Integrating genomic alterations in diffuse large B-cell lymphoma identifies new relevant pathways and potential therapeutic targets.

Genome studies of diffuse large B-cell lymphoma (DLBCL) have revealed a large number of somatic mutations and structural alterations. However, the clinical significance of these alterations is still not well defined. In this study, we have integrated the analysis of targeted next-generation sequencing of 106 genes and genomic copy number alterations (CNA) in 150 DLBCL. The clinically significant findings were validated in an independent cohort of 111 patients. Germinal center B-cell and activated B-cell DLBCL had a differential profile of mutations, altered pathogenic pathways and CNA. Mutations in genes of the NOTCH pathway and tumor suppressor genes (TP53/CDKN2A), but not individual genes, conferred an unfavorable prognosis, confirmed in the independent validation cohort. A gene expression profiling analysis showed that tumors with NOTCH pathway mutations had a significant modulation of downstream target genes, emphasizing the relevance of this pathway in DLBCL. An in silico drug discovery analysis recognized 69 (46%) cases carrying at least one genomic alteration considered a potential target of drug response according to early clinical trials or preclinical assays in DLBCL or other lymphomas. In conclusion, this study identifies relevant pathways and mutated genes in DLBCL and recognizes potential targets for new intervention strategies.

Elastin mutation screening in a group of patients affected by vascular abnormalities.

Supravalvular aortic stenosis is an uncommon but well-characterized congenital form of left ventricular outflow obstruction. The lesion involves the ascending aorta and often occurs in association with pulmonary arterial stenoses or stenoses of other arteries, especially at major branch points. It can occur sporadically, as an autosomal dominant condition, or as one component of Williams-Beuren syndrome. In fact, the clinical and structural characteristics of supravalvular aortic stenosis are identical in both syndromic and nonsyndromic cases. The severity of supravalvular aortic stenosis varies; but if it is left untreated, it may result in heart failure, myocardial infarction, and sudden death. Supravalvular aortic stenosis in Williams-Beuren patients occurs as a consequence of a complete deletion of one copy of the elastin gene on chromosome 7q11.23. However, the underlying genetic cause of isolated supravalvular aortic stenosis has been identified as translations, gross intragenic deletions, and point mutations that disrupt the elastin gene. We report the results obtained in a mutation screening of the elastin gene in 28 patients with supravalvular aortic stenosis and other vascular abnormalities. The aim of the screening was to characterize the molecular cause of this lesion. We have detected 11 changes, including nine polymorphisms and two novel putative missense mutations.

Using videoclips to improve theoretical anatomy teaching

The introduction of multimedia technology intoteaching has brought important changes in universityteaching. This study seeks to evaluatewhether the use of videoclips as an aid in theoreticallessons, improves students’ performance.This study compares the results obtained inthe scores of Locomotive System Anatomy fortwo consecutive groups of students that took theFirst Course of Descriptive Anatomy in thedegree in Biology at the Faculty of Health andLife Sciences at the “Universitat Pompeu Fabra”of Barcelona.In the first group (G1 n=72) theoretical teachingwas performed through conventionallectures supported with Power Point slides. Inthe second group (G2 n=70), during the sameperiod of time, teaching was done by a combinationof theoretical explanations, slides andmultimedia anatomy videos, which were usedin order to reinforce the key issues of all lectures.The evaluation of theoretical knowledgewas achieved through a multiple-choice test of30 questions (70% of final mark), completingwith a test of 15 short questions (30% of thefinal mark). Evaluation was performed doneselectively based on the same items in 2 examinationsusing different questions. Comparison of the results revealed that students receiving video input performed significantly better (G1:76 % vs. G2: 93 %). Results ofstudents opinion performed between two groups find out to be similar in each group (G1:5.7 vs. G2: 5.9). The adequacy of the teaching material was (G1: 7.9 vs. G2: 7.5) and general satisfaction with the teaching methods was (G1: 6.8 vs. G2: 6.8). In conclusion, it was found that using videoclips for teaching Human Anatomy significantly improves students’ comprehension of theoretical contents

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Chromosomal high-polysomies predict tumour progression in T1 transitional cell carcinoma of the bladder.

UNLABELLED: The main prognostic factor generally accepted for tumour progression in T1 transitional cell carcinoma (TCC) of the bladder is histological grade. Despite this fact it is considered inaccurate to make clinical decisions on individuals. It appears that progression from minimally invasive to deeply invasive cancer is concurrent with the acquisition of genomic alterations that increase the malignant potential of cancer cells. The aim of this study is to determine if changes in chromosomes 7, 8, 9 and 17 copy number can be used to predict recurrence and progression in patients with T1 TCC of the urinary bladder. METHODS: Thirty-one T1 TCC samples were analyzed for chromosomal alterations by fluorescence in situ hybridization using centromeric probes for chromosomes 7, 8, 9 and 17. Clinical data were collected from the patients' clinical records and correlated with chromosomal studies. RESULTS: Histological grade was confirmed as a prognostic factor of tumour progression (p=0.01). None of the cytogenetic alterations demonstrated in the studied group could be related to tumour recurrence. The high-polysomies (five or more copies) of chromosomes 8, 9 and 17 showed predictive value (p=0.05, 0.05, 0.03 respectively) for tumour progression since it was observed that patients with high-polysomy of these chromosomes showed more risk of tumour progression towards muscle-invasive disease than those without high-polysomy alteration. CONCLUSION: Our findings suggest a possible prognostic significance of highly aneuploid cells (high-polysomies of chromosomes 8, 9 and 17) in tumour progression of T1 TCC bladder tumours. FISH analysis is a reproducible technique for evaluating cytogenetic alterations and could contribute to the assessment of the individual prognosis of T1 transitional cell carcinoma of the bladder.

Cryptic chromosomal rearrangement screening in 30 patients with mental retardation and dysmorphic features.

Mental retardation affects 1-3% of the general population, and the genetic causes in many cases are unknown. Cytogenetically undetected chromosomal imbalances have been indicated as an explanation. Nowadays, due to the development of molecular cytogenetic techniques, it is possible to identify cryptic rearrangements involving the ends of chromosomes. We report a screening using chromosome-specific telomere fluorescence in-situ hybridization (FISH) probes, in a group of 30 patients with a well-characterized phenotype including mental retardation, dysmorphic features, and a normal karyotype. Among them, two subtelomeric rearrangements have been detected and characterized. One of them is a de novo deletion of 1p36, which has been previously described as a new contiguous gene syndrome. The second is an unbalanced product of a cryptic translocation involving chromosomes 1 and 13, which results in a partial 1q trisomy and partial 13q monosomy. These findings highlight, the importance of searching for cryptic subtelomeric rearrangements in non-syndromic mentally retarded patients.

Smoldering multiple myeloma: natural history and recognition of an evolving type.

Patients with smoldering multiple myeloma (SMM) meet the diagnostic criteria of multiple myeloma (MM) but are asymptomatic. Between January 1978 and July 2001, 53 patients (median age 63 years) were diagnosed with SMM. The median serum M-protein and proportion of bone marrow plasma cells were 36 g/l and 27% respectively. Two subsets of SMM were identified: (i) evolving SMM (n = 22), characterized by a progressive increase in serum M-protein, a previously recognized monoclonal gammopathy of undetermined significance (MGUS) and a significant higher proportion of IgA type and (ii) non-evolving SMM (n = 26) with stable M-protein that abruptly increases when symptomatic MM develops. Thirty-four patients developed symptomatic MM. The median time to progression in the overall series was 3.2 years and the only feature associated with a shorter time to progression was the evolving versus non-evolving type (1.3 vs. 3.9 years respectively, P = 0.007). The pattern of progression consisted of anaemia, lytic bone lesions or both, without renal failure, hypercalcaemia or extramedullary plasmacytomas. Fifty-seven per cent of patients that required chemotherapy showed no or minimal response. The median survival from diagnosis and from progression was 8.2 and 3.5 years respectively.

Large de novo deletion in chromosome 12 affecting the PAH, IGF1, ASCL1, and TRA1 genes.

Phenylketonuria is one of the most common genetic diseases in humans, affecting 1 in 10,000 whites. Deletions are generally uncommon in genes in which no long highly homologous segments are present, and in phenylalanine hydroxylase (PAH) deficiency they represent only 5% of cases. We present the case of a girl affected by classical phenylketonuria who has been screened for mutations in the PAH gene. During the molecular study a large de novo deletion has detected in 12qter, including PAH, and the genes for insulin-like growth factor 1 (IGF1), human achaete-scute homolog 1 (ASCL1), and tumor rejection antigen (TRA1). The patient showed phenylketonuria, short stature, and pathological electro-oculography results in both eyes, with high affectation of the relative electrogenesis of the photoreceptor-pigment epithelium complex. She had previously been misdiagnosed as homozygous for the IVS8nt-7A-G mutation, instead of heterozygous for a mutation and a de novo deletion. As a result incorrect genetic counseling had been given. The deletion of the PAH, IGF1, and ASCL1 genes could explain the patient's phenotype corresponding to a contiguous gene syndrome. We stress the relevance of polymorphic marker haplotype analysis and the importance of family study in genetic recessive diseases, such as phenylketonuria, to avoid incorrect diagnosis and genetic counseling.

High-grade prostate intraepithelial neoplasia shares cytogenetic alterations with invasive prostate cancer.

BACKGROUND: High-grade prostate intraepithelial neoplasia (PIN) is the most likely precursor of prostate adenocarcinoma. However, the relationship between this lesion and prostate cancer has not yet been established. The detection of cytogenetic changes in the lesions prior to prostate adenocarcinoma would be useful in demonstrating such a pathogenic relationship. METHODS: Twenty eight high-grade PIN cases were found among 57 specimens of radical prostatectomy performed for clinically localized prostate cancer. Fluorescence in situ hybridization (FISH) analysis using centromeric probes to enumerate chromosomes 7, 8, 10, and 12 was performed to study the numerical chromosome alterations. FISH analysis was carried out over isolated nuclei obtained from high-grade PIN areas and prostate cancer foci in the same prostatectomy specimen. RESULTS: Of the 28 suitable cases it was possible to complete the study in 26 tumor and 20 PIN areas. The remaining cases were excluded because of insufficient tissue or poor preservation. Cytogenetic alterations (aneuploidy) were found in 16 of the 26 (62%) tumors studied. The most frequent chromosome alteration was trisomy 7, detected in 12 (75%) aneuploid tumors, followed by monosomy 8 present in 5 (31%) aneuploid tumors. Trisomy 7 was also the most frequent isolated chromosome alteration since it was detected in 7 (44%) tumors. Thirteen of 20 (65%) PIN cases were aneuploid when studied by FISH. Trisomy 7, trisomy 8, and monosomy 8 were the most common cytogenetic alterations in the 20 PIN areas studied, being observed in nine (45%), six (30%), and four (20%) cases, respectively. FISH analysis showed a high correlation (75% cases) in ploidy and pattern of cytogenetic alterations between high-grade PIN areas and the paired prostate cancer focus in the same specimen. CONCLUSIONS: The above results show a cytogenetic link between high-grade PIN and prostate cancer, suggesting that the former could be an early form of prostate cancer.

Rapid diagnosis of acute promyelocytic leukemia by analyzing the immunocytochemical pattern of the PML protein with the monoclonal antibody PG-M3.

The fusion protein, promyelocytic leukemia-retinoic acid receptor (PML-RAR)alpha, generated by the t(15;17) translocation has an abnormal cellular distribution with colocalization of RARalpha and PML proteins. We analyzed the immunostaining pattern of PML protein using the PG-M3 monoclonal antibody directed against the amino terminal portion of PML (retained in wild-type PML and PML-RARalpha fusion protein) in the diagnosis of acute promyelocytic leukemia (APL). In addition, we compared this test with other methods for detecting the PML-RARalpha fusion gene. A normal immunostaining pattern was observed in nonmyeloid disorders and in 78 of 111 acute myeloid leukemias (AMLs). A microgranular pattern was observed in 25 AMLs, all corresponding to APL. These results were concordant with the reverse transcriptase-polymerase chain reaction results for PML-RARalpha fusion gene. Only 1 case positive for the PML-RARalpha transcript showed a normal protein pattern by immunocytochemistry. PML immunostaining was helpful to rapidly differentiate 7 cases with borderline characteristics and to obtain the diagnosis in 2 cases with scarce material. The effectiveness and low cost of this technique support its routine use as a first-line procedure in the differential diagnosis of AML.

Blast crisis of Ph-positive chronic myeloid leukemia with isochromosome 17q: report of 12 cases and review of the literature.

Isochromosome 17q [i(17q)] is frequently observed in the blast crisis (BC) of chronic myelogenous leukemia (CML). It has been suggested that this chromosome abnormality is associated with special hematological characteristics of the BC, but the information on this subject is scarce. The clinical, hematological and cytogenetic features of patients with i(17q) were analyzed in a series of 121 patients with BC of Ph-positive CML. Twelve patients (10%) displayed an i(17q), representing the third commonest cytogenetic abnormality, after trisomy 8 and Ph chromosome duplication. In seven of the 12 patients the BC was preceded by an accelerated phase, and 10 had more than 10% blood basophils at BC diagnosis. The blast cells had a myeloid phenotype in the 12 patients. Five patients exhibited cytogenetic abnormalities in addition to i(17q), with trisomy 8 and duplication of the Ph chromosome being the alterations most frequently observed. Median survival of patients with i(17q) was 22 weeks, which was not significantly different from the survival of patients with myeloid BC in the overall series. These results are similar to the findings in 181 patients with i(17q) from 12 series of the literature, and confirm the special hematologic profile of BC of CML with this cytogenetic abnormality.

Paternal isodisomy 13 in a normal newborn infant after trisomy rescue evidenced by prenatal diagnosis.

Maternal and paternal uniparental disomy of chromosome 13 have been associated with normal phenotypes. We report on a new case of paternal isodisomy 13 in a phenotypically normal girl. Prenatal diagnosis had shown a 46,XX,-13,der(13;13) karyotype in chorionic villi and a 45,XX,der(13;13) karyotype in amniocytes and fetal blood. Molecular studies demonstrated that the de novo der(13;13) was an isochromosome 13 of paternal origin. This observation supports the lack of imprinting effects on chromosome 13 and trisomy rescue as a mechanism leading to uniparental disomy in cases involving isochromosomes.

[Clinical characterization, molecular and FISH studies in 80 patients with clinical suspicion of Williams-Beuren syndrome]. / Caracterización clínica y genética de 80 pacientes con sospecha clínica de síndrome de Williams-Beuren.

BACKGROUND: Williams-Beuren syndrome is a developmental disorder affecting vascular and connective tissues and central nervous system. The syndrome is caused by a submicroscopic deletion in the chromosome 7 implicating the 7q11.23 region. Fluorescence in situ hybridization (FISH) and molecular studies allow us to confirm the clinical suspicion of this syndrome. PATIENTS AND METHODS: We report clinical evaluation, FISH using Elastin Williams/D7S427 probe and molecular study with markers: D7S672, D7S653, D7S489B, D7S2476, D7S1870 and D7S489A, in 80 patients referred to test for Williams-Beuren syndrome. RESULTS: We found hemizygosity for the critical region in 36 patients. From 69 cases studied by FISH, 28 showed the deletion. Molecular studies in 78 cases showed loss of heterozygosity (LOH) in 26 patients. The patients presented the deletion from the paternal or maternal chromosome at equal frequency. Clinical evaluation of mental retardation, facial features, esotopia dental, malocclusion, hoarse voice, supravalvular aortic stenosis (SVAS), hernias, join limitation, WBS personality and mental retardation from positive and negative patients showed estatistical significant differences for all items except mental retardation and joint limitation. The most significant item was the presence of SVAS. CONCLUSION: This study confirms the usefulness of genetic studies as a diagnostic tool for William-Beuren Syndrome.

Trisomy/tetrasomy 21 mosaicism in CVS: interpretation of cytogenetic discrepancies between placental and fetal chromosome complements.

Trisomy/tetrasomy 21 mosaicism was found in chorionic villi (semidirect preparation) obtained from a 40 year old pregnant woman. Since both cell lines were abnormal, the couple elected for pregnancy termination. Placenta and fetal tissue samples were obtained for cytogenetic study. Long term cultured villi showed a non-mosaic trisomy 21 karyotype, while other tissues showed either a normal karyotype or normal/trisomy21 mosaicism. These discrepancies could be explained by a modified "bottle neck" embryogenic model with a trisomic zygote and a non-disjunction event taking place in one of the first divisions. Our case emphasises the need for confirmatory studies in other tissues when mosaicism is encountered in chorionic villi, even if all cell lines are abnormal.

Assaying the granulocyte-macrophage colony-stimulating factor (GM-CSF) as a mitogen of immature cells in fetal blood cultures.

Based on the presence of immature cells in fetal blood, and in an attempt to shorten the cytogenetic reporting time, three simultaneous one-day culture regimes were established in 23 fetal blood samples: (a) the standard phytohemagglutinin (PHA)-stimulated lymphocytes culture, (b) a culture using the granulocyte-macrophage colony-stimulating factor (GM-CSF) as an alternative mitogen, and (c) an unstimulated culture. Diagnostic success rates achieved by these three methods were as follows: 43 per cent (95 per cent CI: 23-64) (GM-CSF), 30 per cent (95 per cent CI: 12-49) (PHA) and 9 per cent (unstimulated). These three regimes were also assayed in three-day cultures giving 100 per cent diagnostic success rate for the PHA and GM-CSF, and 62 per cent (95 per cent CI: 41-83) for the unstimulated. A moderate correlation was found between the initial concentration of cultured erythroblasts and the metaphase count in one-day GM-CSF-stimulated (r=0.43, p=0.01) and unstimulated (r=0.35, p=0.05) cultures, suggesting that erythroblasts may be in part responsible for the mitotic index observed in these two regime cultures. In conclusion, our experience suggests that immature cells in fetal blood may be successfully cultured for diagnostic purposes.

Prevalence of Y chromosome microdeletions in oligospermic and azoospermic candidates for intracytoplasmic sperm injection.

OBJECTIVE: To determine the prevalence and type of Y chromosome microdeletions in 136 consecutively seen intracytoplasmic sperm injection (ICSI) candidates and in 50 consecutively seen azoospermic men attending an infertility clinic. DESIGN: Controlled clinical study. SETTING: Genetics laboratory and infertility clinic at a University hospital. PATIENT(S): One hundred eighty-six men who were seen at an infertility clinic and who were referred to a genetics counseling service for genetic assessment before ICSI. INTERVENTION(S): Collection of semen and blood samples. MAIN OUTCOME MEASURE(S): Semen analysis; serum FSH, LH, and T levels; karyotype analysis; and presence or absence of several single tagged site markers along the Y chromosome (sY274, sY238, sY276, sY84, sY102, sY143, sY153, sY254, sY269, sY202, sY158, sY160). RESULT(S): Yq chromosome microdeletions were detected in 10 (5.4%) of 186 consecutively seen ICSI candidates. The number of microdeletions was much higher in azoospermic patients (16%; 8 of 50) than in oligospermic patients (1.5%; 2 of 136). Two of the azoospermic patients with a Yq microdeletion also had sex chromosome aneuploidy mosaicism. No microdeletions were detected in 100 consecutively seen fathers who were included as controls. CONCLUSION(S): The prevalence of Yq microdeletions in the azoospermic group was much higher than in the oligospermic group and was consistent with the prevalence of Yq microdeletions detected in other series of azoospermic men in different geographic areas. All Yq microdeletions found in our patients belong to the AZFc region, indicating that microdeletions of the AZFa and AZFb regions are infrequent among oligospermic ICSI candidates or azoospermic males in our population.

Molecular, cytogenetic, and clinical characterisation of six XX males including one prenatal diagnosis.

Cytogenetic analysis, fluorescent in situ hybridisation (FISH), and molecular amplification have been used to characterise the transfer of Yp fragments to Xp22.3 in six XX males. PCR amplification of the genes SRY, RPS4Y, ZFY, AMELY, KALY, and DAZ and of several other markers along the Y chromosome short and long arms indicated the presence of two different breakpoints in the Y fragment. However, the clinical features were very similar in five of the cases, showing a male phenotype with small testes, testicular atrophy, and azoospermia. All these patients have normal intelligence and a stature within the normal male range. In the remaining case, the diagnosis was made prenatally in a fetus with male genitalia detected by ultrasound and a 46,XX karyotype in amniocytes and fetal blood. Molecular analysis of fetal DNA showed the presence of the SRY gene. FISH techniques also showed Y chromosomal DNA on Xp22.3 in metaphases of placental cells. To our knowledge, this is the second molecular prenatal diagnosis reported of an XX male.

Cytogenetic studies in fetal blood.

In order to assess the effectiveness and reliability of cytogenetic diagnosis provided by fetal blood, we report the first 186 cases of fetal blood sampling performed for rapid karyotype between 19-37 weeks of pregnancy in our Prenatal Diagnosis Unit. The overall diagnostic success rate was 98%, achieving 100% in the last period of the study. Chromosomal anomalies were detected in 16% (29/182) of the fetuses. In malformed fetuses this rate increased from 8-9% in isolated malformation or markers of aneuploidy to 50% in multiple malformations. In pregnancies in which a previous cytogenetic study in amniotic fluid was inconclusive, fetal blood made it possible to obtain a definitive result, with no discrepancies found at phenotypic follow-up examination. Interestingly enough, one of the four previously defined as pseudomosaicisms was found to be a non-mosaic in fetal blood, and only 1 of 4 mosaicisms was confirmed in fetal blood. In conclusion, cytogenetic analysis of fetal blood samples appears to be effective, rapid and reliable to establish the fetal karyotype in selected cases.

Brachycephaly is ineffective for detection of Down syndrome in early midtrimester fetuses.

The aim of this study was to assess the value of fetal brachycephaly in the detection of Down syndrome at 13-18 weeks of pregnancy. The cephalic index (CI) was determined in 555 consecutive chromosomally normal fetuses, and in 38 chromosomal abnormalities, prior to amniocentesis. A CI > 0.85 was observed in 14% (2/14) of the fetuses with Down syndrome and in 11% of the normal fetuses. In conclusion, our data show that brachycephaly is not a useful marker for Down syndrome in early midtrimester fetuses.

Prenatal diagnosis of fragile X syndrome: (CGG)n expansion and methylation of chorionic villus samples.

Fragile X syndrome is the most common form of inherited mental retardation, due to an expansion of the (CGG)n trinucleotide repeat in the FMR-1 gene and hypermethylation of its 5' upstream CpG island. Two major problems remain to be resolved for fragile X prenatal diagnosis: the abnormal methylation patterns of chorionic villus samples (CVS) and the inability to predict the mental status of females with the full mutation. We present here the results of ten prenatal diagnoses of fragile X syndrome using Southern blotting and polymerase chain reaction (PCR) amplification, and the analysis of 50 further CVS to test the methylation status of the CpG island of the FMR-1 gene. In the ten 'at-risk' CVS, eight normal (five males and three females) and two affected male fetuses were detected. Absence of methylation in the CVS was observed in two cases, which was not found upon subsequent examination of the newborn or of fetal tissues. In the 50 CVS not 'at risk' for fragile X syndrome, abnormal fragment patterns for probe StB12.3 were detected in 32 per cent for female and 24 per cent for male fetuses. This abnormal pattern could be due to absent or partial methylation of the CpG island of the FMR-1 gene in chorionic villus tissues.

Chorionic villus sampling by biopsy forceps. Results of 1580 procedures from a single centre.

The results of a prospective series of 1580 chorionic villus sampling (CVS) procedures using biopsy forceps are presented. Most of the procedures (1442), including 11 sets of twins, were performed by the transcervical approach (TC-CVS), using a curved-shank thin forceps, and 138 by the transabdominal approach (TA-CVS), using a trocar-guided straight thin forceps. The mean gestational age for TC-CVS was 10.9 weeks, and in 233 cases (16 per cent) the procedure was carried out between the 12th and 14th weeks. The mean gestational age for TA-CVS was 16.7 weeks. The major indication for CVS was advanced maternal age (92.7 per cent in the TC and 91.8 per cent in the TA approach), and indications for abnormal ultrasound findings were more common in the TA approach (4.5 per cent) than in TC-CVS (0.07 per cent). Although sampling was apparently accomplished in all the procedures, in 3.1 per cent of the TC-CVS and 2.2 per cent of TA-CVS procedures, the samples were less than 1 mg after dissection. A cytogenic report was obtained in 96.1 per cent of the TC-CVS and 90.6 per cent of the TA-CVS. Maternal serum alpha-fetoprotein (MSAFP) was measured before and after TC-CVS and the post-CVS MSAFP was positively correlated with the sample weight. Second-trimester amniocentesis following CVS was required in 5.2 per cent (TC-CVS) and 6.5 per cent (TA-CVS), due to the failure to obtain a cytogenetic report or diagnostic confirmation. The follow-up to the 20th week was 100 per cent by ultrasound scan, and 88.6 per cent from the 21st week to 1 week after delivery. Fetal loss rates within 2 weeks of the procedure were 1.7 per cent (TC-CVS) and 0.8 per cent (TA-CVS) and total fetal loss accumulated to 1 week after delivery was 4.6 per cent (TC-CVS) and 5.9 per cent (TA-CVS). Factors found to increase significantly fetal loss in the TC-CVS series were maternal age and the collection of very small samples, but not the number of forceps insertions.