Faculty Bibliography

Preimplantation genetic diagnosis was performed in 122 embryos obtained by IVF from 11 patients carriers of haemophilia, Duchenne's muscular dystrophy, Barth's syndrome, cystic fibrosis, Pelizaeus-Merzbacher syndrome or Rett's syndrome. After multiplex polymerase chain reaction (PCR) or fluorescence in situ hybridization (FISH) analysis with multiple probes, 28 embryos diagnosed as not affected were replaced. Of these, eight implanted (28%) and produced three ongoing pregnancies, three deliveries of four babies and a biochemical pregnancy. However, one case screened for cystic fibrosis was misdiagnosed and the pregnancy was terminated. In order to evaluate the efficiency of multiplex PCR, 55 non-replaced embryos were reassessed by PCR or by FISH. Identical results were obtained in all cases. However, one embryo which had only X-chromosome specific amplification by PCR was found to be XO in all its cells by FISH. Although multiplex PCR is demonstrated to be reliable for sexing of human embryos, FISH has the additional advantages of supplying ploidy assessment while not being affected by contamination

The purpose of this investigation was to determine the parental origin of the pronucleus furthest from the second polar body (the distal pronucleus) in dispermic human zygotes. Intact dispermic embryos (n = 53) and those from which the distal pronucleus (n = 50) was removed at the zygote stage were biopsied after cleavage. Blastomeres were sexed using either coamplification of X and Y probes using a duplex polymerase chain reaction (PCR), or simultaneous fluorescence in situ hybridisation (FISH) with directly fluorochrome-labelled probes for chromosomes X, Y and 18. The ratio X/Y was determined in both groups of embryos by assessing a minimum of two blastomeres. If the pronuclei in dispermic zygotes are topographically in a fixed position, the X/Y ratio should change from 1:3 in dispermic embryos to 1:1 in enucleated ones. The ratio of embryos containing only an X chromosome and those with X as well as Y chromosomes in the intact dispermic zygotes was 1.0:2.3 which is similar to the theoretical ratio of 1:3. This ratio was 1.0:1.3 in dispermic zygotes from which the distal pronuclei were removed. This ratio is not significantly different from the 1:1 ratio based on a statistical analysis with a sample size of 50. These sex ratios would have been considered different if more than 200 enucleations had been performed. Although the ratio X/Y was altered following removal of distal pronuclei, suggesting frequent targeting of male pronuclei, accidental removal of the female pronucleus could not be excluded. This indicates that enucleation of dispermic zygotes could produce high yields of gynogenetic and androgenetic embryos for research purposes. Clinical application aimed at producing biparental zygotes may be hazardous, since mosaicism was common among enucleated embryos

OBJECTIVE: To use fluorescence in situ hybridization to corroborate the polymerase chain reaction (PCR) preimplantation diagnosis of human embryos in three couples carrying a chromosome X-linked disease. SETTING: Clinical and research IVF laboratories. PATIENTS: Individuals undergoing preimplantation diagnosis. RESULTS: Four ETs were performed in couples undergoing preimplantation diagnosis by multiplex PCR or fluorescence in situ hybridization, resulting in the birth of two normal female twins. The result of another is pending. A total of 22 embryos were analyzed by PCR. Embryos that were diagnosed as being at risk of carrying the genetic abnormality (n = 8), embryos that failed diagnosis (n = 4), and genetically normal embryos that arrested development (n = 4) were further analyzed by fluorescence in situ hybridization. The sex of all 16 embryos was determined and confirmed the previous 12 preimplantation diagnoses by multiplex PCR. In addition, fluorescence in situ hybridization analysis allowed the detection of two aneuploid embryos, one XO and one XXY, previously diagnosed by PCR as a normal female and male. Two mosaics were also detected. CONCLUSION: Polymerase chain reaction and fluorescence in situ hybridization are possible for preimplantation sex determination in cases of genetic sex-linked disease. Fluorescence in situ hybridization, however, supplies additional information about sex chromosome aneuploidy and is not susceptible to contamination or misdiagnosis of monosomy X

A short fluorescence in-situ hybridization (FISH) procedure using fluorochrome and digoxigenin labelled DNA probes was developed for application in human preimplantation embryos in order to analyse the five chromosomes most involved in human aneuploidy (X, Y, 18, 13 and 21). The chromosomes were fluorescent-stained and detected simultaneously in 157 blastomeres from 30 human embryos. Successful FISH analysis was achieved in 93% of the blastomeres. Aberrations for these chromosomes were found in 70% of abnormally developing monospermic embryos. The majority of normally developing monospermic embryos obtained from older patients were also chromosomally abnormal. By analysing all or most of the cells from these embryos, true mosaicism was distinguished from technique failure. Mosaic embryos, polyploid embryos with ploidies as high as 8n, haploid embryos, embryos monosomic for 13/21 and for X, and embryos trisomic for 13/21 and 18, were common in abnormally developing embryos. In contrast, aneuploidy was the main chromosome abnormality found in normally developing monospermic embryos.

PURPOSE: Diploidy in embryos developing from single pronucleated zygotes can occur following parthenogenetic activation or by asynchronous inflation of pronuclei following fertilization. To distinguish between these two mechanisms, sexing was performed. RESULTS: The presence of a Y chromosome in the embryo was considered proof that fertilization had occurred. Twenty-one dividing embryos originating from single pronucleated zygotes were analyzed using the polymerase chain reaction or fluorescence in situ hybridization. In total, 43% (9/21) of the embryos showed Y chromosomes. CONCLUSION: It can be extrapolated that more than 80% of them originated from fertilized eggs

Evidence of sexual dimorphism before fetal gonadal differentiation in mammals has been accumulating, suggesting that male embryos develop faster than female ones. The current investigation was performed to evaluate whether the development rate of precompacted human embryos is controlled by sex chromosomes. Sex was determined by polymerase chain reaction and fluorescence in situ hybridisation in 172 arrested embryos derived from in vitro fertilisation. The sex ratio (1.02:0.98) did not differ significantly from 1:1. Although more males appeared to have greater fragmentation, the difference between the sex ratios of highly fragmented and normal embryos (1.08:0.92) was not significant. Arrested female embryos had a tendency to exhibit more than five nuclei and less than 10% fragmentation, but the trend was not statistically significant. The current results suggest that the first developmental block in human embryos occurs prior to and shortly after genomic activation and is not determined by the presence of the Y chromosome

A new technology called primer extension preamplification (PEP), which has been applied to single spermatozoa, increases the amount of polymerase chain reaction (PCR) templates by amplifying DNA of the whole genome. The current investigation was aimed at applying PEP to single human blastomeres. Two blastomeres with nuclei from arrested embryos were selected for this study. Using three different PEP protocols (experiments I, II and III), DNA from single blastomeres was amplified using 15-base oligonucleotide random primers. The efficiency of the procedure was determined by further amplifications of aliquots of the PEP products with two specific sequences. Three aliquots from each PEP product were used as PCR templates for the human X chromosome (X) or the exon 10 of the cystic fibrosis gene (CF). PCR amplified products were analysed by gel electrophoresis. In experiment I, when X primers were used, positive signals were detected in all 10 embryos (100%), 90.0% (18/20) of the blastomeres, and in 80.0% (96/120) of the replicates. When CF primers were amplified, all embryos (100%, 10/10), 90.9% (18/20) of the blastomeres and 78.3% (47/60) of the replicates were positive. In experiment II, efficiency was significantly reduced when total time for the procedure was minimized from 8 h to 5 h and 45 min. Although the time was further reduced to 4 h and 40 min in experiment III, the efficiency remained the same as in experiment I when the volume of PEP was reduced from 60 microliters (experiments I and II) to 40 microliters. One out of 132 control replicates (0.8%) was contaminated.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE: Our objective was to evaluate the presence of asymptomatic Chlamydia trachomatis infection by means of the polymerase chain reaction in male members of couples with previously undiagnosed infertility. STUDY DESIGN: Twenty-eight infertile-couples who had negative cultures or negative results when tested by deoxyribonucleic acid probe for Chlamydia trachomatis in semen and cervical samples were studied. Semen samples were tested for Chlamydia trachomatis by means of the polymerase chain reaction. Sera from both partners were diluted 1:128 and tested for immunoglobulin M antibodies to Chlamydia. Sera and ejaculated sperm were evaluated for the presence of antisperm antibodies. Semen analyses were also performed. RESULTS: Chlamydia trachomatis was identified in semen from 11 (39.3%) of the male partners. Its detection correlated with the presence in the ejaculate of motile sperm containing antisperm antibodies (p < 0.01). Either antisperm immunoglobulin G, immunoglobulin A, or both were located on sperm only from 5 (45.5%) of the 11 men whose results were positive when tested for Chlamydia trachomatis. Similarly, immunoglobulin G or immunoglobulin A antibodies to sperm were only detected in 5 (45.5%) of the spouses of men with Chlamydia in semen. Immunoglobulin M antibody to Chlamydia trachomatis was identified in only one of the men. However, antichlamydial immunoglobulin M antibodies were present in sera from 6 (54.5%) female partners of men with seminal Chlamydia trachomatis but in none of the other 17 women (p < 0.01). CONCLUSION: Although undetected by culture of deoxyribonucleic acid probe of semen samples, Chlamydia trachomatis was nevertheless identified in semen of some symptom-free men by the polymerase chain reaction. This is probably a result of the increased sensitivity of the polymerase chain reaction to detect Chlamydia trachomatis. The increased prevalence of an autoimmune response to sperm in men with this organism in their semen suggests that a subclinical chlamydial infection may activate an immune response to sperm. A similar association between Chlamydia trachomatis in semen and circulating antisperm antibodies in female partners indicates that Chlamydia may also induce an immune response to sperm in women. Infertility in these couples may be the result of a direct inflammatory response in the cervix or endometrium to repeated Chlamydia exposure or of the ability of Chlamydia to evoke an immune response to spermatozoa

Our objective was to evaluate and compare the efficacy and mechanisms of co-culturing mouse embryos with Vero cells in both Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12) and human tubal fluid (HTF) culture medium. Two-cell CB6F1 mouse embryos were cultured either in the presence of Vero cells (group A) or in culture medium alone (group B). In DMEM/F12 significantly more morulae developed in group A than in group B on day 3 (91 versus 23%; P less than 0.01). In contrast, the mouse embryos grew rapidly in HTF and significant differences were noted only in later embryonic stages (on day 5; 86% and 50%, P less than 0.01 of group A and B respectively, hatching or hatched). Similar experiments using DF1 and ICR mouse strains also revealed enhanced embryo development in the presence of Vero cells. To determine whether the embryo-enhancing effects of Vero cells were due to the removal of toxins or to the secretion of embryotrophic factors, ICR mouse embryos were cultured in fresh media with cells (group A), without cells (group B) and in cell-free conditions using cell-conditioned media which were obtained in the presence (group C) or absence (group D) of embryos. These results demonstrated that completion of hatching was highest (52%; P less than 0.01) in group A after 6 days in culture. There were no significant differences between groups B, C and D (rates of total hatching 18, 17 and 17%, respectively). It is concluded that Vero cells improve the development of mouse embryos and this is likely to be due to removal of substances inhibitory to development.(ABSTRACT TRUNCATED AT 250 WORDS)