Direct comparison of detection systems used for the development of single-cell genetic tests in preimplantation genetic diagnosis.

PURPOSE: Single-cell polymerase chain reaction (PCR) requires efficient amplification and accurate detection. We compare the accuracy of heteroduplex, fluorescent-fragment, and fluorescent single-strand conformation polymorphism (F-SSCP) analysis as detection systems for analysis of a PCR assay developed for preimplantation genetic diagnosis. METHODS: A single-cell, fluorescent multiplex PCR assay was developed for the cystic fibrosis delta F508 mutation and the short tandem repeat, D21S11. Detection systems were compared by analyzing blinded PCR products. RESULTS: Amplification rates for cystic fibrosis were 89% by heteroduplex and 91% by fragment analysis, while it was 72% for D21S11 by fragment analysis. No difference in allele dropout was detected for cystic fibrosis by any method (2%). Overall accuracy was high, > 97%, although SSCP was the least accurate. CONCLUSIONS: Heteroduplex and fragment analysis proved equal in the diagnosis of a single amplified locus. We determined that fragment analysis allows maximal accuracy of detection and permits analysis of a second loci, controlling for DNA contamination and allelic dropout.

The development of preimplantation genetic diagnosis for myotonic dystrophy using multiplex fluorescent polymerase chain reaction and its clinical application.

Preimplantation genetic diagnoses (PGD) for single gene defects require considerable time and resources for the standardization of polymerase chain reactions that are rapid, sensitive and reliable. Developing tests for the trinucleotide repeat diseases, where the expansion of unstable repeats produces the phenotypes, are particularly complex. One of these disorders is myotonic dystrophy where, at present, diagnosis at the single cell level relies on the detection of the normal alleles from both the affected and unaffected parent. The incorporation of short tandem repeat polymorphisms in the assay can give additional information to improve the accuracy of diagnosis. We have developed a multiplex fluorescent reaction for myotonic dystrophy and one of two closely mapped, highly heterozygous, short tandem repeats (D19S219 and D19S559) on chromosome 19 to reduce the possibility of misdiagnosis due to contamination, act as a control for allelic drop-out and maximize the number of embryos genotyped. This protocol was designed as a general diagnosis for myotonic dystrophy, using the most informative of the two polymorphisms for each couple. Subsequently this approach was used in a PGD treatment cycle.

Impact of reducing the number of embryos transferred from three to two in women under the age of 35 who produced three or more high-quality embryos.

OBJECTIVE: To investigate the effect of a reduction in the number of good-quality embryos transferred in patients <35 years of age on pregnancy and multiple pregnancy rate. DESIGN: Prospective observational study with historical controls. SETTING: Academic tertiary referral unit. PATIENT(S): Three hundred eight patients <35 years of age undergoing IVF-ET. INTERVENTION(S): For patients who had three or more good quality embryos available for transfer, those in group 1 were given the option to have either two or three embryos replaced, whereas those in group 2 were allowed a maximum of two embryos transferred. In both groups, patients who had less than three good-quality embryos had the option to have three embryos transferred. MAIN OUTCOME MEASURE(S): Pregnancy and multiple pregnancy rates. RESULT(S): Patients in group 1, compared with those in group 2, had significantly more embryos (3 vs. 2) of significantly higher cumulative embryo score (31 vs. 24) transferred. This resulted in significantly higher multiple (57.8% vs. 30.8%) and triplet (15.6% vs. 1.4%) pregnancy rates in group 1. However, no difference in overall clinical pregnancy rate (37.2% vs. 41.2%) or live birth rate (28.1% vs. 29.4%) was observed between group 1 and 2. CONCLUSION(S): In women <35 years of age, who have three or more good-quality embryos available for transfer, a maximum of two embryos should generally be transferred.

In vitro fertilization and intracytoplasmic sperm injection pregnancies after successful transport of oocytes by airplane.

OBJECTIVE: To determine the feasibility of a transport IVF program involving air transportation of oocytes. DESIGN: Prospective cohort study. SETTING: Regional hospital (Hôpital de Chicoutimi) and University Infertility Center (McGill Reproductive Center, Montreal). PATIENT(S): The first series of patients referred for IVF or IVF and ICSI, for a variety of indications, who opted for inclusion in the transport IVF program. INTERVENTION(S): The IVF-ET with ovarian stimulation and oocyte collection at the peripheral unit and transport of the oocytes by airplane to the McGill Reproductive Center where IVF or ICSI was performed. MAIN OUTCOME MEASURE(S): Clinical pregnancy. RESULT(S): Seven couples, in the first series, underwent nine cycles of transport IVF treatment. Two also underwent ICSI. There were two clinical pregnancies. CONCLUSION(S): Transport IVF using air travel is possible and opens the possibility for this type of program to be implemented in large countries with scattered populations, such as the United States, Canada, and Australia.

Natural spermatoceles in irreversible obstructive azoospermia--reservoirs of viable spermatozoa for assisted conception.

Fine needle aspiration of asymptomatic spermatoceles detected in five men with obstructive azoospermia was evaluated as a minimally invasive method of sperm retrieval for assisted conception. Sperm preparations adequate for in-vitro fertilization were initially obtained from three men, but there was failure of fertilization in all three cycles. However, intracytoplasmic sperm injection (ICSI) in three couples resulted in fertilization of 58% of metaphase II oocytes, with embryo transfers in five of six cycles. A successful pregnancy and delivery resulted from spermatozoa from a man with an irreversible vasectomy, for whom previous epididymal microaspiration and ICSI had been unsuccessful. Spermatoceles were found in 4% of men with irreversible obstructive azoospermia referred for assisted conception. These patients should therefore be examined very carefully for small cysts near the epididymis, because fine needle aspiration, usually without anaesthesia, can instantly locate a ready source of viable spermatozoa for ICSI. This is less invasive than the alternative sperm retrieval procedures, which are more traumatic and require local or general anaesthesia.

Oocyte donation--implications for fertility treatment in the nineties.

In the past decade, oocyte donation has become a widely used assisted reproduction technique with reported pregnancy rates often higher than those for conventional in-vitro fertilization. Recipients of donated oocytes include women with premature ovarian failure or severe genetic disorders, those who respond poorly to ovarian hyperstimulation, women over 40 years of age, and, recently, postmenopausal women. The donation of oocytes to older women raises many medical and social issues which have to be addressed.

Bacterial colonization of ultrasonic nebulizers: implications for frequency of circuit changing.

UNLABELLED: We conducted a prospective study of 50 consecutive postoperative patients over a 5-month period to characterize the bacterial contamination of ultrasonic nebulizers (USNs) after 24, 48, and 72 hours (h) of use. METHODS: Samples of the USN effluent mist and reservoir fluid were cultured and the results were correlated with clinical data from patient records, especially indications of pneumonia or related infections. RESULTS: Two thirds of the USNs were bacteria-free at the three times they were tested. After 24 h, cultures of the mist revealed bacterial growth in only 1 of the 50 patients. After 48 and 72 h, cultures of the mist showed bacterial growth in 6 instances (12%). Colonization of the reservoir fluid was also limited to 1 patient after 24 h. Reservoir-fluid cultures were positive in 10% of the USNs at 48 h and in 18% at 72 h. No clinically significant signs of pneumonia developed in any patient. CONCLUSIONS: These results suggest that colonization of USNs is minimal during the first 72 h of use by postoperative patients with uncomplicated clinical courses and that clinical consequences are unlikely in such patients even when the USN is found to have been colonized. Although the Centers for Disease Control has recommended daily changing of USN circuits, this practice may not be warranted in all clinical settings. Further studies are needed to determine the colonization of USNs used by patients with more complicated clinical courses.

Mechanically assisted ventilation in a community hospital. Immediate outcome, hospital charges, and follow-up of patients.

In 100 consecutive patients undergoing mechanically assisted ventilation, we prospectively determined immediate survival, hospitalization charges, and subsequent one-year outcome. Sixty percent of the patients survived the episode of assisted ventilation. This survival decreased to 50% at the time of hospital discharge and to 33% one year after hospitalization. There were no posthospitalization deaths in patients less than age 50 years. In those patients older than 70 years, however, 51% were dead by the time of hospital discharge and 73% died by one year following discharge. Comparison of features that reflect the magnitude of intensive respiratory care, such as hours of ventilation and intensive care unit (ICU) length of stay, disclosed no statistically significant differences between survivors and nonsurvivors at discharge. Hospitalization charges averaged $10,968 per patient. The total charge for respiratory therapy services (including arterial blood gas determinations) averaged $2,200. Respiratory care service charges were only marginally different between survivors and nonsurvivors. By contrast, total charges and total length of stay were greater for the survivors. Resource use in the intensive care setting as reflected by hours of mechanical ventilation and ICU length of stay was similar in both survivors and nonsurvivors, with the larger total cost for survivors relating primarily to care outside the ICU. Also, we confirm that prognosis is excellent in patients less than 50 years of age who survive mechanically assisted ventilation for acute respiratory failure and that extubation in elderly patients is not necessarily indicative of a good prognosis.

A review of the hospital course of asthmatic children and adults.

We reviewed 82 consecutive hospital admitted patients treated for acute severe asthma over a seven-month period at a University medical center and an additional 25 patients at an affiliated community hospital. Those asthmatics treated on the adult medical service had a significantly longer length of hospitalization when compared to those treated on the pediatric service (t = 5.12; p less than 0.005). In addition, longer hospitalization periods were noted for those asthmatics who smoked (t = 2.98; p less than 0.005) and for those with a history of chronic bronchitis (t = 2.32; p less than 0.025). Drug regimens were frequently suboptimal; 30% of the patients reviewed were receiving no therapeutic agents prior to admission. Of those patients receiving theophylline 53% had serum levels of less than 10 mcg/ml on their post admission assessment. Although frequently suboptimal, inadequate drug regimens prior to admission did not lengthen hospital stay. Poor compliance with medications was recorded as a major cause of decompensation in nine patients whose mean age was 13 years. Finally, occupational exposure to airway irritants was elicited from 24% of the adult population at the University medical center. Increased length of stay in the hospital for adult asthmatics may reflect relatively fixed airway disease among these patients since a significant proportion of them related a history compatible with chronic bronchitis or were smokers. Occupational histories should be evaluated in adult asthmatics to rule out workplace exposures as a cause for severe decompensation. In addition, the importance of educational efforts addressing therapeutic non-compliance is evident from this study.