To save content items to your account,
please confirm that you agree to abide by our usage policies.
If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your account.
Find out more about saving content to .
To save content items to your Kindle, first ensure email@example.com
is added to your Approved Personal Document E-mail List under your Personal Document Settings
on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part
of your Kindle email address below.
Find out more about saving to your Kindle.
Note you can select to save to either the @free.kindle.com or @kindle.com variations.
‘@free.kindle.com’ emails are free but can only be saved to your device when it is connected to wi-fi.
‘@kindle.com’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.
The integrity of chromatin in the spermatozoon is essential for reproductive outcome. The aim of this study was to evaluate the most effective and cost-effective method to reduce the percentage of spermatozoa with defects in chromatin decondensation for use in assisted reproductive technologies (ART) procedures. Sperm samples from 15 sub-fertile males were examined at CFA Naples to determine the sperm decondensation index (SDI), using the aniline blue test, before and after preparation, comparing density gradients with two different swim-up approaches. All three techniques led to a reduction in decondensed spermatozoa with no statistical difference (P > 0.05) between the control and the treated sperm. In contrast, we found a highly significant decrease in SDI (P < 0.01) after the two swim-up methods in all the samples, confirming the efficacy of these methods in lowering the percentage of chromatin compaction damage. There was no statistical difference between the two swim-up methods, however swim-up from the pellet led to improved count, motility and the percentage of normal condensed spermatozoa. We suggest that swim-up from the pellet be used in ART on sub-fertile males, both to reduce cell stress by multiple centrifugation and improve the recovery rate of mature spermatozoa.
The aim of our study was to evaluate the correlation between sperm quality and ploidy status of the derived blastocysts. We performed a retrospective analysis on a restricted pool of patients enrolling only those who had no female factors. Male patients with genetic factors affecting spermatogenesis were also excluded. We chose a maternal age ≤38 years to decrease the female factor, therefore the male factor was the main component of sterility. We divided the patients in four groups based on semen quality and comparing fertilization, pregnancy and euploidy rates above all. In total, 201 intracytoplasmic sperm injection (ICSI) cycles were enrolled in the study. Cycles were divided into four groups, according to semen source: normal semen, oligoasthenoteratozoospermia (OAT), cryptospermia or non-obstructive azoospermia (NOA). An extremely statistically lower fertilization rate was found in NOA patients. Unexpectedly, no differences were detected in blastocyst formation, euploidy, aneuploidy and mosaicism rates among the four groups. Interestingly, we also found a higher abortion rate comparing NOA to normal semen with an odds ratio of 4.67. In our study no statistically significant differences among the analyzed groups were found, showing little or no effect at all using spermatozoa from different semen sources or quality. This may be linked to the oocyte competence of fixing sperm DNA damage and it could be hypothesized that only sperm with a good rate of DNA integrity are able to fertilize the oocyte, explaining why poor quality semen is reflected in a low fertilization rate without effect on ploidy.
Intraventricular lesions are challenging pathologies in neurosurgery. Walter Dandy had a major impact in advancing our understanding of the management of these lesions. Furthermore, the introduction of the microscope and microsurgical techniques have improved the surgical outcomes of these lesions. Several approaches have been described to address ventricular lesions, and can be classified anatomically as anterior, lateral, or posterior. The operative corridor for each of these approaches transgresses unaffected neural tissues. Therefore, tailoring the approach to individual patient lesion characteristics and anatomy is crucial to maximize exposure and minimize morbidity. The majority of open and endoscopic approaches to the third ventricle use the interhemispheric anterior transcallosal, frontal transsulcal, or frontal transcortical corridor to access the lateral ventricle. Once inside the lateral ventricle, the operative corridors to the third ventricle include working through the foramen of Monroe (transforaminal approach) for small lesions located in the anterior superior part of the third ventricle, or through the choroidal fissure (transchoroidal or subchoroidal) which provide access to lesions located in, or extending into, the middle or posterior parts of the roof of the third ventricle. In this chapter, we will discuss the transchoroidal, subchoroidal, and combined transchoroidal and subchoroidal approaches to the third ventricle.
Assisted reproductive technology is today considered a safe and reliable medical intervention, with healthy live births a reality for many IVF and ICSI treatment cycles. However, there are increasing numbers of published reports describing epigenetic/imprinting anomalies in children born as a result of these procedures. These anomalies have been attributed to methylation errors in embryo chromatin remodelling during in vitro culture. Here we re-visit three concepts: (1) the so-called ‘in vitro toxicity’ of ‘essential amino acids’ before the maternal to zygotic transition period; (2) the effect of hyperstimulation (controlled ovarian hyperstimulation) on homocysteine in the oocyte environment and the effect on methylation in the absence of essential amino acids; and (3) the fact/postulate that during the early stages of development the embryo undergoes a ‘global’ demethylation. Methylation processes require efficient protection against oxidative stress, which jeopardizes the correct acquisition of methylation marks as well as subsequent methylation maintenance. The universal precursor of methylation [by S-adenosyl methionine (SAM)], methionine, ‘an essential amino acid’, should be present in the culture. Polyamines, regulators of methylation, require SAM and arginine for their syntheses. Cystine, another ‘semi-essential amino acid’, is the precursor of the universal protective antioxidant molecule: glutathione. It protects methylation marks against some undue DNA demethylation processes through ten-eleven translocation (TET), after formation of hydroxymethyl cytosine. Early embryos are unable to convert homocysteine to cysteine as the cystathionine β-synthase pathway is not active. In this way, cysteine is a ‘real essential amino acid’. Most IVF culture medium do not maintain methylation/epigenetic processes, even in mouse assays. Essential amino acids should be present in human IVF medium to maintain adequate epigenetic marking in preimplantation embryos. Furthermore, morphological and morphometric data need to be re-evaluated, taking into account the basic biochemical processes involved in early life.
Most clinical microbiology laboratories have replaced toxin immunoassay (EIA) alone with multistep testing (MST) protocols or nucleic acid amplification testing (NAAT) alone for the detection of C. difficile.
Study the effect of changing testing strategies on C. difficile detection and strain diversity.
A Veterans’ Affairs hospital.
Initially, toxin EIA testing was replaced by an MST approach utilizing a glutamate dehydrogenase (GDH) and toxin EIA followed by tcdB NAAT for discordant results. After 18 months, MST was replaced by a NAAT-only strategy. Available patient stool specimens were cultured for C. difficile. Restriction endonuclease analysis (REA) strain typing and quantitative in vitro toxin testing were performed on recovered isolates.
Before MST (toxin EIA), 79 of 708 specimens (11%) were positive, and after MST (MST-A), 121 of 517 specimens (23%) were positive (P < .0001). Prior to NAAT-only testing (MST-B), 80 of the 490 specimens (16%) were positive by MST, and after NAAT-only testing was implemented, 67 of the 368 specimens (18%) were positive (P = nonsignificant). After replacing toxin EIA testing, REA strain group diversity increased (8, 13, 13, and 10 REA groups in the toxin EIA, MST-A, MST-B, and NAAT-only periods, respectively) and in vitro toxin concentration decreased. The average log10 toxin concentration of the isolates were 2.08, 1.88, 1.20 and 1.55 ng/mL for the same periods, respectively.
MST and NAAT had similar detection rates for C. difficile. Compared to toxin testing alone, they detected increased diversity of C. difficile strains, many of which were low toxin producing.
In the armory of medical technology available for alleviation of disease and quality of life enhancement, there is nothing to match the unique contribution of assisted reproductive technology (ART). There is no other life experience that matches the birth of a baby in significance and importance. The responsibility of nurturing and watching children grow and develop alters the appreciation of life and health, with a resulting long-term impact upon individuals, families and, ultimately, society. Thus, the combination of oocyte and sperm to create an embryo with the potential to develop into a unique individual cannot be regarded lightly, as merely another form of invasive medical technology, but must be treated with the respect and responsibility merited by the most fundamental areas of human life.
Every cell in an individual has a unique chromosome complement, with 20 000–25 000 genes coded into a DNA sequence of 3 billion base pairs, packed into 23 pairs of chromosomes: a total of 46 chromosomes in each diploid human cell. All of these cells have the same genetic information, copied during mitotic divisions by replicating the DNA during each cell cycle. The pattern of gene activity in each cell (gene expression/transcription) dictates its function and fate, enabling different cells to differentiate and carry out distinct functions.
The first live births following frozen-thawed embryo transfer were reported in 1984 and 1985 by groups in Australia, the Netherlands and the United Kingdom. Since that time, the original protocols have been modified and simplified such that cryopreservation with successful survival of sperm, oocytes and embryos is now an essential component of every routine IVF program. Pregnancy and live birth rates after frozen embryo transfer contribute significantly to cumulative conception rates after fresh transfer. In recent years, traditional methods of freezing and thawing have been increasingly replaced by protocols for vitrification/warming. For both slow freezing and vitrification, an understanding of the basic principles of cryobiology involved is essential to ensure that the methodology is correctly and successfully applied, in order to minimize cell damage during the processes of freezing/vitrification and thawing/warming.
After a blastocyst has implanted in the uterus and begins to differentiate into the three primary germ layers, a special population of cells develops as primordial germ cells (PGCs). These are destined to become the gametes of the new individual: future reproduction of the organism is absolutely dependent upon the correct development of these unique populations of cells. They originate immediately behind the primitive streak in the extraembryonic mesoderm of the yolk sac; toward the end of gastrulation they move into the embryo via the allantois, and temporarily settle in the mesoderm and endoderm of the primitive streak. In humans, PGCs can be identified at about 3 weeks of gestation, close to the yolk sac endoderm at the root of the allantois.
At least 50% of couples referred for infertility investigation and treatment are found to have a contributing male factor. Male factor infertility can represent a variety of defects, which result in abnormal sperm number, morphology or function. Detailed analysis of sperm assessment and function are important for accurate diagnosis, and are described in detail in numerous textbooks of practical andrology and semen analysis. A comprehensive review of semen analysis is beyond the scope of this book, and only details relevant to assisted conception treatment will be described here.
Gametogenesis, embryo development, implantation and in-vitro culture involve numerous complex pathways and interactions at the cellular and molecular level; a true understanding of their significance requires fundamental knowledge of the underlying principles. This chapter therefore provides a condensed overview and review of basic terminology and definitions, with particular emphasis on aspects relevant to reproductive biology and in-vitro fertilization.
During the transition from morula to blastocyst the embryo enters the uterus, where it is sustained by oxygen and a rich supply of metabolic substrates in uterine secretions. The subsequent sequence of events that lead to implantation is a crucial milestone in mammalian embryo development. Carefully orchestrated programs are set into action, which establish diverse cell lines, specify cell fate and major remodeling that will generate the embryo and its extraembryonic tissues: during gastrulation, the three primary germ layers that lead to body formation are formed. The critical conditions that are created in this early stage will pave the way to a successful pregnancy.
Every individual treatment cycle involves a number of different stages and manipulations in the laboratory, and each case must be assessed and prepared for in advance; the afternoon prior to the procedure (the day after hCG administration) is a convenient time to make the preparations. The laboratory staff should ensure that all appropriate consent forms have been signed by both partners, including consent for special procedures and storage of cryopreserved embryos. Details of any previous assisted conception treatment should be studied, including response to stimulation, number and quality of oocytes, timing of insemination, fertilization rate, embryo quality and embryo transfer procedure, and judgments regarding whether any parameters at any stage could be altered or improved in the present cycle can be assessed. The risk of introducing any infection into the laboratory via gametes and samples must be absolutely minimized: screening tests such as human immunodeficiency virus (HIV 1 and 2: Anti-HIV 1, 2) and hepatitis B (HbsAg/Anti-HBc) and C (Anti-HCV-Ab) should be confirmed, as well as any other tests indicated by the patients’ history (e.g., HTLV-I antibody, RhD, malaria, Trypanosoma cruzi, Zika virus). If donor gametes are to be used, additional tests for the donor are required: chlamydia, cytomegalovirus and a validated testing algorithm to exclude the presence of active infection with Treponema pallidum for syphilis testing.
Biologists and physiologists began to micromanipulate cells during the last century, using a variety of manipulator systems to dissect or record from cells. The earliest attempt to inject sperm was recorded in 1914, when G.I. Kite injected sperm cells into starfish oocytes, but with inconclusive results (Lillie, 1914). Experiments in which sperm were injected into eggs around the mid-1960s were primarily designed to investigate the early events of fertilization, i.e. the role of membrane fusion, activation of the oocyte and the formation of the pronuclei. Two series of early experiments by independent groups demonstrated major species differences. Hiramoto showed that microinjection of spermatozoa into unfertilized sea urchin oocytes did not induce activation of the oocyte or condensation of the sperm nucleus (Hiramoto, 1962), whereas others demonstrated the opposite in frog oocytes. Ryuzo Yanagimachi and his group later demonstrated that isolated hamster nuclei could develop into pronuclei after microinjection into homologous eggs, and a similar result was obtained after injecting freeze-dried human spermatozoa into a hamster egg (reviewed by Yanagimachi, 2005). These experiments indicated that membrane fusion events can be bypassed during activation of mammalian oocytes, without compromising the initiation of development. The experiments not only provided information on the mechanism of fertilization, but also led to a new technique in clinical embryology.
After completing fertilization with fusion of the pronuclei during syngamy, the zygote now has a diploid complement of chromosomes, undergoes its first mitotic division and then continues to divide by mitosis into a number of smaller cells known as blastomeres. In humans, the first few cleavage divisions take place in the oviduct, before the embryo reaches its site of implantation in the uterus (Figure 5.1).