates, and IUI, IVF/ICSI, FET pregnancy rates and miscarriage rates. RESULTS: Sorted specimens averaged 87.7 ± 5.0% X-bearing sperm after sorting for X and 74.3 ± 7.0% Y-bearing sperm after sorting for Y. Seventy-three percent of sorts were for girls. For babies born, 93.5% were females and 85.3% were males after sorting for X- and Y-bearing sperm, respectively. IUI, IVF/ICSI, and FET clinical pregnancy rates were 14.7%, 30.8%, and 32.1%, respectively; clinical miscarriage rates were 15.5%, 10.2%, and 12.7%. CONCLUSIONS: Flow cytometric sorting of human sperm shifted the X:Y sperm ratio. IUI, IVF/ICSI and FET outcomes were consistent with unimpaired sperm function. Results provide evidence supporting the effectiveness of flow cytometric sorting of human sperm for use as a preconception method of influencing a baby's sex. TRIAL REGISTRATION: NCT00865735 (ClinicalTrials.gov).

Preimplantation Genetic Diagnosis: Prenatal Testing for Embryos Finally Achieving Its Potential.

Preimplantation genetic diagnosis was developed nearly a quarter-century ago as an alternative form of prenatal diagnosis that is carried out on embryos. Initially offered for diagnosis in couples at-risk for single gene genetic disorders, such as cystic fibrosis, spinal muscular atrophy and Huntington disease, preimplantation genetic diagnosis (PGD) has most frequently been employed in assisted reproduction for detection of chromosome aneuploidy from advancing maternal age or structural chromosome rearrangements. Major improvements have been seen in PGD analysis with movement away from older, less effective technologies, such as fluorescence in situ hybridization (FISH), to newer molecular tools, such as DNA microarrays and next generation sequencing. Improved results have also started to be seen with decreasing use of Day 3 blastomere biopsy in favor of polar body or Day 5 trophectoderm biopsy. Discussions regarding the scientific, ethical, legal and social issues surrounding the use of sequence data from embryo biopsy have begun and must continue to avoid concern regarding eugenic or inappropriate use of this technology.

The accuracy of chromosomal microarray testing for identification of embryonic mosaicism in human blastocysts.

BACKGROUND: Most previous studies of chromosomal mosaicism in IVF embryos were performed by fluorescence in situ hybridization (FISH) methods. While there are reports implicating chromosome aneuploidy in implantation failure following transfer and pregnancy loss by spontaneous miscarriage, the significance of mosaicism for the developmental potential of growing embryos is unknown. However, the low prevalence of chromosomal mosaicism in chorionic villus sampling and amniotic fluid specimens suggests the presence of selection against mosaic embryos for implantation and early pregnancy. The absence of evidence for selective allocation of abnormal cells to the trophectoderm (TE) of mosaic blastocysts permits these cells to be a good proxy for embryonic mosaicism detection by chromosomal microarrays (CMA). The purpose of this study was to establish the limits of detection and the prevalence of chromosome mosaicism in day 5/6 human embryos using CMA with TE biopsies. RESULTS: From reconstitution experiments we established log2 ratio thresholds for mosaicism detection. These studies indicated that chromosomal mosaicism at levels as low as between 25-37% can be consistently identified. Follow-up studies by FISH on non-transferred abnormal embryos confirmed the diagnostic accuracy of CMA testing. The number of cells in a TE biopsy can influence mosaicism detection. CONCLUSIONS: Chromosomal microarrays can detect mosaicism in TE biopsies when present at levels as low as between 25-37% and the prevalence of day 5/6 blastocysts which were mosaic and had no other abnormalities reached 15% among a cohort of 551 embryos examined. Validated protocols for establishing detection thresholds for mosaicism are important to reduce the likelihood of transferring abnormal embryos.

Non-disclosing preimplantation genetic diagnosis for Huntington disease.

OBJECTIVES: Individuals at risk for Huntington disease face difficult decisions regarding their reproductive options. Most do not wish to pass on the gene for Huntington disease to their children, but may not be prepared themselves to undergo presymptomatic testing and learn their genetic status. For these reasons, many at-risk individuals with a family history of HD would choose a method of genetic diagnosis that would assure them that they can have children unaffected with HD without revealing their own genetic status (non-disclosing). We have shown that, with a carefully designed and executed programme of non-disclosing preimplantation genetic testing, one can successfully assist at-risk couples to have their own biological children who are free from Huntington disease, without forcing parents to confront knowledge of their own genetic status. METHODS: Couples where one partner was at 50% risk for Huntington disease underwent in vitro fertilization with preimplantation embryo biopsy and molecular analysis for Huntington disease where appropriate. RESULTS: After extensive counselling and informed consent, 10 couples underwent 13 in vitro fertilization and two frozen embryo transfer cycles in a programme for non-disclosing preimplantation genetic diagnosis for Huntington disease. In 11 cycles, embryos determined to be free of Huntington disease were transferred, resulting in five clinical pregnancies. One set of twins and three singleton pregnancies have delivered. One pregnancy resulted in a first-trimester loss. CONCLUSIONS: The option of non-disclosing preimplantation genetic diagnosis should be reviewed, along with other relevant medical options, when counselling at-risk Huntington disease families.

Principios e aplicaçoes da reaçao da cadeia de polimerase / Principles and applications of the polymerase chain reaction

A invençao da tecnica da reaçao da cadeia de polimerase(PCR) para a amplificaçao de acido nucleico tem tido um impacto maior sobre diversas areas tanto da pesquisa basica como da clinica. Desde seu inicio em 1985, comunicaçoes sobre uma grande variedade de aplicaçoes da PCR foram recebidas com grande atençao na literatura cientifica e medica. Esta tecnologia tem mostrado grande aplicabilidade ao diagnostico de doenças humanas, incluindo areas tao diversas como doenças infecciosas, desordens geneticas e cancer. O presente artigo apresenta uma ampla revisao dos principios da PCR incluindo consideraçoes geneticas que devem ser observadas no uso ou no planejamento de ensaios baseados na PCR. A aplicaçao destes ensaios com base na PCR para o diagnostico de desordens geneticas e para triagem de doenças infecciosas sao tambem discutidas

Principios y aplicaciones de la reacción en cadena de la polimerasa / Principle and application of the chain reaction of the polymerase

El descubrimiento de la reacción en cadena de la polimerasa (PCR), técnica para la amplificación de ácidos nucleicos, ha tenido un enorme impacto sobre áreas diversas, tanto de la investigación básica como de la investigación clínica. Desde su descubrimiento en 1985, los informes sobre una gran variedad de aplicaciones de la PCR han recibido mucha atención en la literatura médica y científica. Esta tecnología ha demostrado tener una gran aplicabilidad para el diagnóstico de enfermedades humanas, incluyendo áreas tan diversas como enfermedades infecciosas, desórdenes genéticos y cáncer. Este artículo presenta una amplia revisión de los principios de la PCR, que incluyen conceptos genéricos que deben manejarse cuando se use o se diseñe un ensayo basado en la PCR. También se discute la aplicación de tales ensayos para el diagnóstico de desórdenes genéticos y para el estudio de enfermedades infecciosas. En los últimos 10 años ha aumentado, de manera notable, la aplicación de herramientas de la biología molecular para el diagnóstico de las enfermedades humanas. Los ensayos con sondas de ADN están, ahora, disponibles comercialmente para la detección e identificación de una gran variedad de patógenos humanos, así como para el diagnóstico de desórdenes genéticos humanos. Una manifestación del rápido crecimiento de la tecnología del ADN, ha sido el desarrollo de técnicas para amplificar secuencias específicas de ácidos nucleicos. En la literatura, han sido descritos muchos métodos, que se enumeran en la tabla i. Una comparación de estos métodos ha sido el tema de una revisión frecuente. Entre ellos se destacan la reacción en cadena de la polimerasa, como la de mayor impacto, tanto como herramienta de investigación como de diagnóstico. El objetivo de este artículo es presentar una breve revisión de los principios y aplicaciones de la amplificación de PCR, que incluyen una discusión de la selección correcta de las secuencias en blanco, el diseño de los "primer" y los medios necesarios para llevar a cabo la técnica