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Infections, Infertility, and AssistedReproductionAssisted reproductive technology (ART) treatmentis vulnerable to the hazard of potential infectionfrom many different sources: patients, samples,staff, and the environment. Culture of gametes andembryos in vitro provides multiple targets fortransmission of potential infection, including thedeveloping embryo, neighbouring gametes andembryos, the couple undergoing treatment, andother couples being treated during the same period.This unique situation, with multifaceted opportuni-ties for microbial growth and transmission, makesinfection and contamination control absolutelycrucial in the practice of assisted reproduction, andin the laboratory in particular. This unique and practical book provides a basicoverview of microbiology in the context of ART,providing an up-to-date guide to infections inreproductive medicine. The relevant facets of thecomplex and vast ﬁeld of microbiology arecondensed and focused, highlighting informationthat is crucial for safe practice in both clinicaland laboratory aspects of ART. This is an essentialpublication for all ART clinics and laboratories.Kay Elder is Director of Continuing Education atBourn Hall Clinic, Bourn, Cambridge, UK.Doris J. Baker is Chair and Professor, Departmentof Clinical Sciences at the University of Kentucky.Julie A. Ribes is Associate Professor of Pathologyand Laboratory Medicine at the University ofKentucky.
Infections,Infertility, andAssistedReproductionKay Elder, M.B., Ch.B., Ph.D.Director of Continuing Education, Bourn Hall Clinic,Cambridge, UKDoris J. Baker, Ph.D.Professor and Chair, Department of Clinical Sciences andDirector of Graduate Programs in Reproductive LaboratoryScience, University of Kentucky, Lexington, KY, USAJulie A. Ribes, M.D., Ph.D.Associate Professor of Pathology and Laboratory Medicine, andDirector of Clinical Microbiology, University of Kentucky,Lexington, KY, USA
To: our families,Robbie and BethanyJohn and JustinPaul and Maxwell JamesWith love and thanks for their patience,tolerance, and support.
ContentsForeword page xiPreface xiiiAcknowledgements xviiPart I Overview of microbiology1 Introduction 3 History of microbiology 3 History of assisted reproduction 7 Artiﬁcial insemination 7 In vitro fertilization 8 Assisted reproductive technology (ART) and microbiology 10 Overview of microbiology 11 References 14 Further reading 15 Appendix: glossary of terms 162 Bacteriology 21 Structure and function of bacteria 21 Bacterial structure 21 Bacterial growth 26 Bacterial metabolism 26 Bacterial classiﬁcation and identiﬁcation 27 Nomenclature 27 Identiﬁcation of bacteria 27 Major groups of organisms 38 Gram-negative bacilli and coccobacilli 38 Gram-negative cocci 42 Gram-positive cocci that are catalase positive 42 vii
viii Contents Gram-positive cocci that are 4 Virology 105 catalase-negative 43 Introduction 105 Gram-positive bacilli that are Virus structure 105 non-branching and catalase Host range and speciﬁcity 106 positive 44 Viral replication 106 Gram-positive bacilli that are Growth characteristics 106 non-branching and catalase Lytic growth 107 negative 46 Lysogenic growth 107 Gram-positive bacilli that are Latent infections 107 branching or partially Virus classiﬁcation 107 acid-fast 47 Double-stranded DNA 107 Anaerobic bacteria 47 Single-stranded DNA 109 Mycobacteria and bacteria with Double-stranded RNA 109 unusual growth requirements 50 Single-stranded RNA 109 Normal ﬂora in humans 54 Single-stranded (+) sense RNA Further reading 61 with DNA intermediate 109 Appendix 62 Double-stranded DNA with RNA 2.1 Media used for isolation of intermediate 109 bacteria 62 Laboratory diagnosis of viral 2.2 Biochemical tests for disease 110 identiﬁcation of bacteria 67 Direct examination 110 2.3 Antibacterial agents 85 Culture 110 Further reading for Appendix 2.3 89 Antigen detection systems 110 Serologic diagnosis 111 3 Mycology: moulds and yeasts 90 Molecular diagnostics 111 Introduction 90 Viruses directly relevant to ART 111 Classes of fungi 91 Double-stranded DNA Zygomycetes 92 viruses 112 Ascomycetes 92 Hepatitis viruses 115 Basidiomycetes 92 Retroviruses 116 Deuteromycetes 92 Human oncornaviruses 117 Laboratory classiﬁcation of Further reading 118 fungi 92 Appendix: antiviral agents 119 Taxonomic classiﬁcation 92 5 Prions 122 Clinical classiﬁcation of fungi 94 Prion protein 122 Infections 94 Prion diseases 122 Contaminants 98 Animal 122 Laboratory identiﬁcation of fungi 99 Human 122 Direct examination 99 Prion structure 122 Culture 99 Replication 124 Microscopic examination for Transmission 124 fungal structures 100 Clinical presentation 126 Mycology in ART 100 Sporadic Creutzfeldt–Jakob Further reading 100 disease (nvCJD) 126 Appendix: antifungal agents 102
x Contents Further reading 259 Treatment of HBV seropositive couples 336 References 259 Treatment of HCV seropositive couples 336 Treatment of HIV seropositive 12 Cytomegalovirus and couples 337 blood-borne viruses 262 Semen washing procedures for Cytomegalovirus (CMV) 262 HBV/HCV/HIV serodiscordant Hepatitis B virus (HBV) 270 couples 339 Hepatitis C virus (HCV) 275 Virus decontamination 340 Hepatitis D virus (HDV) 278 Accidental exposure 340 HIV and AIDS 281 HBV prophylaxis 341 Human T-lymphotrophic viruses HCV prophylaxis 341 (HTLV) 290 HIV prophylaxis 341 HTLV-I 291 Air transport of biohazardous HTLV-II 291 materials 342 References 293 Useful addresses for air transport of Further reading 293 hazardous materials 349 Appendix to Part II: specimen culture by Appendix: general laboratory safety body site 299 issues 350 References 350 Part III Infection and the assisted Further reading 351 reproductive laboratory 15 Prevention: patient screening 13 Infection and contamination and the use of donor gametes 353 control in the ART laboratory 305 Routine screening 353 Sources of infection 305 Prevalence of BBV: geographic Sterilization methods 316 distribution 353 Physical methods of sterilization 316 The use of donor gametes 353 Chemical methods of Recruitment of donors 355 sterilization 318 Screening 356 Disinfection and decontamination 320 Procedures and technical Air quality, classiﬁcation of cleanrooms aspects 357 and biological safety cabinets 321 Use of gametes for donation 357 Biological safety cabinets (BSCs) 322 Treatment evaluation 358 Microbiological testing and Summary of donor testing contamination 325 practices and proposals in the Fungal contamination in the laboratory 325 USA 358 Laboratory cleaning schedules 327 Cryopreservation and transmission of References 330 infection 358 Further reading 331 Tissue banking: ovarian and testicular tissue 361 14 Handling infectious agents in References 362 the ART laboratory 332 Further reading 363 Blood-borne viruses 332 Biosafety levels 333 Biosafety for ART 334 Index 365
ForewordRoger G. GosdenThe Jones Institute for Reproductive MedicineNorfolk, VA.Lucinda L. VeeckWeill Medical College of Cornell UniversityNew York, NYWolbachia are gram-negative, intracellular bacte-ria that shelter in the gonads of invertebrates, andhave profound effects on the fertility of their hosts.In some species, infected hosts can only reproduceparthenogenetically, in others cytoplasmic incom-patibility prevents infected males from breedingwith uninfected females, and in some cases geneti-cally determined male embryos are transformed intofemales. Wolbachia engineers effects, as do all par-asites, for selﬁsh ends. Although this bizarre pathol-ogy is unknown in medical science, the relationshipsbetween microbes and human fertility are nonethe-less complex, fascinating and important for the prac-tice of reproductive medicine. Unfortunately, and usually without advance warn-ing, microbes occasionally enter the clinical lab-oratory through infected semen or vaginal tissue.When this occurs, a patient’s treatment outcome maybe seriously compromised because microbes canquickly deplete nutrients in culture media and alterthe pH, and it would be irresponsible to knowinglytransfer an infected embryo or semen to a patient.Bacterial and fungal growth are often obvious andeasily tested, but how often do infectious agentsgo unrecognized and contribute to the problems ofinfertility, treatment failure and even possibly affectthe child-to-be? This is the ﬁrst book on medical microbiologythat has been written by experts in reproduction forclinical scientists and physicians in their own ﬁeld.They are to be congratulated on ﬁlling a gap in the xi
xii Foreword literature between microbiology and assisted repro- In the ﬁnal section, the practical implications of this duction, which they achieve in three sections. The knowledge are addressed in the context of infertility, ﬁrst serves as a primer of medical microbiology for and especially the setting of the clinical embryology readers who are unfamiliar or rusty on the subject. laboratory. Every embryologist is trained in sterile The second focuses on microbes that have implica- techniques, ﬁltration of media and prudent use of tions for human reproduction, whether by causing antibiotics to keep out the bugs, but a deeper knowl- infertility (a familiar example being Chlamydia) or edge of the foundations of safe and effective practice by jeopardizing reproductive safety (such as HIV). is an undervalued safeguard for patient care.
PrefaceThe world of microbes is intrinsically fascinating.Microbes are abundant in every place on earth wherelarger living creatures exist, and they can also thrivein habitat extremes where no other kind of organ-ism can survive for long: from deep under the sea tothe stratosphere – up to 32 km in the atmosphere,in oil formations and in hot telluric water. It is esti-mated that the total biomass of microbes probablyexceeds that of all the plants and animals in the bio-sphere. This biomass is predominantly composedof bacteria, and these microorganisms play a cru-cial role in recycling much of the organic materialin the biosphere. Despite their minute size, microor-ganisms carry out all the fundamental processes ofbiochemistry and molecular biology that are essen-tial to the survival of all living species. Although theirsize may give them the illusion of being ‘primitive’,their range of biochemical and biophysical capabil-ities is far wider than that of higher organisms. Oneof their most important properties is adaptabilityand versatility, a key feature in their long history ofevolution. Fossil records suggest that at least somemembers of the microbial world, oxygen-producingcyanobacter-like organisms, had evolved 3.46 billionyears ago (Schopf, 1993); a viable fungus, Absidiacorymbifera, was recovered from the right boot thataccompanied the frozen, well-preserved prehistoriccorpse, ‘Ice Man’, aged approximately 5300 years(Haselwandter & Ebner, 1994). Records of microbial disease that probably inﬂu-enced the course of history can be found in archae-ological sites of early civilizations, as well as in later xiii
xiv Preface periods of history. A hieroglyph from the capital of with the help of microscopy. Culture of microor- ancient Egypt dated approximately 3700 BC illus- ganisms and of preimplantation embryos in vitro trates a priest (Ruma) with typical clinical signs of requires special media and growth conditions to a viral infection, paralytic poliomyelitis. The mum- promote cell division, and both are visualized and miﬁed body of the Pharaoh Siptah, who died in 1193 assessed at various stages following cell division. A BC, also shows signs of classic paralytic poliomyelitis, knowledge of microbiology is fundamental to the and the preserved mummy of Rameses V has facial safety and success of assisted reproductive tech- pustular lesions suggesting that his death in 1143 BC niques – but the ﬁeld of microbiology is vast, and con- was probably due to smallpox. This virulent disease tinues to increase in complexity with the discovery was endemic in China by 1000 BC, and had reached of new organisms and implementation of new med- Europe by 710 AD. Hernando Cortez transferred the ical treatments. The ﬁeld of assisted reproductive disease to the Americas in 1520, and it appears that technology also continues to expand and develop, around 3 500 000 Aztecs died of smallpox within the particularly in areas of science and biotechnology. next two years – arguably precipitating the end of the Members of an assisted reproduction team are not Aztec empire. usually also experts in infectious diseases, and may In the early 1330s an outbreak of deadly Bubonic ﬁnd it difﬁcult to identify and follow signiﬁcant plague occurred in China, one of the busiest of the areas of microbiology that can impact upon their world’s trading nations, and rapidly spread to West- practice. ern Asia and Europe. Between 1347 and 1352 this The purpose of this book is to select areas and top- plague, ‘The Black Death’, killed 25 million people – ics in microbiology that are speciﬁcally relevant to one-third of the population of Europe – with assisted reproductive technology (ART), in order to far-reaching social, cultural and economic repercus- provide a very basic background of facts and funda- sions. mental principles. A background of understanding can help prevent contamination and transmission The world of assisted reproduction is equally fas- of disease in ART, and also limit the opportunities cinating, and is one that also has a long history of for microbial survival in embryo culture and cryop- evolution. The concept of assisted procreation by reservation systems. The book is divided into three human artiﬁcial insemination was a topic of dis- Parts: cussion between Jewish philosophers as early as the third century AD, and tales exist of fourteenth- Part I provides an outline of microorganism century Arab horse breeders obtaining sperm from classiﬁcation and identiﬁcation, as a foundation mated mares belonging to rival groups, using the for understanding the relationships and the sperm to inseminate their own mares. Assisted differences between the types of organisms that reproduction explores the fundamental principles may be encountered in routine ART practice. behind the creation of a new life, the intricate bio- The microorganisms that are human pathogens logical mechanisms that are involved when mature or resident ﬂora, and those that are routinely gametes come into contact, combine genetically found in the environment are introduced. Each and set in motion a cascade of events leading to chapter includes an Appendix of antimicrobial the correct expression of genes that form a new drugs and their modes of action. individual. Part II details organisms that cause disease of Microbiology and assisted reproduction both deal the reproductive tract and those that are with a miniature world, magniﬁed for observation blood-borne pathogens, describing their
Preface xvetiology, pathogenesis, diagnosis, pathology and REFERENCEStreatment. Haselwandter, K. & Ebner, M. R. (1994). Microorganisms sur-Part III describes the practical application of viving for 5300 years. FEMS Microbiology Letters, 116(2),microbiology principles within an assisted 189–93.reproduction laboratory. Schopf, J. W. (1993). Microfossils of the early Archean apex chart: new evidence of the antiquity of life. Science, 260, 640–6.
AcknowledgementsDigital images for illustrations were produced withthe expert help of Stephen Welch and Robbie Hughes.We would like to thank all of our colleagues andfriends for their valuable encouragement, input andcomments throughout the preparation of this book,with particular acknowledgement of the contribu-tions made by Marc van den Berg, Charles Cornwell,Rajvi Mehta, Rita Basuray, George Kalantzopoulos,Dimitra Kaftani and Kim Campbell. Special thanksto Professor Bob Edwards for his personal reﬂectionson the ‘History of IVF’ and to Alan Smith for his per-spectives on the development of biotechnology. Barbara and Janet – thank you for your endlesspatience and moral support. We are also grateful for the support of Bourn HallClinic, Cambridge, and the Departments of ClinicalSciences and Pathology and Laboratory Medicine,University of Kentucky, and the University ofKentucky Clinical Microbiology Laboratory. xvii
1 IntroductionHistory of microbiology pike does through the water. The second sort . . . oft-times spun round like a top . . . and these were far more inThe history of microbiology, the scientiﬁc study of number . . . there were an unbelievably great company ofmicroorganisms, had its origins in the second half of living animalcules, a-swimming more nimbly than any I had ever seen up to this time. The biggest sort . . . bent their bodythe seventeenth century, when Anton van Leeuwen- into curves in going forwards . . . Moreover, the otherhoek (1632–1723), a tradesman in Delft, Holland, animalcules were in such enormous numbers, that all thelearned to grind lenses in order to make microscopes water . . . seemed to be alive.that would allow him to magnify and observe a widerange of materials and objects. Although he had no His Letters to the Royal Society also included descrip-formal education and no knowledge of the scientiﬁc tions of free-living and parasitic protozoa, spermdogma of the day, his skill, diligence and open mind cells, blood cells, microscopic nematodes, and aled him to make some of the most important discov- great deal more. In a Letter of June 12, 1716, he wrote:eries in the history of biology. He made simple pow- . . . my work, which I’ve done for a long time, was noterful magnifying glasses, and with careful attention pursued in order to gain the praise I now enjoy, but chieﬂyto lighting and detail, built microscopes that mag- from a craving after knowledge, which I notice resides in meniﬁed over 200 times. These instruments allowed more than in most other men. And therewithal, whenever Ihim to view clear and bright images that he studied found out anything remarkable, I have thought it my duty toand described in meticulous detail. In 1673 he began put down my discovery on paper, so that all ingenious people might be informed thereof.writing letters to the newly formed Royal Society ofLondon, describing what he had seen with his micro- At this time, around the turn of the eighteenth cen-scopes. He corresponded with the Royal Society for tury, diseases were thought to arise by ‘spontaneousthe next 50 years; his letters, written in Dutch, were generation’, although it was recognised that cer-translated into English or Latin and printed in the tain clinically deﬁnable illnesses apparently did notPhilosophical Transactions of the Royal Society. These have second or further recurrences. The ancient Chi-letters include the ﬁrst descriptions of living bacte- nese practice of preventing severe natural small-ria ever recorded, taken from the plaque between his pox by ‘variolation’, inoculating pus from smallpoxteeth, and from two ladies and two old men who had patients into a scratch on the forearm, was intro-never cleaned their teeth: duced into Europe in the early 1800s. The English farmer, Benjamin Justy, observed that milkmaids I then most always saw, with great wonder, that in the said who were exposed to cowpox did not develop small- matter there were many very little living animalcules, very pox, and he inoculated his family with cowpox pus to prettily a-moving. The biggest sort . . . had a very strong and prevent smallpox. Long before viruses had been rec- swift motion, and shot through the water (or spittle) like a ognized as an entity, and with no knowledge of their 3
4 Introduction properties, the physician Edward Jenner (1749–1823) bacteria on solid media such as sterile slices of potato was intrigued by this observation, and started the and on agar kept in the recently invented Petri dish. ﬁrst scientiﬁc investigations of smallpox prevention In 1892 he described the conditions, known as Koch’s by human experimentation in 1796. Jenner used Postulates, which must be satisﬁed in order to deﬁne ﬂuid from cowpox pustules on the hand of a milk- particular bacteria as a cause of a speciﬁc disease: maid to inoculate the 8-year-old son of his gar- (i) The agent must be present in every case of the dener, and later challenged the boy by deliberately disease. inoculating him with material from a real case of (ii) The agent must be isolated from the host and smallpox. The boy did not become infected, having grown in vitro. apparently developed an immunity to smallpox from (iii) The disease must be reproduced when a pure the cowpox vaccination. Jenner’s early experiments culture of the agent is inoculated into a healthy led to the development of vaccination as protection susceptible host. against infectious disease and laid the foundations (iv) The same agent must be recovered once again for the science of immunology, which was further from the experimentally infected host. developed during the nineteenth century by Pasteur, Koch’s further signiﬁcant contributions to microbi- Koch, von Behring and Erlich. ology included work on the tubercle bacillus and The nineteenth century was a ‘Golden Age’ in the identifying Vibrio as a cause of cholera, as well as history of microbiology. The Hungarian doctor, Ignaz work in India and Africa on malaria, plague, typhus, Semmelweiss (1818–1865), observed that puerperal trypanosomiasis and tickborne spirochaete infec- fever often occurred when doctors went directly from tions. He was awarded the Nobel Prize for Physiology the post- mortem room to the delivery room, and sel- or Medicine in 1905, and continued his work on bac- dom occurred when midwives carried out deliveries. teriology and serology until his death in 1910. He thus introduced the notion that infectious agents During the 1800s, agents that caused diseases might be transmitted, and suggested hand washing were being classiﬁed as ﬁlterable – small enough with chlorinated lime water. Signiﬁcant discoveries to pass through a ceramic ﬁlter (named ‘virus’ about bacteria and the nature of disease were then by Pasteur, from the Latin for ‘poison’) – or non- made by Louis Pasteur, Joseph Lister, Paul Ehrlich, ﬁlterable, retained on the surface of the ﬁlter (bac- Christian Gram, R. J. Petri, Robert Koch (1843–1910) teria). Towards the end of the century, a Russian and others. Louis Pasteur and Robert Koch together botanist, Dmitri Iwanowski, recognized an agent developed the ‘germ theory of disease’, disproving (tobacco mosaic virus) that could transmit disease the ‘spontaneous generation’ theory held at the time. to other plants after passage through ceramic ﬁlters Louis Pasteur (1822–1895) developed the scientiﬁc ﬁne enough to retain the smallest known bacteria. basis for Jenner’s experimental approach to vaccina- In 1898 Martinus Beijerinick conﬁrmed and devel- tion, and produced useful animal and human vac- oped Iwanowski’s observations, and was the ﬁrst to cines against rabies, anthrax and cholera. In 1876 describe a virus as contagium vivum ﬂuidum (solu- Robert Koch provided the ﬁrst proof for the ‘germ ble living germ). In 1908 Karl Landsteiner and Erwin theory’ with his discovery of Bacillus anthracis as Popper proved that poliomyelitis was caused by a the cause of anthrax. Using blood from infected ani- virus, and shortly thereafter (1915–1917), Frederick mals, he obtained pure cultures of the bacilli by grow- Twort and Felix d’Herrelle independently described ing them on the aqueous humour of an ox’s eye. viruses that infect bacteria, naming them ‘bacterio- He observed that under unfavourable conditions phages’. These early discoveries laid the foundation the bacilli could form rounded spores that resisted for further studies about the properties of bacteria adverse conditions, and these began to grow again and viruses, and, more signiﬁcantly, about cell genet- as bacilli when suitable conditions were restored. ics and the transfer of genetic information between Koch went on to invent new methods of cultivating cells. In the 1930s, poliovirus was grown in cultured
History of microbiology 5cells, opening up the ﬁeld of diagnostic virology. By drug sales in excess of 10 billion US dollars. The ﬁrstthe 1950s, plasmids were recognized as extranuclear genetically engineered human protein, insulin, wasgenetic elements that replicate autonomously, and available by 1982, and the ﬁrst complete genomeJoshua Lederberg and Norton Zinder reported on sequence of a bacterium, Haemophilus inﬂuenzae,transfer of genetic information by viruses (Zinder & was published in 1995. Hormones and other proteinsLederberg, 1952). manufactured by recombinant DNA technology are Following the announcement of the DNA double now used routinely to treat a variety of diseases, andhelix structure by Watson and Crick in 1953, the prop- recombinant follicle stimulating hormone (FSH),erties of bacteria, bacteriophages and animal viruses luteinizing hormone (LH) and human chorionicwere fully exploited and formed the basis of a new gonadotrophin (hCG) are available for routine usescientiﬁc discipline: molecular biology, the study of in assisted reproductive technology.cell metabolic regulation and its genetic machinery. A parallel line of investigation that was also a keyOver the next 20 years, Escherichia coli and other feature in molecular biology and medicine duringbacterial cell-free systems were used to elucidate the the latter part of the twentieth century came frommolecular steps and mechanisms involved in DNA the study of retroviruses, novel viruses that requirereplication, transcription and translation, and pro- reverse transcription of RNA into DNA for their repli-tein synthesis, assembly and transport. The devel- cation. During the 1960s, Howard Temin and Davidopment of vaccines and antimicrobial drugs began Baltimore independently discovered viral reverseduring the 1950s, and antibiotic resistance that could transcription, and in 1969 Huebner and Todaro pro-be transferred between strains of bacteria was iden- posed the viral oncogene hypothesis, subsequentlytiﬁed by 1959 (Ochiai et al., 1959). In 1967, Thomas expanded and conﬁrmed by Bishop and Varmus inBrock identiﬁed a thermophilic bacterium Thermus 1976. They identiﬁed oncogenes from Rous sarcomaacquaticus; 20 years later, a heat-stable DNA poly- virus that are also present in cells of normal animals,merase was isolated from this bacterium and used including humans. Proto-oncogenes are apparentlyin the polymerase chain reaction (PCR) as a means essential for normal development, but can becomeof amplifying nucleic acids (Brock, 1967; Saiki et al., cancer-causing oncogenes when cellular regulators1988). Another signiﬁcant advance in molecular bio- are damaged or modiﬁed. Bishop and Varmus werelogy came with the recognition that bacteria produce awarded the Nobel Prize for Medicine or Physiol-restriction endonuclease enzymes that cut DNA at ogy in 1989. In 1983, Luc Montagnier discovered aspeciﬁc sites, and in 1972 Paul Berg constructed a retrovirus believed to cause the acquired immunerecombinant DNA molecule from viral and bacterial deﬁciency syndrome (AIDS) – the human immun-DNA using such enzymes (Jackson et al., 1972). The odeﬁciency virus (HIV). By the end of the twentiethconcept of gene splicing was reported by 1977, and in century, the total number of people affected by thisthat same year Frederick Sanger and his colleagues novel virus exceeded 36 million.elucidated the complete nucleotide sequence of the Around this same time, another novel pathogen ofbacteriophage X174, the ﬁrst microorganism to a type not previously described also came to light:have its genome sequenced (Sanger et al., 1977). in 1982 Stanley Prusiner discovered that scrapie, aBerg, Gilbert and Sanger were awarded the Nobel transmissible spongiform encephalopathy (TSE) inPrize for Chemistry in 1980. sheep, could be transmitted by a particle that was These discoveries involving microorganisms apparently composed of protein alone, with no asso-established the foundation for genetic engineer- ciated nucleic acid – the prion protein (Prusiner,ing. Gene cloning and modiﬁcation, recombinant 1982). This was the ﬁrst time that an agent with nei-DNA technology and DNA sequencing established ther DNA or RNA had been recognized as pathogenic,biotechnology as a new commercial enterprise: by challenging previous dogmas about disease patho-2002 the biotechnology industry had worldwide genesis and transmission.
6 Introduction The ﬁeld of microbiology continues to grow and vectors, animal reservoirs, or environmental elicit public concern, both in terms of disease pathol- sources of novel pathogens, e.g. prions. ogy and in harnessing the properties of microbes for (iv) Modern air transportation allows large numbers the study of science, especially molecular genetics. of people, and hence infectious disease, to travel During the past 25–30 years, approximately 30 new worldwide with hitherto unprecedented speed. pathogens have been identiﬁed, including HIV, hem- Other areas that can contribute to pathogen emer- orrhagic viruses such as Ebola, transfusion-related gence include events in society such as war, civil hepatitis C-like viruses, and, most recently, the coro- conﬂict, population growth and migration, as well navirus causing sudden acute respiratory syndrome as globalization of food supplies, with changes (SARS). The ﬁrst SARS outbreak occurred in the in food processing and packaging. Environmental Guangdong province of China in November 2002 changes with deforestation/reforestation, changes and had spread as a major life-threatening penumo- in water ecosystems, ﬂood, drought, famine, and nia in several countries by March 2003. The infec- global warming can signiﬁcantly alter habitats and tious agent was identiﬁed during that month, and exert evolutionary pressures for microbial adapta- a massive international collaborative effort resulted tion and change. Human behaviour, including sex- in elucidating its complete genome sequence only ual behaviour, drug use, travel, diet, and even use 3 weeks later, in mid-April 2003. The genome of child-care facilities have contributed to the trans- sequence reveals that the SARS virus is a novel class mission of infectious diseases. The use of new med- of coronavirus, rather than a recent mutant of the ical devices and invasive procedures, organ or tis- known varieties that cause mild upper respiratory sue transplantation, widespread use of antibiotics illness in humans and a variety of diseases in other and drugs causing immunosuppression have also animals. Information deduced from the genome been instrumental in the emergence of illness due sequence can form the basis for developing targeted to opportunistic pathogens: normal microbial ﬂora antiviral drugs and vaccines, and can help develop such as Staphylococcus epidermidis cause infec- diagnostic tests to speed efforts in preventing the tions on artiﬁcial heart valves, and saprophytic fungi global epidemic of SARS. At the beginning of June cause serious infection in immunocompromised 2003, 6 months after the ﬁrst recorded case, the World patients. Health Organization reported 8464 cases from more Microorganisms can restructure their genomes in than two dozen countries, resulting in 799 deaths. response to environmental pressures, and during These new diseases are now being deﬁned within a replication there is an opportunity for recombina- context of ‘emergent viruses’, and it is clear that new tion or re-assortment of genes, as well as recombi- infectious diseases may arise from a combination of nation with host cell genetic elements. Some viruses different factors that prevail in modern society: (e.g. HIV) evolve continuously, with a high frequency (i) New infectious diseases can emerge from ge- of mutation during replication. Retroviruses are netic changes in existing organisms (e.g. SARS changing extraordinarily rapidly, evolving sporadi- ‘jumped’ from animal hosts to humans, with a cally with unpredictable patterns, at different rates change in its genetic make-up), and this ‘jump’ in different situations. Their genetic and metabolic is facilitated by intensive farming and close and entanglement with cells gives retroviruses a unique crowded living conditions. opportunity to mediate subtle, cumulative evolu- (ii) Known diseases may spread to new geographic tionary changes in host cells. Viruses that are trans- areas and populations (e.g. malaria in Texas, mitted over a long time period (HIV) have a selective USA). advantage even when their effective transmission (iii) Previously unknown infections may appear in rates are relatively low. humans living or working in changing ecologic Assisted reproduction techniques are now being conditions that increase their exposure to insect used to help people who carry infectious diseases
Artificial insemination 7(including those that are potentially fatal and may By the turn of the nineteenth century, the use of arti-have deleterious effects on offspring) to have chil- ﬁcial insemination in rabbits, dogs and horses haddren. This potential breach of evolutionary barri- been reported in several countries. In 1866 an Italianers raises new ethical, policy and even legal issues physician, Paolo Mantegazza, suggested that spermthat must be dealt with cautiously and judiciously could be frozen for posthumous use in humans and(Minkoff & Santoro, 2000). for breeding of domestic animals, and in 1899 the Russian biologist Ivanoff reported artiﬁcial insem- ination (AI) in domestic farm animals, dogs, foxes,History of assisted reproduction rabbits and poultry. He developed semen extenders, began to freeze sperm and to select superior stal-Assisted reproduction may also be said to have its lions for breeding. His work laid the foundation fororigin in the seventeenth century, when Anton van the establishment of artiﬁcial insemination as a vet-Leeuwenhoek ﬁrst observed sperm under the micro- erinary breeding technique.scope and described them as ‘animalcules’. In 1779, Around this same time in Cambridge UK, theLazzaro Spallanzani (1729–1799), an Italian priest reproductive biologist, Walter Heape, studied theand scientist was the ﬁrst to propose that contact relationship between seasonality and reproduc-between an egg and sperm was necessary for an tion. In 1891 he reported the recovery of a pre-embryo to develop and grow. He carried out artiﬁ- implantation embryo after ﬂushing a rabbit oviductcial insemination experiments in dogs, succeeding and transferring this to a foster mother with contin-with live births, and went on to inseminate frogs ued normal development (Heape, 1891). His workand ﬁsh. Spallanzani is also credited with some of encouraged others to experiment with embryo cul-the early experiments in cryobiology, keeping frog, ture; in 1912 Alain Brachet, founder of the Belgianstallion and human sperm viable after cooling in School of Embryology, succeeded in keeping a rabbitsnow and re-warming. The Scottish surgeon, John blastocyst alive in blood plasma for 48 hours. Preg-Hunter (1728–1793), was the ﬁrst to report artiﬁcial nancies were then successfully obtained after ﬂush-insemination in humans, when he collected sperm ing embryos from a number of species, from micefrom a patient who sufferered from hypospadias and and rabbits to sheep and cows. Embryo ﬂushing andinjected it into his wife’s vagina with a warm syringe. transfer to recipients became a routine in domesticThis procedure resulted in the birth of a child in 1785. animal breeding during the 1970s.The next documented case of artiﬁcial inseminationin humans took place in 1884 at Jefferson MedicalCollege in Philadelphia, USA: Artificial insemination A wealthy merchant complained to a noted physician of his By 1949, Chris Polge in Cambridge had developed inability to procreate and the doctor took this as a golden the use of glycerol as a semen cryoprotectant, and opportunity to try out a new procedure. Some time later, his the process of semen cryopreservation was reﬁned patient’s wife was anaesthetised. Before an audience of for use in cattle breeding and veterinary practice. medical students, the doctor inseminated the woman, using The advantages of artiﬁcial insemination were rec- semen obtained from ‘the best-looking member of the class’. ognized: genetic improvement of livestock, decrease Nine months later, a child was born. The mother is reputed in the expense of breeding, the potential to increase to have gone to her grave none the wiser as to the manner of her son’s provenance. The husband was informed and was fertility, and a possible disease control mechanism. delighted. The son discovered his unusual history at the age Almquist and his colleagues proposed that bacte- of 25, when enlightened by a former medical student who rial contaminants in semen could be controlled by had been present at the conception. (Hard, AD, Artiﬁcial adding antibiotics to bovine semen (Almquist et al., Impregnation, Medical World, 27, p. 163, 1909) 1949). The practice of artiﬁcial insemination was
8 Introduction soon established as a reproductive treatment in Alan Parkes and Bunny Austin, continuing to explore humans. Methods for cryopreserving human semen his interest in genetics, mammalian oocytes and the and performing artiﬁcial insemination were reﬁned process of fertilization. During this period he started in the early 1950s (Sherman & Bunge, 1953), and expanding his interests into human oocyte matura- a comprehensive account of Donor Insemination tion and fertilization, and with the help of the gynae- was published in 1954 (Bunge et al., 1954). By the cologist Molly Rose began to observe human oocytes mid-1980s, however, it became apparent that donor retrieved from surgical biopsy specimens. In 1962 he insemination had disadvantages as well as advan- observed spontaneous resumption of meiosis in a tages, including the potential to transmit infectious human oocyte in vitro for the ﬁrst time. After a brief diseases. Before rigorous screening was introduced, period in Glasgow with John Paul, he was appointed HIV, Chlamydia, Hepatitis B and genital herpes as a Ford Foundation Fellow in the Physiological Lab- were spread via donor semen (Nagel et al., 1986; oratory in Cambridge in 1963. By this time Chang Berry et al., 1987; Moore et al., 1989; McLaughlin, (1959) had successfully carried out in vitro fertiliza- 2002). tion with rabbit oocytes and sperm, and Yanagamichi (1964) subsequently reported successful IVF in the golden hamster. Whittingham was working towards In vitro fertilization fertilization of mouse eggs in vitro, and reported suc- cess in 1968. In Cambridge, Bob Edwards began on Advances in reproductive endocrinology, including the slow and arduous road that eventually led to identiﬁcation of steroid hormones and their role in successful human in vitro fertilization. He contin- reproduction, contributed signiﬁcantly to research ued his studies using the limited and scarce material in reproductive biology during the ﬁrst half of the available from human ovarian biopsy and pathology twentieth century. During the 1930s–40s, the pitu- specimens, and published his observations about itary hormones responsible for follicle growth and maturation in vitro of mouse, sheep, cow, pig, rhe- luteinization were identiﬁed, and a combination of sus monkey and human ovarian oocytes (Edwards, FSH and LH treatments were shown to promote 1965). Working with several Ph.D. students, Edwards maturation of ovarian follicles and to trigger ovula- fertilized mouse and cow eggs in vitro, and obtained tion. Urine from postmenopausal women was found mouse offspring. With his students, he studied the to contain high concentrations of gonadotrophins, growth of chimaeric embryos constructed by inject- and these urinary preparations were used to induce ing a single or several inner cell mass cells from ovulation in anovulatory patients during the early donor blastocysts into recipient genetically marked 1950s. blastocysts. The birth of live chimaeras conﬁrmed Parallel relevant studies in gamete physiology the capacity of single stem cells to colonize virtually and mammalian embryology were underway by all organs in the recipient, including germline, but this time, with important observations reported by not trophectoderm. They also removed small pieces Austin, Chang and Yanagimachi. In 1951, Robert of trophectoderm from live rabbit blastocysts and Edwards began working towards his Ph.D. project determined their sex by identifying whether they in Edinburgh University’s Department of Animal expressed the sex chromatin body. Embryos with Genetics headed by Professor Conrad Waddington this body were classiﬁed as females and the others and under the directon of Alan Beatty. Here he began as males. The sex of fetuses and offspring at birth to pursue his interest in reproductive biology, study- had been correctly diagnosed, signifying the onset ing sperm and eggs, and the process of ovulation of preimplantation genetic diagnosis for inherited in the mouse. After 1 year spent in Pasadena at the characteristics. At this time, anomalies observed in California Institute of Technology working on prob- some rabbit offspring caused them some concern, lems in immunology and embryology, in 1958 he but a large study by Chang on in vitro fertiliza- joined the MRC in Mill Hill, where he worked with tion in mice proved that the anomalies were due
In vitro fertilization 9to the segregation of a recessive gene, and not due co-culture allowed the development of stage-speciﬁcto IVF. media optimized for embryo culture to the blastocyst In 1968 Edwards began his historic collabora- stage. Advances in technique and micromanipula-tion with Patrick Steptoe, the gynaecologist who tion technology led to the establishment of assistedpioneered and introduced the technique of pelvic fertilization (intracytoplasmic sperm injection, ICSI)laparoscopy in the UK. Using this new technique in by a Belgian team led by Andr´ Van Steirteghem and ehis clinical practice in Oldham General Hospital near including Gianpiero Palermo (Palermo et al., 1992)Manchester, Steptoe was able to rescue fresh pre- by the mid 1990s. Other microsurgical interventionsovulatory oocytes from the pelvis of patients who were then introduced, such as assisted hatching andsuffered from infertility due to tubal damage. Bob embryo biopsy for genetic diagnosis. Gonadal tis-Edwards and his colleague, Jean Purdy, traveled from sue cryopreservation, in vitro oocyte maturation andCambridge to Oldham in order to culture, observe embryonic stem cell culture are now under develop-and fertilize these oocytes in vitro. The team began to ment as therapeutic instruments and remedies forexperiment with culture conditions to optimize the the future.in vitro fertilization system, and tried ovarian stim- The ﬁrst live calves resulting from bovine IVFulation with drugs in order to increase the number were born in the USA in 1981. This further mile-of oocytes available for fertilization. After observing stone in reproductive biotechnology inspired theapparently normal human embryo development to development of IVF as the next potential commer-the blastocyst stage in 1970, they began to consider cial application of assisted reproduction in domes-re-implanting embryos created in vitro into the uteri tic species, following on from AI and conventionalof patients in order to achieve pregnancies: the ﬁrst transfer of embryos produced in vivo from superovu-human embryo transfers were carried out in 1972. lated donors. The assisted reproductive techniquesDespite the fact that their trials and experiments continued to be reﬁned so that by the 1990s IVF waswere conducted in the face of ﬁerce opposition and integrated into routine domestic species breedingcriticism from their peers at the time, they contin- programmes. Equine IVF has also been introducedued to persevere in their efforts, with repeated failure into the world of horse breeding (although repro-and disappointment for the next 6 years. Finally, their ductive technology procedures cannot be used for10 years of collaboration, persistence and persever- thoroughbreds). China used artiﬁcial inseminationance were rewarded with the successful birth of the to produce the ﬁrst giant panda cub in captivity inﬁrst IVF baby in 1978. 1963, and assisted reproduction is now used in the The modern ﬁeld of assisted reproductive technol- rescue and propagation of endangered species, fromogy (ART) arrived with the birth of Louise Brown on pandas and large cats to dolphins. Artiﬁcial insem-July 25, 1978. After a 2-year lag, when no funds or ination, and, in some cases, in vitro fertilizationfacilities were available to continue their pioneering are used routinely in specialist zoos throughout thework, Steptoe and Edwards opened a private clinic world.near Cambridge: Bourn Hall Clinic, dedicated solely In the ﬁeld of scientiﬁc research, applicationto treating infertility patients using in vitro fertiliza- of assisted reproductive techniques in animal sys-tion and embryo transfer. The ﬁrst babies were con- tems has helped to unravel the fundamental stepsceived within days, and many more within 3 months. involved in fertilization, gene programming andTheir rapid clinical success in achieving pregnancies expression, regulation of the cell cycle and patt-and live births led to the introduction of IVF treat- erns of differentiation. Somatic cell nuclear transferment worldwide throughout the 1980s and 1990s. into enucleated oocytes has created ‘cloned’ animalsThe ﬁrst babies born after transfer of embryos that in several species, the most famous being Dolly thehad been frozen and thawed were born in 1984–85, sheep, who was born in Edinburgh in 1997; Dolly wasand cryopreservation of embryos as well as semen euthanized at an early age in February 2003, afterbecame routine. Experiments with cell cultures and developing arthritis and progressive lung disease.
10 Introduction Advances in molecular biology and biotechnol- cover a wide range of expertise, including micro- ogy continue to be applied in ART. Preimplantation biology. Culture media and systems used to pro- genetic diagnosis (PGD), introduced in 1988, is used cess and culture gametes and embryos are designed to screen embryos for sex-linked diseases or auto- to encourage cell growth, but this system can also somal mutations in order to exclude chromosomally encourage the growth of a wide variety of microbes. abnormal embryos from transfer. Molecular biology Whereas cell division in preimplantation embryos is techniques can identify chromosomes with the use relatively slow, with each cell cycle lasting approxi- of ﬂuorescently labelled probes for hybridization, or mately 24 hours, microbes can multiply very rapidly to amplify DNA from a single blastomere using PCR. under the right conditions. Under constant condi- This technique is also used for gender selection, now tions the generation time for a speciﬁc bacterium used routinely in animal breeding programmes. is reproducible, but varies greatly among species. The generation time for some bacteria is only 15–20 minutes; others have generation times of hours or Assisted reproductive technology (ART) even days. Spore-forming organisms have the ability and microbiology to go into ‘suspended animation’, allowing them to withstand extreme conditions (freezing, high tem- Assisted reproduction is a multidisciplinary ﬁeld that peratures, lack of nutrients). This important prop- relies on close teamwork and collaboration between erty allows the organisms to survive in central heat- different medical and scientiﬁc disciplines that must ing and air conditioning systems or cooling towers Fig. 1.1. Schematic diagram of a typical eukaryotic cell, illustrating characteristic intracellular organelles.
Overview of microbiology 11for indeﬁnite periods: the ‘Ice Man’ fungus survived Overview of microbiologyat least 5300 years, and live bacterial spores werefound in ancient pressed plants at Kew Gardens Naturam primum cognoscere rerumdating back to the seventeenth century. Viruses First . . . to learn the nature of things (Aristotle, 384–322 BC )do not form spores, but some can survive dry on Living things have been traditionally classiﬁedhandkerchiefs, or cleaning or drying cloths if protein into ﬁve biological Kingdoms: Animals (Animalia),is present, e.g. in droplets from a sneeze or cough. Plants (Plantae), Fungi (Fungi), Protozoa (Protista)These properties presents special problems in the and Bacteria (Monera). Animals, plants, fungi andART laboratory since many agents used to inhibit or protozoa are eukaryotic, with nuclei, cytoskele-destroy microorganisms are toxic to sperm, oocytes tons, and internal membranes (see Fig. 1.1). Theirand embryos. An ART laboratory must incorporate chromosomes undergo typical reorganization dur-strict guidelines for maintaining necessary sterile ing cell division. Bacteria are prokaryotes, i.e. theyconditions without compromising the gametes and have no well-deﬁned nucleus or nuclear mem-embryos. brane and divide by amitotic division (binary ﬁs- Patients presenting for infertility treatment often sion) (Fig. 1.2). The world of microbes covers a widehave a background of infectious disease as a factor variety of different organisms within the Kingdomsin their infertility, and it is now acknowledged that of Fungi, Protista and Monera, with a diverse rangesome chronic infectious diseases may be ‘silent’ orinapparent, but transmissible in some patients (e.g.Herpes, Ureaplasma, Chlamydia). Some patientsmay be taking antimicrobial drugs that will have Inner cytoplasmica negative effect on gamete function: for example, membranebacterial protein synthesis inhibitors affect spermmitochondrial function, adversely affecting sperm Cell wallmotility. The trend for worldwide travel also intro-duces new contacts and potentially infectious agents Piliacross previous geographic barriers. Nucleoid There are many factors to be taken into accountwhen assessing the risk from microorganisms, andsome that are unique to ART are complex and mul-tifaceted. Formal assessment for quantifying riskrequires experimental study data, epidemiologicalinformation, population biology and mathematicalmodelling – this is not possible in assisted reproduc-tive practice. Instead, ART laboratory practitionersneed to collate, review and evaluate relevant infor-mation, bearing in mind that ART practises breach Flagellabiological barriers, increasing the risk: Wisdom lies in knowing what one is doing and why one is doing it – to take liberties in ignorance is to court disaster – each fragment of knowledge teaches us how much more we have yet to learn. (John Postgate Microbes and Man, 2000) Fig. 1.2. Schematic diagram of a typical prokaryotic (bacterial) Chance favours the prepared mind (Louis Pasteur, cell, illustrating the nucleoid, inner cytoplasmic membrane, 1822–1895) cell wall, pili and ﬂagella.
12 Introduction Table 1.1. Relative sizes of microbes and cells Microbe Size Cell Size Microbe Cells Prion protein 27–55 kD molecular weight, Red blood cell (yardstick) 7 m 243 amino acids, <15 nm White blood cell ranges 6–25 m Small virus 20 nm Epithelial cells (squamous) 40–60 m (Papillomavirus, Poliovirus) HIV virus 110 nm Human gametes/embryos Large virus (Poxvirus, 250–400 nm Mature spermatozoon 4.0–5.0 m in length; Herpesvirus) head size 2.5–3.5 m in width Bacteria size range 0.25 m to 1 m in width and Mature spermatozoon tail 45 m 1 to 3 m in length length Mycoplasma species 0.3 × 0.8 m Round spermatid 7–8 m Staphylococcus spp. 1–3 m Primary spermatocyte 14–16 m Fungi up to 25 m Human oocyte 100 m–115 m (including ZP) Single-celled yeast 8 m Zygote pronuclei ∼30 m Protozoa 10–60 m Cleaving embryo approx. 175 m, including ZP Amoeboid cyst 10–20 m diameter Trichomonas vaginalis 13 m long Parasite eggs 50–150 m diameter Parasitic adult worms 2 cm–1 m long 1 m = 1 × 10−6 metre = 1 × 10−3 mm; 1 m = 1 × 10−9 metre = 1 × 10−6 mm; ZP: zona pellucida. Fig. 1.3. Size of microorganisms and microscope resolution. Bacteria, protozoa and fungi all can be viewed with light microscopy. The average diameter for protozoa is 12–60 m; the largest parasitic protozoan, Balantidium coli is 60 m × 40 m. Bacteria range in size from 0.25 to 1 m in width and 1 to 3 m in length. Viruses are larger than macromolecules, but smaller than the smallest bacteria. Viruses can be visualized by electron microscopy.
Overview of microbiology 13Fig. 1.4. Overview of microorganisms, in order of size and complexity.
14 Introduction of properties. This microcosm is further classiﬁed an organism to sustain itself. The authors speculate into basic ‘families’ of phylogenetic classiﬁcation that these nanoorganisms may represent an earlier based upon evolutionary sequence and character- intermediate form of life (Huber et al., 2002). Their istic properties. ﬁnding suggests that there may be other similarly (i) Prokaryotes: Bacteria, Cyanobacteria, Ricket- unusual groups of microbes yet to be discovered. tsiae Pathogenic microbiology primarily deals with gen- (ii) Fungi: (eukaryotes) moulds, mushrooms, yeasts era and species within families. Microorganisms that (iii) Viruses are important in clinical ART practice can be found (iv) Eukaryotic protists (protozoa): unicellular or in all the categories, and they cover a wide range of multicellular sizes. Table 1.1 describes the size range of microbes A virus is a submicroscopic infectious particle com- relative to blood cells, gametes and embryos; posed of a protein coat and a nucleic acid core. Viru- Fig. 1.3 illustrates the relative sizes of protozoa, bac- ses, like cells, carry genetic information encoded in teria, viruses and macromolecules. An overview of their nucleic acid, and can undergo mutations and the microorganisms discussed in Part I, in order of reproduce; however, they cannot carry out meta- size and complexity, is presented in Fig. 1.4. bolism, and thus are not considered ‘alive’ by the classical deﬁnition. Viruses are classiﬁed by the type of nucleic acid they contain, and the shape of their REFERENCES protein capsule. Prions are a recent addition to the list of ‘micro- Almquist, J. O., Glantz, P. J. & Shaffer, H. E. (1949). The effect organisms’. They do not ﬁt into any of the ‘classical’ of a combination of penicillin and streptomycin upon the families, and present a further challenge to accepted viability and bacterial content of bovine semen. Journal of deﬁnitions of ‘life’: although they apparently show Dairy Science, 32: 183–90. multiplication, heredity and variation, they do not Berry, W. R., Gottesfeld, R. L., Alter, H. J. & Vierling, J. M. have the ability to react and adjust to environments. (1987). Transmission of hepatitis B virus by artiﬁcial insem- In the late 1970s, a new group of organisms was ination. Journal of the American Medical Association, 257: 1079–81. added to this classiﬁcation: the Archaea, a group of Brock, T. D. (1967). Micro-organisms adapted to high tempera- bacteria that live at high temperatures or produce tures. Nature (London), 214: 882–5. methane. Because these organisms are biochemi- Bunge, R. G., Keeteel W. C. & Sherman J. K. (1954). Clinical use cally and genetically different from usual bacteria, it of frozen semen. Fertility and Sterility, 5: 520–9. was proposed that life be divided into three domains: Chang, M. C. (1959). Fertilization of rabbit ova in vitro. Nature Eukaryota, Eubacteria and Archaea – rather than (London), 184: 406. the classical ﬁve kingdoms. Archaeans have been Edwards, R. G. (1965). Maturation in vitro of mouse, sheep, cow, found in the most extreme environments on the pig, rhesus monkey and human ovarian oocytes. Nature planet, and also in plankton of the open sea and as (London), 208: 349–51. methane-producing organisms inside the digestive (1989). Life Before Birth: Reﬂections on the Embryo Debate. tracts of cows, termites, and marine life. They live London: Hutchinson. Heape, W. (1891). Preliminary note on the transplantation and in the anoxic muds of marshes and at the bottom growth of mammalian ova within a uterine foster-mother. of the ocean, and even thrive in petroleum deposits Proceedings of the Royal Society, 48: 457. deep underground. In May 2002, the discovery of Huber, H., Hohn, M. J., Rachel, R., Fuchs, T., Wimmer, V. C. & another new member of the Archaea was reported, Stetter, K. O. (2002). A new phylum of Archaea represented the Nanoarchaeota. This microbe has one of the by a nanosized hyperthermophilic symbiont. Nature, 417: smallest genomes known, with 500 000 nucleotide 63–7. bases. The sequence of these nucleotide bases may Jackson, D. A., Symons, R. H. & Berg, P (1972). Biochemical . point to the minimum number of genes needed for method for inserting new genetic information into DNA of
Further reading 15 simian virus 40: circular SV 40 DNA molecules containing Advisory Committee on Dangerous Pathogens (2002). Microbi- lamda phage genes and the galactose operon of Escherichia ological risk assessment – an interim report, HMSO Publi- coli. Proceedings of the National Academy of Science, USA, cations. 69: 2904–9. Austin, C. R. (1951). Observations on the penetration of theMcLaughlin, E. A. (2002). Cryopreservation, screening and stor- sperm into the mammalian egg. Australian Journal of Sci- age of sperm – the challenges for the twenty-ﬁrst century. entiﬁc Research, 4: 581–96. Human Fertility, 5 (Suppl.): S61–5. Balen, A. H. & Jacobs, H. S. (1997). Infertility in Practice. Edin-Minkoff, H. & Santoro, N. (2000). Ethical considerations in the burgh: Churchill Livingstone. treatment of infertility in women with human immunode- Bloom, B. R. (2003). Lessons from SARS. Science, 300: 701. ﬁciency virus infection. New England Journal of Medicine, Brackett, R. G., Bousquet, D., Boice, M. L. et al. (1982). Normal 343: 1748–50. development following in vitro fertilization in the cow. Biol-Moore, D. E., Ashley, R. L., Zarutskie, P. W. et al. (1989). Trans- ogy of Reproduction, 27: 147–58. mission of genital herpes by donor insemination. Journal Cole, H. & Cupps, P. (1977). Reproduction in Domestic Animals. of the American Medical Association, 261: 3441–3. New York: Academic Press.Nagel, T. C., Tagatz, G. E. & Campbell, B. F. (1986). Transmission of Dobell, C. (ed.) (1960). Antony van Leeuwenhoek and his ‘Little Chlamydia trachomatis by artiﬁcial insemination. Fertility Animals’. New York: Dover Publications. and Sterility, 46: 959–62. Edwards, R. G. (1972). Control of human development. In Arti-Ochiai, K., Yamanda, K., Kimura, K. & Sawada, O. (1959). Stud- ﬁcial Control of Reproduction; Reproduction in Mammals, ies on the inheritance of drug resistance between Shigella Book 5, ed. Austin C. R. & Short R. V., pp. 87–113. Cambridge, strains and Escherichia coli strains. Nippon Iji Shimpo, UK: Cambridge University Press. 1861: 34–46. Edwards, R. G., Bavister, B. D. & Steptoe, P. C. (1969). Early stagesPalermo, G., Joris, H., Devroey, P & Van Steirteghem, A. C. (1992). . of fertilization in vitro of human oocytes matured in vitro. Pregnancies after intracytoplasmic injection of single sper- Nature (London), 221: 632–5. matozoon into an oocyte. Lancet, 340: 17–18. Epidemiologic Notes and Reports: HIV-1 infection and artiﬁcialPrusiner, S. B. (1982). Novel proteinaceous particles cause insemination with processed semen (1990). Morbidity and scrapie. Science, 216: 136–44. Mortality Weekly Report, 39(15): 249,255–6.Saiki, R. K., Gelfand, D. H., Stoffel, S. et al. (1988). Primer-directed Ford, B. J. (1991). The Leeuwenhoek Legacy. Bristol: Biopress and enzymatic ampliﬁcation of DNA with a thermostable DNA London: Farrand Press. polymerase. Science, 239: 487–91. Gosden, R. G. (1999). Cheating Time: Sex, Science and Aging. NewSanger, F., Air, G. M., Barrell, B. G. et al. (1977). Nucleotide York: W. H. Freeman & Co. sequence of bacteriophage phi X174 DNA. Nature, 165: 687– Haselwandter, K. & Ebner, M. R. (1994). Microorganisms surviv- 95. ing for 5300 years. FEMS Microbiology (Lett). 116: 189–94.Sherman, J. K. & Bunge, R. G. (1953). Observations on preserva- Medvei, V. V. (1993). The History of Clinical Endocrinology. tion of human spermatozoa at low temperatures. Proceed- Carnforth, Lancs. Parthenon. ings of the Society for Experimental Biology and Medicine, Morice, P., Josset, P., Charon, C. & Dubuisson, J. B. (1995). History 82(4), 686–8. of infertility. Human Reproduction Update, 1(5): 497–504.Yanagamachi, R. & Chang, M. C. (1964). IVF of golden hamster Parkes, A. S. (1966). The rise of reproductive endocrinology, ova. Journal of Experimental Zoology, 156: 361–76. 1926–40. Journal of Endocrinology, 34: 19–32.Zinder, N. & Lederberg, J. (1952). Genetic exchange in Polge, C. (1972). Increasing reproductive potential in farm ani- Salmonella. Journal of Bacteriology, 64: 679–99. mals. In Artiﬁcial Control of Reproduction, Reproduction in Mammals, Book 5, ed. Austin C. R. & Short R. V., pp. 1–32. Cambridge, UK: Cambridge University Press. Postgate, J. (2000) Microbes and Man, 4th edition. Cambridge,FURTHER READING UK: Cambridge University Press. Robert G. Edwards at 75 (2002). Reproductive BioMedicinehttp://www.ucmp.berkeley.edu/archaea/archaea.html Online 4, suppl. 1.http://www-micro.msb.le.ac.uk/Tutorials/Time/Machine.html Schopf, J. W. (1993). Microfossils of the early Archean apexhttp://www.tulane.edu/∼dmsander/WWW/224/ chart: new evidence of the antiquity of life. Science, 260: Classiﬁcation224.html 640–6.
16 Introduction Steptoe, P. C. & Edwards, R. G. (1970). Laparoscopic recovery of BBV Blood-borne viruses. pre-ovulatory human oocytes after priming of ovaries with biotype biologic or biochemical type of an organ- gonadotrophins. Lancet, i: 683–9. ism. Organisms of a given biotype have identical (1976). Reimplantation of a human embryo with subsequent tubal pregnancy. Lancet, i: 880–2. biologic or biochemical characteristics. Key mark- (1978). Birth after the re-implantation of a human embryo ers are used to recognise biotypes in tracing spread (letter). Lancet, ii: 366. of organism during epidemics. Yanagamachi, R. & Chang, M. C. (1964). IVF of golden hamster candle jar a jar with a lid providing a gas-tight seal ova. Journal of Experimental Zoology, 156: 361–76. in which a small white candle is placed and lit after Whittingham, D. G. (1968). Fertilization of mouse eggs in vitro. culture plates are added. The candle will only burn Nature, 200: 281–2. until the oxygen is reduced to a point that burning is no longer supported; at that point the atmosphere in the jar will have a lower oxygen content than room Appendix: air and ∼3% carbon dioxide. glossary of terms capnophile an organism that requires increased CO2 (5–10%) and approximately 15% O2 . Neisse- abscess local collection of pus. ria gonorrhoeae and Haemophilus inﬂuenzae are aerobe a bacterium that grows in ambient air, which capnophilic bacteria. These organisms will grow in a contains 21% oxygen (O2 ) and a small amount candle jar with 3% CO2 or a CO2 incubator. (0.03%) of carbon dioxide (CO2 ). carrier a healthy person who is carrying, and usu- aerosol suspension of small solid or liquid particles ally excreting an infectious agent, but who generally in air; ﬁne spray of droplets. has no symptoms of the infection. anaerobe a bacterium that usually cannot grow catalase bacterial enzyme that breaks down hydro- in the presence of oxygen, but which grows in gen peroxide with release of oxygen. atmosphere composed of 5–10% hydrogen (H2 ), CD4 an antigen found on the surface of T-helper 5–10% CO2 , 80–90% nitrogen (N2 ) and 0% oxygen cells and certain other types of cell such as the mono- (O2 ). cyte, that acts as a receptor for attachment of HIV. antibiotic substance produced by a microorganism CFU Colony forming units; numbers of bacterial that suppresses the growth of, or kills, other microor- colonies on agar cultures, each of which started as ganisms. a single bacterium in the original specimen. antigenaemia the presence of an antigen in the chlamydyospore thick-walled fungal spore formed blood. from vegetative cell. antimicrobial chemical drug produced by a chronic infection persistence of a replicating infec- microorganism that is growth suppressive or tious agent in the host for longer than 6 months. microbiocidal in activity. commensalism a symbiotic relationship in which antiseptic compound that inhibits bacterial growth one organism beneﬁts without harming the host without necessarily killing the bacteria. organism. bacteriostatic agent that inhibits the replication of community-acquired pertaining to outside the target bacteria, but does not kill the organism. hospital (community-acquired infection). bacteriocidal agent that kills the target organism. conidia asexual fungal spores. bacteriophage virus that infects a bacterium. conjugation passing genetic information between bacteriuria bacteria in the urine. bacteria via pili.
Appendix: glossary of terms 17contaminant an agent that causes contamination EMB eosin-methylene blue.or pollution. In laboratory cultures, contaminants enterotoxin toxin that affects the intestinalgenerally arise from skin ﬂora or from environmental mucosa.sources. endotoxin substance containing lipopolysaccha-CSF cerebrospinal ﬂuid. ride found in the gram-negative cell bacterial wall;culdocentesis aspiration of cul-de-sac ﬂuid requir- plays an important role in complications of sep-ing puncture of the vaginal wall to enter into the sis (shock, disseminated intravascular coagulation,retroperitoneal space. thrombocytopenia).cystitis inﬂammation of the urinary bladder. epidemiology the study of occurrence and distribu-cytopathic effect (CPE) changes in cell morphol- tion of disease in populations and factors that contrology resulting from viral infection of a cell culture the presence or absence of disease.monolayer. etiology causative agent or cause of disease.dark-ﬁeld microscopy technique used to visual- fastidious an organism with very stringent growthize very small or thin microorganisms such as requirements. Certain anaerobes will not grow in thespirochaetes; light is reﬂected or refracted from the presence of even traces of oxygen.surface of viewed objects. eugonic growing luxuriantly (refers to bacterial cul-decontamination process of rendering an object or tures).area safe by removing microbes or rendering them fermentation anaerobic decomposition of carbo-harmless using biologic or chemical agents. hydrate.deﬁnitive host the host in which sexual reproduc-tion of a parasite takes place. ﬁmbrae ﬁngerlike proteinaceous projections that act as a bacterial adherence mechanism.dematiaceous pigmented (dark coloured) moulds,as in those that produce melanin. When examining ﬂagella structures composed mostly of proteinthese moulds, the reverse side of the culture plate responsible for microbe motility.appears dark, indicating a pigmented mycelium. FTA ﬂuorescent treponemal antibody A.dermatophyte parasitic fungus on skin, hair or fulminant a condition or symptom that is of verynails. sudden onset, severe, and of short duration, oftenDFA direct ﬂuorescent antibody test. leading to death.dimorphic fungi fungi with both mould and yeast hyaline non-pigmented, used to refer to fungalphases. organisms that do not produce melanin. Whendisinfectant agent that destroys or inhibits micro- examining the reverse side of the culture plate, theseorganisms. organisms appear tan–white.dysgonic bacteria that grows poorly in culture. iatrogenic caused by medical or surgical interven- tion, induced by the treatment itself.dysuria painful urination. IFA indirect ﬂuorescent antibody.edema excessive accumulation of tissue ﬂuid. immunocompromised a state of reduced resis-Eh oxidation–reduction potential. tance to infection and other foreign substancesendemic occuring in a particular region or popula- that results from drugs, radiation illness, congenitaltion. defect or certain infections (e.g. HIV).elementary body infectious stage of Chlamydia. immunoglobulin antibody; there are ﬁve classes:ELISA enzyme-linked immunosorbent assay. IgG, IgM, IgA, IgE and IgD.