sperm chromatin assessment

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  • Introduction

    Semen analysis is the basic and most commonlyused test for predicting fertility; however, the stan-dard measurements of sperm concentration, percent-age motility, and morphology may not reveal subtlesperm defects. In this context, sperm chromatinabnormalities have been studied extensively in thepast decade as a cause for male infertility.1 The focuson the genomic integrity of the male gamete hasbeen further intensified by the growing concern oftransmission of genetic diseases through assistedreproductive techniques (ART), specifically intracy-toplasmic sperm injection (ICSI). Accumulating evi-dence indicates that a negative correlation existsbetween disturbances in the organization of thegenomic material in sperm nuclei and the fertilitypotential of spermatozoa, whether in vivo orin vitro.2,3 This emphasizes that stable DNA, which iscapable of decondensation at the appropriate time inthe fertilization process, is one of the criteria neededto consider a spermatozoon fertile.4 Conventionalsemen analysis per se cannot cover the diverse arrayof biological properties that the spermatozoonexpresses as a highly specialized cell.5,6 In addition,the results of semen analyses can be very subjectiveand prone to intra- and interobserver variability.7

    At the present time, it is clear that a sperm chro-matin structure of poor quality may be indicative ofmale subfertility, regardless of the number, motility,and morphology of spermatozoa. Sperm chromatinstructure evaluation is an independent measure ofsperm quality that provides good diagnostic andprognostic capabilities. Therefore, it may be consid-ered a reliable predictor of a couples inability tobecome pregnant,8 and may also have an impact onthe offspring, resulting in infertility.9

    Many techniques have been described for evalua-tion of the chromatin status. In this chapter, wedescribe the normal sperm chromatin architectureand the causative factors leading to its aberrations.We also provide the rationale and the different

    methodologies employed for sperm chromatinassessment.

    Human spermchromatin structure

    The nuclear status of sperm cells is determined bytwo major events that occur during spermiogenesis:acquisition of the final nuclear shape and thereplacement of somatic-type histones by protamines(sperm-specific basic nuclear proteins) leading tohighly packaged chromatin. Sperm DNA is orga-nized in a specific manner to keep the chromatin inthe nucleus compact and stable. It is packed into atight, almost crystalline status that is at least sixtimes more condensed than mitotic chromosomes. Itoccupies nearly the entire nucleus volume, whereassomatic cell DNA only partly fills the nucleus.10

    This DNA organization not only permits the verytightly packaged genetic information to be trans-ferred to the egg, but also ensures that the DNA isdelivered in a physical and chemical form thatallows the developing embryo to access the geneticinformation.11

    Sperm nuclei do not have the volume required forthe type of packaging present in somatic cells,because packing the DNA in even a single, closelypacked nucleosome would require 9.9 m3, which ismore than twice the volume of an average spermnucleus. Thus, a completely different type of DNApackaging must be present in mammalian spermnuclei.12 Organization of chromatin for packaging inthe spermatozoon takes place at four different levels:chromosomal anchoring, which refers to the attach-ment of the DNA to the nuclear annulus; formationof DNA loop domains as the DNA attaches to thenewly added nuclear matrix; replacement of histonesby protamines, which condense the DNA into com-pact doughnuts; and chromosomal positioning.12 Thehistones are first displaced by transition proteins

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  • (TNPs), which are removed from the condensingchromatin at later stages and replaced by protamines.It is of interest to note that the condensation of chro-matin begins in the posterior pole and proceedsapically, which is a unique feature in humans thatis not present in other mammalian species.13 Spermepididymal maturation implies a final stage of chro-matin organization involving protamine cross-linkingby disulfide bond formationa step that is supportedby the fact that protamines contain a significantnumber of cysteine residues that participate insperm chromatin compaction by forming multipleinter- and intraprotamine disulfide cross-links. All ofthese interactions make mammalian DNA the mostcondensed eukaryotic DNA.14

    Origin of spermchromatin abnormalities

    Sperm nuclear chromatin abnormalities/DNA dam-age could occur at the time of, or be the result of,DNA packing at spermiogenesis.15 Environmentalstress, gene mutations, and chromosomal abnormali-ties can disturb the highly refined biochemicalevents that occur during spermatogenesis. This canultimately lead to an abnormal chromatin structurethat is incompatible with fertility. However, the exactmechanism(s) by which chromatin abnormalities/DNA damage arise in human spermatozoa are notprecisely understood. Three main theories have beenproposed: defective sperm chromatin packaging,apoptosis, and oxidative stress (OS).

    Contributing factors

    The most important factor contributing to spermchromatin damage is leukocytospermia. It may resultin reactive oxygen species (ROS)-induced cross-damage of sperm,16 and is associated with proin-flammatory mediators such as cytokines, causingalterations in the regulation of spermiogenesis andsubsequently chromatin aberrations. Similarly, OSmay be the underlying reason why sperm DNA fromsmokers contains more strand breaks than that fromnon-smokers.17 High levels of sperm DNA damagecan be seen following exposure to irradiation andchemotherapeutic agents and may persist for severalmonths.18 Finally, sperm preparation techniquesinvolving repeated high-speed centrifugation andthe isolation of spermatozoa from the protectiveantioxidant environment provided by seminalplasma may contribute to increased sperm DNAdamage via mechanisms that are mediated by theenhanced generation of ROS.19

    Indications of spermchromatin assessment

    Diagnosis of male infertility

    The positive relationship between poor sperm para-meters and DNA damage in spermatozoa points toinherent problems.20 In general, infertile patientshave higher levels of DNA strand breaks than seen infertile males.21 Moreover, spermatozoa from infertilepatients are generally more susceptible to the effectsof DNA-damaging agents such as H2O2 and radi-ographic exposure.22 Thus, the sperm DNA integritymay be considered an objective marker of spermfunction that serves as a significant prognostic factorfor male infertility. Recently, we discovered a signif-icant increase in sperm chromatin structure assay(SCSA)-defined DNA damage in sperm from infertilemen with normal semen parameters, leading us tospeculate that sperm DNA damage analysis mayreveal a hidden abnormality of sperm DNA in infer-tile men classified with idiopathic infertility basedon apparently normal standard semen parameters.23

    Assisted reproductive techniques

    During ICSI, the sperm cell is injected directly intothe cytoplasm of the mature oocyte. Because thesperm membraneoocyte interaction is no longerrelevant, increased emphasis is placed on the qualityof the sperm chromatin and the ability of the oocyteto initiate decondensation and pronuclear forma-tion. In general, the ICSI fertilization rate does notexceed 6580%24 despite the mechanical injection ofone sperm into a mature oocyte. The fertilizationrate is lower than expected possibly because spermthat are selected from semen of patients with malefactor infertility may have defects in their DNA.Therefore, although the most normal-appearing andmotile spermatozoa are selected, there is always asmall percentage of sperm used in in vitro fertiliza-tion (IVF)/ICSI that contain varying degrees of DNAdamage.2 Semen samples characterized by increasedDNA fragmentation levels are often associated withdecreased fertilization rates and/or embryo cleavagefollowing IVF and ICSI, and may be linked to anincrease in early embryo death.25

    In a recent study (Fig 7.1), we tested the correla-tion of sperm DNA damage with different ART out-comes. We reported that the percentage of DNAfragmentation index (DFI) was significantly higherin infertile men who did not achieve a clinical preg-nancy with ART (38 (interquartile range 2843))than in those who did (21 (1325); p = 0.001). Inaddition, we found no clinical pregnancy when the

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  • DFI was higher than 28%.26 Thus, assessment ofsperm chromatin may help to predict the successrates of ART.

    Cancer patients

    Patients with cancer are often referred to sperm banksbefore chemotherapy, radiation therapy, or surgery isinitiated. Although pregnancies and births have beenreported using cryopreserved sperm from patientswith cancer, these semen samples have decreased fer-tilization potential. The extent of DNA damage mayhelp to determine how semen should be cryopre-served before therapy begins. Specimens with high

    sperm concentration and motility and low levels ofDNA damage should be preserved in relatively largealiquots that are suitable for intrauterine insemina-tion (IUI). If a single specimen of good quality isavailable, then it should be preserved in multiplesmall aliquots suitable for IVF or ICSI.27

    Evaluation of spermnuclear DNA damage

    Different methods may be used to evaluate thestatus of the

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