Kenneth C. Drury

Results on single cell PCR for Huntington's gene and WAVE™ product analysis for preimplantation genetic diagnosis

Triple repeat base pair amplification is the basis for a number of prevalent genetic diseases such as Huntingtons, Fragile X, Myotonic Dystrophy and others. We have chosen to investigate the use of PCR to amplify a portion of the Huntingtons gene in single cells in order to develop a clinical test system for preimplantation genetic diagnosis (PGD). Amplification of CAG triple repeat sequences poses difficulties due to resistance of GC melting for amplification. Special PCR modifications are necessary to carry out the amplification of GC rich areas found in most triple base pair expansions. We have used a modified polymerase chain reaction (PCR) protocol to amplify the expanded repeat sequence of the Huntingtons gene with satisfactory efficiency. Detection of the amplified expanded CAG repeats is shown to be possible using both agarose gel electrophoresis and high definition denaturing high pressure liquid (DHPLC) chromatography. The incidence of allele dropout (ADO) is documented.

Primer System for Single Cell Detection of Double Mutation for Tay-Sachs Disease

AbstractPurpose: Nearly 100% of infantile Tay-Sachs disease isproduced by two mutations occurring in the alpha chain ofthe lysosomal enzyme beta-N-acetylhexosaminidase (HEXA)in the Ashkenazi Jewish population. Although others havedescribed primer systems used to amplify both sitessimultaneously, few discuss the allele dropout problems inherent inthis test. Our goal was to construct a more robust testenabling stronger signal generation for single cellpreimplantation genetic diagnosis and to investigate theoccurrence of allele dropout. Methods: New nested primers were designed to optimizedetection of both major Tay-Sachs mutations. Four hundredfifty-seven single cells, including normal cells and thosecarrying mutations of either the 4bp insertion exon 11 orsplice-site intron 12 defects, were used to screen a newprimer system. Results: Based on PCR amplified product analysis, totalefficiency of amplification was 85.3%, (390/457). The alleledropout rate for the 4bp insertion mutation in exon 11 andsplice-site mutation in intron 12 was 4.8% and 5.8%,respectively. Conclusons: Multiple mutation detection and analysiswithin the Tay-Sachs disease gene (HEXA) is possible usingsingle cells for clinical preimplantation genetic diagnosis.Alternative PCR primers and conditions offer variousmethods for developing systems compatible to specificprogram requirements.

Validity of a rapid assay for antisperm antibodies in semen

OBJECTIVE To determine the validity of a rapid assay for antisperm antibodies in semen. DESIGN Prospective comparison of the results of standard and rapid antisperm antibody assays performed simultaneously. SETTING Tertiary care infertility center. PATIENT(S) Couples who presented for infertility evaluation. INTERVENTION(S) Semen analysis and measurement of antisperm antibodies in semen using a standard and a rapid immunobead binding test (IBT). MAIN OUTCOME MEASURE(S) [1] Comparison of sperm parameters between semen-containing antisperm antibodies and semen free of antisperm antibodies. [2] Validation of the rapid test by calculation of sensitivity, specificity, positive and negative predictive values of the rapid assay using the standard assay as a gold standard. [3] Cost comparison of the standard and rapid test. RESULT(S) [1] Nine semen specimens with antisperm antibodies had a significantly lower sperm concentration, motility, and total motile fraction compared to 44 specimens without antisperm antibodies. Also, specimens with antisperm antibodies had a significantly higher percentage of vibratory sperm and percent of bound antisperm antibodies. The strict morphology, liquefaction time, semen volume, and white blood cell concentration were no different between the two groups. [2] Using a threshold of > or =12% of bound antisperm antibodies in the rapid assay, the sensitivity, specificity, positive and negative predictive values of the test are 100% when correlated with a threshold of > or =20% in the standard assay. Increasing the threshold in the standard assay decreases the specificity and positive predictive value of the rapid assay but not the sensitivity and the negative predictive value. [3] The cost of the rapid assay was 16% that of the standard test and its performance took 20% of the time it took to set and perform the standard test. CONCLUSION(S) A rapid test for antisperm antibodies is valid, reliable, and more cost and labor effective than a standard IBT.

Simultaneous Single-Cell Detection of Two Mutations for Cystic Fibrosis

AbstractPurpose: A single-cell diagnosis procedure using polymerase chain reaction (PCR) technology was developed to simultaneously detect two cystic fibrosis (CF) mutations (DF-508, W1282X). Methods: Human viable, arrested, and nonviable embryos and immature, and nonfertilized oocytes donated by our patients were used to detect apoptosis by Tunel labeling, annexin staining, and single-cell reverse transcriptase–polymerase chain reaction (RT-PCR). Results: Using cells carrying the DF-508 mutation, the PCR signal efficiency for the affected homozygous, normal homozygous, and carrier heterozygote cell populations were 91%, 81%, and 92%, respectively. The total combined PCR efficiency was 87.7% and the ADO rate was 5.7%. For W1282X carrier heterozygote cells, the PCR signal efficiency was 82.0% and the ADO rate was 8.7%. Conclusions: Methods have been developed to detect two common mutations simultaneously for CF in single-cell assays. The high signal efficiencies and low ADO rates obtained in these tests allow those embryos from couples wishing to avert the transmission of this serious genetic disease to their offspring to be screened by preimplantation genetic diagnosis.

ART and Its Impact on Male Infertility Management

Approximately 12–15% of sexually active couples are infertile. The etiology of infertility is likely multifactorial. Previous work has estimated that 50% of infertility is attributed to the female, 30% to the male, and 20% to both the male and female. Recent advances, mainly in the assisted reproductive technologies, have allowed some couples with severe male factor infertility to establish a pregnancy. The most significant advance is in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI). Previously, these couples had at best only a remote chance of establishing a pregnancy due to severely reduced sperm concentration, compromised sperm function, or uncorrectable obstruction. As a result of the high success of IVF/ICSI, and as a by-product of the fact that fertility visits are often initiated by the female partner, the practice of modern assisted reproductive technology (ART) can often times proceed without a complete evaluation of the male partner. Instead, almost any couple even those with a severe male factor, after being evaluated by only a reproductive endocrinologist, can theoretically become pregnant using IVF/ICSI.

Integrating Preimplantation Genetic Diagnosis into the ART Laboratory

Technology has become the driving force in the field of assisted reproductive infertility medicine. The ability to conduct in vitro fertilization, embryo micromanipulation, and cryopreservation allows the clinical embryologist to obtain an ever increasing ability to observe and test the viability of the human embryo in the laboratory. At the same time, molecular genetics and molecular biology have developed to the point of allowing the laboratory to detect and determine various aspects of the embryonic genome and knowledge of the detailed structure of human chromosomes. These technological abilities are now used not only to identify single gene mutations linked to specific diseases, but identify chromosomal abnormalities residing in the in vitro cultured human embryo as well. Selection of mutation-free and chromosomally normal embryos is now a reality that brings new hope to couples at risk for giving birth to affected offspring. There are also mounting indications that these technologies are also able to benefit couples facing other infertility issues such as failure to conceive during ART procedures. Ethical issues, concerning the use of these new technologies, have arisen from a number of quarters where it is troubling to think of, no less accept, the possible outcomes derived from their applications. One long sought after outcome, being requested by prospective parents, is to select the gender of the desired offspring for purely non-medical motives. Infertility programs throughout the United States and the world are now capable of utilizing these advanced technologies to make gender selection a reality for interested couples, as well as, enhance the ability to treat a variety of problems relating to infertility. Reproductive genetic programs may want to consider early on how they wish to utilize these technical abilities within their individual programs. There should be serious consideration of the pros and cons of developing these highly technical, time consuming and expensive procedures within a given facility. This chapter is designed to consider and help answer those questions.

Twelve-well culture plate for the efficient collection and culture of human oocytes and embryos

AbstractObjective: Our goal was to assess a 12-well oocyte collection and embryo culture plate for use in the IVF laboratory. Design: This was a prospective nonrandomized study. Setting: The setting was a university in vitro fertilization program. Patients: Eighty-four consecutive infertility couples presenting for IVF were studied. Main Outcome Measures: The main outcome measure was pregnancy (delivered). Results: A 34% delivered pregnancy rate per retrieval was attained using the 12-well collection and culture plate without the use of expensive culture media and special serum supplementation. Conclusions: The use of a 12-well plate for oocyte collection and embryo culture provides a simple, economical, efficient, and effective means of producing human embryos during in vitro fertilization. This system is capable of supporting high rates of ongoing and delivered pregnancies.