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PubMedDate not supplied

Monozygotic twinning and IVF/ICSI treatment: a report of 11 cases and review of literature.

Monozygotic twinning is a relatively rare event in in-vivo conception, being estimated to occur in 0.42% of all births. The underlying mechanism for monozygotic twin formation is the division of the embryo early in its development. Separation of cells may theoretically occur before or after inner cell mass formation. In this analysis we report 11 cases of monozygotic twins resulting from IVF or intracytoplasmic sperm injection (ICSI) treatment cycles performed between 1991 and 1998 at St James's University Hospital, Leeds, and Bourn Hall Clinic, Cambridge, UK. Six cases (55%) followed treatment with ICSI and seven cases (64%) were in women aged > or = 35 years. This article also reviews the scientific literature discussing information pertaining to frequency, aetiology and potential complications of the monozygotic twinning phenomenon. We conclude that patients at risk of monozygotic twinning are those aged >35 years of age and those who had artificial opening in the zona pellucida by application of micromanipulation techniques. Women undergoing assisted conception treatment, particularly those with these two risk factors, must be informed of the low but definite risk of monozygotic twinning when counselled regarding the number of embryos to be transferred and chances of multiple births.

Adult↗
PubMedDate not supplied

Genetic and teratogenic effects of cancer treatments on gametes and embryos.

Male and female germ cells vary in their sensitivity to the mutagenic effects of chemotherapy and radiotherapy, depending on their stage of maturation and the agent used. Although sperm DNA damage exists following treatment, no increase in genetic defects or congenital malformations was detected among children conceived to parents who have previously undergone chemotherapy or radiotherapy. The use of assisted reproductive technologies and micromanipulation techniques might increase this risk; hence caution should be exercised. In female cancer patients, miscarriage and congenital malformations are not increased following chemotherapy. However, when IVF and embryo cryopreservation is practised between or shortly after treatment, possible genetic risks to the growing oocytes exist, and hence the babies should be screened. During pregnancy, the potential teratogenic effects of chemotherapy influence the choice and timing of therapy. Termination is usually recommended in the first trimester. Second- and third-trimester exposure does not usually increase the teratogenic risk and cognitive development, but it may increase the risk of poor obstetric outcome and fetal myelosuppression. During the first two weeks after fertilization of the embryo, radiation is lethal but not teratogenic. High doses of radiation during pregnancy induce anomalies, impaired growth and mental retardation, and there may be an increased risk of childhood leukaemia and other tumours in the offspring.

Abnormalities, Drug-Induced↗
PubMed1995

Sliding and ATPase.

Sliding machines composed of F-actin and myosin or microtubules and kinesin or dynein convert the free energy of ATP hydrolysis into sliding movements and mechanical work. Development of optical microscopy with micromanipulation techniques has made possible direct observation of single events of sliding exhibited by single sliding machines. The experimental data and theoretical consideration suggest that the influx-efflux coupling in these machines may be loose. Specific characters of sliding machines are summarized and the problems we have for understanding of the coupling mechanism are discussed.

Actins↗
PubMed1995

Allele-specific loss in chromosome 9p loci in preneoplastic lesions accompanying non-small-cell lung cancers.

BACKGROUND: Carcinogenesis is a multistep process, which may begin as a consequence of chromosomal changes. Deletions in the short arm of chromosome 9 (9p) have been observed in lung carcinomas. In addition, morphologically recognizable preneoplastic lesions, frequently multiple in number, precede onset of invasive carcinomas. PURPOSE: We tested for deletions and loss of heterozygosity (LOH) at 9p loci in preneoplastic and neoplastic foci in lungs of patients with non-small-cell lung carcinomas (NSCLCs). METHODS: Seven archival, paraffin-embedded, surgically resected NSCLC specimens were selected. They were predominantly from patients with adenocarcinomas and contained multiple preneoplastic lesions, including hyperplasia, metaplasia, dysplasia, and carcinoma in situ (CIS). Fifty-three histologically identified preneoplastic and malignant lesions present in bronchi, bronchioles, and alveoli were precisely microdissected from stained tissue sections with a micromanipulator. Stromal lymphocytes were used to determine constitutional heterozygosity. The specimens were analyzed for LOH using polymerase chain reaction-based assays for polymorphism in dinucleotide repeats (microsatellite markers) in interferon alfa (IFNA) and D9S171 loci on 9p. RESULTS: All seven cases were constitutionally heterozygous for one or both microsatellite markers. Five of seven cases had LOH at one or both 9p loci in the invasive primary cancers (doubly informative cases). Four of these five cases also revealed LOH in preneoplastic foci. In the doubly informative cases, LOH was detected in five (38%) of 13 foci of hyperplasia, four (80%) of five foci of dysplasia, and three (100%) of three CIS lesions. LOH was detected in preneoplastic lesions from all regions of the respiratory tract, including bronchi, bronchioles, and alveoli, and involved five different cell types. The identical allele was lost from both the preneoplastic lesions and the corresponding tumors (12 of 12 lesions, 17 of 17 comparisons), a phenomenon we have referred to as "allele-specific mutation." Statistical analyses employing a cumulative binomial test demonstrated that the probabilities of such findings occurring by chance are 2.4 x 10(-4) and 7.6 x 10(-6), respectively. From comparisons with the previously published data on other chromosomal abnormalities in the same tissue specimens, it appears that LOH at 3p and 9p loci occurred early in the hyperplasia stage, but the ras gene point mutations were relatively late, at the CIS stage. CONCLUSIONS: LOH at 9p loci occurs at the earliest stage in the pathogenesis of lung cancer and involves all regions of the respiratory tract. LOH in NSCLC is not random but targets a specific allele in individuals. Studying preneoplastic lesions may help identify intermediate markers for risk assessment and chemoprevention.

Adenocarcinoma↗
PubMed1996

Effect of gastrin-releasing peptide on sperm functions.

Male infertility can be related to defects in motility, capacitation, acrosome reaction, binding and penetration of the zona pellucida. While different in-vitro techniques (such as micromanipulation which is complicated and expensive) are available for the treatment of male infertility, several pharmacological agents have been shown to increase fertilizing capacity under accurate experimental conditions. Gastrin-releasing peptide (GRP, the mammalian homologue of the amphibian skin peptide bombesin) is present in the reproductive tract and expressed by the pregnant ovine endometrium prior to attachment and throughout the pregnancy. A bombesin-like peptide resulting from alternate splicing of the GRP gene in testis has been detected in primates. In this study, we have tested the ability of GRP to enhance human sperm functions such as motility, capacitation, zona binding and acrosome reaction. Analysis of sperm motility was performed with the ATS 20 computer-assisted semen analysis (CASA) system. Zona binding was analysed using intact human unfertilized oocytes and selective labelling of spermatozoa with two fluorochromes. Our results did not show any positive effect of GRP on these parameters under our experimental conditions. However, when GRP at the concentration of 100 nM was added after ionophore treatment, the percentage of reacted cells increased. significantly (P < 0.05) compared with situations where each agent was used alone. This led us to suppose that the role of bombesin in the different stages of fertilization might not exclude other unknown factors.

Acrosome↗
PubMed1996

Identification of a Gly862 to Ser substitution in the type I collagen gene from a single spermatozoon.

Individual spermatozoa from the father of two affected infants with osteogenesis imperfecta were separated by dilution and micromanipulation. A segment of the type I collagen gene containing the mutant region was amplified by nested polymerase chain reaction and sequenced. Among 40 individual spermatozoa, 15 specimens were identified as mutants with a substitution of guanine3208 to adenine (glycine862 to serine) while 18 and seven specimens were of the wild and mixed types respectively. Through this study, we have established a molecular procedure that can be considered as prerequisite for preimplantation diagnosis of genetic disorders with a single point mutation.

Collagen↗
PubMed1997

Paternal X-chromosome inactivation in human trophoblastic cells.

Dosage compensation for X-chromosome-linked genes between male and female mammals occurs by inactivation of one of the two X chromosomes in the female. In somatic cells, either the paternal or the maternal X chromosome is randomly inactivated in a given cell. In contrast, in the extra-embryonic tissues of mice, the paternally-derived X chromosome is preferentially inactivated. The evidence for paternal X-chromosome inactivation in humans is controversial and remains to be clarified. In this study, we have developed a sensitive polymerase chain reaction (PCR) technique to investigate the methylation pattern of the X-linked androgen receptor (AR) gene. The 5' CpG island of this gene is methylated on the inactive X chromosome and hypomethylated on the active X chromosome in somatic cells. The paternal and the maternal alleles of the AR gene may be distinguished by a polymorphism in the number of CAG triplet repeats within the CpG island. As a source of human extra-embryonic tissue, we used chorionic villus (CV) samples from female conceptuses of 10-12 weeks gestation. From a tiny branch of a CV sample, two distinct cell lineages, the trophoblastic and mesodermal lineages, were dissected apart by trypsin digestion and micromanipulation and DNA was extracted separately from these purified tissues. Digestion of the DNA with the methylation-sensitive restriction enzyme, Hpall, followed by PCR amplification revealed that the paternal allele is preferentially methylated in trophoblastic cells, but not in mesodermal cells. These results strongly suggest that the paternal X chromosome is preferentially inactivated in the human extra-embryonic tissues early in development.

Animals↗
PubMed1998

Ooplasmic transfer in mature human oocytes.

Ooplasmic transplantation aimed at restoring normal growth in developmentally compromised oocytes and embryos was evaluated in seven couples (eight cycles) with multiple implantation failures. Two approaches were investigated to transfer ooplasm from donor eggs at metaphase II (MII) stage into patient MII eggs: (i) electrofusion of a ooplasmic donor fragment into each patient egg (three cycles), and (ii) direct injection of a small amount of ooplasm from a donor egg into each patient egg (five cycles). Some donor eggs were used multiple times. Donor eggs were divided into two groups, one being used for ooplasmic extraction and the other one for egg donation. Cleaved embryos resulting from the latter were cryopreserved, where numbers and satisfactory development permitted. A second control group consisted of embryos derived from patient eggs after intracytoplasmic sperm injection without ooplasmic transfer. This was performed when sufficient number of eggs were available (n = 5). Donor eggs (n = 40) were evaluated cytogenetically after micromanipulation in order to confirm the presence of chromosomes. One egg was anuclear and the recipient embryos were not transferred. Normal fertilization was significantly higher after injection of ooplasm (63%) in comparison with fusion (23%). Pronuclear anomalies appeared enhanced after fusion with ooplasts. Embryo morphology was not improved in the three cycles with electrofusion and patients did not become pregnant. An improvement in embryo morphology was noted in two patients after injection of ooplasm and both became pregnant, but one miscarried. A third pregnancy was established in the repeat patient, without obvious embryo improvement. One baby was born and the third pregnancy is ongoing with a normal karyotype. Two other patients with male factor infertility had poor embryos after ooplasmic injection, but the donor embryo controls were also poor. The patients did not become pregnant and had no donor embryos frozen. Ooplasmic transfer at the MII stage may be promising in patients with compromised embryos; however, evaluation of ooplasmic anomalies and optimization of techniques will require further investigation prior to widescale application.

Cell Culture Techniques↗
PubMed1999

Approximately half of the erythroblasts in maternal blood are of fetal origin.

The enrichment of fetal erythroblasts from the peripheral blood of pregnant women is currently actively pursued for the development of a non-invasive means of prenatal diagnosis. Since erythroblasts in maternal blood are not all of fetal origin, and currently no reliable method exists to distinguish between the maternal and fetal erythroblasts, their use for prenatal diagnosis is not without uncertainty. The purpose of this study was to determine the percentage of fetal erythroblasts in maternal blood at the single cell level and to what extent such cells can reproducibly be used for polymerase chain reaction (PCR)-based prenatal diagnostic analyses. Erythroblasts were enriched from the peripheral blood of rhesus negative pregnant women using magnetic cell sorting (MACS). Single erythroblasts identified morphologically were individually micromanipulated and analysed by a multiplex PCR reaction for the fetal SRY and rhesus D genes. As a control for the PCR reaction the beta-globin gene was used. The PCR results were validated by the results obtained by invasive procedures. In all instances where single erythroblasts were examined, the correct fetal genotype for the two fetal specific loci was detected. Furthermore, our results indicate that approximately 50% of the enriched erythroblasts are of fetal origin.

DNA-Binding Proteins↗
PubMed2000

Prenatal diagnosis of beta-thalassaemia using fetal erythroblasts enriched from maternal blood by a novel gradient.

We have assessed a new technique for the isolation of fetal erythroblasts from maternal blood for the non-invasive prenatal diagnosis of pregnancies at risk of beta-thalassaemia. This method relies on the separation of erythroblasts from maternal nucleated cells by a novel step gradient and high speed centrifugation. In four of the six cases examined, single erythroblasts were identified by immunohistochemistry for zeta (zeta) globin. These were individually micromanipulated and analysed by single cell polymerase chain reaction (PCR) and subsequent sequencing of the region of beta-globin locus where the mutations most common to the region of Puglia, Italy, are clustered. In each of the four instances where fetal erythroblasts were identified by antibody staining, the fetal beta-globin genotype was correctly determined. To date, this represents the largest series of non-invasive prenatal diagnoses performed for this haemoglobinopathy.

Centrifugation, Density Gradient↗
PubMed1992

Mitotic recombination of yeast artificial chromosomes.

Large regions of human DNA can be cloned and mapped in yeast artificial chromosomes (YACs). Overlapping YAC clones can be used in order to reconstruct genomic segments in vivo by meiotic recombination. This is of importance for reconstruction of a long gene or a gene complex. In this work we have taken advantage of yeast protoplast fusion to generate isosexual diploids followed by mitotic crossing-over, and show that it can be an alternative simple strategy for recombining YACs. Integrative transformation of one of the parent strains with the construct pRAN4 (containing the ADE2 gene) is used to disrupt the URA3 gene contained within the pYAC4 vector arm, providing the markers required for forcing fusion and detecting recombination. All steps can be carried out within the commonly used AB1380 host strain without the requirement for micromanipulation. The method was applied to YAC clones from the human MHC and resulted in the reconstruction of a 650 kb long single clone containing 18 known genes from the MHC class II region.

Blotting, Southern↗
PubMed1997

Microamplification of specific chromosome sequences; an improved method for genome analysis.

An improved method was developed for microdissection and cloning of metaphase as well as pachytene chromosomes. The protocol incorporates efficient ligation of chromosomal DNA with linker adaptors, abolishment of microcloning steps and the reduction of micromanipulation. The threshold for amplifying genomic DNA template was in the range of 2-20 femtogram. The amplification products had a size distribution between 200 and 1300 bp (average 500 bp). Using pachytene chromosomes of maize the selectivity for segment-specific libraries can be increased between 10- and 20-fold. The approach described here is being applied to the fine mapping and isolation of genes conveying resistance against plant pathogens.

Chromosomes↗
PubMed1998

Representational difference analysis using minute quantities of DNA.

Representational difference analysis (RDA) is a differential hybridization method which can effectively isolate unique DNA sequences from complex and highly related genomes or cDNA libraries. A major drawback of the RDA analysis is the requirement for pure driver and relatively pure tester samples, ruling out the analysis of whole tissue biopsies. To circumvent this problem, we have modified the technique for the analysis of very small quantities of DNA so that pure cell populations isolated by micromanipulation from tissue sections can be analyzed. Using this modified technique, as few as 50 diploid cells ( approximately 500 pg of DNA) can be analyzed.

DNA↗
PubMed2003

Sequence-specific fluorescent labeling of double-stranded DNA observed at the single molecule level.

Fluorescent labeling of a short sequence of double-stranded DNA (dsDNA) was achieved by ligating a labeled dsDNA fragment to a stem-loop triplex forming oligonucleotide (TFO). After the TFO has wound around the target sequence by ligand-induced triple helix formation, its extremities hybridize to each other, leaving a dangling single-stranded sequence, which is then ligated to a fluorescent dsDNA fragment using T4 DNA ligase. A non-repeated 15 bp sequence present on lambda DNA was labeled and visualized by fluorescence microscopy after DNA combing. The label was found to be attached at a specific position located at 4.2 +/- 0.5 kb from one end of the molecule, in agreement with the location of the target sequence for triple helix formation (4.4 kb from one end). In addition, an alternative combing process was noticed in which a DNA molecule becomes attached to the combing slide from the label rather than from one of its ends. The method described herein provides a new tool for the detection of very short sequences of dsDNA and offers various perspectives in the micromanipulation of single DNA molecules.

Bacteriophage lambda↗
PubMed1977

Dual infection by "+" and "-" mating types of Ajellomyces (Blastomyces) dermatitidis.

A culture of Blastomyces dermatitidis isolated via bronchial washing from a patient with blastomycosis was studied intensively. The primary isolate was shown to have contained both the "+" and "-" mating type determiners of Ajellomyces dermatitidis. This was evidenced by the observations that both a first and a second subculture, when paired with itself produced fertile ascocarps typical of A. dermatitidis. Both these subcultures determined the production of ascospores when paired individually with each of six testers, these testers not producing fertile fruiting bodies when selfed. Isolation by micromanipulation of "+" and "-" ascospores from ascocarps produced by selfing a subculture established the mixed ("+", "-") nature of the clinical isolate and of the infection. The mixed infection was considered to have resulted from inhalation of both the "+" and "-" ascospores or conidia of A. dermatitidis or from the inhalation of one spore that contained both the "+" and "-" mating type determiners.

Adult↗
PubMed1990

Development of assisted conception.

Advances in assisted human conception in the last twenty years were founded on the detailed knowledge of the mammalian reproductive cycle gained in the earlier years of this century. Several lines of study coincided in the 1960's, including the use of ovarian stimulants to regulate follicle growth, oocyte maturation and ovulation, fertilization and embryonic growth in vitro, and the introduction of safer and better methods of laparoscopy. A combination of these techniques with embryo replacements in the uterus permitted the introduction of IVF for the alleviation of infertility. In turn, these methods were extended to other forms of assisted conception, including GIFT, which gave a wider choice of methods for the treatment of specific causes of infertility. Advances during the last decade include rapid progress in the use of ultrasound, the introduction of the LHRH agonists in new forms of stimulation, embryo cryopreservation, and micromanipulative techniques on oocytes and embryos for the alleviation of male infertility and the diagnosis of genetic disease in preimplantation embryos.

Female↗
PubMed1996

Evaluation of a cut-off value for normal sperm morphology using strict criteria to predict fertilization after conventional in-vitro fertilization and embryo transfer in asthenozoospermia.

The predictive value of sperm morphology evaluation using strict criteria (MEUSC) on fertilization and pregnancy rates was evaluated in 60 couples undergoing in-vitro fertilization (IVF) and embryo transfer. In all, 47 of the male partners had a progressive sperm motility < 50% with a density > 20 x 10(6)/ml (asthenozoospermia) and 13 had normozoospermia. MEUSC was performed on the same semen sample that was used for IVF on at least 100 spermatozoa after Papanicolaou staining at x 1250 magnification. Defects in the head, mid-piece and tail were counted separately, borderline forms were classified as abnormal and the teratozoospermia index was calculated for each sample. Of the spermatozoa in the asthenozoospermic group, 13.0% were judged as normal on MEUSC versus 19.8% in normozoospermia (P = 0.0013). The number of normal spermatozoa on MEUSC correlated with the progressive motility in asthenozoospermia (rho = 0.41, P = 0.0043). Defects in the mid-piece (P = 0.0004) and tail (P = 0.025) were more common, and the teratozoospermia index (P = 0.015) was higher in asthenozoospermic than normozoospermic samples. The parameters of MEUSC did not correlate with the fertilization rate. In asthenozoospermia, differences in fertilization rates were calculated for each cut-off value between 4 and 10% normal spermatozoa on MEUSC. A cut-off value of 6% gave the best statistical power. If > 6% of spermatozoa were normal, a median of 66.7% of ova were fertilized, compared with a median fertilization rate of 35.4% if < or = 6% of spermatozoa were normal (P = 0.022). The highest cut-off value still giving significant discrimination was 8% (normal MEUSC > 8%, median fertilization rate 66.7%; < or = 8%, median fertilization rate 35.7%, P = 0.028), and this may be used as the critical value for normal morphology in asthenozoospermia. It is concluded that despite a significant linear relationship between morphology using strict criteria and progressive motility, MEUSC is still useful as a predictor for fertilization in asthenozoospermia. In cases of asthenozoospermia with < or = 8% normal spermatozoa on MEUSC, micromanipulation should be considered.

Adult↗
PubMed1996

Ethical and legal aspects of assisted reproduction practice in Asia.

This report describes the ethical and legal aspects of assisted reproduction technology (ART) that have been instituted in Asian countries. The data were collected by a questionnaire circulated to ART units in Asia. These are Taiwan, Singapore, Korea, Indonesia, Thailand, Japan, Iran, India, Jordan, Malaysia, China, Israel, Hong Kong, Pakistan, Lebanon, Saudi Arabia, and Persian Gulf countries. According to the survey, there are approximately 260 ART centers in Asia (half of which are in Japan). On a global basis each ART centre in Asia serves an average population of 13 million people. On the other hand, in those Asian countries where the standards of living are relatively high, the availability of ART services, including the more sophisticated and costly ART procedures like micromanipulation, is similar to that in the Western world. In most of the Asian countries practising ART, however, no state registry exists. Taiwan is the only country that has specific legislation, and in six other countries some kind of ministerial regulations are practised. We conclude that ART is now practised in 20 countries in Asia. The prevailing rules and cultural heritage in many of these Asian countries has a major influence on the implementation of ART in Asia. However, in view of the complicated and sensitive issues involved, and as no supervision on ART clinics exists in most of the Asian countries, we advocate that some kind of quality control should be urgently instituted in all centres practising ART. In this way, it is hoped that the highest standards be attained for all parties concerned.

Asia↗