Anthony Rutherford

Allelic drop-out and preferential amplification in single cells and human blastomeres: implications for preimplantation diagnosis of sex and cystic fibrosis.

Abstract Previously the diagnosis of sex and cystic fibrosis status has been studied on single cells using the polymerase chain reaction (PCR). It has been suggested that allelic drop-out (PCR failure of one allele) and/or preferential amplification (hypo-amplification of one allele) may contribute to poor reliability and misdiagnosis, although this remains controversial as some reports suggest that allelic drop-out does not occur. We investigated an improved method of diagnosing sex and cystic fibrosis in single cells using a new technology (fluorescent PCR) to determine the base level of PCR artefacts (allelic drop-out and preferential amplification) which, in combination with improved sensitivity, should improve PCR reliability and accuracy. Fluorescent PCR gives high reliability (approximately 97%) and accuracy rates (approximately 97%) in somatic cells for both sex and cystic fibrosis diagnosis and its lower detection threshold allows allelic drop-out and preferential amplification to be easily distinguished. We also achieved high reliability and accuracy in diagnosing cystic fibrosis in human blastomeres. This study confirms earlier reports of both allelic drop-out and preferential amplification in single cell analysis. We demonstrate that both allelic drop-out and preferential amplification occur in somatic cells and suggest these are separate phenomena. Preferential amplification appeared common in single cell PCR while allelic drop-out apparently occurred at random in each allele. Preferential amplification was mainly amplification of the larger allele. We suggest that some inaccuracy/misdiagnosis may be due to both preferential amplification as well as allelic drop-out. Other findings were variability in drop-out between PCR and that amplification of signals from human blastomeres may be linked to embryo quality. We suggest that allelic drop-out is dependent on the number of cells within the sample.

Elective Single Embryo Transfer: Guidelines for Practice British Fertility Society and Association of Clinical Embryologists

Assisted conception treatment is the single most important cause in the increase in multiple pregnancy and births over the last 25 years. Multiple births are associated with significant peri natal morbidity and mortality. Europe has led the way in reducing multiple births by widespread adoption of an elective single embryo policy, which in Belgium is linked to an increase in state funding. Randomized controlled trials suggest that an eSET policy must include the ability to cryopreserve and transfer any remaining quality embryos to obtain parity with a double embryo transfer. This document provides a review of the available evidence with guidelines for practice, to help facilitate the introduction of an eSET policy in the UK.

Association between amino acid turnover and chromosome aneuploidy during human preimplantation embryo development in vitro

This study investigated the relationship between human preimplantation embryo metabolism and aneuploidy rates during development in vitro. One hundred and eighty-eight fresh and cryopreserved embryos from 59 patients (33.9 +/- 0.6 years) were cultured for 2-5 days. The turnover of 18 amino acids was measured in spent media by high-performance liquid chromatography. Embryos were either fixed for interphase fluorescent in situ hybridization analysis of chromosomes 13, 18, 19, 21, X or Y, or were assayed for mitochondrial activity. Amino acid turnover was different (P < 0.05) between stage-matched fresh and cryopreserved embryos due to blastomere loss following warming. The proportion of embryos with aneuploid cells increased as cell division progressed from pronucleate- (23%) to late cleavage stages (50-70%). Asparagine, glycine and valine turnover was significantly different between uniformly genetically normal and uniformly abnormal embryos on Days 2-3 of culture. By Days 3-4, the profiles of serine, leucine and lysine differed between uniformly euploid versus aneuploid embryos. Gender significantly (P < 0.05) affected the metabolism of tryptophan, leucine and asparagine by cleavage-stage embryos. Pronucleate zygotes had a significantly higher proportion of active:inactive mitochondria compared with cleavage-stage embryos. Furthermore, mitochondrial activity was correlated (P < 0.05) with altered aspartate and glutamine turnover. These results demonstrate the association between the metabolism, cytogenetic composition and health of human embryos in vitro.

Expression of genes involved in early cell fate decisions in human embryos and their regulation by growth factors

Little is understood about the regulation of gene expression in human preimplantation embryos. We set out to examine the expression in human preimplantation embryos of a number of genes known to be critical for early development of the murine embryo. The expression profile of these genes was analysed throughout preimplantation development and in response to growth factor (GF) stimulation. Developmental expression of a number of genes was similar to that seen in murine embryos (OCT3B/4, CDX2, NANOG). However, GATA6 is expressed throughout preimplantation development in the human. Embryos were cultured in IGF-I, leukaemia inhibitory factor (LIF) or heparin-binding EGF-like growth factor (HBEGF), all of which are known to stimulate the development of human embryos. Our data show that culture in HBEGF and LIF appears to facilitate human embryo expression of a number of genes: ERBB4 (LIF) and LIFR and DSC2 (HBEGF) while in the presence of HBEGF no blastocysts expressed EOMES and when cultured with LIF only two out of nine blastocysts expressed TBN. These data improve our knowledge of the similarities between human and murine embryos and the influence of GFs on human embryo gene expression. Results from this study will improve the understanding of cell fate decisions in early human embryos, which has important implications for both IVF treatment and the derivation of human embryonic stem cells.

Trends in the incidence of ectopic pregnancy in England and Wales from 1966 to 1996

Objective To examine the incidence of ectopic pregnancy over the period 1966 to 1996.

Fluorescent PCR: A new technique for PGD of sex and single-gene defects

AbstractBackground: For single-cell diagnosis, particularly preimplantation genetic diagnosis to be successful four main criteria must be achieved: sensitivity, reliability, accuracy, and identification/elimination of contamination. Methods and Results: Fluorescent PCR achieves all four necessary criteria and, in addition, currently allows genes on up to nine chromosomes to be simultaneously investigated. Fluorescent PCR has high sensitivity (∼1000× conventional analysis systems), high reliability (97%), and high accuracy (97%) rates for both sex and CF diagnosis in single somatic cells. The low detection threshold allows allelic dropout (one of the main causes of misdiagnosis) to be easily distinguished from PCR phenomena such as preferential amplification. High reliability (90%) and accuracy (97–100%) have been achieved in sex and CF diagnosis in human blastomeres. Fluorescent PCR can also be used to DNA fingerprint (STR profiling) single cells to identify the source/origin of the cell and determine if contamination has occurred. Conclusion: Fluorescent PCR is therefore a suitable method for PGD.

Comparison of effects of zona drilling by non-contact infrared laser or acid Tyrode's on the development of human biopsied embryos as revealed by blastomere viability, cytoskeletal analysis and molecular cytogenetics

Use of a non-contact infrared laser (IRL) or acid Tyrodes for zona drilling before embryo biopsy was compared by assessing blastomere viability using various fluorescent markers or culture of the single biopsied blastomere, and, by cytoskeletal and molecular cytogenetic analysis of the biopsied embryos following culture to the blastocyst stage. There was no significant difference in the proportion of biopsied embryos that showed no damage in both the biopsied blastomere and in the remaining embryo (acid Tyrodes: 75% versus IRL: 68%), or in the proportion of single biopsied blastomeres that divided in culture (P > 0.05). However, single biopsied blastomeres from laser drilled embryos showed a greater tendency to form miniblastocysts. The proportion of laser or acid Tyrodes biopsied embryos that reached the blastocyst stage by day 6 was similar, although evident earlier (day 5) in the laser biopsied embryos. Spindle abnormalities at the blastocyst stage included tripolar and tetrapolar spindles, but their incidence was not significantly different from controls. In addition, no significant difference was observed in the incidence of chromosomal abnormalities and mosaicism between the two groups. It is concluded that using an IRL at a safe working distance does not cause adverse immediate or longer term effects on the development of human biopsied embryos, although damage can occur if drilling within this distance is unavoidable. Acid Tyrodes drilling can also cause damage, and tended to retard blastocyst development.

Implementation of the NICE guideline – Recommendations from the British Fertility Society for national criteria for NHS funding of assisted conception

Assisted conception providers in England were surveyed to establish the uptake of NICE guideline for infertility particularly in respect of assisted conception and the criteria used to accept patients for NHS funded treatment. Detailed information on selection criteria was obtained from a group of commissioning consortia at an advanced stage in their arrangements. While there was an overall increase in the number of NHS IVF cycles purchased in England, implementation is stalled at one fresh cycle in the vast majority of Primary Care Trusts (PCTs). There is little consensus about the criteria used for acceptance into an NHS programme. This is particularly so in respect of social criteria which are often arbitrary and used as a rationing tool. This information complements that provided by the survey of Primary Care Trusts performed in March 2005 by the All Party Parliamentary Group on Infertility (APPGI) in partnership with the National Infertility Awareness Campaign (NIAC) which together provide a basis for recommendations for NHS funding. The recommendations presented should be applied across England and Wales to ensure consistency, fairness and equity of access.

Demonstration of a mechanism of aneuploidy in human oocytes using Multifluor fluorescence in situ hybridization

OBJECTIVE To evaluate the potential of Multifluor fluorescence in situ hybridization (M-FISH) for karyotyping the human oocyte and first polar body. DESIGN Prospective case study. SETTING Research laboratories, university hospital. PATIENT(S) A 33-year-old woman with polycystic ovary syndrome who was undergoing ovarian stimulation and ICSI. MAIN OUTCOME MEASURE(S) Karyotyping of all chromosomes within an oocyte and first polar body, using GV stage oocytes matured to metaphase II in vitro. RESULT(S) Oocyte hyperploidy was diagnosed by M-FISH to be 23, X +15 cht +19 cht +22 cht. The correspond- ing polar body was hypoploid, with a karyotype of 23, X -15 cht -19 cht -22 cht. This was due to unbalanced predivision at meiosis I. Reprobing confirmed karyotype assignments for chromosomes X, 13, 18, and 21. CONCLUSION(S) The mechanism involved in maternally derived aneuploidy can be defined by using M-FISH to simultaneously karyotype both oocyte and first polar body chromosomes at metaphase II. Multifluor FISH may be useful for investigative studies of maternally derived aneuploidy, which is a major cause of preimplantation waste in natural and assisted reproduction.

Managing anovulatory infertility and polycystic ovary syndrome

In this second overview of the current management of infertility we discuss anovulatory infertility and polycystic ovary syndrome. This syndrome (formerly known as Stein-Leventhal syndrome) is the most common hormonal disturbance in women—around one fifth of women in the United Kingdom are affected. It is also the most common reason for women not to ovulate, and the combination of being overweight and having polycystic ovary syndrome can have a profound effect on reproductive health. #### Summary points We referred to the Cochrane database of systematic reviews, The National Institute for Health and Clinical Excellence (NICE) guidelines for the investigation and management of infertility (2004), and our knowledge of the current literature. Anovulation is the cause of infertility in about a third of couples attending infertility clinics, and polycystic ovary syndrome accounts for 90% of such cases.1 Once tests have excluded other causes of androgen excess and menstrual disturbance, the syndrome can be confirmed by the presence of two of the following criteria— biochemical or clinical hyperandrogenism (hirsutism, acne, or alopecia); menstrual irregularity; and polycystic ovaries (figure⇓).2 Symptoms, signs, and biochemical features vary greatly among affected women and may change over time in individual women.3 This review will concentrate on the management of infertility. The general practitioner should be able to start investigations and formulate a diagnosis before referral to a specialist in reproductive medicine. Three dimensional ultrasound scan of a polycystic ovary (centre) showing multiple cysts …