Before the beginning: the genetic risk of a couple aiming to conceive.

Disorders of genetic etiology exist in 2%-3% of live-born infants. Identifying couples with increased susceptibility for offspring with anomalies or genetic disorders is increasingly effective as a result of molecular advances. Preimplantation genetic testing (PGT) with the use of trophectoderm biopsy, 24-chromosome testing, and molecular testing have allowed wider applicability for avoiding a clinical pregnancy termination. Cell-free DNA in maternal blood is another targeted option, although invasive prenatal genetic diagnosis provides the greatest amount of genetic information. DNA-based methods to detect subtle chromosomal abnormalities are much more sensitive than traditional karyotypes and do not require cultured cells. Aneuploidy and structural chromosomal abnormalities can be readily detected with the use of small amounts of DNA, if necessary amplified, as in PGT. Novel approaches exist for detecting perturbations in single-gene disorders. Not only has the molecular basis for many monogenic disorders been elucidated, but modest costs for DNA sequencing has made testing feasible. As the number of testable genetic disorders has increased, principles underlying screening have advanced. Genetic screening for disorders of high incidence in certain ethnic groups was initiated decades ago; however, limitations exist, and reduction in live-born incidence is not infrequently small. Expanded carrier screening is now offered in panethnic fashion, extending surveillance to couples of mixed ethnicities and involving many more genetic conditions. Targeted gene panels (e.g., adult-onset cancer genes) further increase the number of genetic disorders amenable to screening, often leading to PGT.

Overview of Preimplantation Genetic Diagnosis (PGD): Historical Perspective and Future Direction.

Preimplantation genetic diagnosis (PGD) can be considered the earliest form of prenatal testing. It was first used in humans over 26 years ago. At its inception, PGD could only be performed for a limited number of genetic disorders. Technological advances in molecular biology and cytogenomics have been utilized in the field of PGD to greatly expand the spectrum of genetic disorders that can now be detected in early human embryos.

Preimplantation diagnosis and other modern methods for prenatal diagnosis.

Prenatal treatment of congenital adrenal hyperplasia (CAH) has long involved prenatal treatment with dexamethasone, administered to the pregnant woman to prevent genital masculinization of an affected female fetus. Although it is unnecessary to treat unaffected or affected males because their genital development would not be disturbed, there has only been incremental progress in determining fetal gender sufficiently each to avoid treating males and unaffected females. Invasive procedures were initially necessary, with first-trimester amniocentesis at 15-20 weeks and then chorionic villus sampling (CVS) at 10-12 weeks gestation. Two approaches now allow personalized treatment of affected female fetuses prior to female genital differentiation. Only preimplantation genetic diagnosis (PGD) is available prior to clinical pregnancy. Recent technological advances have further allowed both single gene diagnosis (e.g., CAH) and aneuploidy detection concomitantly, resulting in far better pregnancy rates than heretofore possible in assisted reproduction technology.

CSB-PGBD3 Mutations Cause Premature Ovarian Failure.

Premature ovarian failure (POF) is a rare, heterogeneous disorder characterized by cessation of menstruation occurring before the age of 40 years. Genetic etiology is responsible for perhaps 25% of cases, but most cases are sporadic and unexplained. In this study, through whole exome sequencing in a non-consanguineous family having four affected members with POF and Sanger sequencing in 432 sporadic cases, we identified three novel mutations in the fusion gene CSB-PGBD3. Subsequently functional studies suggest that mutated CSB-PGBD3 fusion protein was impaired in response to DNA damage, as indicated by delayed or absent recruitment to damaged sites. Our data provide the first evidence that mutations in the CSB-PGBD3 fusion protein can cause human disease, even in the presence of functional CSB, thus potentially explaining conservation of the fusion protein for 43 My since marmoset. The localization of the CSB-PGBD3 fusion protein to UVA-induced nuclear DNA repair foci further suggests that the CSB-PGBD3 fusion protein, like many other proteins that can cause POF, modulates or participates in DNA repair.

Ethnic specificity of variants of the ESR1, HK3, BRSK1 genes and the 8q22.3 locus: no association with premature ovarian failure (POF) in Serbian women.

OBJECTIVE: To identify whether variants found in a large Han Chinese cohort - 8q22.3 SNPs rs3847153 and rs3108910; and one SNP each in HK3 (rs2278493), ESR1 (rs2234693) and BRSK1 (rs12611091) - are associated with premature ovarian failure (POF) in a different ethnic group (Serbian). DESIGN: Case-control genetic association study in 197 Serbian POF cases and 552 matched controls. RESULTS: None of the SNPs found associated with POF in Chinese cohort were found to be associated in the Serbian sample. CONCLUSIONS: In contrast to Han Chinese, no association was found between POF in Serbian women and any of the four tested loci: 8q22.3, HK3, ESR1 and BRSK1. This indicates that ethnically distinct populations may show differences in gene-regulating pathways and genes causing POF.

Preventing preterm births: analysis of trends and potential reductions with interventions in 39 countries with very high human development index.

BACKGROUND: Every year, 1·1 million babies die from prematurity, and many survivors are disabled. Worldwide, 15 million babies are born preterm (<37 weeks' gestation), with two decades of increasing rates in almost all countries with reliable data. The understanding of drivers and potential benefit of preventive interventions for preterm births is poor. We examined trends and estimate the potential reduction in preterm births for countries with very high human development index (VHHDI) if present evidence-based interventions were widely implemented. This analysis is to inform a rate reduction target for Born Too Soon. METHODS: Countries were assessed for inclusion based on availability and quality of preterm prevalence data (2000-10), and trend analyses with projections undertaken. We analysed drivers of rate increases in the USA, 1989-2004. For 39 countries with VHHDI with more than 10,000 births, we did country-by-country analyses based on target population, incremental coverage increase, and intervention efficacy. We estimated cost savings on the basis of reported costs for preterm care in the USA adjusted using World Bank purchasing power parity. FINDINGS: From 2010, even if all countries with VHHDI achieved annual preterm birth rate reductions of the best performers for 1990-2010 (Estonia and Croatia), 2000-10 (Sweden and Netherlands), or 2005-10 (Lithuania, Estonia), rates would experience a relative reduction of less than 5% by 2015 on average across the 39 countries. Our analysis of preterm birth rise 1989-2004 in USA suggests half the change is unexplained, but important drivers include non-medically indicated labour induction and caesarean delivery and assisted reproductive technologies. For all 39 countries with VHHDI, five interventions modelling at high coverage predicted a 5% relative reduction of preterm birth rate from 9·59% to 9·07% of livebirths: smoking cessation (0·01 rate reduction), decreasing multiple embryo transfers during assisted reproductive technologies (0·06), cervical cerclage (0·15), progesterone supplementation (0·01), and reduction of non-medically indicated labour induction or caesarean delivery (0·29). These findings translate to roughly 58,000 preterm births averted and total annual economic cost savings of about US$3 billion. INTERPRETATION: We recommend a conservative target of a relative reduction in preterm birth rates of 5% by 2015. Our findings highlight the urgent need for research into underlying mechanisms of preterm births, and development of innovative interventions. Furthermore, the highest preterm birth rates occur in low-income settings where the causes of prematurity might differ and have simpler solutions such as birth spacing and treatment of infections in pregnancy than in high-income countries. Urgent focus on these settings is also crucial to reduce preterm births worldwide. FUNDING: March of Dimes, USA, Eunice Kennedy Shriver National Institute of Child Health and Human Development, and National Institutes of Health, USA.

Circulating trophoblastic cells provide genetic diagnosis in 63 fetuses at risk for cystic fibrosis or spinal muscular atrophy.

This study sought to determine whether a reliable non-invasive prenatal diagnosis (NI-PND) of cystic fibrosis (CF) or spinal muscular atrophy (SMA) can be achieved through analysis of circulating fetal trophoblastic cells (CFTC). The kinetics of CFTC circulation were also studied. CFTC were isolated by isolation by size of epithelial tumour/trophoblastic cells at 9-11 weeks of gestation, before chorionic villus sampling (CVS), from the blood of 63 pregnant women at 25% risk for having a child affected by either CF (n=32) or SMA (n=31). Collected cells were laser-microdissected, short tandem repeat-genotyped to determine fetal origin and blindly assessed for mutation analysis. CFTC were independently analysed weekly (4-12 weeks of gestation) in 14 women who achieved pregnancy following IVF. Diagnostic results were compared with those obtained by CVS. All seven CF and seven SMA pregnancies carrying an affected fetus were correctly identified as well as non-affected pregnancies. CFTC provided 100% diagnostic sensitivity (95% CI 76.8-100%) and specificity (95% CI 92.7-100%) in these 63 consecutive pregnancies at risk for CF or SMA. CFTC were found to circulate from 5 weeks of gestation and can be used to develop an early and reliable approach for NI-PND. We sought to determine whether a reliable non-invasive prenatal diagnosis (NI-PND) of two rare genetic diseases - cystic fibrosis (CF) and spinal muscular atrophy (SMA) - can be achieved through analysis of circulating fetal trophoblastic cells (CFTC) in blood of pregnant women. We also studied the time of appearance and circulation of CFTC in maternal blood. CFTC were isolated from maternal blood by isolation by size of epithelial tumour/trophoblastic cells (ISET; an approach for cell isolation from blood) at 9-11 weeks of gestation before chorionic villus sampling (CVS) from the blood of 63 pregnant women at 25% risk for having a child affected by either CF (n=32) or SMA (n=31). Collected cells were analysed by genetic test to determine fetal origin and blindly assessed for mutation analysis. We independently analysed CFTC in maternal blood samples taken weekly (4-12 weeks of gestation) from 14 women who achieved pregnancy following IVF. Diagnostic results were compared with those obtained by CVS. All seven CF and seven SMA pregnancies carrying an affected fetus were correctly identified as well as non-affected pregnancies. CFTC provided 100% diagnostic sensitivity and specificity in these 63 consecutive pregnancies at risk for CF or SMA. CFTC were found to circulate from 5 weeks of gestation and can be used to develop an early and reliable approach for NI-PND.

Invasive procedures for prenatal diagnosis: any future left?

Invasive diagnostic procedures (e.g chorionic villus sampling and amniocentesis) remain essential to the complete prenatal genetic diagnosis armamentarium. Both procedures are relatively safe in experienced hands, carrying procedure-related losses of about 1 in 400. Sensitivity of aneuploidy detection with either invasive test is near 100%, 10-15% higher than non-invasive protocols that use maternal serum analyte and fetal nuchal translucency screening. Application of cell-free fetal DNA for aneuploidy screening may or may not narrow this difference. Irrespective, invasive procedures are currently required for application of array comparative genome hybridisation.

Micronutrients and women of reproductive potential: required dietary intake and consequences of dietary deficiency or excess. Part II--vitamin D, vitamin A, iron, zinc, iodine, essential fatty acids.

Part II of this review considers additional micronutrients. Vitamin D is a fat soluble vitamin found in foods of animal origins (fatty fish, liver oil) or fortified products (milk, cheese). Vitamin D deficiency is common in African-American women living in northern latitudes. Vitamin D supplementation may be needed to reach desired 25-(OH)D3 concentrations of >50 nmol/L. In foods of animal origin, preformed Vitamin A is present; in plants (fruits and vegetables) vitamin A precursors (ß-carotenoids) are present. Vitamin A supplementation is usually not warranted, and in developing countries should not exceed 3000 µg (10,000 IU)/day. Iron in the form of haem-iron is found in meat, fish and poultry; non-haem (inorganic) iron is found in vegetables, fruits and grains. Iron supplementation may be necessary in the third trimester, earlier in pregnancy or in non-pregnant states if serum ferritin is <20 µg/L or haemoglobin <10.9 g/dL. Zinc is available in red meat, seafood including oysters and unpolished grains; supplementation is not necessary. To assure adequate iodine, food is fortified worldwide with iodated salt. If urinary iodine levels are low, supplementation is needed. Essential fatty acids requirements can be met by one to two portions of fish per week.

Preimplantation genetic diagnosis at 20 years.

First reported in 1990, PGD has evolved into a complementary form of prenatal diagnosis offering novel indications. DNA for PGD can be recovered with equal safety and facility from polar bodies I and II, blastomere (8 cell embryo) and trophectoderm (5-6 day blastocyst). Diagnostic accuracy is very high (>99%) for both chromosomal abnormalities and single gene disorders. Traditional application of FISH with chromosome specific probes for detecting aneuploidy and translocations may be replaced or complemented by array comparative genome hybridization (array CGH); biopsied embryos can now be cryopreserved (vitrification) while analysis proceeds in orderly fashion. PGD has been accomplished for over 200 different single gene disorders. Novel indications for PGD not readily applicable by traditional prenatal genetic diagnosis include avoiding clinical pregnancy termination, performing preconceptional diagnosis (polar body I), obtaining prenatal diagnosis without disclosure of prenatal genotype (nondisclosure), diagnosing adult-onset disorders particularly cancer, and identifying HLA compatible embryos suitable for recovering umbilical cord blood stem cells.

What next for preimplantation genetic screening? Randomized clinical trial in assessing PGS: necessary but not sufficient.

The randomized clinical trial (RCT) is a powerful experimental design that when properly executed produces generalizable results. Conducting a RCT becomes complex when technical skills are required. Without requisite skills, a RCT may yield misleading results, an elegant RCT unwittingly generating spurious results due to technical inexperience. This pitfall is applicable to procedures used to evaluate assisted reproductive technologies. RCTs assessing the value of preimplantation genetic screening, also called preimplantation genetic diagnosis for aneuploidy testing--require three general prerequisites--proper study design, skilled operators (embryo biopsy), and skilled laboratory cytogeneticists (diagnosis). Lacking either of the latter two, even an elegantly designed RCT is not necessarily valid.

Causes of fetal wastage.

Fetal wastage has many causes, but genetic factors are by far the most common. The earlier the pregnancy loss occurs, the greater the likelihood of genetic causation. Among first trimester abortions, 50% to 80% show chromosomal abnormalities, usually aneuploidy. This is greater than all other causes combined. Chromosomal numerical abnormalities can be recurrent and sporadic; failure to take this into account is a major pitfall in many reports addressing causation. Moreover, many causes of fetal wastage that are traditionally considered to be "nongenetic" are actually the result of perturbations of gene products-proteins. Among nongenetic causes of first trimester fetal wastage, the best established are thyroid abnormities; antifetal antibodies; and the inherited and acquired thrombophilias. The latter are more established in the second trimester. Uterine anomalies can lead to second trimester losses. Infections seem uncommon, and alloimmune causes are not validated.

Recovery and amplification of placental RNA from dried maternal blood spots: utility for non-invasive prenatal diagnosis.

Methods utilizing circulating cell-free RNA in plasma have clinical applications for cancer and prenatal genetic analysis. Given these potential roles, the feasibility of detecting placental specific RNA in dried maternal blood spots after storage at room temperature for varying lengths of time was investigated. Using real-time polymerase chain reaction (PCR), positive amplification of placental-specific beta-human chorionic gonadotrophin transcripts was demonstrated in nine of 11 dried blood samples from first and second trimester pregnancies stored at room temperature for up to 4 weeks. This work demonstrates feasibility in isolation and amplification of placental mRNA using dried maternal blood spots. With the development of fetal and placental RNA markers, this approach would allow simplified collection, transport, and storage of samples for prenatal genetic diagnosis and pregnancy related complications.

Endocervical fetal trophoblast for prenatal genetic diagnosis.

PURPOSE OF REVIEW: For over a decade, methods of first-trimester, noninvasive prenatal genetic diagnosis have been actively pursued by many investigators. Isolation of fetal trophoblast from endocervical specimens remains an attractive approach, given the greater numbers of fetal cells than in maternal blood and the better potential for fetal-cell identification based on markers specific for a single cell type (trophoblasts). RECENT FINDINGS: Current studies demonstrate feasibility in identification and molecular analysis of fetal trophoblast cells for prenatal genetic testing. Sampling methods involving lavage, cytobrush, or aspiration of cervical specimens, however, have limitations in the recovery of trophoblasts. SUMMARY: Clinical applications await further systematic studies to determine safety and accuracy in recovery.

Quantitative DNA perturbations of p53 in endometriosis: analysis of American and Icelandic cases.

OBJECTIVE: To investigate quantitative aberrations involving p53 copy numbers in eutopic endometrial and endometriotic tissue from two populations. DESIGN: Comparative analysis of normal and diseased tissue. SETTING: Tissue specimens collected in Iceland and USA. PATIENT(S): Subjects with moderate/severe endometriosis (Iceland, n = 26; USA, n = 45). Paraffin-embedded tissue from 19 matched Icelandic cases and seven unaffected controls. American cases were fresh surgical tissue from 17 matched cases and 28 unaffected controls. DNA isolation and real-time polymerase chain reaction (PCR) with TaqMan assay were performed. MAIN OUTCOME MEASURE(S): The frequency of p53 loss and/or gain based on quantitative differences for copy numbers of p53 located on chromosome (17p) and GAPDH on a control locus (chromosome 12p). RESULT(S): Among American cases, significant p53 gain (n = 13) or loss (n = 4) was observed in 17 of 21 cases. In Icelandic cases this was not seen to the same degree. Mean normalized p53 values were 3.46 and 1.16 copies per reaction, respectively. Significant differences were observed between normalized p53 in the control blood and affected tissue for the American and Icelandic cases compared to standard GAPDH control but not in normal Icelandic and American endometrium. CONCLUSION(S): The results continue to support a role for nonrandom somatic p53 locus alterations in the pathogenesis of late or severe-stage endometriosis. Differences between Icelandic and American subjects have implications for generalization of genome-wide approaches.