Preimplantation genetic diagnosis and screening.

Preimplantation genetic diagnosis (PGD) identifies genetic abnormalities in preimplantation embryos prior to embryo transfer. PGD is an exciting technology that may improve the likelihood of a successful pregnancy and birth for five distinct patient groups: (1) those with infertility related to recurrent miscarriages or unsuccessful in vitro fertilization (IVF) cycles, (2) those with unexplained infertility, (3) advanced maternal age, (4) severe male factor infertility, and (5) couples at risk for transmitting a hereditary disease to their offspring. PGD is always performed following an IVF cycle where multiple oocytes are retrieved and fertilized. Sophisticated techniques such as multiprobe, multicolor fluorescence in situ hybridization are used to test single cells for structural or numerical chromosome abnormalities, whereas the polymerase chain reaction, linkage analysis, and DNA sequencing are used to analyze single cells for disease-specific DNA mutations. PGD allows one to transfer only those embryos identified as being free of genetic abnormalities, thus potentially increasing the implantation rate and decreasing the miscarriage rate. These technologies identify embryos free of specific genetic abnormalities and may increase the likelihood of achieving the patient's goal: the birth of a healthy infant.

Aneuploidy frequencies in semen fractions from ten oligoasthenoteratozoospermic patients donating sperm for intracytoplasmic sperm injection.

OBJECTIVE: To determine aneuploidy frequencies in pellet and swim-up semen fractions from 10 infertile men with severe oligoasthenoteratozoospermia (OAT) who were donating sperm for intracytoplasmic sperm injection and to determine whether the swim-up isolation method would successfully separate aneuploid from haploid sperm. DESIGN: Prospective study. SETTING: Infertility clinic and molecular genetics laboratory. PATIENT(S): Ten patients with severe OAT. INTERVENTION(S): Cytogenetic analyses by fluorescence in situ hybridization to determine aneuploidy frequencies for chromosomes 1, 13, 18, 21, X, and Y in sperm from swim-up and pellet fractions. MAIN OUTCOME MEASURE(S): Gametic aneuploidy was scored in sperm fractions separated by the swim-up technique and clinical results after intracytoplasmic sperm injection were tabulated. RESULT(S): In all cases, chromosome aneuploidy levels in patients were significantly greater than in controls. The type and percentage of aneuploid sperm for all patients with OAT found in both swim-up and pellet fractions were not different, with the exception of diploid sperm, which remained in the pellet fraction. After ET, 2 (20%) of 10 couples achieved successful pregnancies. CONCLUSION(S): The data show significantly higher rates of diploidy, autosomal disomy and nullisomy, sex chromosome disomy and nullisomy, and total aneuploidy in sperm from all separated fractions obtained from all patients with OAT versus controls. This patient population with OAT may be at increased risk of producing aneuploid offspring.

Detection of aneuploidy for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X and Y by fluorescence in-situ hybridization in spermatozoa from nine patients with oligoasthenoteratozoospermia undergoing intracytoplasmic sperm injection.

Recent evidence suggests that infertile males donating semen for intracytoplasmic sperm injection (ICSI) may be at an increased risk of transmitting numerical (predominantly sex chromosome) abnormalities to their offspring. The present study was designed to determine aneuploidy in spermatozoa from oligoasthenoteratozoospermic (OAT) patients undergoing ICSI. Aneuploidy frequencies of 12 autosomes and the sex chromosomes were determined by fluorescence in-situ hybridization (FISH) on spermatozoa from fresh ejaculate of nine severe OAT patients and four proven fertile donors. FISH, using directly labelled (fluorochrome-dUTP) satellite or contig DNA probes specific for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X, and Y, was performed on decondensed spermatozoa. Per chromosome disomy frequencies for autosomes and sex chomosomes in OAT males were 0-5. 4%. In contrast, the disomy frequencies in controls were 0.05-0.2%. The frequency of diploid spermatozoa in OAT patients was 0.4-9.6%; controls showed a mean of 0.04%. Using recently developed formulae, the total aneuploidy in our OAT patient population was estimated to be 33-74%. In contrast, estimates of mean total aneuploidy in the spermatozoa of controls ranged from 4.1 to 7.7%, depending upon method of calculation. Six series of ICSI were performed on five of the OAT patients. Four resulted in no establishment of pregnancy; the others failed to establish ongoing pregnancies. Our cytogenetic data show significantly elevated frequencies of diploidy, autosomal disomy and nullisomy, sex chromosome aneuploidy, and total aneuploidy in OAT patients, which may contribute to the patients' infertility.

Cytogenetics of somatic cells and sperm from a 46,XY/45,X mosaic male with moderate oligoasthenoteratozoospermia.

OBJECTIVE: To determine aneuploidy frequencies in sperm from a patient with normal phenotype and 46,XY/45,X mosaicism in somatic cells (peripheral lymphocytes). DESIGN: Case report. SETTING: Infertility clinic and genetics laboratory. PATIENT: A 30-year-old male with primary infertility and moderate oligoasthenoteratozoospermia. INTERVENTION(S): Cytogenetic analysis of somatic cells and determination by fluorescence in situ hybridization of aneuploidy frequencies for the gonosomes (sex chromosomes) and chromosome 18 in sperm from whole and Percoll-separated semen. MAIN OUTCOME MEASURE(S): Somatic and gametic aneuploidy were scored. RESULT(S): Analysis of lymphocyte metaphase cells showed a mosaic 46,XY (90%)/ 45,X (10%) karyotype. Significantly higher frequencies of gonosomal (semen, 1.92% versus 0.70%; Percoll, 1.12% versus 0.46%), and chromosome 18 (semen, 0.89% versus 0.28%; Percoll, 0.26% versus 0.10%) disomy were detected in the sperm of the patient compared with those observed in spermatozoa from a proved fertile control. CONCLUSION(S): Significantly higher frequencies of aneuploid sperm suggest that the patient is at elevated risk of producing offspring with numerical chromosome abnormalities.

Association of the Robin sequence with the fragile X syndrome.

We report on 4 individuals with the fragile X [fra(X)] syndrome and the Robin sequence (or elements of that sequence). To our knowledge, this association has been described in only one other boy. However, males with the fra(X) syndrome have been reported to have an increased incidence of cleft palate. We recommend that children with a cleft palate or the Robin sequence be assessed for developmental delays and a family history of mental retardation. The fra(X) syndrome may be one of the genetic causes of the Robin sequence and, when indicated, children with the sequence should be tested for fra(X).

Guidelines for the preparation and analysis of the fragile X chromosome in lymphocytes.

The following guidelines were adopted by an Ad Hoc Committee convened at the Fourth International Workshop on the Fragile X Syndrome and X-Linked Mental Retardation to establish minimum cytogenetic standards for the preparation and analysis of the fragile X chromosome. The intention of the committee was to develop and provide practical standards for the routine cytogenetic detection of the fragile X. The guidelines describe reasonable criteria for effective tissue culture methods for eliciting the Xq27.3 fragile site in vitro and for the analysis of such chromosome preparations.

Speculation on the role of transposable elements in human genetic disease with particular attention to achondroplasia and the fragile X syndrome.

We suggest that mutations for fragile X-positive Martin-Bell syndrome, and perhaps also for achondroplasia, may result from the insertion of transposable elements (TE's). Loss of genetic function could result from either the insertion of TE's within or adjacent to a normal chromosomal gene or, in the case of fragile X, from the loss of genes distal to the site of TE insertion following subsequent TE excision without ligation of the resulting discontinuity. The phenotypically and often cytogenetically normal transmitting males in fragile X pedigrees are interpreted not as "nonpenetrant" transmitters of a fully formed fragile X but rather as transmitters of some or all of the factors necessary for TE insertion at Xq27. We consider it likely that such insertion frequently first occurs, both in soma and especially in the germline, in their daughters. Our models predict that father to son transmission of causative factors would be a common occurrence in fragile X pedigrees. The absence of documented father to son transmission either points to a flaw in the models or reflects systematic bias in the collection of pedigree information.

Chromosome damage in peripheral lymphocytes from American thorium workers.

An analysis of the frequency of chromosome aberrations in lymphocytes from 47 thorium workers and three external controls is presented. Thirty-seven of these cases were divided into three age-matched groups based upon the means of the sums of their emanating radium-224 and bismuth-212 (Ra + Bi) burdens. The low burden group (mean Ra + Bi burden = -0.06 +/- 0.03 nCi) had two two-break chromosome aberrations in 1200 cells, the moderate burden group (mean Ra + Bi burden = 0.21 +/- 0.03 nCi) had three such aberrations in 1000 cells and the high burden group (mean Ra + Bi burden = 0.99 +/- 0.21 nCi) had five such aberrations in 1500 cells. While there is a two-fold increase in the two-break aberration frequency in pooled data from the two higher exposure groups (8/2500 cells) as compared with the lowest exposure group (2/1200 cells), the difference between these subsamples is not statistically significant (p = 0.32). The frequency of dicentrics and centric ring chromosomes (8/3300 cells) in the pooled higher body burden groups from the total sample (mean Ra + Bi burden = 0.56 +/- 0.09 nCi) does however show a very highly significant increase over the historical control frequency. A similar analysis based on total months of employment in the thorium-processing industry did not show a positive relationship between duration of employment and aberration frequency. In broad outline, our results are compatible with those from similar studies on Brazilian thorium workers and Thorotrast patients.